DEVELOPMENT AND VALIDATION OF
ANALYTICAL METHODS FOR THE ANTIDIABETIC
POLYHERBAL FORMULATION
A Thesis submitted to Gujarat Technological University
For the Award of
Doctor of Philosophy
in
Pharmacy
by
Megha Ashesh Shah
Enrollment No.: 149997390007
Under supervision of
Dr. Harsha U. Patel
GUJARAT TECHNOLOGICALUNIVERSITY
AHMEDABAD
[November, 2019]
i
© [Megha Ashesh Shah]
ii.
DECLARATION
I declare that the thesis entitled “DEVELOPMENT AND VALIDATION OF
ANALYTICAL METHODS FOR THE ANTIDIABETIC POLYHERBAL
FORMULATION.” Submitted by me for the degree of Doctor of Philosophy is the
record of research work carried out by me during the period from May 2014 to
November 2019 under the supervision of Dr. Harsha U. Patel and this has not formed
the basis for the award of any degree, diploma, associateship, fellowship, titles in this
or any other University or other institution of higher learning.
I further declare that the material obtained from other sources has been duly
acknowledged in the thesis. I shall be solely responsible for any plagiarism or other
irregularities, if noticed in the thesis.
Signature of the Research Scholar: Date:
Name of Research Scholar: Mrs. Megha Ashesh Shah
Place:
iii.
CERTIFICATE
I certify that the work incorporated in the thesis “DEVELOPMENT AND VALIDATION
OF ANALYTICAL METHODS FOR THE ANTIDIABETIC POLYHERBAL
FORMULATION.” Submitted by Mrs. Megha Ashesh Shah was carried out by the
candidate under my supervision/guidance. To the best of my knowledge: (i) the candidate has
not submitted the same research work to any other institution for any degree/diploma,
Associateship, Fellowship or other similar titles (ii) the thesis submitted is a record of original
research work done by the Research Scholar during the period of study under my supervision,
and (iii) the thesis represents independent research work on the part of the Research Scholar.
Signature of Supervisor: Date:
Name of Supervisor: Dr. Harsha U. Patel
Place:
iv
Course-Work Completion Certificate
This is to certify that Mrs. Megha Ashesh Shah Enrollment no.
149997390007 is a PhD scholar enrolled for PhD program in the branch
Pharmacy of Gujarat Technological University, Ahmedabad.
(Please tick the relevant option(s))
He/She has been exempted from the course-work (successfully
completed during M.Phil Course)
He/She has been exempted from Research Methodology Course
only(successfully completed during M.Phil Course)
He/She has successfully completed the PhD course work for the partial
requirement for the award of PhD Degree. His/ Her performance in the
course work is as follows-
Grade Obtained in Research
Methodology (PH001) Grade Obtained in Self Study Course
(Core Subject) (PH002)
Supervisor’s Sign
(Dr. Harsha U. Patel)
v.
Originality Report Certificate
Annexure – V
It is certified that PhD Thesis titled “Development and Validation of Analytical methods
for the Antidiabetic Polyherbal Formulation” by Mrs. Megha Ashesh Shah has been
examined by us. We undertake the following:
a. Thesis has significant new work / knowledge as compared already published or are
under consideration to be published elsewhere. No sentence, equation, diagram,
table, paragraph or section has been copied verbatim from previous work unless it
is placed under quotation marks and duly referenced.
b. The work presented is original and own work of the author (i.e. there is no
plagiarism). No ideas, processes, results or words of others have been presented as
Author own work.
c. There is no fabrication of data or results which have been compiled / analysed.
d. There is no falsification by manipulating research materials, equipment or
processes, or changing or omitting data or results such that the research is not
accurately represented in the research record.
e. The thesis has been checked using Urkund (copy of originality report attached) and
found within limits as per GTU Plagiarism Policy and instructions issued from time
to time (i.e. permitted similarity index <10%).
Signature of the Research Scholar: …………………………… Date: ….………
Name of Research Scholar: Mrs. Megha Ashesh Shah
Place: …………………………………
Signature of Supervisor: ……………………………… Date: ………………
Name of Supervisor: Dr. Harsha U. Patel
Place: …………………
Urkund Analysis Result Analysed Document: Thesis Megha Shah.docx (D59422921)Submitted: 11/22/2019 2:33:00 PM Submitted By: [email protected] Significance: 6 %
Sources included in the report:
https://www.semanticscholar.org/paper/Simultaneous-Determination-of-Gallic-Acid%252C-Ellagic-Kim-Seo/a98584c626a0e897c96d9741639600e4d988ff56/figure/2 https://www.researchgate.net/publication/257302709_Simultaneous_estimation_of_gallic_acid_ellagic_acid_and_ascorbic_acid_in_emblica_officinalis_and_in_unani_polyherbal_formulations_by_validated_HPLC_method https://www.researchgate.net/figure/Comparison-of-the-contents-of-gallic-acid-ellagic-acid-and-eugenol-in-Syzygium_fig7_251236112 https://www.ncbi.nlm.nih.gov/pubmed/23878761 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329618/ https://www.researchgate.net/publication/286019668_Characterization_of_phenolic_compounds_in_Pseudarthria_viscida_root_extract_by_HPLC_and_FT-IR_analysis https://link.springer.com/chapter/10.1007/978-1-4615-3476-1_15 https://www.researchgate.net/publication/266344838_Simultaneous_estimation_of_Gallic_acid_Curcumin_and_Quercetin_by_HPTLC_method https://www.researchgate.net/publication/276090533_Development_and_Validation_of_HPTLC_Method_to_Detect_Curcumin_and_Gallic_Acid_in_Polyherbal_Microencapsulated_Formulation https://www.ncbi.nlm.nih.gov/pubmed/27032211 https://www.longdom.org/open-access/simultaneous-quantification-of-pharmacologically-active-markersquercetin-kaempferol-bergenin-and-gallic-acid-from-cuscuta-campestr-2153-2435-1000490.pdf https://www.researchgate.net/publication/282465791_Development_and_validation_of_Stability_Indicating_HPLC_method_for_determination_of_Ellagic_and_Gallic_acid_in_Jambul_seed https://nepjol.info/index.php/IJASBT/article/view/12908 https://www.researchgate.net/publication/333035623_Development_and_validation_of_absorbance_correction_method_and_first_order_derivative_spectrophotometric_method_for_simultaneous_estimation_of_gallic_acid_ellagic_acid_and_curcumin_in_polyherbal_antid
U R K N DU
vii
PhD THESIS Non-Exclusive License to
GUJARAT TECHNOLOGICAL UNIVERSITY
In consideration of being a PhD Research Scholar at GTU and in the interests of the
Facilitation of research at GTU and elsewhere, I, Mrs. Megha Ashesh Shah
having (Enrollment No. 149997390007) hereby grant a non-exclusive, royalty
Free and perpetual license to GTU on the following terms:
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Archives.
Any abstract submitted with the thesis will be considered to form part of the thesis.
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others, including privacy rights, and that I have the right to make the grant
conferred by this non-exclusive license.
If third party copyrighted material was included in my thesis for
which, under the terms of the Copyright Act, written permission from
viii
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from the copyright owners to do the acts mentioned in paragraph (a)
above for the full term of copyright protection.
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the copyright in my thesis, in any way consistent with rights granted by me to
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i) I further promise to inform any person to whom I may hereafter assign or
license my copyright in my thesis of the rights granted by me to my
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j) I am aware of and agree to accept the conditions and regulations of PhD
including all policy matters related to authorship and plagiarism.
Signature of the Research Scholar:
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Seal.
ix
(Briefly specify the modifications suggested by the panel)
(The panel must give justifications for rejecting the research work)
Thesis Approval Form
The viva-voce of the PhD Thesis submitted by Smt. Mrs. Megha Ashesh Shah
(Enrollment No.: 149997390007) entitled “DEVELOPMENT AND VALIDATION OF
ANALYTICAL METHODS FOR THE ANTIDIABETIC POLYHERBAL
FORMULATION.” was conducted on …………………….………… (Day and date) at
Gujarat Technological University.
(Please tick any one of the following option)
The performance of the candidate was satisfactory. We recommend that he/she be
awarded the PhD degree.
Any further modifications in research work recommended by the panel after 3
months from the date of first viva-voice upon request of the Supervisor or request of
Independent Research Scholar after which viva-voice can be re-conducted by the
same panel again.
The performance of the candidate was unsatisfactory. We recommend that he/she
should not be awarded the PhD degree.
---------------------------------------------------
-------------------------------------------------
Name and Signature of Supervisor with Seal
1) (External Examiner 1) Name and
Signature
---------------------------------------------------
------------------------------------------------- 2) (External Examiner 2) Name and Signature
3) (External Examiner 3) Name and Signature
ix
ABSTRACT
Diabetes mellitus is the most common endocrine disorder, affecting 16 million individuals in
the United States and 200 million worldwide. Despite the use of advanced synthetic drugs for
the treatment, use of herbal remedies is gaining higher importance because of synthetic drugs
have drawbacks and limitations. Antidiabetic herbal formulations (AHF) are considered to be
more effective for the management of diabetes. In recent days, different formulations are
available which are used for management of Diabetes. So, present investigation was undertaken
with a view to develop Different Analytical methods for simultaneous estimation of Gallic acid,
Ellagic acid and Curcumin, markers present in different antidiabetic Polyherbal formulations so
as we can measure them in very precise manner. Most sensitive RP-HPLC method by applying
QbD concept was developed and further LC-MS/MS study was performed to differentiate
Curcumin from other Curcuminoids. HPTLC Method was developed by using less amount of
solvents and with good resolution of peaks which is very helpful for early phase of formulation
development. Chemometric Methods (CLS & ILS) were developed which are capable to
determine constituents in presence of matrix very accurately. UV spectrophotometric Methods
(Absorbance correction method and First order derivative) were developed as they are very
much convenient and less time consuming and also possible for small scale industries. All
developed methods were validated according to ICH guideline and statistical comparison was
done by ONE WAY ANOVA method. Developed Analytical methods can be widely used in
recent era as this is a prime requirement for dossier submission and commercial acceptability.
x
ACKNOWLEDGEMENT
Every big endeavor in the life is the outcome of joint efforts. I want to convey my Gratitude
to all those who directly or indirectly contributed to accomplish the work successfully. The
first and foremost I am thankful to almighty for driving me through the consistent eternal
forces throughout the journey. Really a long one!!
It gives me profound pleasure to express deep gratitude to my esteemed guide Dr. Harsha U.
Patel, Principal, Shri Satsangi Saketdham “Ram Ashram”, Group of Institutes, Faculty of
Pharmacy, Vadasma, Mehsana, for her selfless support throughout the work. I have been
blessed and will always be proud to have her as my guide. I had always felt divine vibrations
being in her proximity. The Sacredness, Dedication, Patience, Politeness, Forgiveness,
Simplicity and Working Ethics are some of her virtues I came across.
I am honorably thankful to my DPC members of GTU Dr. B. N. Suhagia, Dean, Faculty of
Pharmacy, Dharmsinh Desai University, Nadiad and Dr. Nehal shah, Principal, Indubhai Patel
College of Pharmacy and Research Centre, Dharmaj for worthy motivation during my PhD
work.
I am very much thankful to Dr. Arindam Paul, Principal, ROFEL, Shri G. M. Bilakhia College
of pharmacy for providing me opportunity to advance in career and allowing me to utilize
the resources at the college. I heartily thankful to Dr. Hasumati A. Raj, Principal,
Laxminarayan Dev College of Pharmacy, Bholav for her consistent support and
encouragement without which this could never have been attained. She has always extended
a helping hand ever since the start of my professional career.
I would appreciate the efforts of My Parents, My beloved Husband cum Motivator Ashesh
Shah, my heart Pranshu, My sweet brother Dhwanil and sister Dipal along with remaining
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Family members for supporting me in this work by setting me socially free. I apologize all
for the time that I couldn’t spend for them.
I am discretely indebted to my colleague Dr. Hitesh Dalvadi for being with me always for his
altruistic support. I also thank to whole ROFEL Parivar for providing continuous support
throughout my Journey of PhD. I am also thankful to Dr. Sonal Desai for helping me
throughout my Research work.
Last but not least, I am heartily thankful to all my Master Students (Heta, Ankita, Priya,
Ridhdhi and Zalak) for providing Last minute support in all cases.
I am grateful to Natural Remedies, Bangalore for providing gift samples of Gallic acid and
Curcumin.
I heartily regret if I missed any names to mention. Thanks to everyone.
Place:
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Table of Contents
1. Introduction 1.1 Introduction to diabetes Mellitus………………………………………………………01
1.1.1 Epidemiology……………………………………………………………………01
1.1.2 Types of diabetes…………………......................................................................02
1.1.3 Long term effect of Diabetes mellitus…………………………………………..04
1.1.4 Different anti diabetic Polyherbal formulations available in market [7]………...05
1.2 A Challenge: Development of Analytical Methods for Polyherbal Formulation……...06
1.2.1 Difficulties in Analysis of Herbal Drugs………………………………………..07
1.2.2 Adulteration may takes place by two ways……………………………………..08
1.3 Selection of Formulation……………………………………………………………….08
1.4 Introduction to HPLC......................................................................................................09
1.4.1 Introduction to Method Development ………………………………………….10
1.5 Introduction to LCMS………………………………………………………………….15
1.6 Introduction to HPTLC………………………………………………………………...15
1.7 Chemometric Methods ………………………………………………………………...17
1.7.1 Classical Least Squares…………………………………………………………18
1.7.2 Inverse Least Squares…………………………………………………………...18 1.8 Introduction to UV Spectrophotometric Methods…………………………………….18
1.8.1 Absorbance Correction Method…………………………………………………20
1.8.2 Derivative Spectroscopy………………………………………………………...21
1.9 Validation of Analytical Method according to ICH Q2(R1)Guideline…………..…...22
1.10 Profile for selected markers…………………………………………………………...24
References…………………………………………………………………………….……26
2. Literature Review
2.1 Gallic acid………………………………………………………………………………29
2.2 Ellagic acid……………………………………………………………………………..43
2.3 Curcumin……………………………………………………………………………….48
References…………………………………………………………………………...……..60
3. Aim and Objective
3.1 Aim of Research………………………………………………………………………..69
3.2 Objective……………………………………………………………………………….70
4. Materials and Methods
5. Preliminary work
5.1 Melting point determination………………………………………………………….....75
5.2 Solubility study……………………………………………………………………….....75
5.3 Infrared Spectroscopic study……………………………………………………………76
5.3.1 Gallic acid………………………………………………………………………..76
5.3.2 Ellagic acid………………………………………………………………………77
5.3.3 Curcumin………………………………………………………………………...78
References…………….…………………………………………………………...………..79
xiii
6. RP-HPLC Method Development
6.1 Experimental work………………………………………………………………………81
6.1.1 Materials and instruments………………………………………………………...81
6.1.2 Solvents and Reagents……………………………………………………………81
6.1.3 Preparation of Stock solution and working standard solution……………………81
6.1.4 Preparation of Calibration curve………………………………………………….82
6.1.5 Determination of wavelength for Measurement………………………………….82
6.1.6 Determination of Formulations…………………………………………………...83
6.1.7 Method Validation………………………………………………………………..84
6.2 Results and Discussion…………………………………………………………………. 88
6.2.1 Selection of wavelength………………………………………………………… .88
6.2.2 Trials for HPLC Method development…………………………………………...89
6.2.3 Selection of critical factors and responses for further optimization……………...92
6.2.4 Optimization of chromatographic condition using DOE approach……………....93
6.2.5 Optimized chromatographic condition of HPLC Method……………………….139
6.2.6 LC-MS/MS Analysis for Curcuminoids……… ………………………………..141
6.2.7 Applicability of the Method……………………………………………………..148
6.2.8 Validation Parameters………………………………………………………...…149
6.3 Summary of the Developed RP-HPLC Method…………………………………...…...157
References………………………………………………………………………………….158
7. HPTLC Method Development
7.1 Experimental work……………………………………………………………………..159
7.1.1 Materials and instruments……………………………………………………….159
7.1.2 Solvents and Reagents…………………………………………………………..159
7.1.3 Preparation of Stock solution and working standard solution…………………..159
7.1.4 Preparation of Calibration curve……………………………………………...…160
7.1.5 Determination of wavelength …………………………………………………..160
7.1.6 Preparation of Mobile phase…………………………………………………….160
7.1.7 Determination of Formulations………………………………………………….160
7.1.8 Method Validation……………………………………………………………….160
7.2 Result and Discussion…………………………………………………………………..164
7.2.1 Trials for HPTLC Method development………………………………………...164
7.2.2 Selection of wavelength…………………………………………………………166
7.2.3 Optimized chromatographic condition for HPTLC Method………………….…167
7.2.4 Applicability of the Method……………………………………………………..167
7.2.5 Validation Parameters………………………………………………………...…169
7.3 Summary of developed HPTLC Method……………………………………………….176
References………………………………………………………………………………….176
8. Chemometric Method Development
8.1 Experimental work………………………………………………………………...…..177
8.1.1 Materials and instruments……………………………………………………….177
8.1.2 Solvents and Reagents…………………………………………………………..177
xiv
8.1.3 Preparation of Stock solution and working standard solution…………………..177
8.1.4 Preparation of Calibration and Validation set…………………………………...177
8.1.5 Determination of wavelength range for measurement…………………………..177
8.1.6 Determination of Formulations………………………………………………….178
8.1.7 Validation Parameters……………………………………………………...……178
8.2 Result and Discussion…………………………………………………………………..179
8.2.1 Determination of wavelength range for measurement…………………………..179
8.2.2 Measurement of Absorbance……………………………………………………179
8.2.3 Equation for CLS Method……………………………………………………….183
8.2.4 Equation for ILS Method………………………………………………………..184
8.2.5 Validation Parameters…………………………………………………………...186
8.2.6 Applicability of the Method……………………………………………………..193
8.3 Summary of Chemometric Methods……………………………………………………194
References…………………………………………………………………………………..195
9. UV Spectrophotometric Methods Development
9.1 Experimental work……………………………………………………………………..197
9.1.1 Materials and instruments……………………………………………………….197
9.1.2 Solvents and Reagents…………………………………………………………..197
9.1.3 Preparation of Stock solution and working standard solution…………………..197
9.1.4 Preparation of Calibration curve………………………………………...………197
9.1.5 Determination of wavelength for Measurement…………………………………198
9.1.6 Determination of Formulations………………………………………………….198
9.1.7 Validation Parameters…………..……………………………………………….198
9.2 Result and Discussion (Absorbance correction Method)………………………………201
9.2.1 Determination of wavelength for measurement…………………………………201
9.2.2 Applicability of Method…………………………………………………………201
9.2.3 Validation Parameters…………………………………………………...………202
9.3 Result and Discussion (First order derivative Spectroscopy)………………………..…207
9.3.1 Determination of wavelength for measurement……………………………..…..207
9.3.2 Applicability of Method…………………………………………………..…….208
9.3.3 Validation Parameters…………………………………………………….....….208
References………………………………………………………………………………….213
10. Statistical Analysis
10.1 ANOVA Test (Analysis of Variance)……………..……………………………….215
10.1.1 Types of tests………………………………………………………………...…..215
10.2 ANOVA, statistical comparison between developed methods……………………..216
References…………………………………………………………………………………..217
11. Summary and Conclusion
11.1 Summary……………………………………………………………………………219
11.2 Conclusion……………………………………………………………………...…...220
List of Publications………………………………………………………………………..223
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LIST OF ABBREVIATIONS
Abbreviation Full Form
Abs. Absorbance
ACN Acetonitrile
Ag Asymmetry of Gallic acid
Ae Asymmetry of Ellagic acid
Ac Asymmetry of Curcumin
ANOVA Analysis of Variance
API Active Pharmaceutical Ingredients
AUC Area Under Curve
CAS No. Chemical Abstract Service Number
CCD Central Composite Design
Conc. Concentration
CLS Classical least squares
cm Centimetre
DAD Diode Array Detector
DOE Design of Experiment
EMA European Medicines Agency
ESI Electrospray Ionization
F1 Proportion of Aqueous Phase at Starting of Separation
F2 Flow Rate
F3 pH of Mobile Phase
F.A. Formic Acid
FDA Food and Drug Administration
Fig. Figure
FRAP Ferric reducing antioxidant power
FTIR Fourier- transform Infrared
GC Gas Chromatography
GCMS Gas chromatography Mass Spectroscopy
HPLC High Performance Liquid Chromatography
HPTLC High Performance Thin Liquid Chromatography
ICH International Council on Harmonization
ILS Inverse least squares
IP Indian Pharmacopoeia
IR Infrared
IUPAC International Union of Pure and Applied Chemistry
LC-MS Liquid Chromatography –Mass Spectrometry
LOD Limit of Detection
LOQ Limit of Quantitation
mL Millilitre
mm Millimetre
mg Milligram
MOA Mechanism of Action
min Minute
MS Mass Spectrometry
MW Molecular Weight
ng Nano gram
NMR Nuclear Magnetic Resonance
nm Nanometer
ODS Octa decyl silane
OPA Ortho Phosphoric Acid
PCR Principle component regression
PLSR Partial least squares regression
PPM Parts Per Million
PRESS Predicted Residual Error Sum of Squares
xvi
QbD Quality by Design
R1 Asymmetry of Gallic acid
R2 Asymmetry of Ellagic acid
R3 Asymmetry of Curcumin
R4 Resolution between Gallic acid and Ellagic acid
R5 Resolution between Ellagic acid and Curcumin
Rf Retardation factor
RP Reverse Phase
Rt Retention time
RtG Retention time of Gallic acid RtE Retention time of Ellagic acid RtC Retention time of Curcumin Rs Resolution
SD Standard Deviation
Ref. No. Reference Number
RMSEP Root mean square error of prediction
RSD Relative Standard Deviation
RSM Response Surface Methodology
Sr. No. Serial Number
TLC Thin Layer Chromatography
UPLC Ultra-Performance Liquid Chromatography
USP United States Pharmacopoeia
UV Ultraviolet
UV- VIS Ultraviolet visible
v/v volume/volume
WHO World Health Organization
w/w Weight/weight
λ Wavelength
λmax Maximum wavelength
% Percentage
°C Degree Celsius
µg Microgram
µL Microlitre
ZCP Zero Crossing Point
xvii
List of Figures
FIGURE 1. 1 Prevalence of diabetes worldwide in 2000 (per 1,000 inhabitants) - world average was
2.8% ...................................................................................................................................................... 1 FIGURE 1. 2 Disability-adjusted life year for diabetes mellitus per 1,00,000 inhabitants in 2004. .......... 2 FIGURE 1. 3 Selected formulations for Research. ..................................................................................... 8 FIGURE 1. 4 HPLC method development step ........................................................................................ 11 FIGURE 1. 5 Analytical Method development in QbD ............................................................................ 14 FIGURE 1. 6 Block diagram of LC-MS. ................................................................................................... 15 FIGURE 1. 7 HPTLC Method development Steps ................................................................................... 17 FIGURE 1. 8 First, Second, third and fourth derivative Spectrum of Gaussian peak ............................ 22
FIGURE 4. 1 Antidiabetic Polyherbal Formulations for Research ......................................................... 71
FIGURE 5. 1 IR graph for Gallic acid (a) Reference standard (b) Sample ............................................. 76 FIGURE 5. 2 IR graph for Ellagic acid (a) Reference standard (b) Sample ........................................... 77 FIGURE 5. 3 IR graph of Curcumin (a) Reference standard (b) Sample ............................................... 78
FIGURE 6.1 Selection of Wavelength for HPLC Method, Overlain spectrum of Gallic acid (4 µg/ml), 88 FIGURE 6.2 Chromatogram using above trials ....................................................................................... 91 FIGURE 6.3 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (-1), F2(1), F3(-1) ......... 96 FIGURE 6.4 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (-1.68), F2(0), F3(0) ...... 97 FIGURE 6.5 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(0), F3(0) ............ 98 FIGURE 6.6 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(1.68), F3(0) ....... 99 FIGURE 6.7 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (1), F2(-1), F3(-1) ....... 100 FIGURE 6.8 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(0), F3(1.68) ..... 101 FIGURE 6.9 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(0), F3(-1.68) .... 102 FIGURE 6.10 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(0), F3(0) ....... 103 FIGURE 6.11 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (1), F2(1), F3(-1) ....... 104 FIGURE 6.12 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (-1), F2(1), F3(1) ....... 105 FIGURE 6.13 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2 (0), F3(0) ....... 106 FIGURE 6.14 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (1), F2(-1), F3(1) ....... 107 FIGURE 6.15 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(0), F3(0) ........ 108 FIGURE 6.16 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (1.68), F2(0), F3(0) ... 109 FIGURE 6. 17 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (-1), F2(-1), F3(-1) ... 110 FIGURE 6.18 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (-1), F2(-1), F3(1)...... 111 FIGURE 6.19 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(-1.68), F3(0) .. 112 FIGURE 6.20 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (1), F2(1), F3(1) ........ 113 FIGURE 6.21 Perturbation graph showing the effect of each factor A, B, and C on (1) Asymmetry of
Gallic Acid (2)Asymmetry of Ellagic Acid (3) Asymmetry of Curcumin (4) Resolution between
Gallic Acid-Ellagic Acid and (5) Resolution between Ellagic Acid- Curcumin. ............................ 121 FIGURE 6.22 (A-O) Three-dimensional plots of the RSM for five responses ....................................... 122 FIGURE 6.23 Predicted vs. Actual Responses for Asymmetry of Gallic Acid. ..................................... 127 FIGURE 6.24 Predicted vs. Actual Responses for Asymmetry of Ellagic Acid. .................................... 128 FIGURE 6.25 Predicted vs. Actual Responses for Asymmetry of Curcumin. ....................................... 128 FIGURE 6.26 Predicted vs. Actual Responses for Resolution between Gallic Acid & Ellagic Acid. .... 129 FIGURE 6.27 Predicted vs. Actual Responses for Resolution between Ellagic Acid & Curcumin ...... 129 FIGURE 6.28 (A-O) Contour Plots for five responses ............................................................................ 131 FIGURE 6.29 Optimized Graphical representation for selection of Factors......................................... 137
xviii
FIGURE 6.30 Bar graph showing individual values of various responses and their Association as a
geometric mean (D Combine) .......................................................................................................... 138 FIGURE 6.31 Final Optimized Chromatogram ..................................................................................... 139 FIGURE 6.32 Synthetic Pathway of Curcumin [10] ................................................................................. 140 FIGURE 6.33 Separation of Bisdesmethoxycurcumin, desmethoxycurcumin and Curcumin by UPLC
[11]. ..................................................................................................................................................... 141 FIGURE 6.34 LC-MS/MS Chromatogram for Mass confirmation (1st Peak) ....................................... 142 FIGURE 6.35 Fragmentation Pattern in LC-MS/MS for a peak on Rt 13.68 min ................................ 143 FIGURE 6.36 LC-MS/MS Chromatogram for Mass confirmation (2nd Peak) ...................................... 144 FIGURE 6. 37 Fragmentation Pattern in LC-MS/MS for a peak on Rt 13.80 min ............................... 145 FIGURE 6.38 LC-MS/MS Chromatogram for Mass confirmation (3rd Peak) ...................................... 146 FIGURE 6.39 Fragmentation Pattern in LC-MS/MS for a peak on Rt 13.97 min ................................ 147 FIGURE 6.40 Chromatograph of formulation (Glysikot) ...................................................................... 148 FIGURE 6.41 Chromatogram of formulation (Diasol)........................................................................... 148 FIGURE 6.42 Chromatogram of Formulation (Diabeta plus) ............................................................... 148 FIGURE 6 43 Chromatogram of placebo ............................................................................................... 150 FIGURE 6.44 Chromatogram for formulation (Glysikot) ..................................................................... 150 FIGURE 6.45 Chromatogram of Gallic acid (4 µg/ml) .......................................................................... 150 FIGURE 6.46 Chromatogram of Ellagic acid (10 µg/ml) ....................................................................... 150 FIGURE 6.47 Chromatogram of Curcumin (2 µg/ml) ........................................................................... 151 FIGURE 6.48 Overlain Chromatogram of Gallic Acid (2-14 μg/ml), Ellagic Acid (5-35 μg/ml) and
Curcumin (1-7 μg/ml) ...................................................................................................................... 151 FIGURE 6.49 Calibration curve of Gallic Acid (2-14 μg/ml) ................................................................. 152 FIGURE 6.50 Calibration curve of Ellagic Acid (5-35 μg/ml)................................................................ 152 FIGURE 6.51 Calibration curve of Curcumin (1-7 μg/ml)..................................................................... 153
FIGURE 7. 1 Selection of Wavelength for HPTLC Method, Overlain spectrum of Gallic acid (4 µg/ml),
Ellagic acid (10 µg/ml) and Curcumin (2 µg/ml). ........................................................................... 166 FIGURE 7. 2 Final Optimized Densitogram ........................................................................................... 167 FIGURE 7. 3 Densitogram of Glysikot granules .................................................................................... 168 FIGURE 7. 4 Densitogram of Diasol Capsule ......................................................................................... 168 FIGURE 7. 5 Densitogram of Diabeta plus Capsule ............................................................................... 168 FIGURE 7. 6 Chromatogram for Specificity. ......................................................................................... 169 FIGURE 7. 7 Overlay Densitogram to confirm markers from mixture ................................................ 169 FIGURE 7. 8 Overlain spectra for Linearity of Gallic Acid (20-400 ng/band), Ellagic Acid (50-1000
ng/band) and Curcumin (10-200 ng/band). ..................................................................................... 170 FIGURE 7. 9 Calibration curve and Peak Purity data of Gallic Acid (20-400 ng/band) ...................... 170 FIGURE 7. 10 Calibration curve and Peak purity data of Ellagic Acid (50-1000 ng/band) ................. 171 FIGURE 7. 11 Calibration curve and Peak purity data of Curcumin (10- 200 ng/band) ..................... 171
FIGURE 8.1 Overlay spectra of Gallic Acid, Ellagic Acid and Curcumin showing spectral region
241nm- 279 nm (20 wavelengths range) .......................................................................................... 179 FIGURE 8. 2 Linearity plots for Gallic Acid, Ellagic acid and Curcumin by CLS & ILS method ..... 189 FIGURE 8. 3 Residual vs. predicted concentration plot for Gallic Acid, Ellagic Acid & Curcumin. . 192 FIGURE 8. 4 Overlain spectra of formulations for Assay calculation (Glysikot, Diasol and Diabeta
Plus) .................................................................................................................................................. 194
FIGURE 9.1 Selection of Wavelength for Absorbance Correction Method, Overlain spectrum of Gallic
acid (4 µg/ml), Ellagic acid (10 µg/ml) and Curcumin (2 µg/ml) .................................................... 201 FIGURE 9.2 Overlain spectra of formulations for Assay calculation (Glysikot, Diasol and Diabeta
Plus) .................................................................................................................................................. 202
xix
FIGURE 9.3 Overlay Spectra of Gallic Acid showing Linearity (2-20 µg/mL) ..................................... 203 FIGURE 9.4 Overlay Spectra of Ellagic Acid showing Linearity (5-50 µg/mL) ................................... 203 FIGURE 9.5 Overlay Spectra of Curcumin showing Linearity (1-10 µg/mL) ....................................... 203 FIGURE 9. 6 Calibration Curve of Curcumin FIGURE 9.7 Calibration Curve of Ellagic Acid
at 364.5 nm (5 – 50 µg/mL) at 421 nm (1-10 µg/mL) .............................................................. 204 FIGURE 9.8 Calibration Curve of Gallic Acid at (266 – 246) nm (2- 20 µg/mL) .................................. 204 FIGURE 9.9 First order derivative Overlain spectrum of Gallic Acid (4 µg/ml ), Ellagic Acid (10 µg/ml
) and Curcumin (2 µg/ml) For Selection of ZCP ............................................................................. 207 FIGURE 9.10 First order overlain spectra of formulations for Assay calculation (Glysikot, Diasol and
Diabeta Plus) .................................................................................................................................... 208 FIGURE 9.11 First Order overlay Spectra of Gallic Acid showing Linearity (2-20 µg/mL) (ZCP of
Gallic Acid is at 343 nm and 452 nm) .............................................................................................. 209 FIGURE 9.12 First Order overlay Spectra of Ellagic Acid showing Linearity (5- 50 µg/Ml (ZCP of
Ellagic Acid is at 255nm and 452 nm) ............................................................................................. 209 FIGURE 9.13 First Order overlay Spectra of Curcumin showing Linearity (1 -10 µg/mL) (ZCP of
Curcumin is at 255 nm and 272.5 nm) ............................................................................................ 210 Figure 9.14 Calibration Curve of Gallic Acid Figure 9.15 Calibration Curve of Ellagic Acid ..... 211 Figure 9.16 Calibration curve of Curcumin at 452 nm (1-10 µg/mL) .................................................... 211
xx
List of Tables
TABLE 1. 1 Types of oral anti diabetic agents currently available in India [6] ......................................... 5 TABLE 1. 2 Different anti diabetic Polyherbal formulations .................................................................... 5 TABLE 1. 3 List of regulatory guidance or other QbD related activities ................................................ 12 TABLE 1. 4 Region and wavelength for electromagnetic spectrum ........................................................ 19 TABLE 1. 5 Chemical and Physical Properties of Gallic acid ................................................................. 24 TABLE 1. 6 Chemical and Physical Properties of Ellagic acid ................................................................ 24 TABLE 1. 7 Chemical and Physical Properties of Curcumin .................................................................. 25
TABLE 2.1 TLC Methods for Gallic acid. ................................................................................................ 29 TABLE 2.2 HPLC Methods for Gallic acid. ............................................................................................. 31 TABLE 2. 3 HPTLC Methods for Gallic acid. .......................................................................................... 34 TABLE 2.4 GC-MS Methods for Gallic acid ............................................................................................ 37 TABLE 2. 5 LC-MS Methods for Gallic acid............................................................................................ 39 TABLE 2. 6 IR Methods for Gallic acid. ................................................................................................... 40 TABLE 2.7 UV Methods for Gallic acid. .................................................................................................. 41 TABLE 2.8 NMR Methods for Gallic acid. ............................................................................................... 42 TABLE 2.9 HPLC Methods for Ellagic acid ............................................................................................. 43 TABLE 2.10 HPTLC Method for Ellagic acid .......................................................................................... 46 TABLE 2.11 HPLC Method for Curcumin............................................................................................... 48 TABLE 2.12 HPTLC Method for Curcumin ............................................................................................ 54 TABLE 2.13 Ultraviolet Method for Curcumin ....................................................................................... 59
TABLE 4. 1 Markers used in Research work .......................................................................................... 71 TABLE 4. 2 Marketed Formulations ........................................................................................................ 71 TABLE 4.3 Instruments used in Research work ...................................................................................... 72 TABLE 4.4 Solvents and Reagents used in Research work ...................................................................... 72 TABLE 4.5 Optimized condition for HPLC Method Development ......................................................... 72 TABLE 4.6 Optimized condition for LC-MS/MS Method Development................................................. 73 TABLE 4.7 Optimized condition for HPTLC Method Development ....................................................... 73
TABLE 5.1 Determination of melting point .............................................................................................. 75 TABLE 5. 2 Solubility testing .................................................................................................................... 75 TABLE 5.3 IR Interpretation for Gallic Acid........................................................................................... 76 TABLE 5.4 IR Interpretation for Ellagic Acid ......................................................................................... 77 TABLE 5.5 IR Interpretation for Gallic Acid........................................................................................... 78
TABLE 6.1 Steps for Accuracy Measurement for Gallic Acid................................................................. 86 TABLE 6.2 Steps for Accuracy Measurement for Ellagic Acid ............................................................... 86 TABLE 6.3 Steps for Accuracy Measurement for Curcumin .................................................................. 86 TABLE 6.4 Trials for RP-HPLC Method Development........................................................................... 89 TABLE 6.5 Combined Data for Identification of Critical Factor and Response for further
Optimization of Chromatogram ........................................................................................................ 92 TABLE 6.6 Final Selected Factors & Responses ...................................................................................... 92 TABLE 6.7 Finalization of Independent variables with different levels and Dependent variables ........ 93 TABLE 6.8 Central composite rotatable design arrangement and responses (Coded Value) ................ 94 TABLE 6.9 Central composite rotatable design arrangement and responses (Actual value) ................. 95 TABLE 6.10 Summary of results of regression analysis for models and response 1. ............................ 114
xxi
TABLE 6.11 Summary of results of regression analysis for models and response 2. ............................ 114 TABLE 6.12 Summary of results of regression analysis for models and response 3. ............................ 114 TABLE 6.13 : Summary of results of regression analysis for models and response 4. ......................... 115 TABLE 6.14 Summary of results of regression analysis for models and response 5. ............................ 115 TABLE 6.15 ANOVA table for response surface quadratic model for Response 1. ............................. 115 TABLE 6.16 ANOVA table for response surface quadratic model for Response 2. ............................. 116 TABLE 6.17 ANOVA table for response surface quadratic model for Response 3. ............................. 117 TABLE 6.18 ANOVA table for response surface quadratic model for Response 4. ............................. 118 TABLE 6.19 ANOVA table for response surface quadratic model for Response 5. ............................. 119 TABLE 6.20 Criteria for optimization of individual responses and factors with Targeted values....... 136 TABLE 6.21 Suggested best solutions having desirability scores nearer to 1.00 for the optimization . 137 TABLE 6.22 Comparison of experimental and predictive value of different experimental runs under
optimum conditions.......................................................................................................................... 138 TABLE 6.23 Assay for formulations by RP-HPLC Method. ................................................................. 149 TABLE 6.24 Observed values for system suitability test *(n=6) ............................................................ 149 TABLE 6.25 Linearity of Gallic Acid (2-14 μg/ml) by RP-HPLC .......................................................... 152 TABLE 6.26 Linearity of Ellagic Acid (5-35 μg/ml) by RP-HPLC ........................................................ 152 TABLE 6.27 Linearity of Curcumin (1-7 μg/ml) by RP-HPLC ............................................................. 153 TABLE 6.28 Repeatability of Gallic Acid, Ellagic Acid and Curcumin by RP-HPLC ......................... 153 TABLE 6.29 Intraday precision of Gallic Acid, Ellagic Acid & Curcumin by RP-HPLC .................... 154 TABLE 6.30 Interday precision of Gallic Acid, Ellagic Acid & Curcumin by RP-HPLC .................... 154 TABLE 6.31 Accuracy data for Gallic Acid, Ellagic Acid and Curcumin by RP-HPLC. ..................... 155 TABLE 6.32 Robustness data for change in flow rate by RP-HPLC. .................................................... 156 TABLE 6.33 Robustness data for change in pH by RP-HPLC. ............................................................. 156 TABLE 6.34 Robustness data for change in Wavelength by RP-HPLC. ............................................... 157 TABLE 6.35 Summary of Validation Parameters for RP-HPLC Method. ........................................... 157
TABLE 7. 1 Steps for Accuracy study for Gallic Acid by HPTLC Method. ......................................... 162 TABLE 7. 2 Steps for Accuracy study for Ellagic Acid by HPTLC Method. ........................................ 162 TABLE 7. 3 Steps for Accuracy study for Curcumin by HPTLC Method. ........................................... 162 TABLE 7. 4 HPTLC TRIALS ................................................................................................................. 164 TABLE 7. 5 Optimized condition for Densitogram. ............................................................................... 167 TABLE 7. 6 Assay of Formulations by HPTLC method. ....................................................................... 169 TABLE 7. 7 Linearity of Gallic Acid (20-400 ng/band) by HPTLC Method. ........................................ 170 TABLE 7. 8 Linearity of Ellagic Acid (50-1000 ng/band) by HPTLC Method. .................................... 171 TABLE 7. 9 Linearity of Curcumin (10 - 200 ng/ band) by HPTLC Method ....................................... 171 TABLE 7. 10 Repeatability of Gallic Acid, Ellagic Acid and Curcumin by HPTLC Method .............. 172 TABLE 7. 11 Intraday precision of Gallic Acid, Ellagic Acid & Curcumin by HPTLC Method ......... 172 TABLE 7. 12 Interday precision of Gallic Acid, Ellagic Acid & Curcumin by HPTLC Method ......... 173 TABLE 7. 13 Accuracy data for Gallic Acid, Ellagic Acid and Curcumin by HPTLC Method ........... 174 TABLE 7. 14 Robustness data for change in Wavelength by HPTLC Method ..................................... 175 TABLE 7. 15 Robustness data for Change in Preconditioning Time by HPTLC Method.................... 175 TABLE 7. 16 Summary of Validation parameters for HPTLC Method. .............................................. 176
TABLE 8. 1 Composition of Calibration set for three constituents used in CLS Techniques ............. 180 TABLE 8. 2 Absorbance data for the Calibration set at wavelength range (241-279 nm). ................... 181 TABLE 8. 3 Composition of Validation set for all three constituents used in CLS Techniques. .......... 182 TABLE 8. 4 Absorbance data for the above Validation set at wavelength range (241-279 nm). .......... 182 TABLE 8. 5 Recovery results obtained for the determination of Gallic Acid, Ellagic Acid & ............. 186 TABLE 8. 6 Recovery results obtained for the determination of Gallic Acid, Ellagic Acid & Curcumin
by ILS Method. ................................................................................................................................ 186
xxii
TABLE 8. 7 Data for precision studies for Gallic Acid, Ellagic Acid and Curcumin by one way
ANOVA ............................................................................................................................................ 187 TABLE 8. 8 LOD and LOQ for Gallic Acid, Ellagic Acid & Curcumin by CLS & ILS method ........ 187 TABLE 8. 9 Actual, Predicted and Residual values by CLS method..................................................... 188 TABLE 8. 10 Actual, Predicted and Residual values by ILS method. .................................................. 188 TABLE 8.11 RMSEP values for Gallic Acid, Ellagic Acid and Curcumin for CLS & ILS method ..... 193 TABLE 8.12 Assay Result of Formulations ............................................................................................ 194 TABLE 8.13 Summary of Validation Parameters for CLS and ILS Methods ...................................... 194
TABLE 9.1 Steps for Accuracy study for Gallic Acid by UV Spectrophotometric Methods. ............... 199 TABLE 9.2 Steps for Accuracy study for Ellagic Acid by UV Spectrophotometric Methods. ............. 199 TABLE 9.3 Steps for Accuracy study for Curcumin by UV Spectrophotometric Methods. ................ 200 TABLE 9.4 Assay result of Formulations by Absorbance Correction Method ..................................... 202 TABLE 9.5 Linearity data for Gallic Acid, Ellagic Acid & Curcumin by Absorbance correction
Method.............................................................................................................................................. 204 TABLE 9.6 Repeatability data for Gallic acid, Ellagic acid and Curcumin by Absorbance correction
Method.............................................................................................................................................. 205 TABLE 9.7 Intraday Precision data for Gallic acid, Ellagic acid and Curcumin by Absorbance
correction Method. ........................................................................................................................... 205 TABLE 9.8 Interday Precision data for Gallic acid, Ellagic acid and Curcumin by Absorbance
correction Method ............................................................................................................................ 205 TABLE 9.9 Accuracy data of Gallic acid, Ellagic acid and Curcumin by Absorbance correction
Method ............................................................................................................................................. 206 TABLE 9. 10 Summary of Validation Parameters by Absorbance correction Method. ....................... 206 TABLE 9.11 Assay result of Formulations by First order derivative Method ...................................... 208 Table 9.12 Linearity data for Gallic Acid, Ellagic Acid & Curcumin by first order derivative
spectroscopy Method. ...................................................................................................................... 210 TABLE 9.13 Repeatability data for Gallic Acid, Ellagic Acid & Curcumin by first order derivative
spectroscopy Method. ...................................................................................................................... 211 TABLE 9.14 Intraday Precision data for Gallic Acid, Ellagic Acid & Curcumin by first order
derivative spectroscopy Method ...................................................................................................... 212 TABLE 9.15 Interday Precision data for Gallic Acid, Ellagic Acid & Curcumin by first order
derivative spectroscopy Method ...................................................................................................... 212 TABLE 9.16 Accuracy data of Gallic acid, Ellagic acid and Curcumin by First order derivative
Method.............................................................................................................................................. 213 TABLE 9.17 Summary of Validation Parameters by First order derivative Spectroscopy. ................. 213
TABLE 10.1 ANOVA Test for Glysikot .................................................................................................. 216 TABLE 10.2 ANOVA Test for Diasol ..................................................................................................... 217 TABLE 10.3 ANOVA Test for Diabeta Plus ........................................................................................... 217
Chapter 1. Introduction to diabetes mellitus
1
CHAPTER 1.
Introduction
1.1 Introduction to Diabetes Mellitus
Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia with
disturbances in carbohydrate, fat and protein metabolism resulting from defects in insulin
secretion, insulin action, or both [1]. Diabetes mellitus taking its place as one of the main
threats to human health in the 21st century. The total number of people with diabetes is
projected to rise from 171 million in 2000 to 366 million in 2030[2]. India accounts for the
largest number of people - 61.3 million - suffering from diabetes in the world, followed by
China and the United States. Gujarat is having the second highest number of diabetics in the
country after Tamil Nadu. Guajarati’s are genetically prone to this disease. Furthermore, our
less physical work environment and bad eating habits are responsible for the current high
prevalence of type 2 diabetes in the state.
1.1.1 Epidemiology
no data
≤ 7.5
7.5–15
15–22.5
22.5–30
30–37.5
37.5–45
45–52.5
52.5–60
60–67.5
67.5–75
75–82.5
≥ 82.5
FIGURE 1. 1 Prevalence of diabetes worldwide in 2000 (per 1,000 inhabitants) - world
average was 2.8%
Chapter 1. Introduction
2
.
No data
<100
100–200
200–300
300–400
400–500
500–600
600–700
700–800
800–900
900–1,000
1,000–1,500
>1,500
FIGURE 1. 2 Disability-adjusted life year for diabetes mellitus per 1,00,000 inhabitants
in 2004.
Globally, as of 2010, an estimated 285 million people had diabetes, with type 2 making up
about 90% of the cases. Its incidence is increasing rapidly, and by 2030, this number is
estimated to almost double [3] Diabetes mellitus occurs throughout the world, but is more
common (especially type 2) in the more developed countries. The greatest increase in
prevalence is, however, expected to occur in Asia and Africa, where most patients will
probably be found by 2030.The increase in incidence in developing countries follows the
trend of urbanization and lifestyle changes, perhaps most importantly a "Western-style"
diet[4]. This has suggested an environmental (i.e., dietary) effect, but there is little
understanding of the mechanism(s) at present, though there is much speculation, some of it
most compellingly presented.
1.1.2 Types of Diabetes [5]
TYPE 1 DIABETES:
In Type 1 diabetes, the pancreas (a large gland behind the stomach) fails to produce insulin.
Without insulin, the body’s cells cannot use glucose (sugar), which the body needs for
energy. It begins to burn its own fats as a substitute. Unless treated with daily injections of
insulin, a person with type 1 diabetes accumulates in the blood dangerous chemical
substances from the burning of fat. This can cause a condition known as ‘keto-acidosis’. To
stay alive, people with type 1 diabetes depend on up to four insulin injections every day of
Chapter 1. Introduction to Diabetes Mellitus
3
their lives. They must test their blood glucose levels several times daily. This is vital to
monitor the complex interaction of food and exercise with their insulin injections.
SYMPTOMS:
The symptoms may occur suddenly. If they occur, see a doctor.
Feeling constantly thirsty
Passing urine frequently, including bedwetting
Excessive hunger Blurred vision
Unexplained weakness and fatigue
Weight loss
Vaginal discharge or itch in young girls
Nausea and vomiting
Through a simple test, a doctor can find out if diabetes is present. Cause The exact cause is
not known. Some people are predisposed to developing type 1 diabetes. In these people, the
diabetes is possibly triggered by a virus. This destroys the part of the pancreas which
produces insulin. Treatment It aims to do what a normal body does naturally - maintain a
proper balance of insulin and glucose. Diabetes "control" means keeping the level of glucose
in the blood as close to normal as possible. The three elements of "control" for type 1
diabetes:
Food
Exercise
Insulin
TYPE 2 DIABETES:
In Type 2 diabetes, the body cells are unable to use insulin properly (insulin resistance). This
causes glucose (sugar) to accumulate in the blood stream. Symptoms The symptoms come
on gradually but many people with type 2 diabetes have no symptoms and are diagnosed
after a blood glucose test. It occurs more frequently in people who have a family history of
diabetes, are over 50 years, are overweight and rarely exercise. Symptoms include:
Feeling tired
Passing urine frequently Feeling constantly thirsty
Blurred vision Itching of the skin or genital area
Slow healing infections
Chapter 1. Introduction
4
Treatment In many people with type 2 diabetes, healthy eating and regular exercise can
control blood glucose levels. The eating and exercise plan a doctor or dietitian may suggest
depends on the person’s age, lifestyle and overall health. In some cases, tablets or insulin
injections may also be necessary.
GESTATIONAL DIABETES:
What is Gestational diabetes? Gestational Diabetes is a form of diabetes that occurs during
pregnancy and usually goes away after the baby is born. This common condition refers to a
high blood glucose level recognized for the first time during pregnancy. It develops towards
the middle \of the pregnancy as a result of the changes in the mother’s hormones. If this
condition is left untreated, it can cause complications for the mother and the baby. Who is
at risk of developing gestational diabetes? Women:
over 30 years of age
with a family history of type 2 diabetes
who are overweight
from certain ethnic groups e.g. India, Asia, Pacific Islands, Middle East
1.1.3 Long–Term Effects Of Diabetes Mellitus
Nephropathy
Neuropathy
Cardiovascular
Peripheral vascular
Cerebrovascular and
Retinopathy
Currently available therapies for diabetes include insulin and various oral antidiabetic agents,
which are used as monotherapy or in combination to achieve better glycemic Regulation.
Many of these oral antidiabetic agents have a number of serious adverse effects; thus,
managing diabetes without any side effects is still a challenge.
Chapter 1. Introduction to Diabetes Mellitus
5
TABLE 1. 1 Types of oral anti diabetic agents currently available in India [6]
Daily
dosage(mg)
Frequency
per day
Duration of
action (hrs)
Mode of Excretion
1. Sulphonylureas (SU)
a. First generation
Chlorpropamide 100-500 1 24-60 Urine
Tolbutamide 500-2500 2-3 6-12 Urine
b. Second generation
Glibenclamide 2.5-20 1-2 16-24 Urine(50),Bile(50)
Glipizide 2.5-20 1-3 8-12 Urine(80), Bile(20)
Gliclazide 80-320 1-2 8-12 Urine(80), Bile(20)
Glimepiride 1-8 1 16-24 Urine(60), Bile(40)
Gliclazide XL 5-20 1 24 Urine(80), Bile(20)
Gliclazide MR 30-120 1 24 Urine(80), Bile(20)
II. Non Sulphonylurea Agents
a. Meglitinide analogs
Repaglinide 1.0-6 2-3 2-4 Bile
Nateglinide 120-360 2-3 2-4 Bile
b. Biguanides
Metformin 250-2500 2-3 8-12 Urine(90),Faeces (10)
Metformin SR* 1-2 1 24 Urine(90), Faeces (10)
Phenformin 25-100 1-3 4-6 Urine
Phenformin TD 100-200 1-2 8-14 Urine
c. Alpha Glucosidase
inhibitor
Acarbose 25-150 1-3 4 Faeces
d. Thiazolidinediones
Rosiglitazone 2-8 1-2 12-24 Urine
Pioglitazone 15-45 1 24 Urine
1.1.4 Different anti diabetic Polyherbal formulations available in market [7]
TABLE 1. 2 Different anti diabetic Polyherbal formulations
Sr.
No.
Formulation Plants
1 Dihar Syzygiumcumini, Momordicacharantia, Emblica officinalis,
Gymnemasylvestre, Enicostemm, Azadirachtaindiaca, Tinosporacordifolia and
Curcuma longa
2 Diabet Curcuma longa, Cosciniumfenestratum, Strychnospotatorum,
Phyllanthusreticulatus. Tamarindusindica, Tribulusterrestris
3 Diasol Eugenia jambolana, Foenumgraceum, Terminalia chebula, Quercus,
infectoria, Cuminumcyminum, Taraxacumofficinale, Emblica officinalis,
Gymneasylvestre, Phyllanthusnerui and Enicostemmalittorale
4 Dianex Gymnemasylvestre, Eugenia jambolana,
MomordicacharantiaAzadirachtaindica, Cassia auriculata, Aegle marmelose,
Withaniasomnifera and Curcuma longa
5 Diashis Syzygiumcumuni, Gymnemasylvestrae, Holarrhenaantidysenterica,
Tinosporacordifolia, Pongamiapinnata, Asphaltum, Psoraleacorylifolia and
Momordicacharantica
6 Diabrid Gymnemasylvestre, Eugenia jambolana, Momordicacharantia,
Trigonellafoenumgraecum
Chapter 1. Introduction
6
7 Diakyur Cassia javanica,Cassiaauriculata, Salacia reticulate, Gymnemasylvestre,
Mucunapruriens, Syzygiumjambolaum,Terminaliaarjuna
8 Diasulin Cassia auriculata, Cocciniaindica, Curcuma longa, Emblica officinalis,
Gymnemasylvestre, Momordicacharantia, Scopariadulcis, Syzygiumcumini,
Tinosporacordifolia, Trigonellafoenumgraecum
9 Diabecon Gymnemasylvestre, Pterocarpus marsupium, Glycyrrhizaglabra,
Caseariaesculenta, Syzygiumcumini, Asparagus racemosus, Boerhaviadiffusa,
Sphaeranthusindicus, Tinosporacordifolia, Swertiachirata, Tribulusterrestris,
Phyllanthusamarus, Gmelinaarborea, Gossypiumherbaceum, Berberisaristata,
Aloe vera, Commiphorawightii, Momordicacharantia, Piper nigrum, Ocimum
sanctum, Abutilon indicum, Curcuma longa, Rumexmaritimus
10 Dia-Care Selaginellabryopteris; chebulicmyrobalan, Syzygiumcumini, Cucurbita pep,
Azadirachtaindica
11 Diabetes-
Daily Care
Cinnamomumzeylanicum, Vanadium, Trigonellafoenumgraecum,
Gymnemasylvestre, Momordicacharantia, Glycyrrhizaglabra
12 Diabecure Juglansregia, Berberis vulgaris, Erythereacentaurium, Achilleamillefolium,
Taraxacumofficinale
13 Diabeta Gymnemasylvestre, Vincarosea, Curcuma longa, Azadirachtaindica,
Pterocarpus marsupium, Momordicacharantia, Syzygiumcumini, Acacia
arabica,Tinosporacordifolia, Zingiberofficinale
14 Diabet Guard Gymnemasylvestre, Eugenia jambolana, TinosporaCordifolia, Curcuma longa ,
FicusRacemosa, Momordicacharantia , Acacia catechu, Indian Gooseberry,
Pterocarpusmarsupium,Cinnamomumtamala, PicrorrhizaKurroa,
Azadirachtaindica, Trigonellafoenumgraecum
15 Glyoherb Bellis perennis, Picrorhizakurroa, SwertiaChirata, Momordicacharantia
,Holarrhenapubescens , phyllanthusemblica,Tribulusterrestris, Jambubij Ext.,
Methi Ext., Neem Patti Ext., Chandraprabha, Arogyavardhini, Haridra Ext.,
Devdar Ext., Nagarmoth Ext., galo
16 Glysikot ChebulicMyrobalan, Tinosporacordifolia, Indian Gooseberry, Salacia
reticulate, Curcuma longa
17 Karmin Plus Momordicacharantia, Azadirachta
indica, Picrorrhizakurroa, Ocimum sanctum and Zinziberofficinale
18 Okudiabet stachytarphetaangustifolia, Alstoniacongensis bark and
Xylopiaacthiopicafruits extract
1.2 A Challenge: Development of Analytical Methods for Polyherbal Formulation.
Plants synthesize substances that are useful for the maintenance of health in humans and
other animals. Due to low toxicity and known pharmacological activity, herbal drugs have
been popularly and extensively used for many centuries.
Chapter 1. A Challenge
7
Plants synthesize a variety of phytochemicals most of them are derivatives of a few
biochemical motifs. All plants produce Chemical compounds as part of their normal
metabolic activities. These include primary and secondary metabolites. [8] “Health for All”
is a dream and goal of WHO in which he gets successes somewhat and strives for more; but
at the moment it has been proven that present pharmaceuticals are not successful in a
satisfactory manner to offer general health benefits. “Quality can be defined as the condition
of a drug that is determined by its characteristics, purity, content, and supplementary
chemical, physical and biological properties or by the built-up processes.” [9] The term
“herbal drugs” denotes plants or plant parts that have been converted into
phytopharmaceuticals by means of simple processes involving harvesting, drying, and
storage. Hence they are capable of variation. This variability is also caused by differences in
growth, geographical location, and time of harvesting. A practical addition to the definition
is also to include other crude products derived from plants, which no longer show any organic
structure, such as essential oils, fatty oils, resins, and gums.
In general, analysis is based on three important Pharmacopoeial definitions:
• Identity - The Condition of Being Specific Herb.
• Purity – The condition of being free from contaminants or adulterant.
• Content – The amount of the active constituents present within the defined Limit. [10]
1.2.1 Difficulties in Analysis of Herbal Drugs
Analysis of herbal drugs is a difficult task as compared to analysis of synthetic drugs because
several problems not applicable to synthetic drugs influence the quality of herbal drugs and
this is as given below. [11- 16]
Herbal drugs are generally combination of many components.
The active principle(s) is (are), in the majority cases mysterious.
Selective analytical technique or reference compound could not exist commercially.
Plant materials are chemically and naturally unpredictable.
Chemo-varieties and chemo cultivars exist.
The source and quality of the raw material is inconsistent.
Adulteration and substitution is a burning problem.
Chapter 1. Introduction
8
Adulteration may be defined as mixing or substituting the original drug material with other
spurious, inferior, defective, spoiled, useless other parts of same or different plant or harmful
substances or drug which do not confirm with the official standards.[17]
1.2.2 Adulteration may takes place by two ways:
DIRECT OR INTENTIONAL ADULTERATION
I. With artificially manufactured materials
II. With inferior quality materials
III. With exhausted material
IV. With foreign matter
V. With harmful / Fictitious substances
VI. Adulteration of powders
INDIRECT OR UNINTENTIONAL ADULTERATION
I. Faulty collection
II. Imperfect preparation
III. Incorrect storage
IV. Gross substitution with plant material
V. Substitution with exhausted drugs
1.3 Selection of Formulation
From the above listed Antidiabetic Herbal Formulations, as of now 3 formulations considered
for further research work in which constituents like Gallic acid, Ellagic acid and Curcumin will
be determine.
FORMULATIONS:
FIGURE 1. 3 Selected formulations for Research.
Chapter 1. Selection of Formulation
9
Risk base criteria for selection
1. Total no. of plants available in particular formulation
2. Type of formulation
1.4 Introduction to HPLC[18-19]
Chromatography is an analytical method that finds wide application for the separation,
identification and determination of chemical components in complex mixtures. This technique
is based on the separation of components in a mixture (the solute) due to the difference in
migration rates of the component through a stationary phase by a gaseous or liquid mobile phase.
HPLC was derived from classical column chromatography and has found an important place in
analytical technique. Most of the drugs in multicomponent dosage forms can be analyzed by
HPLC method because of the several advantages like rapidity, specificity, accuracy, precision
and ease of automation in this method. HPLC method eliminates tedious extraction and isolation
procedures.
Some of the advantages are:-
1. Tends itself to automation and quantitation (less time and less labor),
2. Precise and Reproducible,
3. Speed (Analysis can be accomplished in 20 minutes or less),
4. Greater Sensitivity (Various detectors can be employed),
5. Improved resolution (Wide variety of stationary phases),
6. Reusable columns (Expensive columns but can be used for many analysis),
7. Ideal for the substances of low volatility
8. Easy sample recovery, handling and maintenance,
9. Instrumentation Calculations are done by integrator itself,
10. Suitable for preparative liquid chromatography on a much larger scale.
Chapter 1. Introduction
10
1.4.1 Introduction to Method Development[20-23]
GENERAL CONSIDERATION
Everyday many chromatographers need to develop a HPLC separation method development and
optimization in liquid chromatography is still an attractive field of research for theoretician and
attracts also a lot of interest from practical analysts. Complex mixtures or samples required
systematic method development involving accurate modeling of the retention behavior of the
analytes. Among all the liquid chromatographic methods, the reversed phase systems based on
modified silica offers the highest probability of successful results. However, a large no. of
variables affect the selectivity and the resolution. HPLC method development follows a series
of steps which are summarized as below:-
Chapter 1. Introduction to HPLC
11
FIGURE 1. 4 HPLC method development step
QbD APPROACH IN HPLC METHOD DEVELOPMENT [24-25]
Quality means customer satisfaction in terms of service, product, and process. Customer
demands the perfection in quality, reliability, low cost and timely performance. The concept of
quality by design was summarized by a well-known quality expert Joseph Moses Juran; he
believed that quality could be planned and that most quality associated problems have their
origin in the way which quality was planned in the first place. During the drug development
process, the aspects like drug substances, excipients, container closure systems, manufacturing
processes and quality control tests are critical to product quality.
Chapter 1. Introduction
12
This scientific and knowledge rich understanding will help industry to manufacture quality
products and ultimately flourish industry by means of fame as well as financial Assets. ICH
guidance Q8 (R2) describes QbD as, “A systematic approach to pharmaceutical development
that begins with predefined objectives and emphasizes product and process understanding and
process control, based on sound science and quality risk management”.
‘‘QbD does not necessarily mean less analytical testing’’ rather, it means the right analysis at
the right time, and is based on science and risk assessment. Implementation of QbD helps to
develop rugged and robust method which helps to comply with ICH guideline hence for that
reason pharmaceutical industries are adopting this concept of QbD.
TABLE 1. 3 List of regulatory guidance or other QbD related activities
Agency Guideline/Activity Month Year
USFDA Pharmaceutical cGMP for the 21stCentury - A Risk-Based Approach:
Second Progress Report and Implementation Plan
Sep 2003
USFDA Guidance for Industry: PAT - A Framework for Innovative
Pharmaceutical Development, Manufacturing, and Quality Assurance
Sep 2004
USFDA Pharmaceutical cGMP for the 21st Century - A Risk-Based Approach:
Final Report
Sep 2004
EMA The European Medicines Agency Road Map to 2010: Preparing the
Ground for the Future
March 2005
ICH Pharmaceutical Development (Q8) Nov 2005
ICH Quality Risk Management (Q9) Nov 2005
ICH Pharmaceutical Quality System (Q10) June 2008
ICH Pharmaceutical Development (Q8(R2)) Aug 2009
WHO Quality Risk Management Aug 2010
EMA Road map to 2015 Dec 2010
USFDA Guidance for Industry: Process Validation: General Principles and
Practices
Jan 2011
EMA-
USFDA
EMA-FDA pilot program for parallel assessment of Quality by Design
applications
March 2011
ICH ICH-Endorsed Guide for ICH Q8/Q9/ Q10 Implementation Dec 2011
EMA ICH Quality IWG Points to consider for ICH Q8/Q9/Q10 guidelines Feb 2012
EMA Guideline on Real Time Release Testing (formerly Guideline on
Parametric Release)
March 2012
EMA Guideline on Process Validation (draft) March 2012
USFDA Quality by Design for ANDAs: An Example for Immediate-Release
Dosage Forms
April 2012
ICH Development and Manufacture of Drug Substances (Chemical Entities
and Biotechnological/Biological entities) (Q11)
May 2012
EMA-
USFDA
EMA-FDA pilot program for parallel assessment of Quality-by-Design
applications: lessons learnt and Q&A resulting from the first parallel
assessment
Aug 2013
EMA Guideline on process validation for finished products - information and
to be provided in regulatory submissions
Feb 2014
Chapter 1. Introduction to HPLC
13
BENEFITS OF ANALYTICAL QBD:
Increased understanding and control
Beyond traditional ICH procedure of method validation
Flexibility in analysis of API, impurities in dosage forms, stability samples, and
metabolites in biological samples
Reduction in variability in analytical attributes for improving the method robustness.
To keep the values of analytical attributes within the Pharmacopoeial monographs, and
away from Out Of Specification (OOS) limits
Smooth process of method transfer to the production level
No requirement of re-validation within MODR (Method Operable Design Region).
TERMINOLOGIES EMPLOYED DURING ANALYTICAL QUALITY BY
DESIGN:
1. Analytical Target Profile (ATP)
Prospective summary of objectives of tests/methods and quality requirements.
2. Potential Method Attributes (PMAs)
Characteristics of an analytical method that should be within an appropriate limit or
range, to ensure the desired method performance, e.g., system suitability criteria
3. Critical Method Attributes (CMAs)
Potential method attributes which are influenced by critical method variables and have
the probability to go beyond appropriate limit or range
4. Potential Method Variables (PMVs)
All the possible variables involved in an analytical method
5. Critical Method Variables (CMVs)
Potential analytical variables which have influence on critical method attributes
6. Experimental Runs or Trials
Analytical experiments carried out under defined conditions, i.e., combinations of
factors at varied levels for each of the to be measured
7. Method Operable Design Region (MODR) or Analytical Design Space
Multidimensional explorable space enclosed by upper and lower levels of the coded
variables demonstrated to provide assurance of method performance
Chapter 1. Introduction
14
8. Analytical Control Space or Normal Operating Range (NOR)
Part of the design space usually employed for setting in-house specifications within the
working environment of the company
9. Control Strategy: A schematic set of various controls to surmount all possible sources
of variability to meet ATP requirement during analytical method transfer
FIGURE 1. 5 Analytical Method development in QbD
Chapter 1. Introduction to LCMS
15
1.5 Introduction to LC-MS [26]
LC-MS is a hyphenated technique, combining the separation power of HPLC, with the detection
power of mass spectrometry. Even with a very sophisticated MS instrument, HPLC is still useful
to remove the interferences from the sample that would impact the ionization. In this case, there
is the need for an interface that will eliminate the solvent and generate gas phase ions, and then
transferred to the optics of the mass spectrometer. Most instruments now atmospheric pressure
ionization (API) technique where solvent elimination and ionization steps are combined in the
source and take place at atmospheric pressure. The interface is a particle beam type, which
separates the sample from the solvent, and allows the introduction of the sample in the form of
dry particles into the high vacuum region.
FIGURE 1. 6 Block diagram of LC-MS.
1.6 Introduction to HPTLC[27]
HPTLC is superior to other analytical techniques in terms of total cost and time for analysis. It
is an offline process in which various stages are carried out independently. Important features
of HPTLC include the ability to analyze crude samples containing multi-components,
application of large number of sample and a series of standards using the spray-on technique,
choice of solvents for the HPTLC development is wide as the mobile phases are fully evaporated
before the detection step, processing of standards and samples identically on the same plate
leading to better accuracy and precision of quantification, different and universal selective
Chapter 1. Introduction
16
Detection methods, and in situ spectra recording in sequence to obtain positive identification
of fractions, storage of total sample on layer without time constrains. In addition, HPTLC
method may help to minimizes exposure risk of toxic organic effluents and significantly reduces
its disposal problems, consequently, reducing environment pollution. Therefore, it can be
considered as an environment friendly method. Various stages of HPTLC method development
are fully automated by use of available commercial instruments, and the entire process can be
controlled using software compliant with requirements of drug regulatory agencies. Taking the
above facts together, HPTLC-based methods could be considered as a good alternative as they
are being explored as an important tool in routine analysis. Various steps involved in research
and development to bring any pharmaceutical substance/product to the market are supported by
effective and efficient analysis and therefore, effectual method development and comprehensive
analytical validation are of fundamental importance.
Method development demands primary knowledge about the physicochemical characteristics of
sample, nature of the sample, such as structure, polarity, volatility, stability and solubility. It
involves considerable trial and error procedures. Steps involved in HPTLC method development
are as follow.
Sample Preparation
Selection of Stationary Phase
Layer Prewashing
Selection and Optimization of Mobile Phase
Sample Application
Chromatogram Development
Plate Labeling
Derivatization
Documentation
Detection
Quantitation
Chapter 1. Introduction to HPTLC
17
FIGURE 1. 7 HPTLC Method development Steps
1.7 CHEMOMETRIC METHODS [28]
The utmost difficulties with multi determination methods (HPLC and UV-Vis methods) come
up when the analytes to be determined give partially or completely overlapped spectra.
Multivariate calibration is a valuable tool in the analysis of multicomponent mixtures as it
allows rapid and simultaneous determination of each and every component in the mixture with
sensible accuracy and precision and devoid of the need of lengthy separation procedures. With
the aid of modern instrumentation to acquire and digitize spectral information and dominant
computers to process huge amounts of data, multivariate methods such as classical least squares
(CLS), inverse least squares (ILS), partial least squares regression (PLSR) and principle
component regression (PCR) are finding increasing use in quantitative analysis of complex
mixtures, offering an interesting substitute to chromatographic techniques.
Chapter 1. Introduction
18
1.7.1Classical Least Squares
This method assumes that Beer’s law model with the absorbance at each frequency being
Comparative to the component concentrations. Beer’s law model for m calibration standards
containing l chemical components with the spectra of n digitized absorbance’s is given by:
A = C* K + E1
Where A is the m × n matrix of calibration spectra, C is the m × l matrix of component
concentration, K is the l × n matrix of absorptivity-path length products, and E1 is the m × n
matrix of spectral errors.
Analysis based on the spectrum of unknown components concentration (samples) is given by
below equation
C0 = (KKT)^-1 K*A
WhereC0is vector of predicted concentrations and KT is transpose of the matrix K.
1.7.2 Inverse Least Squares
This method treats these concentrations as a function of absorbance. The inverse of Beer’s law
model for m calibration standards with spectra of n digitized absorbance is given by:
C0 = aT * P
Where C0 and a represents concentration and spectrum of unknown analytes respectively.
Since in ILS, the number of frequencies cannot exceed the total number of calibration
mixtures used, stepwise multiple linear regressions have been used for the selection of
frequencies.
1.8 Introduction to UV Spectrophotometric Methods[29-31]
For treating various complicated diseases new drugs and combinations of drugs are routinely
been introduced in the market. These drugs and combinations of drugs are needed to be
analyzed qualitatively and quantitatively. For analysis of these drugs, different analytical
methods are routinely being used. These analytical methods are classified as the classical and
instrumental. The classical methods are further classified as Gravimetric, titrimetric etc. As
these methods are simple but less precise and more time consuming so the nowadays these
methods are not suggested for routine analysis. The instrumental methods are also subdivided
Chapter 1. Introduction to UV spectrophotometry
19
into electrical and optical methods. The electrical methods include voltammetry, coulometry
and optical methods which consist of absorption and emission methods. The absorption method
includes visible spectrophotometry, ultraviolet spectrophotometry, infrared spectrophotometry,
atomic absorption spectrophotometry, while emission includes methods emission
spectroscopy, flame photometry, fluorimetry, etc. The other prominent method includes
isotopes, radioactivity, X-ray fluorescence and separation methods as various chromatographic
principles viz. HPLC, GC and HPTLC etc. Analytical methods developed by using
sophisticated instruments such as spectrophotometer, HPLC, GC and HPTLC have wide
applications to assuring the quality and quantity of raw materials and finished products. These
methods are easy to perform, precise and show reproducible results as compared to any other
methods. One of the most exploited methods for the analysis of drugs is spectrophotometry;
which may be defined as a method of analysis that embraces the measurement of absorption by
chemical species of the radiant energy at definite and narrow wavelength, approximating
monochromatic radiation. The electromagnetic spectrum is divided into following regions on
basis of wavelength.
TABLE 1. 4 Region and wavelength for electromagnetic spectrum
Region Wavelength
Far (or vacuum) UV 100-200 µm
Near UV 200-400 µm
Visible 400-780 µm
Near infrared 0.78-5 µm
Infrared 5-40 µm
Spectrophotometric method is simple, rapid, moderately specific and applicable to small
quantity of the compound. The fundamental law that governs the quantitative
spectrophotometric analysis is the
Beer’s -Lambert’s law which is stated as:
Chapter 1. Introduction
20
“When a beam of monochromatic light is allowed to pass through a transparent cell containing
a solution of an absorbing substance, reduction of intensity of the light may occurs; the rate of
reduction in intensity is proportional to the thickness of the medium and the concentration of
the absorbing substances”
Mathematically Beer-Lamberts law expressed as:
A = abc
Where,
A = absorbance or optical density
a = absorptivity or extinction coefficient
b = path length of radiation through sample (cm)
c = concentration of solute in solution.
For assay of substance in multicomponent samples, following methods are routinely being used.
Simultaneous equation method
Absorbance ratio method
Absorbance correction method
Dual wavelength method
Derivative Spectrophotometric method
Ratio Spectra derivative method
Difference Spectrophotometry
Chemical derivatization method
Area under curve method
Multi component mode of Analysis
1.8.1 ABSORBANCE CORRECTION METHOD [32]
Absorbance correction method is modification of simultaneous equation method. It uses the
absorbances at two different wavelengths, one at λmax of first drug where second drug also
Chapter 1. Introduction to UV spectrophotometry
21
shows considerable absorbance (λ2) and other being the wavelength at which the first drug has
practically nil absorbance (λ1).
The concentration of two drugs (X and Y) in sample solution was calculated by using
following equations:
Cy = A2 / ay2
Cx = A1-ay1* Cy/ax1
Where, A1 and A2 are the absorbances of mixture at λ1 and λ2 respectively,
ay1 and ay2 are absorptivities of y at λ1 and λ2 respectively,
ax1 is absorptivity of X at λ2,
CX is concentration of X,
CY is concentration of Y.
1.8.2 DERIVATIVE SPECTROSCOPY[33,34]
For the purpose of spectral analysis in order to relate chemical structure to electronic transitions,
and for analytical situations in which mixture contribute interfering absorption, a method of
manipulating the spectral data is called derivative spectroscopy. Derivative spectrophotometry
involves the conversions of a normal spectrum to its first, second or higher derivative spectrum.
(As shown in figure 1.2). In the context of derivative spectrophotometry, the normal absorption
spectrum is referred to as the fundamental, zero order, or D0 spectrum. The first derivative D1
spectrum is a plot of the rate of change absorbance with wavelength against wavelength i.e. a
plot of the slope of the fundamental spectrum against wavelength or a plot of dA/dλ λ. The
maximum positive and maximum negative slope respectively in the D0 spectrum corresponds
with a maximum and a minimum respectively in the D1 spectrum. The λmax in D0 spectrum is
a wavelength of zero slope and gives dA/dλ= 0 in the D1 spectrum. The second derivative D2
spectrum is a plot of the curvature of the D0 spectrum against wavelength or a plot of d2A/ dλ2
vs λ. The maximum negative curvature in the D0spectrum gives a minimum in the D2 spectrum,
and the maximum positive curvature in the D0 spectrum gives two small maxima called satellite
bands in the D2 spectrum. The wavelength of maximum slope and zero curvature in the D0
spectrum correspond with cross-over points in the D2 spectrum. These spectral transformations
confer two principal advantages on derivative spectrophotometry. Firstly, an eve order spectrum
is of narrower spectral bandwidth than its fundamental spectrum. A derivative spectrum
Chapter 1. Introduction
22
Therefore shows better resolution of overlapping bands than the fundamental spectrum and may
permit the accurate determination of the λmax of the individual bands.
FIGURE 1. 8 First, Second, third and fourth derivative Spectrum of Gaussian peak
1.9 Validation of Analytical Method according to ICH Q2 (R1) Guideline. [35]
Analytical Procedure
The analytical procedure refers to the way of performing the analysis. It should describe in detail
the steps necessary to perform each analytical test. This may include but is not limited to: the
sample, the reference standard and the reagents preparations, use of the apparatus, generation of
the calibration curve, use of the formulae for the calculation, etc.
SPECIFICITY: It is the ability to assess unequivocally the analyte in the presence of
components which may be expected to be present. Typically these might include impurities,
degradants, matrix, etc. Lack of specificity of an individual analytical procedure may be
compensated by other supporting analytical procedure(s).
LINEARITY: It expresses ability (within a given range) to obtain test results which are
directly proportional to the concentration (amount) of analyte in the sample.
RANGE: It expresses the interval between the upper and lower concentration (amounts) of
analyte in the sample (including these concentrations) for which it has been demonstrated
that the analytical procedure has a suitable level of precision, accuracy and linearity.
Chapter 1. Validation of Analytical Methods
23
ACCURACY: It expresses the closeness of agreement between the value which is accepted
either as a conventional true value or an accepted reference value and the value found. This
is sometimes termed trueness.
PRECISION: It expresses the closeness of agreement (degree of scatter) between a series
of measurements obtained from multiple sampling of the same homogeneous sample under
the prescribed conditions. Precision may be considered at three levels: repeatability,
intermediate precision and reproducibility. Precision should be investigated using
homogeneous, authentic samples. However, if it is not possible to obtain a homogeneous
sample it may be investigated using artificially prepared samples or a sample solution. The
precision of an analytical procedure is usually expressed as the variance, standard deviation
or coefficient of variation of a series of measurements.
Repeatability: It expresses the precision under the same operating conditions over a
short interval of time. Repeatability is also termed intra-assay precision.
Intermediate precision: It expresses within-laboratories variations: different days,
different analysts, different equipment, etc.
Reproducibility: It expresses the precision between laboratories (collaborative studies,
usually applied to standardization of methodology).
DETECTION LIMIT: It is the lowest amount of analyte in a sample which can be
detected but not necessarily quantitated as an exact value.
QUANTITATION LIMIT: It is the lowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and accuracy. The quantitation limit is a
parameter of quantitative assays for low levels of compounds in sample matrices, and is
used particularly for the determination of impurities and/or degradation products.
ROBUSTNESS: It is a measure of its capacity to remain unaffected by small, but
deliberate variations in method parameters and provides an indication of its reliability
during normal usage.
SYSTEM SUITABILITY TESTING: System suitability testing is an integral part of
many analytical procedures. The tests are based on the concept that the equipment,
electronics, analytical operations and samples to be analyzed constitute an integral system
that can be evaluated as such.
Chapter 1. References
24
1.10 Profile For Selected Markers [36-38]
1.10.1 Gallic Acid
TABLE 1. 5 Chemical and Physical Properties of Gallic acid
Parameter Gallic acid
Structure
Synonym Gallic acid
Gallate
3,4,5-Trihydroxybenzoate
Formula C7H
6O
5
Log P 0.70
Solubility soluble in alcohol, ether, glycerol, acetone
negligible in benzene, chloroform, petroleum ether
MP 260- 262 °C
Density 1.694 g/cm
3
(anhydrous)
Mol. Wt. 170.12 g/mol
PKa 10
1.10.2 Ellagic Acid
TABLE 1. 6 Chemical and Physical Properties of Ellagic acid
Parameter Ellagic acid
Structure
Synonym 2,3,7,8-Tetrahydroxy-chromene[5,4,3-cde]chromene-5,10-dione
Formula C14H6O8
Log P 1.59
Solubility Soluble in alcohol and alkalis, in pyridine. Practically insoluble in ether
MP 358 - 362°C
Density 1.67 g/cm³
Mol. Wt. 302.197 g/mol
PKa 5.54
Chapter 1. References
25
1.10.3 Curcumin
TABLE 1. 7 Chemical and Physical Properties of Curcumin
Parameter Curcumin
Structure
Synonym Diferuloylmethane; curcumin I; C.I. 75300; Natural Yellow 3
Formula C21H20O6
Log P 3.29
Solubility Insoluble in water and ether; soluble in alcohol, glacial acetic acid
MP 183 - 185 °C
Mol. Wt. 368.38 g·mol−1
PKa 9.06
Chapter 1. References
26
References:
1. WHO, “Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and
classification of diabetes mellitus.” Report of a WHO Consultation, 2006, 1–59.
2. Zimmet PZ, 1997, “The global epidemiology of non-insulin dependent diabetes mellitus and the metabolic
syndrome”. Journal of Diabetes and its complications, 11(2), 60-68, ISSN No. 1056-8727.
3. Sarah wild, 2004, “Global Prevalence of Diabetes Estimates for the year 2000 and projections for 2030.” Diabetes
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extract for bacillary dysentery.” Scholars Research Library, 3(1), 63-72, ISSN No. 0975-5071.
5. ICMR Guidelines for Management of Type 2 Diabetes- 2005, Pharmacological Treatment For Diabetes, section 7,
16-31.
6. National diabetes Services scheme, diabetes information sheet, Diabetes Australia, 1-37.
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Journal of Medicinal Chemistry, 6, 226-240, ISSN No. 0223-5234.
8. Mohammad A, Bhavani SA & Sharma S, 2010, “Analysis of Herbal Products by Thin-layer Chromatography: A
Review”, International Journal of Pharma and Biosciences, 1(2), 1-50, ISSN No. 0975-6299.
9. Kalyankar TM, 2014, “Analysis of Herbal Drugs: a Review.” Asian Journal of Medicinal and Analytical Chemistry,
1 (1), 12-20, ISSN No. 2456-6217.
10. Garg C, Khan SA, Ansari SH, Garg M, 2010, “Efficacy and Safety Studies of Foeniculum Vulgare through
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Pharmaceutical Sciences, 2(2), 40-43, ISSN No. 2656-0097.
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Series WHO Regional office for the Western Pacific, Manila, 1998, 3, 35.
(http://apps.who.int/medicinedocs/en/d/Jh2945e/)
12. WHO Quality Control Methods for Herbal Materials Updated edition of Quality control methods for medicinal
plant materials 1998, World Health Organization, Geneva, 1998, 1-9.
13. WHO. WHO Monographs on Selected Medicinal Plants, World Health Organization, Geneva, 1999, 1, 34.
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Lab, 5, 1- 23.
16. WHO Guidelines on Safety Monitoring Of Herbal Medicines in Pharmacovigilance Systems, World Health
Organization Geneva, 2004, 1-5.
17. Kamboj A. 2012, “Analytical Evaluation of Herbal Drugs.” Drug discovery Research in Pharmacognosy. 23-57,
ISSN No. 9789-5351.
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19. O’Haver, D.Y., 1983, “First-Derivative Spectrophotometric Determination of a Mixture of Pirbuterol
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No. 0004-5756.
20. Lough WJ.,Wainer IW. In High performance liquid chromatography, Fundamental principle and practice; Blackie
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36. Compound summary, Gallic acid. Available: https://pubchem.ncbi.nlm.nih.gov/compound/Gallic-acid[accessed
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May, 2019]
28
Chapter 2. Gallic acid
29
CHAPTER 2
Literature Review
2.1 GALLIC ACID
TABLE 2.1 TLC Methods for Gallic acid.
Sr.
No.
Drug Method specification Detection
wavelength
Ref. no
1. Gallic acid Estimation of Gallic acid in herbal drugs by ferric
reducing antioxidant power (FRAP) assay
Stationary phase:-
Silica gel 60 F254 plates
Mobile phase: - Chloroform: ethyl formate:
formic acid (5:4:1)
Sample:- Extracts of powder drug with
hexane and water (1:6)
593nm 1
2. Gallic acid
Eugenol
Estimation of gallic acid and eugenol from
Syzygium aromaticum (L) Merr and Perry (clove)
Stationary phase :- TLC plates precoated with
silica gel 60 F254
Mobile phase:- Toluene : Ethyl acetate :
Formic acid :- (3:2:0.4)
Sample :extracts of flower bud of Syzygium
aromaticum and Perry (clove)
The average percentage recovery of gallic and eugenol
are 97.90% and 99.79% respectively.
280nm 2
3. Esters of
gallic acid Estimation of esters of gallic acid from tannase
Aspergillus Niger
Stationary phase :- Silica gel G TLC plates
Mobile phase :- Chloroform : Methanol
(80:20)
Sample :- Cultures of Aspergillus Niger
273nm 3
Chapter 2. Literature Review
30
4. Gallic acid
Bergenin
Catechin
Estimation of gallic acid , Berginin and catechin in
Bergenia ciliata and Bergenia ligulata
Stationary phase :- HPTLC plates precoated
with silica gel 60F254
Mobile phase :- Toluene : Ethyl acetate :
formic acid (4:6:1)
Sample :- extracts of B. ciliata and B.ligulata
The average percentage recovery of gallic acid
:- 99.23%
The average percentage recovery of catechin
:- 98.66%
The average percentage recovery of bergenin
:- 99.29%
254nm and
366nm
4
5. Gallic acid
Pyrogallol
and tannic
acid
Estimation of gallic acid ,pyrogallol and tannic acid
Stationary phase :- Silica gel G TLC plates
Mobile phase :- Ethyl formate : chloroform :
formic acid
Sample : Methanolic extract of tannic acid
Rf value of gallic acid :0.40
254nm 5
6. Gallic acid
Gallicin
Lupeol
β -
Sitosterol
(B.
suffruticosa)
Estimation of gallic acid , gallicin , lupeol and β –
Sitosterol from Bergia suffruticosa
Stationary phase :- Silica gel G TLC plates
Mobile phase :- Toluene: Ethyl acetate :
Methanol : formic acid (6:3:1:0.5)
Sample :- extracts of B. suffruticosa
Rf value of all fractions at a single spot using
1% methanol in chloroform :- 0.58
Rf value of all fractions at single spot using 2-
10 % methanol in chloroform :-0.40
The average percentage recovery of gallic
acid, gallicin, lupeol and β –Sitosterol are
100.58%, 99.89%,99.79% and 100.11%
respectively.
- 6
7. Gallic acid
Theogallin
and Quinic
acid
Estimation of gallic acid , Theogallin and Quinic
acid in Kombucha beverage
Tlc was performed by two chromatographic systems:
Stationary phase:- Microcrystalline cellulose
and Silica gel G(7)
Mobile phase:- ethyl acetate:formic
acid:acetic acid:water (100:11:11:26)
Sample : vapourization of Kombucha leaf
Stationary phase:- Silica gel GF254
Mobile phase:- Chloroform:ethyl
acetate:formic acid (5:4:1)
Sample : ether extract of Kombucha leaf
- 7
Chapter 2. Gallic acid
31
TABLE 2.2 HPLC Methods for Gallic acid.
Sr.
No.
Drug Method Specification Detection
Wavelength
Ref.no.
1. Gallic acid Estimation of Gallic acid in Symplocos racemosa
(Roxb)
Stationary phase: Inertsil C8-4
column(LCGC)
Mobile phase:
Solvent A: 0.1% orthophosphoric acid in
water of ph 2.5
Solvent B: Acetonitrile
Sample : extracts of S.racemosa(ROxb) with
water ,alcohol (1:1)
Retention time of gallic acid: 8.5 min
280nm 8
2. Gallic acid Estimation of Gallic acid in dendrophthoe falcate
Linn.
Stationary phase:TherMOS 2 HYPERSIL C18
column
Mobile phase: 0.1% Orthophosphoric
acid:Acetonitrile(400cm3:600cm3)
Sample:extracts of stem bark of D.falcate
Linn. With methanol
Recovery of gallic acid :98.94%
271nm 9
3. Gallic acid Gallic acid estimation in the rind of Punica
granatum-Pomegranate
Stationary phase: Cosmosil C18 column
Mobile phase: Ethyl acetate: ethanol:
water(1:5:4)
Sample: Extracts of pomegranate rind
Retention time of gallic acid: 3.496 min
366nm 10
4. Gallic acid Estimation of gallic acid in Terminalia chebula
Stationary phase: Inertsil ODS -3 column
Mobile phase :
Solvent A: 0.2% formic acid in water
Solvent B : Acetonitrile
Sample : extracts of dried fruits of T.chebula
215, 271nm 11
5. Gallic acid
& Ascorbic
acid
Estimation of ascorbic acid and gallic acid in
Phyllanthus emblica
Stationary phase: C18 reverse phase column
Mobile phase:
Solvent A: 0.1% v/v in water
Solvent B: Acetonitrile / methanol
Sample: extracts of fresh fruits of p.emblica
Retention time of ascorbic acid : 3.60min
Retention time of gallic acid : 10.77min
278nm 12
Chapter 2. Literature Review
32
6. Gallic acid
Caffeic acid
Rutin
Quercetin
Ferulic acid
Estimation of gallic acid ,caffeic acid, rutin
,quercetin and ferulic acid in Pseudarithria
viscida root
Stationary phase : C18 reverse phase column
Mobile phase :
Solvent A :water : acetic acid (25:1 v/v)
Solvent B: Methanol
Sample : extracts of the fresh roots of
Pseudarthria viscida root
Retention time of gallic acid : 5.6
Retention time of caffeic acid:9.3
Retention time of rutin: 10.2
Retention time of quercetin: 12.3
Retention time of ferulic acid: 23.8
280nm 13
7. Gallic acid
Caffeic acid
Rutin
Quercetin
Ferulic acid
Estimation of gallic acid, cafeic acid , rutin ,
quercetin and ferulic acid in Amaranthus caudatus
Stationary phase :- C18 reverse phase column
Mobile phase :-
Solvent A:-Water: Acetic acid (25:1)
Solvent B:- Methanol
Sample :- Extracts of leaves of Amaranthus
caudatus
Retention time of gallic acid:- 5.5
Retention time of caffeic acid:-9.4
Retention time of rutin :-10.4
Retention time of quercetin:-12.3
Retention time of ferulic acid :-24.5
280nm 14
8. Gallic acid
and Ellagic
acid
Development and Validation of Stability indicating
HPLC method for determination of Ellagic and
Gallic acid in Jambul seeds (Syzygium cumin)
Stationary phase:- Hypersil C18 column
Mobile phase:- 1% Orthophosphoric acid:
Acetonitrile (70:30 v/v)
Sample:- Extract of s. cumini seeds and
formulation
Retention time of Ellagic acid:- 3.1±0.05
min
Retention time of Gallic acid:- 4.1± 0.05
min
271nm 15
9. Gallic acid
and
Protocatechuic
acid
Development and Validation of a RP-HPLC
method for Identification and Estimation of Gallic
acid and Protocatechuic acid in trigasornmas
recipe
Stationary phase:- C18 reverse phase column
Mobile phase:-
Solvent A:- Acetonitrile
Solvent B:- 0.1% acetic acid acid in water
Retention time of Gallic acid:- 9.28 ±0.05
min
Retention time of protocatechuic acid:- 17.67
± 0.03min
280 nm 16
Chapter 2. Gallic acid
33
10. Gallic acid Development of a rapid and simple HPLC-UV
method for determination of gallic acid in
Schinopsis brasiliensis
Stationary phase:- Phenomenex Gemini NX
C18 column
Mobile phase:- 0.05% Orthophosphoric acid:
Methanol
Retention time of gallic acid:- 8.5min
271nm 17
11. Gallic acid Development and Validation of stability indicating
RP-HPLC method for gallic acid
Stationary phase:- Thermo Hypersil BDS-
C18 column
Mobile phase:- Water acidified with
phosphoric acid (0.01%): Methanol (95: 5
v/v)
Retention time of gallic acid:- 6.4min
271nm 18
12. Gallic acid
and Ellagic
acid
Method development and validation of gallic acid
and Ellagic acid in Argwadharistam
Stationary phase:- Phenomenx- Luna C18
column
Mobile phase:-
Solvent A:- Acetonitrile
Solvent B:- Buffer solution
Retention time of gallic acid:- 4.8min
Retention time of ellagic acid:- 8.46min
254nm 19
13. Gallic acid High- performance Liquid Chromatographic
method for the Quantification of gallic acid in
Simhanada guggulu
Stationary phase:- RP C18 column
Mobile phase:-
Solvent A:- Acetonitrile
Solvent B:- Water: 0.3% O- Phosphoric acid
Retention time of gallic acid:- 5.29min
254nm 20
14. Gallic acid By using RP- HPLC Technique Quantitative and
Qualitative analysis of gallic acid from Industrial
waste
Stationary phase:- Phenomenax C18 column
Mobile phase:- Acetonitrile: 0.01%
Orthophosphoric acid (80:20 v/v)
Retention time of gallic acid:- 1.82min
271nm 21
15. Gallic acid,
Caffeine and
Catechins
Development of an Improved Isocratic HPLC
method for the determination of gallic acid,
caffeine and catechins in tea
Stationary phase:- C6 phenyl column
Mobile phase:- Water: Acetonitrile:
Methanol: ortho phosphoric acid: Ethyl
acetate (77.5: 18: 2.0: 0.5: 2.0 v/v)
Retention time of gallic acid:- 12.5min
278nm 22
Chapter 2. Literature Review
34
TABLE 2. 3 HPTLC Methods for Gallic acid.
Sr. No. Drug Method specification Detection
wavelength
Ref.
no.
1. Gallic acid Estimation of gallic acid in Myrica esculenta
Stationary phase: 60 F254 HPTLC plates
Mobile phase:Toluene:ethyl acetate : formic
acid :methanol(3:3:0.6:0.4)
Sample: extracts of stem bark powder of M.
esculenta
Amount of gallic acid in free form: 0.276%
Amount of gallic acid in compound form:
0.541%
Rf value of gallic :0.51
280nm 24
2. Gallic acid Estimation of gallic acid in Phyllanthus emblica
Linn.
Stationary phase: 60F254 aluminium HPTLC
plates precoated with silica gel
Mobile phase : Toluene:ethyl acetate : formic
acid : methanol (3:3:0.8:0.2)
Sample : methanolic extracts of dried fruit
powder of P. emblica Linn.
Migration distance :80mm
Rf value of gallic acid :0.40
278nm 25
3. Gallic acid Estimation of gallic acid in 34tellate34l
formulations
Stationary phase: 60 F254 pre-coated TLC
plates with silica gel
Mobile phase : Toluene : Methanol :
Ethylacetate : Formic acid (30:5:55:10)
Sample : 20 capsules were weighed and
average weight is calculated and extracted
with methanol
Rf value of gallic acid : 0.57
280nm 26
16. Gallic acid
and Oleanolic
acid
RP – HPLC method development and validation of
simultaneous estimation of gallic acid and oleanic
acid in antihyperlipidemic Polyherbal tablets
Stationary phase:- C18 column
Mobile phase:- 0.1% Orthophosphoric acid:
Methanol (5: 95 v/v)
Retention time of gallic acid:- 2.8min
Retention time of oleanolic acid: 9.9min
222nm 23
Chapter 2. Gallic acid
35
4. Gallic acid Estimation of gallic acid in Nymphaea 35tellate
Wild
Stationary phase : 60F254 aluminium plates
pre-coated with silica gel
Mobile phase : chloroform : ethyl acetate :
formic acid (7.5: 6:0.5)
Sample : hydroalcoholic extracts of dried
flowers of Nymphae 35tellate Wild
Rf value of gallic acid :0.24
292nm 27
5. Gallic acid
Quercetin,
Lupeol
Estimation of Gallic acid and quercetin in Acacia
leucophloea:
Stationary phase :Silica gel GF254 plates
Mobile phase : Toluene:ethyl
acetate:Formic acid (6:4:0.8)
Sample :Methanolic extract of
A.leucphloea flower (10mg/ml)
Migration distance :80mm
Rf value of gallic acid and quercetin are 0.22
and 0.37 respectively.
254nm-280nm 28
6. Gallic acid,
rutin,
Quercetin
Estimation of Gallic acid in Terminalia chebula
Stationary phase: Silica gel F254 plates
Mobile phase : Toluene : Acetone: Glacial
acetic acid (3:1:2)
Sample :T. chebula extract (10mg/ml)
Migration distance :80mm
Rf value of gallic acid:0.30
254nm 29
7. Gallic acid,
Curcumin
Quercetin
Estimation of gallic acid ,Curcumin and Quercetin
Stationary phase:Silica gel 60 F254 plates
Mobile phase = Toluene:ethyl acetate :formic
acid(4.5:3:0.2)
Rf value of gallic acid:0.40
Rf value of curcumin:0.73
Rf value of quercetin:0.55
366nm 30
8. Gallic acid
Ascorbic acid Estimation of Gallic acid in Terminalia chebula
and Terminalia belerica
Stationary phase : Silica gel 60 F254 plates
Mobile phase : Ethyl
acetate:Toluene:Acetate (4.5:4:1)
Sample : 1g/50ml of triphala
churna (each T. chebula , T. belerica
,E.officinalis)
Migration distance :80mm
Rf value of gallic acid:0.54
254nm 31
Chapter 2. Literature Review
36
9. Gallic acid,
Curcumin,
Quercetin and
Trigonelline
HPTLC method development and validation of
antidiabetic marker compound from Polyherbal
formulation
Stationary phase : Silica gel 60 F254 plates
Mobile phase : Isopropyl alcohol:
Ammonia: Acetone (1:1:1 v/v/v)
Migration distance :80mm
Rf value of gallic acid: 0.42
Rf value of Curcumin: 0.81
Rf value of Quercetin: 0.66
Rf value of Trigonelline: 0.34
32
10. Gallic acid,
Ellagic acid
and Corilagin
Simultaneous estimation of Corilagin, gallic acid
and Ellagic acid by HPTLC method
Stationary phase : Silica gel 60 F254 plates
Mobile phase : n-butanol: Water:
Methanol: Formic acid (6:1:0.1:0.8 v/v/v/v )
Migration distance :80mm
Rf value of corilagin: 0.44
Rf value of gallic acid: 0.80
Rf value of Ellagic acid: 0.64
283nm 33
11. Gallic acid
and Piperine
Development and validation of simultaneous
estimation for piperine and gallic acid in zeal
herbal granules by HPTLC method
Stationary phase : Silica gel 60 F254 plates
Mobile phase :Toluene: Ethyl acetate:
Formic acid (11:15:1 v/v/v)
Migration distance :80mm
Rf value of gallic acid: 0.50
Rf value of piperine: 0.70
254nm 34
12. Gallic acid
and quercetin Validated high performance thin layer
chromatography method for simultaneous
determination of quercetin and gallic acid in leea
indica
Stationary phase : Silica gel 60 F254 plates
Mobile phase :Toluene: Ethyl acetate:
Formic acid (5:4:1 v/v/v)
Migration distance :80mm
Rf value of gallic acid: 0.45
Rf value of quercetin: 0.63
254nm 35
Chapter 2. Gallic acid
37
13. Gallic acid
and Ellagic
acid
Comparative HPTLC estimation and antibacterial
effect of Ellagic acid, gallic acid and ethanolic
extract of syzygium cumini seeds under accelerated
storage condition
Stationary phase : Silica gel 60 F254 plates
Mobile phase :Toluene: Ethyl acetate:
Formic acid (6:6:1.2 v/v/v)
Migration distance :80mm
Rf value of gallic acid: 0.57 ±0.02
Rf value of Ellagic acid: 0.47±0.02
271nm 36
TABLE 2.4 GC-MS Methods for Gallic acid
Sr.No Drug Method specification Ref.
No
1. Gallic acid Estimation of gallic acid in Terminalia Bellerica
Column :- Silica gel open column
Sample:- Acetone extract of Terminalia Bellerica fruit
rind powder
Solvents:- Petroleum ether, Chloroform , Ethyl acetate ,
Acetone and Methanol.
37
2. Gallic acid
(V.Negudo) Estimation of gallic acid in Vitex Negudo
Gas chromatograph:- GC CLARUS 500 Perkin Elmer
system
Column :- column Elite 1 fused silica capillary
AOC -20i Autosampler is used
Sample:- Ethanolic extracts of Vitex Negudo leaves
Carrier gas:- Helium gas (99.999%)
Temperature program :- 250 to 280⁰C
Total phenol content :- 27.72mg/100 of gallic acid
equivalent (GE)
38
3. Gallic acid
Benzoic acid
and its
monohydroxy
dihydroxy and
trihydroxy
derivatives
Estimation of various acids in Paeonia 37rotocate and Paeonia
tenuifolia roots
Gas chromatograph:- Hewlett Packard 5890 gas
chromatograph
Column:- HP-5 fused silica capillary column
Sample:- Methanolic extracts of the roots of P.peregrina
and P.tenufolia
Detector: HP 5972 MSD
Carrier gas : Helium gas
Temperature program :-80-240⁰ C
39
4. Gallic acid
Caffeic acid
Rutin
Quercetin
Ferulic acid
(A.caudat)
Estimation of gallic acid , caffeic acid , rutin , quercetin and
ferulic acid in Amaranthus caudatus
Gas chromatograph:- GC CLARUS 500 PerkinElmer
system
Column:-Elite 1 fused silica capillary column
Sample: Ethanolic extract of the leaves of A.caudatus
Carrier gas:- Helium gas (99.999%)
Temperature program :-110⁰C to 200⁰C
40
Chapter 2. Literature Review
38
5. Gallic acid
Ascorbic acid Estimation of gallic acid and ascorbic acid in Bougain Villea
Glabra choicy leaves
Gas chromatograph :- GC CLARUS 500 PerkinElmer
system
AOC-20i Autosampler is used
Sample:- Ethanolic extract of Bougain Villea Glabra
leaves
Carrier gas :-Nitrogen gas for removing and
concerntrating sediments.
Total phenol content :- 30.00mg/100 of gallic acid
equivalent (GE )
41
6. Gallic acid
Pyrogallol
Caffeic acid
Estimation of gallic acid , Pyrogallol and caffeic acid in
Labisia paucifolia
Gas chromatograph :- Shimadzu QP2010PLUS system
Column :- thin capillary column
Sample :- Methanolic extracts of dried leaves ,stem and
root
Carrier gas :- Helium gas
Temperature program :- 50⁰C to 80⁰C
Eletron impact mode :- 70eV
42
7. Gallic acid
Pyrogallol
Estimation of gallic acid and pyrogallol in Emblica officinalis
Gaertn
Gas chromatograpgh :- GC-MS CLARUS 500
PerkinElmer system
AOC-20i Autosampler is used
Sample:- Methanolic extract of leaves of Emblica
officinalis Gaertn
Carrier gas :- Helium gas
Tempeature program :- 40⁰C to 280⁰C
Electron impact mode :- 70eV
Total GC run time :- 60 minutes
Scan interval :- 0.5 seconds
43
8. Gallic acid
Ascorbic acid Estimation of gallic acid in Phyllanthus emblica
Gas chromatograpgh: -GC-MS CLARUS 500
PerkinElmer system
Column :- Elite-1 fused silica capillary column
AOC-20i Autosampler is used
Sample:- Ethyl acetate extract of Phyllanthus emblica
Carrier gas :- Helium gas
Temperature program :- 40⁰C to 300⁰C
Electron impact mode:- 70eV
Total GC run time:- 34 minutes
Scan interval :-0.5 seconds
44
Chapter 2. Gallic acid
39
TABLE 2. 5 LC-MS Methods for Gallic acid.
Sr
No.
Drug Method Specification Ref.
No.
1. Gallic acid
Ethyl gallate Estimation of gallic acid and ethyl gallate in Lagerstroemia
39rotocat (Linn) Pers
Stationary phase :- Zorbax SB-C18 column
Mobile phase :- Methanol: Acetonitrile: 10mM ammonium
acetate (10:25:65)
Flow rate of mobil phase:- 0.25mL/min
System used: Agilent G6410A triple quadruple
The lower limit of quantification of gallic acid and ethyl
gallate of the method were :- 0.5 and 1 mg/mL
The intra-day and inter-day accuracy and precision were
less than 8.0%
45
2.
Gallic acid Estimation of gallic acid in Carica Papaya
Stationary phase :- Hypersil C-18 column
Mobile phase :-
Solvent A : 0.1% acetic acid
Solvent B: 100% Methanol
Sample:- Methanolic extract of air dried and finely
powdered parts of C.papaya
System used :- EXSIGENT UPLC system
The total phenolic content can be determined by :- The
Folin Ciocalteu Spectrophotometric system
Desolvation gas :- Nitrogen gas
46
3. Gallic acid Estimation of gallic acid in rat plasma
Stationary phase :- Shimazdu Shim pack VP-ODS C-18
column
Mobile phase :- Methanol: Formic acid (40:60)
Flow rate of mobile phase:- 2mL/min
Sample:- Pretreatment involves extraction using ethyl
acetate with 39rotocatechuic acid
System used :- Finnigan TSQ quantum discovery MAXTM
LC-MS system with negative ion mode
The mass spectrometric conditions were as follows:-
1. Spray voltage : -3,800 V
2. Heated capillary temperature:- 300⁰C
3. Sheath gas:- Nitogen gas
4. Collision gas:- Argon gas
47
4. Gallic acid Estimation of gallic acid in Spruce (Picea Abies)
Sample:- Pre-extracted with petroleum ether and methanol
Analysis was done using LC-MS in negative ion mode
(MRM mode used)
Retention time of gallic acid:- 2.3 min
48
Chapter 2. Literature Review
40
5. Gallic acid,
Corilagin,
Ascorbic acid,
Chebulagic acid
and chebulinic
acid
HPLC-MS profiles and quantitative analysis of triphala
formulation
Stationary phase :- Generix C18 column
Mobile phase :-
Solvent A-: 1% acetic acid in water, pH 2.65
Solvent B:- Acetonitrile or Methanol
Flow rate of mobile phase:- 0.25mL/min
System used :- Bruker Amazon SL mass spectrometer
coupling with HPLC, Dionex
The mass spectrometric conditions were as follows:-
1. Capillary voltage : -4,500 V
2. Heated capillary temperature:- 220⁰C
3. Collision gas:- Nebulizer gas
49
TABLE 2. 6 IR Methods for Gallic acid.
Sr
No.
Drug Method specification Ref.no
1. Gallic acid Estimation of gallic acid in diospyrus ferrea (willd.) Bakh root
by FTIR
Spectrometer :- Shimadsu Prestige 2 FTIR spectrometer
The fourier Transform Infrared spectrum (FTIR) of each
extract at the IR region :- 4000 to 500 cm-1
Finger print region :- 500-3500 cm-1
Number of scans :-27
Device used for scanning :- Horizontal Attenuated Total
Reflection (HATR)
The FTIR spectrum of standard gallic acid contained 8
major peaks at the range :- 1022.27 ,1234.44 ,1448.54
,1622.13 ,1714.22 ,3043.67 ,3280.92 ,3365.78
50
2. Gallic acid Estimation of gallic acid in pyragallol
Spectrometer :- Perkin Elmer system 2000 FTIR
spectrometer
Diffused reflectance Infrared Fourier Transform Sector
(DRIFTS) of polycrystalline compounds was measured in :-
KBr matrix
Number of scans:- 512
51
3. Gallic acid Estimation of X-ray structure of gallic acid
Infrared spectra of gallic acid was recorded in two forms:-
crystalline and dry
Infrared intensities was calculated by :- post HF-DFT
method with the Becke3LYP functional and 6-31G* basis
set
52
4. Gallic acid Estimation of gallic acid in Acetonitrile clusters
Gallic acid acetonitrile clusters were investigated using :-
B3LYP/6-311++G (2d,2p) method
Conformers of gallic acid by conformational analysis :-
GA-I , GA-II ,GA- III and GA –IV
Mole ratios of this conformers :- 1:1 ,1:2 and 1:4
The bands were collected in the range of 1800-1000 cm-1
53
Chapter 2. Gallic acid
41
TABLE 2.7 UV Methods for Gallic acid.
Sr
No.
Drug Method specification Detection
wavelength
Ref.No.
1. Gallic acid Estimation of gallic acid in Diospyrus ferrea(willd.)
Bakh root
Spectrometer : Jasco V 530 spectropho-
tometer
Extraction yield was calculated by extraction
factor
The formula for extraxtion factor : EF= A
(λmax)xd
Where (λmax)= absorption values
D = dilution factor
Most effiecient solvent : Ethanol
Absorption maxima for ethanol (λmax)=
271nm and 227nm
Extraction factor of ethanol at 271nm=99± 45
Extraction factor of ethanol at 227nm=20±34
Extraction factor for gallic acid at 272nm
=150± 22
Extraction factor for gallic acid at 220nm= 23±
11
54
2. Gallic acid
Rutin Estimation of gallic acid and rutin in Triphala
churna
Spectrometer: Shimazdu 1800 UV/visible
soectrometer
Solvent: Methanol
Linearity range of gallic acid :5-30µg/mL
Linearity range of rutin :5-30 µg/mL
The coefficient of correlation for gallic acid at
273nm :0.9941
The coefficient of correlation for rutin at
359nm: 0.999
Percentage estimation of gallic acid :
101.35±0.947
Percentage estimation of rutin : 99.78±0.326
273nm and
359nm
respectively
55
3. Gallic acid Validation of UV – Spectrophotometric method with
stress degradation study for gallic acid in ayurvedic
formulation of amla capsule
Spectrometer: Shimazdu 1800 UV/visible
soectrometer
Linearity range of gallic acid :1-6µg/mL
The coefficient of correlation for gallic acid at
274nm: 0.999
Percentage estimation of gallic acid :
99.19%w/v
274nm 56
Chapter 2. Literature Review
42
TABLE 2.8 NMR Methods for Gallic acid.
Sr
No.
Drug Method Specification Ref. No.
1. Galloyltyrosine
(inga Laurina) Estimation of galloyltyrosine from Inga Laurina
Spectra : 1HNMR (400 mHz) and 13 C NMR
(100mHz)
Spectrometer : Bruker 400 mHz NMR spectrometer.
57
2. Gallic acid
(Carob)
Estimation of gallic acid in Carob leaves
Spetra : 1 H NMR (300 and 500 mHz) and 13 C NMR (75.46 mHz)
Spectrometer : Varian Mercury VX-300 NMR
spectrometer
58
Chapter 2. Ellagic acid
43
2.2 ELLAGIC ACID
TABLE 2.9 HPLC Methods for Ellagic acid
Sr.No. Drug Method specification Detection
wavelength
Ref.no
1. Taxol and
ellagic acid Validated HPLC method for the simultaneous
determination of taxol and ellagic acid in a Punica
granatum fruit extract containing combination
formulation
Stationary phase :- 25 x 4.6 mm, 5 µm, C18
RP (Luna)
Mobile phase :- methanol and 0.05% H3PO4,
in gradient elution mode
flow rate of 1 mL/min
retention times of 13.75 min. and 11.6 min.
for paclitaxel and ellagic acid, respectively
230nm 59
2. Ellagic Acid Determination of Free Ellagic Acid Content in
Guava Leaves by HPLC
Stationary phase :- inertsil ODS-SP column
(250 mm × 4.5 mm, 5μm), 35 ℃
Mobile phase :- 3% glacial acetic acid (phase
A) and net methanol (phase B) with the
following gradient elution program
Injection volume:- 10µl
254nm 60
3. Ellagic acid Determination of Ellagic Acid in Pomegranate
Seeds by RP-HPLC
Stationary phase :- Arcus EP-C18 column
(250 mm × 4.6 mm, 5 μm)
Mobile phase :- methanol and 0.1% TFA at a
flow rate of 1.0 mL/min by gradient elution
flow rate of 1 mL/min
254nm 61
4. Ellagic acid Determination of ellagic acid in pseudofruits of
some species of roses
Stationary phase :- Hypersil 200 × 4.6 mm
I.D., 5 μm
Mobile phase :-mobile phase A, methanol n
water n
phosphoric acid (49,5:49,5:1, v/v/v), in
gradient elution
phases: B, methanol n water n phosphoric acid
(199,5:799,5:1, v/v/v), C, methanol n water
Phosphoric acid (599,5:399,5:1, v/v/v)
flow rate of 1 mL/min
Injection volume:- 20µl
254nm &
360nm
62
Chapter 2. Literature Review
44
5. Ellagic acid Antioxidant Assay-Guided Purification and LC
Determination of Ellagic Acid in Pomegranate Peel
Stationary phase :- TSK-gel ODS-80Tm
column
Mobile phase :-2% aqueous acetic acid and
methanol (gradient elution mode
flow rate of 1 mL/min
retention times -7.7 min
254nm 63
6. Ellagic acid Ellagic acid content in berries: Influence of
domestic processing and storage
Stationary phase :- LichroCART
(125*3mm)RP C18, 5µm.
Mobile phase :-1% formic acid and
acetonitrile in gradient elution mode.
flow rate of 1 mL/min
260nm 64
7. Ellagic &
Gallic acid Development and Validation of Improved RP-
HPLC method for Identification and Estimation of
Ellagic and Gallic acid in Triphala churna
Stationary phase :- RPHPLC C18 column
Mobile phase :-acetonitrile as
solvent A and O-Phosphoric acid in Water (0.3%)
as solvent B using gradients elution
flow rate of 0.8 mL/min
254nm 65
8. Ellagic &
Gallic acid HPLC Analysis of Gallic and Ellagic Acids in
European Oakwood (Quercus robur L.) and
Eucalyptus (Eucalyptus globulus)
Stationary phase :- Lichrospher RP 18 E 5µm,
10 cm
Mobile phase :-Water : Methanol: Phosphoric
acid in different proportion
- 66
9. Ellagic acid A Simple method for the Extraction of Phenolic
compound (Ellagic acid) from strawberry using
ultrasound and analyze it by HPLC
Stationary phase :- Shimadzu ODS column
Mobile phase :-
Solvent A:- Water (0.1% TFA, v/v)
Solvent B:- Acetonitrile
Flow rate:- 1 ml/min
375nm 67
10. Ellagic acid Stability indicating RP- HPLC method
development and force degradation studies of
Ellagic acid
Stationary phase :- Enable C-18 column
Mobile phase :- Phosphate buffer:
Acetonitrile: Methanol 10mM pH 2.6
(5:55:40 v/v/v)
Flow rate:- 0.6 ml/min
Retention time of Ellagic acid:- 4.91±0.02min
254nm 68
Chapter 2. Ellagic acid
45
11. Ellagic acid Development and Validation of a RP- HPLC
method for the determination of Ellagic acid in
Terminalia bellirica extract and single herb capsule
of Terminalia bellirica
Stationary phase :- Inertsil ODS column
Mobile phase :-
Solvent A:- n- Hexane sulfonic acid (20 mM)
Solvent B:- 100% Methanol
Flow rate:- 0.7 ml/min
Retention time of Ellagic acid:- 21min
254nm 69
12. Ellagic acid Development and Validation of a HPLC- UV
Method for the Evaluation of Ellagic acid in liquid
extracts of Eugenia uniflora L. (Myrtaceae) leaves
and its Ultrasound- Assisted extraction
optimization
Stationary phase :- Supelco C18 column
Mobile phase :- Water/acetonitrile or
methanol
Flow rate:- 0.5 to 1.2 ml/min
Retention time of Ellagic acid:- 12.22min
254 and
280nm
70
13. Ellagic acid High Performance Liquid Chromatography
method for Quantification of Ellagic acid in IN vivo
and IN vitro plant parts of Oroxylum indicum (L.)
vent
Stationary phase :- Symmetry C18 column
Mobile phase :- Water: Methanol:
Acetonitrile: Orthophosphoric acid
(60:30:38:1 v/v/v/v)
Flow rate:- 1ml/min
Retention time of Ellagic acid:- 3.29min
262nm 71
14. Ellagic acid
and Quercetin
Development and Validation of Novel RP- HPLC
method for the simultaneous estimation of Ellagic
acid and Quercetin in an Ayurvedic formulation
Stationary phase :- Shim- pack HPLC C18
column
Mobile phase :- 0.02 M potassium dihydrogen
buffer (pH 3.5 with OPA) and Acetonitrile
(60:40 v/v)
Flow rate:- 1.2 ml/min
Retention time of Ellagic acid:- 1.65min
Retention time of Quercetin:- 2.94min
255nm 72
Chapter 2. Literature Review
46
15. Ellagic acid,
Quercetin and
Rutin
Development and Validation of RP- HPLC method
for the simultaneous estimation of Quercetin,
Ellagic acid and Rutin in hydroalcoholic extract of
triphala churna
Stationary phase :- Shim- pack HPLC C18
Mobile phase :- 0.02 M potassium dihydrogen
buffer (pH 3 with OPA) and Methanol (55:45
v/v)
Flow rate:- 1 ml/min
Retention time of Quercetin:- 7.52min
Retention time of Ellagic acid:- 9.10min
Retention time of Rutin:- 12.47min
254nm 73
16. Ellagic acid Development and Validation of a HPLC Analytical
method for determination of Ellagic acid in
Epilobium Angustifolium extract
Stationary phase :- Sunfire C18 column
Mobile phase :- 0.1% Orthophosphoric acid
and acetonitrile
Flow rate:- 1 ml/min
Retention time of Ellagic acid:- 35min
280nm 74
TABLE 2.10 HPTLC Method for Ellagic acid
Sr.No. Drug Method specification Detection
wavelength
Ref.no
1. rubiadin,
sennoside and
ellagic acid
Simultaneous Analysis and Quantification of
Markers of Manjisthadi Churna Using High
Performance Thin Layer Chromatography
Stationary phase :- TLC plates precoated with
0.2-mm layers of silica gel 60F254
Mobile phase:- toluene:ethyl
acetate:methanol:formic acid (10:9:6:5 v/v)
plate was dried in hot air oven at 105° for 5
min
Scanning: Camag thin layer chromatography
(TLC) scanner-III linked to Wincats software
280nm 75
2. Ellagic acid Analysis of Ellagic acid in Fresh and processed fruit
products by High Performance Thin Layer
Chromatography
Stationary phase :- TLC plates precoated with
0.2-mm layers of silica gel 60F254
Mobile phase:- Toluene: Ethyl acetate:
Formic acid= 5:5:2.5 v/v
Rf Value: 0.35
254nm 76
3. Ellagic acid,
Gallic acid and
Picroside-I
Quantification of Ellagic acid, Gallic acid and
Picroside-I from Phalatrikadi kvatha churna
by HPTLC
Stationary phase :- TLC plates precoated with
0.2-mm layers of silica gel 60F254
Mobile phase:- ethyl acetate-formic acid
:methanol (6:0.6:0.4 v/v)
280nm 77
Chapter 2. Ellagic acid
47
4. Gallic acid &
Ellagic acid HPTLC Method for Estimation of Ellagic Acid and
Gallic Acid in Triphala churanam Formulations.
Stationary phase :- TLC plates precoated with
0.2-mm layers of silica gel 60F254
Mobile phase:-
280nm 78
5. Gallic acid &
Ellagic acid
Quantification of gallic acid and ellagic acid in
arjunarishta by validtaed hptlc densitometry.
Stationary phase :- 20 x 10 cm HPTLC plates
coated with 0.25 mm layers of silica gel 60
F254
Mobile phase:- toluene- ethyl acetate- formic
acid- methanol, 6+6+1.2+0.25 (v/v) (Gallic
acid)
toluene-ethyl acetate-formic acid-methanol,
9+9+3+0.6 (v/v)(Ellagic acid)
Rf Value: 0.49±0.02 (Gallic acid)
0.46±0.02 (Ellagic acid)
290 &
285nm
79
6. Gallic acid &
Ellagic acid
Comparison & Quantification of Marker compound
of Triphala Guggulu by using HPTLC method.
Stationary phase :- TLC aluminium Plates
precoated with silica gel 60F254
Mobile phase:- toluene:ethyl acetate:formic
acid:methanol(3:3:0.8:0.5v/v/v/v)
Rf Value: 0.57±0.02 (Gallic acid)
0.48±0.02 (Ellagic acid)
280nm 80
7. Ellagic acid HPTLC method for the quantification of Ellagic
acid in different Eucalyptus species
Stationary phase :- Polamide F254 TLC Plate
Mobile phase:- Ethyl acetate: Formic acid:
water (17: 2: 3 v/v/v)
Rf Value: 0.26 (Ellagic acid)
394nm 81
8. Ellagic acid Development and Validation of HPTLC method for
estimation of Ellagic acid in antidiabetic herbal
formulation
Stationary phase :- Precoated Silica F254 TLC
Plate
Mobile phase:-Toluene: Ethyl acetate: Formic
acid: Methanol (3: 3: 8: 2 v/v/v)
Rf Value: 0.90 (Ellagic acid)
280nm 82
Chapter 2. Literature Review
48
2.3 CURCUMIN
TABLE 2.11 HPLC Method for Curcumin
Sr.
No.
Drug Method specification Detection
wavelengt
h
Ref.no
1. curcumin,
demethoxycur
cumin, and
bisdemethoxyc
urcumin
Improved HPLC Method for the Determination of
Curcumin, Demethoxycurcumin, and Bis
demethoxycurcumin
Stationary phase :- C18 column
Mobile phase :-methanol, 2% AcOH, and
acetonitrile in different proportion
flow rate of 1 mL/min
425nm 83
2. curcumin,
demethoxycur
cumin, and
bisdemethoxyc
urcumin
Development and validation of Improved Reverse
Phase HPLC method for simultaneous determination
of curcumin, demethoxycurcumin, and
bisdemethoxycurcumin
Stationary phase :- C18 column
Mobile phase :- Acetonitrile:0.1%trifluoro
acetic acid(50:50)
flow rate of 1.5 mL/min
420nm 84
3. Curcumin and
piperine
Development and validation of simultaneous
estimation method for curcumin and piperine by RP-
UFLC
Stationary phase :- Phenomenex C8 column
(250 x 4.6 mm, 5μ i.d.)
Mobile phase :-25 mM potassium dihydrogen
ortho phosphate buffer (pH 3.5) and acetonitrile
(30: 70 v/v)
flow rate of 1 mL/min
Retention time: 4.4 min and 5.2 min for
curcumin & piperine
280nm 85
4. Curcumin and
piperine
A Liquid Chromatography Method for the
Simultaneous Determination of Curcumin and
Piperine in Food Products Using Diode Array
Detection
Stationary phase :- C18 column (250 X 4.6 mm)
Mobile phase :-50mM potassium dihydrogen
orthophosphate (pH 3.5): Acetonitrile (40:60)
flow rate of 1 mL/min
424nm &
340nm
86
Chapter 2. Curcumin
49
5. Curcuminoid Determination of Curcuminoid pigments in Turmeric
by Reverse Phase High Performance Liquid
Chromatography
Stationary phase :- Styrene Divinyl Benzene
copolymer column
Mobile phase :-Acetonitrile: Water (55:45 %
v/v)
flow rate of 1 mL/min
Ambient temperature
425nm 87
6. sinomenine,
paeoniflorin,
paeonol, and
curcumin
Combinative method using HPLC quantitative and
qualitative analyses for quality consistency
assessment of a herbal medicinal preparation
Stationary phase :- Phenomenex ODS column
Mobile phase :-acetonitrile and aqueous phase
(containing 0.1% phosphoric acid, adjusted with
triethylamine to pH 3.5 ± 0.2) with gradient
elution
flow rate of 1 mL/min
88
7. Curcumin A Sensitive Reversed Phase HPLC Method for the
Determination of Curcumin
Stationary phase :- Merck C15 (250 cm X 4.6
mm)
Mobile phase :-acetonitril: tetrahydrofuran: 2%
acetic acid 50:30:20 (2%)
flow rate of 0.7 mL/min
Retention Time: 4.587 minutes
425nm 89
8. Curcumin &
piperine
Application of validated RP-HPLC-PDA method for
the
simultaneous estimation of curcumin and piperine in
Eudragit E 100 nanoparticles
Stationary phase :- Luna C18 column (Reversed
phase, 150 mm _ 4.6 mm with 5 µm
Mobile phase :-0.1% ortho phosphoric acid
aqueous solution and acetonitrile (45:55, v/v)
flow rate of 1.2 mL/min
Retention Time: curcumin at 8.685 min and
piperine at 5.969 min
262nm 90
9. Quercetin and
curcumin UV spectrophotometric and HPLC method
development of Quercetin and curcumin in
polyherbal churna and it’s validation.
Stationary phase :- HiQ Sil C-18 column (150
mm x 4.6 mm with 5 micron)
Mobile phase :-methanol: acetonitrile:
phosphate buffer (pH 5) in the ratio of 42.5 :
42.5: 15 % v/v/v
flow rate of 1.2 mL/min
Retention Time: 3.220 min. & 4.287 min. for
quercetin and curcumin respectively.
265nm 91
Chapter 2. Literature Review
50
10 curcumin,
desmethoxycurcu
min and
bisdesmethoxycurc
umin
A simple isocratic HPLC method for the
simultaneous determination of curcuminoids in
commercial turmeric extracts.
Stationary phase :- Reverse-phase
chromatography on an Alltima C18 column
Mobile phase :-acetonitrile and 2% v/v
acetic acid (40:60, v/v)
flow rate :- 2 mL/min
Retention Time: 3.220 min. & 4.287 min.
for quercetin and curcumin respectively.
425nm 92
11 Curcumin Stability-indicating RP-HPLC determination of
Curcumin in Vicco Turmeric cream and
Haridrakhand churna
Stationary phase :- Lachrom HPLC with
Lichrospher, ODS, (250× 4.6) mm, 5 μm
Mobile phase :-ACN: THF: 2%Aceticacid:
Water (35: 30: 20:15 v/v/v/v)
flow rate :- 0.5 mL/min
Retention Time: 6.2 min.
429nm 93
12. Curcumin and
Cinnamaldehyde
Development and Validation of RP- HPLC
method for the simultaneous determination of
Cinnamaldehyde and Curcumin in
Pharmaceutical Formulation of Lozenge
Stationary phase :- C18 (250 × 4.6 mm)
5µm
Mobile phase :-ACN: Methanol: Water (32:
36: 32 v/v/v)
flow rate :- 1 mL/min
Retention time of Cinnamaldehyde:-
5.656min
Retention time of Curcumin:- 9.213min
280nm 94
13. Curcumin A simple, sensitive and rapid isocratic reversed-
phase high- performance liquid chromatography
method for the determination and stability study
of curcumin in pharmaceutical samples
Stationary phase :- C18 (150 × 4.6 mm)
5µm
Mobile phase :-ACN: Ammonium acetate
(pH 3.5) (45:55 v/v)
flow rate :- 1 mL/min
Retention time of Curcumin:- 17min
425nm 95
Chapter 2. Curcumin
51
14. Curcumin A New Stability indicating RP – HPLC method
for determination of curcumin: An application to
nanoparticulate formulation
Stationary phase :- Jasco HPLC- MD2010-
PDA C18 column
Mobile phase :-
Solvent A:- ACN
Solvent B:- Phosphate Buffer pH 3
Flow rate:- 1 mL/min
Retention time of Curcumin:- 16.10min
422nm 96
15. Curcumin and
Piperine
Development and Validation of stability
indicating RP – HPLC method for simultaneous
estimation of curcumin and piperine in bulk
mixture
Stationary phase :- Synchronis C18 (250 ×
4.6 mm) 5µm column
Mobile phase :-ACN: Water (0.1% Acetic
acid, pH 3.2) (60:40 v/v)
Flow rate:- 1mL/min
Retention time of curcumin:- 8.14min
Retention time of Piperine:- 9.04min
343nm 97
16. Curcumin and β-
Boswellic acid
Novel Validated HPLC method development for
simultaneous analysis of curcumin and β-
Boswellic acid
Stationary phase :- C18 (250 × 4.6 mm)
5µm column
Mobile phase :-ACN: Water (90:10 v/v)
Flow rate:- 1mL/min
Retention time of curcumin:- 3.32min
Retention time of β- Boswellic acid:-
8.44min
425nm and
242nm
98
17. Curcumin,
demethoxycurcumi
n and
bisdemethoxycurc
umin
A simple binary reverse phase high performance
liquid chromatographic method for the
determination of Curcumin, demethoxycurcumin
and bisdemethoxycurcumin
Stationary phase :- C18 (ODS-3, 250 × 4.6
mm) 5µm column
Mobile phase :-0.1% w/v Orthophosphoric
acid: Acetonitrile (1:1 v/v)
Flow rate:- 1 mL/min
Retention time of curcumin:- 19.22min
Retention time of demethoxycurcumin:-
17.62min
Retention time of bisdemethoxycurcumin:-
16.14min
420nm 99
Chapter 2. Literature Review
52
18. Curcumin Stability indicating RP- HPLC determination of
Curcumin in vicco turmeric cream and kasturi
turmeric churna
Stationary phase :- Agilent – TC C18 (250 ×
4.6 mm) 5µm column
Mobile phase :- Methanol : Acetonitrile: 5%
Acetic acid (35: 50: 15 v/v/v)
Flow rate:- 1 mL/min
Retention time of curcumin:- 4.92min
420nm 100
19. Tetrahydrocurcumi
n
RP- HPLC method development and validation of
tetrahydrocurcumin using multilevel full -
factorial design in bulk, nanoemulsion and
liposomes
Stationary phase :- C18 Qualisil BDS (250
× 4.6 mm) 5µm column
Mobile phase :- Acetonitrile: Methanol
(53:47 v/v/v) and 0.26% v/v with glacial
acetic acid
Flow rate:- 0.6 mL/min
Retention time of tetrahydrocurcumin:-
4.5min
280nm 101
20. Curcumin and
Catechin
Analytical method development and validation
for simultaneous estimation of catechin and
curcumin by HPLC in an Ayurvedic formulation
Stationary phase :- Hemochrom Intsil C18
(250 × 4.6 mm) 5µm column
Mobile phase :- Methanol: 0.1%
Orthophosphoric acid
Flow rate:- 0.8 mL/min
Retention time of curcumin:- 7.66min
Retention time of catechin:- 3.16min
269nm 102
21. Curcumin Curcuminoid content of curcuma longa L. and
Curcuma xanthorrhiza rhizome based on drying
method with NMR and HPLC – UVD
Stationary phase :- C18 (150 × 4.6 mm)
7µm column
Mobile phase :- Methanol
Flow rate:- 1 mL/min
Retention time of curcuminoid:- 2.27min
420nm 103
Chapter 2. Curcumin
53
22. Curcumin and
Acyclovir Development and validation of high- performance
liquid chromatography method for simultaneous
determination of acyclovir and curcumin in
polymeric microparticles
Stationary phase :- Phenomenex C18 (150
× 4.6 mm) 5µm column
Mobile phase :- Acetonitrile: 0.1%
Phosphoric acid: Methanol (50: 40: 10
v/v/v)
Flow rate:- 0.8 mL/min
Retention time of curcumin:- 10.01min
Retention time of acyclovir:- 2.93min
254nm 104
23. Curcumin and
Gefitinib Simultaneous Estimation of Curcumin and
Gefitinib in bulk by using RP- HPLC technique
with PDA detector
Stationary phase :- Qualisil BDS C18 (250
× 4.6 mm) 5µm column
Mobile phase :- Acetonitrile: Water with 0.1
% formic acid (30:70 v/v)
Flow rate:- 0.2 mL/min
242nm 105
24. Curcumin and
Cyclosporine Analytical method development and validation
for simultaneous estimation of curcumin and
cyclosporine by RP- HPLC
Stationary phase :- Eclipse XDB-C18 (150
× 4.6 mm) 5µm column
Mobile phase :- Acetonitrile: Water:
Methanol (50: 10: 40 v/v/v)
Flow rate:- 0.5 mL/min
Retention time of curcumin:- 3.04min
Retention time of cyclosporine:- 6.58min
214nm 106
Chapter 2. Literature Review
54
TABLE 2.12 HPTLC Method for Curcumin
Sr.No. Drug Method specification Detection
wavelength
Ref.no
1. Curcumin,
Dimethoxy
curcumin &
bisdemethoxy
curcumin
Extraction & Purification of curcuminoids from
turmeric.
Stationary phase :- TLC plates precoated with
0.2-mm layers of silica gel 60F254
Mobile phase:- Chloroform :Methanol
(95:5v/v)
Rf Value: 0.67, 0.6 and 0.506 Curcumin,
Dimethoxy curcumin & Bis demethoxy
curcumin
420nm 107
2. Curcumin A HPTLC Method for chemotaxonomic evaluation
of some curcuma species and their commercial
samples
Stationary phase :- TLC plates precoated with
0.2-mm layers of silica gel 60F254
Mobile phase:- Chloroform: Ethanol:Acetic
acid(95:4:5 v/v)
Rf Value: 0.75 for Curcumin.
260nm 108
3. Camphor
Curcumin,
dimethoxy
Curcumin
& bis
demethoxy
Curcumin
Comparison of Curcuma caesia Roxb. with
other Commonly Used Curcuma Species by HPTLC
Stationary phase :- Merck TLC plates
precoated
with silica gel 60 F254 (10 cm X 10 cm with
250 μm layer thickness)
Mobile phase:- toluene: ethyl acetate:
methanol (18:1:1) up to 80 mm distance
Anisaldehyde sulfuric acid reagent is used as
derivatizing agent for visualization
Rf Value: Camphor at 0.6, curcumin at 0.38,
demethoxycurcumin at 0.3 and bis-demethoxy
curcumin at 0.24.
Spraying
Reagent
109
4. Curcumin and
Gallic acid Development and Validation of HPTLC Method to
Detect Curcumin and Gallic Acid in Polyherbal
Microencapsulated Formulation.
Stationary phase :- silica gel 60 F254
Mobile phase:- chloroform:ethyl
acetate:formic acid:methanol
(7.5 mL + 6 mL + 0.5 mL + 0.5 mL)
Rf Value:- curcumin at 0.59 ± 0.02, Gallic
acid at 0.25 ± 0.03.
322nm 110
5. Curcumin Validated method for estimation of curcumin from
different varieties of curcuma longa.
Stationary phase :- precoated aluminium
backed HPTLC plates of 0.2 mm layer
thickness with silica gel 60 F254
Mobile phase:- chloroform: methanol (9.5:0.5)
plate was developed up to 80 mm at
temperature of 20 ± 4oC for 10 min.
421nm 111
Chapter 2. Curcumin
55
6. Curcumin Validated HPTLC analysis method for
quantification of variability in content of curcumin
in Curcuma longa L (turmeric) collected from
different geographical region of India
Stationary phase :- TLC aluminum plates
precoated with silica gel 60F254
Mobile phase:- toluene-chloroform-methanol
(5:4:1, v/v/v)
Rf Value:- curcumin at 0.31±0.02
430nm 112
7. Curcumin Validated method for estimation of curcumin in
turmeric powder.
Stationary phase :- 0.2 mm layer thickness
with silica gel 60 F254
Mobile phase:- dichloromethane and methanol
(99:1)
Rf Value:- curcumin at 0.43
427nm 113
8. Curcumin and
Gallic acid
Development and validation of HPTLC method to
detect Curcumin and Gallic acid in polyherbal
formulation.
Stationary phase :- TLC aluminum plates
precoated with silica gel 60 F254
Mobile phase:- chloroform:ethyl
acetate:formic acid (7.5 mL + 6 mL + 0.5 mL)
Rf Value :- curcumin at 0.55 ± 0.02, gallic
acid at 0.26 ± 0.03
254nm 114
9. Curcuminoids Improved HPTLC Method for Determination of
Curcuminoids from Curcuma longa.
Stationary phase :- precoated HPTLC
LiChrosphere aluminium plates Si 60F254
Mobile phase:- chloroform‐methanol (98∶2
v/v)
366nm 115
10 Curcumin &
Ellagic acid
Development and Validation of HPTLC Method for
Estimation of Curcumin, Ellagic acid in Gel
Formulation.
Stationary phase :- silica gel 60 F254 TLC
plate
Mobile phase:- toluene: ethyl acetate:
methanol: formic acid (2.5: 2.5: 0.2: 0.8)
Rf Value :- curcumin at 0.6 , Ellagic acid at
0.5.
- 116
Chapter 2. Literature Review
56
11. Curcumin,
Metanil
Yellow, and
Sudan Dye
A Simple 2-Directional High-Performance Thin-
Layer Chromatographic Method for the
Simultaneous Determination of Curcumin, Metanil
Yellow, and Sudan Dyes in Turmeric, Chili, and
Curry Powders
First Direction:
Stationary phase :- silica gel 60 F254 TLC
plate
Mobile phase:- chloroformmethanol (9 1, v/v)
Rf Value :-curcumin (0.77),
demethoxycurcumin (0.69),
bis(demethoxy)curcumin (0.61), and the
synthetic dye metanil yellow (0.05).
Second Direction:
Stationary phase :- silica gel 60 F254 TLC
plate
Mobile phase:- , toluene: hexane: acetic acid
(50 :50 :1, v/v/v)
Rf Value :- sudan I (0.30) and sudan IV (0.23)
- 117
12. Curmin &
piperine
Simultaneous Estimation of Curcumin and Piperine
in Their Crude Powder Mixture and Ayurvedic
Formulation Using High Performance Thin Layer
Chromatography.
Stationary phase :- TLC aluminium plates
precoated with silica gel G60 F254.
Mobile phase:- Chloroform: Methanol (9.6:0.4
v/v)
Rf Value :-curcumin at 0.57 and piperine at
0.82
373nm 118
13. Curmin,
piperine &
thymol
Rapid HPTLC method for identification and
quantification of curcumin, piperine and thymol in
an ayurvedic formulation.
Stationary phase :- silica gel 60 F 254 plates
Mobile phase:- toluene-ethyl acetate-
methanol, 9 + 1 + 0.5 ( v/v )
Rf Value : curcumin at 0.23, piperine at 0.30,
and thymol were at 0.64.
420, 333 and
277nm
119
14. Curcumin,
Piperine &
Boswellic acid
Development and validation of HPTLC method to
detect curcumin, piperine, and boswellic acid in
polyherbal transdermal patch.
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- chloroform: ethyl acetate:
formic acid (7.5 mL + 6 ml + 0.2 mL)
Rf Value : curcumin at 0.48 ± 0.02, piperine at
0.52 ± 0.03, and boswellic acid at 0.61 ± 0.03)
540 nm 120
Chapter 2. Curcumin
57
15 Curcumin,
demethoxycur
cumin &
bisdemethoxyc
urcumin
High-performance thin layer chromatographic
method for quantitative determination of
curcuminoids in Curcuma longagermplasm
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- chloroform: methanol
(48:2, v/v)
Rf Value : curcumin, demethoxycurcumin and
bisdemethoxycurcumin (RF value of
0.66 ± 0.02, 0.48 ± 0.02 and 0.30 ± 0.02)
425nm 121
16. Curcumin,
demethoxycur
cumin &
bisdemethoxyc
urcumin
Development of HPTLC Method and Its Validation
for the Estimation of Curcuminoids from
Polyherbal Mouth Ulcer Gel Formulation.
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- Chloroform: methanol: Glacial
acetic acid (7.5: 2.0: 0.5 v/v/v)
Rf Value : curcumin at 0.56,
demethoxycurcumin at 0.31 and
bisdemethoxycurcumin at 0.18.
430nm 122
17. Curcumin,
demethoxycur
cumin &
bisdemethoxyc
urcumin
Occurrence of curcuminoids in Curcuma longa : A
quality standardization by HPTLC.
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- chloroform:methanol (48:2,
v/v)
Rf Value : curcumin at 0.67,
demethoxycurcumin at 0.47 and
bisdemethoxycurcumin at 0.29.
425nm 123
18. Gallic acid,
Curcumin &
Quercetin
Simultaneous estimation of Gallic acid, Curcumin
and Quercetin by HPTLC method.
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- toluene: ethyl acetate: forminc
acid (4.5:3.0:0.2 v/v/v)
Rf Value :gallic acid at 0.40, curcumin at 0.73
and quercetin at 0.55.
366nm 124
19. Curcumin Stability-indicating HPTLC determination of
curcumin in bulk drug and pharmaceutical
formulations.
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- chloroform:methanol
(9.25:0.75 v/v)
Rf Value :Curcumin at 0.48 ± 0.02
430nm 125
Chapter 2. Literature review
58
20 Curcumin Validated HPTLC method for estimation of
curcumin content in dietary supplement
formulation.
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- n-hexane: ethyl acetate:
methanol: formic acid (8: 2: 1: 2-3 drops v/v)
Rf Value :Curcumin 0.29
421nm 126
21. Curcumin Standardization of an Ayurvedic Formulation-
Kalyanavleha and estimation of curcumin using
HPTLC.
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- toluene : ethyl acetate (8:2)
and toluene: ethyl acetate: methanol (9: 1: 1)
as mobile phase for hexane soluble and
chloroform soluble fractions respectively
Rf Value :different for different species.
366nm 127
22. Curcumin,
demethoxycur
cumin &
bisdemethoxyc
urcumin
Development and standardization of turmeric
cream by HPTLC.
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- chloroform: ethanol: acetic
acid (48:2:0.1 v/v/v)
Rf Value : curcumin at 0.38 ,
demethoxycurcumin at 0.23 and
bisdemethoxycurcumin at 0.16.
300nm 128
23. Curcumin and
galangin
Development and validation of HPTLC method for
simultaneous estimation of curcumin and galangin
in Polyherbal capsule dosage form
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- n-hexane: ethyl acetate : acetic
acid: methanol (7: 2: 0.5: 0.5 v/v/v)
Rf Value : curcumin at 0.28 , galangin: 0.48
404nm 129
24. Curcumin HPTLC method development and quantification of
curcumin content in different extracts of rhizomes
of curcuma longa L.
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- Chloroform: Methanol:
Formic acid (9.6: 0.4: 0.1 v/v/v)
Rf Value : curcumin at 0.70
366nm 130
25. Curcumin and
Catechin Analytical method development and its validation
for simultaneous estimation of catechin and
curcumin by HPTLC from ancho lean tablets
Stationary phase :- TLC aluminium plates
precoated with silica gel 60F254
Mobile phase:- Toluene: Ethyl acetate: formic
acid (7: 2.5: 0.5 v/v/v)
Rf Value : curcumin at 0.58 and catechin:-
0.23
269nm 131
Chapter 2. Curcumin
59
TABLE 2.13 Ultraviolet Method for Curcumin
Sr.No. Drug Method specification Detection
wavelength
Ref.no
1. Curcumin &
Quercetin Simultaneous estimation of Curcumin and
Quercetin in Ayurvedic Proprietary Medicine by
UV Spectrophotometry
Spectrometer: Shimazdu 1800 UV/visible
spectrophotometric
Solvent: Methanol
λmax of Quercetin is 256 nm and λmax of
Curcumin is 263 nm.
Linearity (Quercetin) :2-20 µg/ml
(Curcumin) : 4-36 µg/ml
256nm
&263nm
132
2. Curcumin UV- Visible spectrophotometric estimation of
curcumin in nano-formulation
Spectrometer: Shimazdu UV/visible
spectrophotometric
Solvent: Methanol
Linearity (curcumin):- 5-25 µg/ml
421nm 133
3. Curcumin Development and validation of UV
spectrophotometric method for the estimation of
curcumin in an ayurvedic formulation
haridrakhand
Spectrometer: Shimazdu 1650 UV/visible
spectrophotometric
Solvent: Methanol
Linearity (curcumin):- 2-10 µg/ml
421nm 134
4. Curcumin Development and validation of UV-
Spectrophotometric method for the estimation of
curcumin in standardized Polyherbal formulation
Spectrometer: Jasco double beam UV/visible
spectrophotometric
Solvent: Ethyl acetate
Linearity (curcumin):- 1-5 µg/ml
418nm 135
5. Curcumin and
Capsaicin Development and validation of analytical method
for simultaneous determination of curcumin and
capsaicin in bulk
Spectrometer: Sican 2301 UV
spectrophotometer
Solvent: Methanol
Linearity (curcumin):- 1-5 µg/ml
(Capsaicin)- 25-125 µg/ml
421nm and
280nm
136
Chapter 2. Literature Review
60
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88. Ying Xie, Zhi-Hong Jiang, Hua Zhou, 2007, “Combinative method using HPLC quantitative and
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89. Yadav VR, Sarasija S, 2009, “A Sensitive Reversed Phase HPLC Method for the Determination of
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90. C. Moorthi a, C. Senthil Kumar b, S. Mohan b, Kiran Krishnan c, K. Kathiresan , 2013, “Application of
validated RP-HPLC-PDA method for the simultaneous estimation of curcumin and piperine in Eudragit
E 100 nanoparticles.” Journal of Pharmacy Research, 7, 224-229, ISSN No. 0974-6943.
91. Salunkhe VR, Patil SJ, 2014, “UV spectrophotometric and HPLC method development of Quercetin and
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92. Wisut Wichitnithad et al, 2009, “A simple isocratic HPLC method for the simultaneous determination of
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1565.
93. ChittoraNawal Kishore et al, 2010, “Stability-indicating RP-HPLC determination of Curcumin in Vicco
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94. Gavhad G, Sirsath V, Chaware V and Biyani K. 2015, “Development and Validation of RP- HPLC method
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Lozenge.” International Journal of Chemical and Pharmaceutical Sciences, 4(3), 311-316, ISSN No. 0976-
9390.
95. Amanolahi F, Mohammadi A, Oskuee RK, Nassirli H and Nokouei BM. 2017, “A simple, sensitive and
rapid isocratic reversed- phase high- performance liquid chromatography method for the determination
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444-453, ISSN No. 2228-7930.
96. Panigrahi S and hirlekar R. 2016, “A New Stability indicating RP – HPLC method for determination of
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Pharmaceutical Science, 8(12), 149-155, ISSN No. 2656-0097.
97. Chahar MY and Mashru R. 2016, “Development and Validation of stability indicating RP – HPLC method
for simultaneous estimation of curcumin and piperine in bulk mixture.” World Journal of Pharmaceutical
Research, 5(5), 1262-1276, ISSN No. 2277-7105.
Chapter 2. References
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98. Nighojkar PA, Momin M and Jain V. 2016, “Novel Validated HPLC method development for
simultaneous analysis of curcumin and β- Boswellic acid.” American Journal of PharmTech Research,
6(02), 201-208, ISSN No. 2249-3387.
99. Yedugani Lingam et al. 2016, “A simple binary reverse phase high performance liquid chromatographic
method for the determination of Curcumin, demethoxycurcumin and bisdemethoxycurcumin.” Natural
Products: An Indian Journal, 12(1), 26-30, ISSN No. 0974-7508.
100. Yoshasri J, Venkata SP and Ramarao N. 2017, “Stability indicating RP- HPLC determination of
Curcumin in vicco turmeric cream and kasturi turmeric churna.” International Journal of Development
Research, 7(8), 14692-14714, ISSN No. 2230-9926.
101. Chhabra N, Solanki A, Athawale R and Mahajan S. 2017, “RP- HPLC method development and
validation of tetrahydrocurcumin using multilevel full - factorial design in bulk, nanoemulsion and
liposomes.” European Journal of Pharmaceutical and Medical Research, 4(9), 275-281, ISSN No. 2394-
3211.
102. Joshi S, Dr. Jadhav VM and Dr. Kadam VJ. 2018, “Analytical method development and validation for
simultaneous estimation of catechin and curcumin by HPLC in an Ayurvedic formulation.” World
Journal of Pharmacy and Pharmaceutical Sciences 7(4), 1693-1702, ISSN No. 2656-0097.
103. Hadi S, Artanti AN. Rinanto Y and Wayuni DSC. 2018, “Curcuminoid content of curcuma longa L. and
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Series: Materials sciences and Engineering, 349, ISSN No. 1757-899X.
104. Reolon et al. 2018, “Development and validation of high- performance liquid chromatography method
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Applied Pharmaceutical Science, 8(1), 136-141, ISSN No. 2231-3354.
105. Savale SK. 2018, “Simultaneous Estimation of Curcumin and Gefitinib in bulk by using RP- HPLC
technique with PDA detector.” Hygeia Journal of Drugs and Medicines, 10(1), 1-8, ISSN No. 2229-
3590.
106. Desai N, Momin M, Singh U, Khan T and Sherje A. 2019, “Analytical method development and
validation for simultaneous estimation of curcumin and cyclosporine by RP- HPLC.” International
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107. Kulkarni SJ, Maske KN, Budre MP, R.P. Mahajan, 2012 “Extraction & Purification of Curcuminoid
from turmeric”. International Journal of Pharmacology and Pharmaceutical Technology, (2), 81-84,
ISSN No. 2166-7276.
108. Sharad Srivastava, 2009, “A HPTLC Method for chemotaxonomic evaluation of some curcuma species
and their commercial samples.” Journal of Scientific and Industrial Research, 68, 876-880, ISSN No.
0975-1084.
109. Reshma V. Lawand, Santosh V. Gandhi, 2013, “Comparison of Curcuma caesia Roxb. With other
Commonly Used Curcuma Species by HPTLC”, Journal of Pharmacognosy and Phytochemistry. 2 (4),
126-131, ISSN No. 2349-8234.
110. A. K. Chavan, S. A. Nirmal & S. R. Pattan, 2015, “Development and Validation of HPTLC Method to
Detect Curcumin and Gallic Acid in Polyherbal Microencapsulated Formulation.” Journal of Liquid
Chromatography and Related Technology, 38(12), 1213-1217, ISSN No. 1520-572X.
111. Jothiven katachalam Kandasamy, 2013, “Validated method for estimation of curcumin from different
varieties of curcuma longa.” International Journal of Pharma and Biosciences, 4(1), 1004 – 1010, ISSN
No. 0975-6299.
112. Kamran Ashraf, 2012, “Validated HPTLC analysis method for quantification of variability in content
of curcumin in Curcuma longa L (turmeric) collected from different geographical region of India”,
Asian Pacific Journal of Tropical Biomedicine, 2(2), 584-588, ISSN No. 2221-1691.
113. Arunava Gantait, Topu Barman & Pulok K. Mukherjee, 2011, “Validated method for estimation of
curcumin in turmeric powder.” Indian Journal of Traditional Knowledge, 10(2), 247-250, ISSN No.
0975-1068.
114. S. D. Sonawane, S. A. Nirmal, A. N. Patil & S. R. Pattan, 2011, “Development and validation of HPTLC
method to detect Curcumin and Gallic acid in Polyherbal formulation”, Journal of Liquid
Chromatography and Related Technology, 34 (20), 2664-2673, ISSN No. 1520-572X.
115. Vijaylata Pathania, Ajai Prakash Gupta & Bikram Singh, 2006, “Improved HPTLC Method for
Determination of Curcuminoids from Curcuma longa.” Journal of Liquid Chromatography and Related
Technology, 29(6), 877-887, ISSN No. 1520-572X.
Chapter 2. Literature Review
66
116. Bele AA, Jadhav VM, Kadam VJ, 2011, “Development and Validation of HPTLC Method for
Estimation of Curcumin, Ellagic acid in Gel Formulation.” International Journal of Pharmaceutical
chemistry and Analysis, 2(6), ISSN No. 2394-2797.
117. Dixit, Sumita; Khanna, Subhash K; Das, Mukul , 2008, “A Simple 2-Directional High-Performance
Thin-Layer Chromatographic Method for the Simultaneous Determination of Curcumin, Metanil
Yellow, and Sudan Dyes in Turmeric, Chili, and Curry Powders.” Journal of AOAC International,
91(6), 1387-1396, ISSN No. 1060-3271.
118. Niraj Vyas, Kanan Gamit, Mohammad Y. Khan, Siddharth Panchal and K. Pundarikakshudu, 2011,
“Simultaneous Estimation of Curcumin and Piperine in Their Crude Powder Mixture and Ayurvedic
Formulation Using High Performance Thin Layer Chromatography”, International Journal of Research
in pharmacy and Biosciences. 2(1), 231-236, ISSN No. 2321-3272.
119. Jayant Verma, Anil Joshi, 2007, “Rapid HPTLC method for identification and quantification of
curcumin, piperine and thymol in an Ayurvedic formulation.” Journal of Planer Chromatography,
19(111), 52-62, ISSN No. 1789-0993.
120. Vaykole AM, Nirmal SA, Jadhav RS & Pattan SR, 2014, “Development and validation of HPTLC
method to detect curcumin, piperine, and boswellic acid in polyherbal transdermal patch.” Journal of
Liquid Chromatography and Related Technology 37(3), 367-378, ISSN No. 1520-572X.
121. M. Paramasivam, R. Poi, H. Banerjee, A. Bandyopadhyay, 2009, “High-performance thin layer
chromatographic method for quantitative determination of Curcuminoids in Curcuma
longagermplasm.”, Food Chemistry, 113(2), 640-644, ISSN No.0308-8146.
122. Safeena Sheikh, Suhail Asghar, Showkat Ahmad, 2013, “Development of HPTLC Method and Its
Validation for the Estimation of Curcuminoids from Polyherbal Mouth Ulcer Gel Formulation.” IOSR
Journal of Pharmacy, 3(1), 29-34, ISSN No. 2250-3013.
123. M. Paramasivam, Md. WasimAktar, R. Poi, H. Banerjee and A. Bandyopadhyay, 2008, “Occurrence of
Curcuminoids in Curcuma longa, A quality standardization by HPTLC.” Bangladesh Journal of
Pharmacology. 3, 55-58, ISSN No. 1991-007X.
124. Thakker VY, Shah VN, Shah UD, Suthar MP, 2011, “Simultaneous estimation of Gallic acid, Curcumin
and Quercetin by HPTLC method.” Journal of Advanced Pharmacy Education and Research, 1, 70-80,
ISSN No. 2249-3379.
125. Ansari MJ , Ahmad S,Kohli K,Ali J, Khar RK, 2005, “Stability-indicating HPTLC determination of
curcumin in bulk drug and pharmaceutical formulations.” Journal of Pharmaceutical and Biomedical
Analysis, 39(1-2), 132-138, ISSN No. 0731-7085.
126. Kekre VA and Walode SG, 2012, “Validated HPTLC method for estimation of curcumin content in
dietary supplement formulation.” Indian Journal of Pharmaceutical Sciences and Research, 3(10),
3796-3800, ISSN No. 0975-8232.
127. Sayyada Khatoon, 2014, “Standardization of an Ayurvedic Formulation- Kalyanavleha and estimation
of curcumin using HPTLC.” Indian Journal of Traditional Knowledge, 13(3), 535-542, ISSN No. 0975-
1068.
128. Saleemulla Khan, Inder K. Makhija, Devang Khamar, Sandhya Rani, 2010, “Development and
standardization of turmeric cream by HPTLC.” International Journal of Biomedical and Advance
Research, 01(04), 109-116, ISSN No. 2229-3809.
129. Kharat S, Namdeo A and Mehta P. 2017, “Development and validation of HPTLC method for
simultaneous estimation of curcumin and galangin in Polyherbal capsule dosage form.” Journal of
Taibah University for Science, 775-781, ISSN No. 1658-3655.
130. Rasheed NMA, Srividya GS and Nagaiah K. 2017, “HPTLC method development and quantification
of curcumin content in different extracts of rhizomes of curcuma longa L.” Annals of Phytomedicine,
6(2), 74-81, ISSN No. 2393-9885.
131. Joshi S, Jadhav VM and Kadam VJ. 2018, “Analytical method development and its validation for
simultaneous estimation of catechin and curcumin by HPTLC from ancho lean tablets.” Indian Journal
of Pharmaceutical and Biological Research, 6(2), 23-30, ISSN No. 2320-9267.
132. Patil Snehal J., Salunkhe Vijay R. 2012, “Simultaneous estimation of Curcumin and Quercetin in
Ayurvedic Proprietary Medicine by UV Spectrophotometry”. International Journal of Research in
Ayurveda and Pharmacy, 3(2), 267-271, ISSN No. 2277-4343.
133. Hazra et al. 2015, “UV- Visible spectrophotometric estimation of curcumin in nano-formulation.”
International Journal of Pharmacognosy, 2(3), 127-130, ISSN No. 2348-3962.
Chapter 2. References
67
134. Warule PS, Patel VP and Gosavi SA. 2017, “Development and validation of UV spectrophotometric
method for the estimation of curcumin in an ayurvedic formulation haridrakhand.” International Journal
of Pharmaceutics and Drug Analysis, 5(5), 193-197, ISSN No. 2348-8948.
135. Singh A and Avupati VR. 2017, “Development and validation of UV- Spectrophotometric method for
the estimation of curcumin in standardized Polyherbal formulation.” Journal of Young Pharmacist, 9(4),
491-495, ISSN No. 0975-1483.
136. Hulaswar PR and Patil KS. 2019, “Development and validation of analytical method for simultaneous
determination of curcumin and capsaicin in bulk.” Journal of Pharmacognosy and Phytochemistry, 8(1),
904-909, ISSN No. 2349-8234.
68
Chapter 3. Aim of Research
69
CHAPTER-3
Aim and Objective
3.1 Aim of Research
Rationale for selection of Project work:
Literature Survey reveals that several methods such as
Three HPTLC methods for simultaneous estimation of Gallic acid and Curcumin.
Four HPLC and Four HPTLC methods for simultaneous estimation of Gallic and Ellagic
Acid.
One HPTLC method for estimation of Ellagic Acid and Curcumin have been reported.
But, not a single UV, HPLC or HPTLC method is reported so far for simultaneous estimation
of Gallic Acid, Ellagic Acid and Curcumin in Polyherbal Formulation. So Aim of present
research was to develop and validate new Analytical Methods for simultaneous estimation of
Gallic acid, Ellagic acid and Curcumin in Polyherbal formulation.
Chapter 3. Aim and Objective
70
3.2 Objective:
To develop most sensitive RP-HPLC Method for simultaneous estimation of Gallic acid,
Ellagic acid and Curcumin in Polyherbal antidiabetic formulations which can also separate
Curcuminoids.
To develop HPTLC Method for simultaneous estimation of Gallic acid, Ellagic acid and
Curcumin in Polyherbal antidiabetic formulations which is important tool in standardization
of Herbal Medical product in initial stage of Research.
To develop Chemometric Methods, multivariate statistical technique for simultaneous
estimation of Gallic acid, Ellagic acid and Curcumin in Polyherbal antidiabetic formulations
in any dose ration availability.
To develop cost effective UV spectrophotometric Methods for simultaneous estimation of
Gallic acid, Ellagic acid and Curcumin in Polyherbal antidiabetic formulations.
All developed methods to be validated according to ICH Q2 (R1) Guideline.
To perform statistical comparison of developed methods as Method development is prime
requirement for dossier submission and commercial availability.
Chapter 4. Materials and Methods
71
CHAPTER 4
Materials and Methods
TABLE 4. 1 Markers used in Research work
Markers Batch No. CAS No. Company
Gallic acid 3520219V 5995-86-8 Natural Remedies, Bangalore
Ellagic acid 27481064453V 476-66-4 Yucca Enterprise, Mumbai
Curcumin 09118-03V 458-37-7 Natural Remedies, Karnataka
TABLE 4. 2 Marketed Formulations
Formulations Mfg. Lic. No. Batch No. Manufacturing date Expiry Date
Glysikot AUS-800 812463 NOV./2015 OCT./2018
Diasol 559-AY-PB DS 060 MAR/ 2015 FEB/ 2018
Diabeta Plus 350-ISM 2014 AUG/2016 JULY/2019
FIGURE 4. 1 Antidiabetic Polyherbal Formulations for Research
Chapter 4. Materials and Methods
72
TABLE 4.3 Instruments used in Research work
Instrument Company
UV-visible spectrophotometer Shimadzu, model 1800 (double beam)
Electronic analytical balance Reptech (0.1 mg sensitivity)
HPLC System Agilent Technologies 1220 infinity LC(Open Lab Control Panel,
software)
HPTLC instrument CAMAG, Switzerland (win CATS Planar Chromatography
Manager, software)
pH meter Systronics
Ultrasonicator Athena technology
LC-MS/MS system Thermofisher scientific (LCQ Fleet)
TABLE 4.4 Solvents and Reagents used in Research work
Name of Chemical Grade Company
Methanol Analytical Reagent Merck
Methanol HPLC Merck
Acetonitrile HPLC Merck
Water HPLC Merck
Ortho Phosphoric Acid HPLC Merck
Formic Acid HPLC Merck
Ethyl Acetate HPLC Merck
Tri ethyl amine HPLC Merck
Toluene HPLC Merck
TABLE 4.5 Optimized condition for HPLC Method Development
HPLC Instrument Agilent Technologies 1220 infinity LC
Software Open Lab Control Panel
Stationary Phase Agilent C18 column (250 mm, 4.6 mm, 5m)
Mobile Phase Gradient Program
Time (Min) 0.1 %F.A. Acetonitrile
0 70 30
6 50 50
12 10 90
15 10 90
15.1 70 30
18 70 30
Flow Rate 0.95 ml/min
Detection 257 nm ( DAD)
Injection Volume 5 L
Diluent Mobile phase
Column Oven Temperature 30°C
QbD (Central Composite Design) Design Expert 10 (software)
Chapter 4. Materials and Methods
73
TABLE 4.6 Optimized condition for LC-MS/MS Method Development
LC-MS/MS Instrument Thermofisher scientific
Software LCQ Fleet
Stationary Phase Thermosynchronis C18 column (250 mm, 4.6 mm, 5m)
Ionization technique ESI Ionization
Mobile Phase Gradient Program
Time (Min) 0.1 %F.A. Acetonitrile
0 70 30
6 50 50
12 10 90
15 10 90
15.1 70 30
18 70 30
Flow Rate 0.95 ml/min
Detection 257 nm( Ion Trap Detector)
Injection Volume 5 L
Run Time 18 min
Diluent First dilution in methanol and further in mobile phase.
Column Oven Temperature 30°C
TABLE 4.7 Optimized condition for HPTLC Method Development
HPTLC Instrument CAMAG, Switzerland.
Software winCATS Planar Chromatography Manager
Sample Applicator Linomat V, CAMAG, Switzerland.
TLC scanner TLC Scanner_171010
Hemilton syringe Having 100 l capacity
Twin trough chamber 20 * 10 cm
TLC Plates TLC Silica gel 60 F254 ( 20 * 20 cm), Merck
Mobile Phase Toluene: Ethyl Acetate : Formic Acid
3 : 3.5 : 1 v/v
Detection 300 nm (D2 lamp)
Temperature 50°C
Chamber Saturation time 15 minutes
74
Chapter 5. Melting Point/Solubility study determination
75
CHAPTER 5
Preliminary Work
Melting point study, Solubility Study and Infrared spectroscopic study were performed for
further confirmation of Markers (Gallic acid, Ellagic acid and Curcumin).
5.1 Melting Point determination
TABLE 5.1 Determination of melting point
Sr. No. Markers Standard value [36-38, Ch-1] Observed value
1 Gallic Acid 260-262 ˚C 258-259 ˚C
2 Ellagic Acid 358-362 ˚C 359-363 ˚C
3 Curcumin 183-185 ̊ C 184-187˚C
5.2 Solubility Study:
The solubility of Gallic Acid, Ellagic Acid and Curcumin in some solvents were practically
determined. Solubility was determined by taking separately 10 mg of Gallic Acid, Ellagic
Acid and Curcumin in 10 ml volumetric flasks, adding required quantity of solvent at room
temperature and shaking for few minutes. Solubility data for each compound were obtained
and shown following Table 5.2
TABLE 5. 2 Solubility testing
Marker Methanol Ether Water
Gallic Acid Freely Soluble Soluble Slightly soluble
Ellagic Acid Soluble Insoluble Insoluble
Curcumin Freely Soluble Insoluble Insoluble
Chapter 5. Preliminary Work
76
5.3 Infrared Spectroscopic Study: IR spectroscopic study were performed at Centre of
Excellence (Div. of Vapi Green Enviro Limited).
Model: PerkinElmer Frontier FT-IR.
5.3.1 Gallic Acid
(a) Reference standard [1]
(b) Sample
FIGURE 5. 1 IR graph for Gallic acid (a) Reference standard (b) Sample
TABLE 5.3 IR Interpretation for Gallic Acid
Functional Group Standard Range cm-1 Observed Value cm-1
O-H (Stretch, H-bonded) 3200-3600 3286.01, 3371.25
C=O (stretch) (Acid) 1700-1725 1702.77
C=C stretching (Aromatic) 1400-1600 1541.94
C-O (stretch) (Acid) 1210-1320 1247.80
Chapter 5. Infrared Spectroscopy study
77
5.3.2Ellagic Acid
(a) Reference Standard [2]
(b) Sample
FIGURE 5. 2 IR graph for Ellagic acid (a) Reference standard (b) Sample
TABLE 5.4 IR Interpretation for Ellagic Acid
Functional Group Standard Range cm-1 Observed Value cm-1
O-H (Stretch, H-bonded) 3200-3600 3556.04
C-H (Stretch) (Aromatic) 3000-3100 3081.88
C=O (Aryl Ketone) 1680-1700 1699.23
C=C stretching (Aromatic) 1400-1600 1508.61, 1583.58
C-O (stretch) (ester) 1000-1300 1112.86
Chapter 5. Preliminary Work
78
5.3.3Curcumin
(a)Reference Standard [3]
(a) Sample
FIGURE 5. 3 IR graph of Curcumin (a) Reference standard (b) Sample
TABLE 5.5 IR Interpretation for Gallic Acid
Functional Group Standard Range cm-1 Observed Value cm-1
O-H (stretch, Free) 3500-3700 3511.70
C=O (Stretch, Ketone) 1620-1680 1628.11
C-O (Ether) 1000-1300 1281.87
C=C (Aromatic) 1400-1600 1429.83
Chapter 5 References
79
References
1. S. Meenakshi et al, 2009, “Total Flavanoid and in vitro Antioxidant Activity of Two Seaweeds of
Rameshwaram Coast”, Global Journal of Pharmacology, 3 (2): 59-62, ISSN No. 1992-0075.
2. Subrahmanyam G et al, 2013, “Ellagic acid – A Novel Organic Electrode Material for High Capacity Lithium
Ion Batteries”, Electronic Supplementary Material (ESI) for Chemical Communications, ISSN No. 1364-548X
3. Esmail EH et al, 2014, “Synthesis and Characterization of some Ternary Metal Complexes of Curcumin with
1, 10-phenanthroline and their Anticancer Applications, Journal of Scientific Research, 6 (3), 509-519, ISSN
No.2070-0237.
80
Chapter 6 Experimental Work
81
CHAPTER - 6
RP- HPLC Method Development
6.1 Experimental work [1,2]
6.1.1 Materials and Instruments: Refer Table 4.1, 4.2 and 4.3.
6.1.2 Solvents and Reagents: Refer Table 4.4
6.1.3 Preparation of Stock solution and working standard solution
PREPARATION OF STANDARD STOCK SOLUTION OF GALLIC ACID:
Accurately weighed 10 mg of Gallic Acid was transferred into 10 mL volumetric flask,
dissolved and diluted up to mark with methanol to get 1000 μg/mL solution of Gallic acid.
PREPARATION OF WORKING STANDARD SOLUTION OF GALLIC ACID:
100 μg/mL of Gallic Acid solution was prepared by diluting 1 mL of stock solution in 10
mL with methanol.
PREPARATION OF STANDARD STOCK SOLUTION OF ELLAGIC ACID:
Accurately weighed 10 mg of Ellagic Acid was transferred into 10 mL volumetric flask,
dissolved and diluted up to mark with methanol to get 1000 μg/mL solution of Ellagic acid.
PREPARATION OF WORKING STANDARD SOLUTION OF ELLAGIC ACID:
100 μg/mL of Ellagic Acid solution was prepared by diluting 1 mL of stock solution in
10 mL with methanol.
PREPARATION OF STANDARD STOCK SOLUTION OF CURCUMIN:
Accurately weighed 10 mg of Curcumin was transferred into 10 mL volumetric flask,
dissolved and diluted up to mark with methanol to get 1000 μg/mL solution of Curcumin.
Chapter 6 RP-HPLC Method Development
82
PREPARATION OF WORKING STANDARD SOLUTION OF CURCUMIN:
100 μg/mL of Curcumin solution was prepared by diluting 10 mL of stock solution in 100
mL with methanol.
6.1.4 Preparation of Calibration curve
CALIBRATION CURVE FOR GALLIC ACID:
The solutions of Gallic Acid ranging from 2-14 μg/mL were prepared by pipetting out 0.2, 0.4,
0.6, 0.8, 1.0, 1.2 & 1.4 mL from the working standard solution of Gallic Acid (100 μg/mL) into
series of 10 mL volumetric flasks and the volume was adjusted to mark with mobile phase to
get concentration of 2, 4, 6, 8, 10, 12 & 14 μg/mL of Gallic Acid.
CALIBRATION CURVE FOR ELLAGIC ACID:
Solutions of Ellagic Acid ranging from 5-35 μg/mL were prepared by pipetting out 0.5, 1.0, 1.5,
2.0, 2.5, 3.0 & 3.5 mL from the working standard solution of Ellagic Acid (100 μg/mL) into
series of 10 mL volumetric flasks and the volume was adjusted to mark with mobile phase to
get concentration of 5, 10, 15, 20, 25, 30 & 35 μg/mL of Ellagic Acid.
CALIBRATION CURVE FOR CURCUMIN:
Solutions of Curcumin ranging from 1-7 μg/mL were prepared by pipetting out 0.1, 0.2, 0.3,
0.4, 0.5, 0.6 and 0.7mL from the working standard solution of Curcumin (100 μg/mL) into series
of 10 mL volumetric flasks and the volume was adjusted to mark with mobile phase to get
concentration of 1, 2, 3, 4, 5, 6 & 7μg/mL of Curcumin.
6.1.5 Determination of Wavelength for Measurement
0.4 mL of working standard solution of Gallic Acid (4 μg/mL), 1 mL of working standard
solution of Ellagic Acid (10 μg/mL) and 0.2 mL of working standard solution of Curcumin
were diluted to 10 mL with mobile Phase individually to get 4 μg/mL of Gallic Acid, 10
μg/mL of Ellagic Acid and 2 μg/mL of Curcumin.
Each solution was scanned between 200-800 nm in UV Spectrophotometry.
At wavelength of 257 nm, all three markers show absorbance. So, it is selected for further
determination of markers.
Chapter 6 Experimental Work
83
6.1.6 Determination of Formulations
EXTRACTION PROCEDURE FOR FORMULATIONS:
Diasol and Diabeta capsules
The content of Twenty five capsules were removed, weighed and mixed properly.
Accurately weighed 10 g of capsule content was transferred to 100 ml volumetric flask
containing 70 ml of methanol and sonicated for 20 mins. The solution was filtered and the
solvent was evaporated to obtain dry residue. Methanol extract thus obtained (10 mg) was
dissolved in 10 ml of methanol, sonicated for 15 min, filtered and used for further analysis.
Glysikot Granules
10 gram of granules was weighed accurately and the whole content was transferred to 100
ml volumetric flask containing 70 ml of methanol and sonicated for 20 mins. The solution
was filtered and the solvent was evaporated to obtain dry residue. Methanol extract thus
obtained (10 mg) was dissolved in 10 ml of methanol, sonicated for 15 min, filtered and
used for further analysis.
CALCULATION OF % ASSAY FROM FORMULATIONS:
% Gallic acid = AT1
AS1 X
WS1
10 X
1
10 X
0.2
10 X
10
WT1
X 𝑃1 ……………………………… (1)
% Ellagic acid = AT2
AS2 X
WS2
10 X
1
10 X
0.5
10 X
10
WT2
X 𝑃2 ……………………………... (2)
% Curcumin = AT3
AS3
X WS3
10 X
1
10 X
0.1
10 X
10
WT3
X 𝑃3……………………………….. (3)
Where, AT is Average Absorbance/ Area of Test solution;
As is Average Absorbance/ Area of standard solution;
WT is weight of Test substance;
Ws is weight of standard substance;
P is for Purity
1 2 and 3 is for Gallic acid, Ellagic acid and Curcumin respectively.
Chapter 6 RP-HPLC Method Development
84
PREPARATION OF MOBILE PHASE:
1 ml formic acid was added and the volume was made 1 L with water (HPLC Grade) (0.1
% v/v F.A. Mobile phase A).The pH of the solution was measured and it was 2.55.
Acetonitrile was selected as another solvent (Mobile phase B). The solution was then
filtered through nylon filter (0.45 μ). The filtrate was sonicated for 15 min. Gradient elution
was performed using Mobile phase A and B.
6.1.7 METHOD VALIDATION
Parameters to be considered for the validation of method are
SYSTEM SUITABILITY STUDIES:
The system suitability was evaluated by six replicate analyses of Gallic acid, Ellagic acid and
Curcumin in a mixture at concentration of 4 μg/ml of Gallic Acid, 10 μg/ml of Ellagic Acid and
2 μg/ml of Curcumin. Retention time, Tailing Factor, Theoretical plates and resolution were
calculated for the standard solutions.
SPECIFICITY:
Specificity is a procedure to detect quantitatively the analytes in the presence of components
that may be expected to be present in the sample matrix. While Selectivity is the procedure to
detect qualitatively the analytes in the presence of components that may expect to be present in
the sample matrix. Specificity of Developed method was established by spiking of Gallic Acid,
Ellagic Acid & Curcumin in Polyherbal formulation and expressing that analytes peak were not
interfered from other constituents.
LINEARITY:
The linearity response was determined by analyzing different concentration for calibration curve
in the range of 2-14 μg/mL, 5-35 μg/mL and 1-7 μg/mL for Gallic Acid, Ellagic Acid and
Curcumin respectively. Plot the calibration curve of Peak Area vs concentration and determine
correlation coefficient and regression line equations for Gallic Acid, Ellagic Acid and
Curcumin.
Chapter 6 Experimental Work
85
PRECISION:
Repeatability
0.8mL of working standard solutions of Gallic Acid, 2.0 mL of Ellagic Acid and 0.4mL of
Curcumin were transferred into separate 10 mL volumetric flasks and diluted up to mark with
mobile phase to get 8 μg/mL, 20 μg/mL and 4 μg/mL for Gallic Acid, Ellagic Acid and
Curcumin respectively. The absorbance of the each solution was measured at selected
wavelengths 6 times and % RSD was calculated.
Intraday Precision
Mixed solutions containing 6, 8, 10 μg/mL Gallic Acid; 15, 20, 25 μg/mL Ellagic Acid and 3,
4, 5 μg/mL of Curcumin were analyzed three times on the same day and % RSD was calculated.
Interday Precision
Mixed solutions containing 6, 8, 10 μg/mL Gallic Acid; 15, 20, 25 μg/mL Ellagic Acid and 3,
4, 5 μg/mL of Curcumin were analyzed on three different days and %RSD was calculated.
ACCURACY:
Accuracy is the closeness of the test results obtained by the method to the true value. Recovery
studies were carried out by addition of standard drug to the sample at 3 different concentration
levels (80%, 100%, 120%) taking into consideration percentage recovery of added bulk drug
samples.
Formulation (A): 0.1 gm of Formulation ( It contains 1.32 mg of Gallic Acid, 2.49 mg of Ellagic
acid and 0.51 mg of Curcumin)
Standard samples: Gallic acid, Ellagic acid and Curcumin
Chapter 6 RP- HPLC Method Development
86
TABLE 6.1 Steps for Accuracy Measurement for Gallic Acid
Sr.
No. Step 1 Step 2 Step 3
Total Gallic Acid
(mg)
1 0.1 gm of Formulation Make up to 100 mL with
mobile phase and filter
Dilute 1 mL solution
from 2nd step to 10 mL
with mobile phase
1.32
2
0.1 gm of formulation
+ 1.05 mg of Gallic
acid
Make up to 100 mL with
mobile phase and filter 2.37
3
0.1 gm of formulation
+ 1.32 mg of Gallic
acid
Make up to 100 mL with
mobile phase and filter 2.64
4
0.1 gm of formulation
+ 1.58 mg of Gallic
acid
Make up to 100 mL with
mobile phase and filter 2.9
TABLE 6.2 Steps for Accuracy Measurement for Ellagic Acid
Sr.
No. Step 1 Step 2 Step 3
Total Ellagic
Acid (mg)
1 0.1 gm of Formulation Make up to 100 mL with
mobile phase and filter
Dilute 1 mL solution
from 2nd step to 10
mL with mobile
phase
2.49
2
0.1 gm of formulation
+ 1.99 mg of Ellagic
acid
Make up to 100 mL with
mobile phase and filter 4.48
3
0.1 gm of formulation
+ 2.49 mg of Ellagic
acid
Make up to 100 mL with
mobile phase and filter 4.98
4
0.1 gm of formulation
+ 2.98 mg of Ellagic
acid
Make up to 100 mL with
mobile phase and filter 5.47
TABLE 6.3 Steps for Accuracy Measurement for Curcumin
Sr.
No.
Step 1 Step 2 Step 3 Total Curcumin
(mg)
1 0.1 gm of
Formulation
Make up to 100 mL with
mobile phase and filter Dilute 1 mL solution
from 2nd step to 10 mL
with mobile phase
0.51
2 0.1 gm of formulation
+ 0.41 mg of
Curcumin
Make up to 100 mL with
mobile phase and filter 0.92
3 0.1 gm of formulation
+ 0.51 mg of
Curcumin
Make up to 100 mL with
mobile phase and filter 1.02
4 0.1 gm of formulation
+ 0.61 mg of
Curcumin
Make up to 100 mL with
mobile phase and filter 1.12
Chapter 6 Experimental Work
87
LIMIT OF DETECTION (LOD):
The LOD was estimated from the set of six calibration curves used to determine method
linearity. The LOD may be calculated as
LOD = 3.3 x (SD / Slope)
Where, SD = the standard deviation of Y- intercept of six calibration curves.
Slope = the mean slope of the six calibration curves.
LIMIT OF QUANTITATION (LOQ):
The LOQ was estimated from the set of six calibration curves used to determine method
linearity. The LOQ may be calculated as
LOQ = 10 x (SD / Slope)
Where, SD = the standard deviation of Y- intercept of six calibration curves.
Slope = the mean slope of the six calibration curves.
ROBUSTNESS:
Robustness of the method was determined by subjecting the method to slight change in the
method condition, individually, the:
Pump flow rate, pH, Change in wavelength.
Three replicates were made for the same concentration (8 μg/ml of Gallic Acid, 20 μg/ml of
Ellagic Acid and 4 μg/ml of Curcumin). % RSD was calculated.
Chapter 6 RP-HPLC Method Development
88
6.2 Results and Discussion
6.2.1 Selection of Wavelength
At wavelength of 257 nm, all three markers showed absorbance. So, it is selected for
further determination of markers.
FIGURE 6.1 Selection of Wavelength for HPLC Method, Overlain spectrum of Gallic acid (4 µg/ml),
Ellagic acid (10 µg/ml) and Curcumin (2 µg/ml)
Chapter 6 Result and Discussion
89
6.2.2 Trials for HPLC Method Development
TABLE 6.4 Trials for RP-HPLC Method Development
Sr.
No
.
Trials
Taken
Observation Remark
s
1 Methanol
: Water
(50:50
v/v)
(Gallic Acid 20 PPM, Ellagic Acid 10 PPM, Curcumin 20 PPM)
Only one
Peak
2 ACN :
Water
(70:30
v/v)
(Gallic Acid 20 PPM, Ellagic Acid 10 PPM, Curcumin 20 PPM)
One
sharp
Peak
along
with
merging
of other
peaks
3 Water (
PH 4
with
OPA)
:ACN
(70:30
v/v)
(Gallic Acid 20 PPM, Ellagic Acid 10 PPM, Curcumin 20 PPM)
Two
separate
peaks but
splitting
Chapter 6 RP-HPLC Method Development
90
4 Water
(0.1
%Formic
Acid) :
ACN :
Methanol
(70:15:15
v/v/v)
Gallic
Acid
Sharp and Symmetric Peak
Gallic Acid 10 ppm
Combine
result will
be used
for
further
separation
Ellagic
Acid
Peak was broad and tailing
Ellagic Acid 10 ppm
Curcumin No Peak was observed up to 15 min
Curcumin 10 ppm
5 Water
with 0.1
% Formic
Acid :
ACN (30
: 70 v/v)
Curcumin
Good peak at early retention time
Curcumin 10 ppm
Chapter 6 Result and Discussion
91
3 consequent peaks of bisdesmethoxy curcumin, dimethoxy curcumin and curcumin were
observed on RT of 3.9, 4.2 and 4.7 min respectively.
CHROMATOGRAM OF TRIALS:
So, from the above trials, it seems that three peaks can be separated by using mobile phase in
gradient mode of elution. From the above chromatograph, we can conclude that, In the High
ratio of aqueous mobile phase, Gallic Acid and Ellagic Acid will separate and as the ratio of
Organic mobile phase increase, Curcumin will leave the Stationary phase and elute out from the
column. We can also conclude that, Mobile Phase pH also impact more on separation.
FIGURE 6.2 Chromatogram using above trials
Chapter 6 RP- HPLC Method Development
92
6.2.3 Selection of Critical factors and Responses for further Optimization [3,4]
Based on above trials, we can decide highly affected Factors and Responses which are
critical for Method development. It is also utilize for determine the levels for
Experimental design.
TABLE 6.5 Combined Data for Identification of Critical Factor and Response for further Optimization of
Chromatogram
Factor RtG RtE RtC Ag Ae Ac Rs2 Rs3
MeOH conc. Low Low Low - - - - -
ACN Conc. Low Low Low High High High High High
Water Conc. Low Low Low High High High Medium Medium
Acidic pH High High High High Low High - -
Basic pH Low Low Low Low Low Low Low Low
Flow Rate Low Low Low Mediu
m
Mediu
m
Mediu
m High High
TABLE 6.6 Final Selected Factors & Responses
Factor 1 (F1) Composition of Aqueous phase at starting (As Gradient Mode)
Factor 2 (F2) Flow Rate
Factor 3 (F3) pH of Mobile phase (% of Formic Acid)
Response 1 (R1) Asymmetry of Gallic Acid
Response 2 (R2) Asymmetry of Ellagic Acid
Response 3 (R3) Asymmetry of Curcumin
Response 4 (R4) Resolution between Gallic acid & Ellagic Acid
Response 5 (R5) Resolution between Ellagic acid & Curcumin
Chapter 6 Result and Discussion
93
TABLE 6.7 Finalization of Independent variables with different levels and Dependent variables
Translation of coded value in Actual units
Independent Variables
Variable Level
Very low
(-2)
Low
(-1)
Medium
(0)
High
(1)
Very High
(2)
Proportion of Aqueous Phase at
Starting of Separation (F1) 60 65 70 75 80
Flow Rate (F2) 0.8 0.9 1 1.1 1.2
pH of Mobile Phase (% of F.A.)
(F3)
2.65
(0.08%)
2.63
(0.09%)
2.55
(0.1%)
2.47
(0.11%)
2.43
(0.12%)
Dependent Variables
R1 Asymmetry of Gallic Acid
R2 Asymmetry of Ellagic Acid
R3 Asymmetry of Curcumin
R4 Resolution between Gallic Acid & Ellagic Acid
R5 Resolution between Ellagic Acid & Curcumin
6.2.4 Optimization of chromatographic conditions using DOE approach [5,6]
For decades HPLC development was based on a trial and error methodology, but employing
a time-consuming trial-and-error approach resulting only in an apparent optimum and
information concerning the sensitivity of the factors on the analytes separation and
interaction between factors is not available. To achieve this objective, any one of the
Chemometrics methods which includes the overlapping resolution maps, factorial design
and response surface methodology can be applied. The best experimental design approach
for the purpose of modelling and optimization are the response surface design.
Chapter 6 RP-HPLC Method Development
94
CCD (Central Composite Design) is chosen due to its flexibility and can be applied to
optimize an HPLC separation by gaining better understanding of factor’s main and
interaction effects. CCD is more advantageous than Box-Behnken design because in CCD,
design space is larger due to axial point. The selection of key factors examined for
optimization was based on preliminary experiments. The factors selected for optimization
process were % of Aqueous phase at starting (A), Flow Rate (B) and pH of Mobile phase
(C). Asymmetry of all three drugs and Resolution between them were selected as Response.
All experiments were conducted in randomized order to minimize the effects of uncontrolled
variables that may introduce a bias on the measurements. A three factor CCD requires 18
experiments, including four center points using rotatable CCD design. The design matrix
and experimental results are summarized in Table 6.8 and 6.9 depicts the illustration of
experimental chromatograms of experimental run at three selected factors according to
below CCD model.
TABLE 6.8 Central composite rotatable design arrangement and responses (Coded Value)
Run F1 F2 F3 R1 R2 R3 R4 R5
1 -1 1 -1 0.18 0.423 0.855 78.54 31.54
2 -
1.68179 0 0 1.754 0.526 0.923 32.76 52.91
3 0 0 0 0.58 0.795 0.79 32.73 37.53
4 0 1.68179 0 0.18 1.143 0.778 83.89 61.58
5 1 -1 -1 1.18 1.252 0.793 6.43 29.06
6 0 0 1.68179 0.892 1.632 1.595 45.87 83.97
7 0 0 -1.68179 1.423 1.175 0.582 15.9 12.75
8 0 0 0 0.89 0.52 0.85 29.43 41.94
9 1 1 -1 0.39 0.986 1.03 83.45 49.33
10 -1 1 1 0.39 0.798 1.016 65.8 73.29
11 0 0 0 0.63 0.974 0.95 34.43 36.96
12 1 -1 1 1.228 0.995 1.219 6.89 74.91
13 0 0 0 0.96 0.621 0.675 43.21 31.29
14 1.68179 0 0 1.54 0.84 0.911 30.7 43.12
15 -1 -1 -1 1.534 1.291 0.823 5.64 34.83
16 -1 -1 1 0.73 1.426 1.423 7.01 67.83
17 0 -1.68179 0 0.29 1.28 0.893 3.83 23.85
18 1 1 1 0.94 0.813 1.07 73.2 78.93
Chapter 6 Result and Discussion
95
TABLE 6.9 Central composite rotatable design arrangement and responses (Actual value)
Run F1 F2 F3 R1 R2 R3 R4 R5
1 65 1.1 0.09 0.18 0.423 0.855 78.54 31.54
2 62.4 1 0.1 1.754 0.526 0.923 32.76 52.91
3 70 1 0.1 0.58 0.795 0.79 32.73 37.53
4 70 1.17 0.1 0.18 1.143 0.778 83.89 61.58
5 75 0.9 0.09 1.18 1.252 0.793 6.43 29.06
6 70 1 0.117 0.892 1.632 1.595 45.87 83.97
7 70 1 0.083 1.423 1.175 0.582 15.9 12.75
8 70 1 0.1 0.89 0.52 0.85 29.43 41.94
9 75 1.1 0.09 0.39 0.986 1.03 83.45 49.33
10 65 1.1 0.11 0.39 0.798 1.016 65.8 73.29
11 70 1 0.1 0.63 0.974 0.95 34.43 36.96
12 75 0.9 0.11 1.228 0.995 1.219 6.89 74.91
13 70 1 0.1 0.96 0.621 0.675 43.21 31.29
14 78.4 1 0.1 1.54 0.84 0.911 30.7 43.12
15 65 0.9 0.09 1.534 1.291 0.823 5.64 34.83
16 65 0.9 0.11 0.73 1.426 1.423 7.01 67.83
17 70 0.83 0.1 0.29 1.28 0.893 3.83 23.85
18 75 1.1 0.11 0.94 0.813 1.07 73.2 78.93
Chapter 6 RP-HPLC Method Development
96
Trial 1
FIGURE 6.3 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (-1), F2(1), F3(-1)
Chapter 6 Result and Discussion
97
Trial 2
FIGURE 6.4 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (-1.68), F2(0), F3(0)
Chapter 6 RP-HPLC Method Development
98
Trial 3
FIGURE 6.5 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(0), F3(0)
Chapter 6 Result and Discussion
99
Trial 4
FIGURE 6.6 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(1.68), F3(0)
Chapter 6 RP-HPLC Method Development
100
Trial 5
FIGURE 6.7 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (1), F2(-1), F3(-1)
Chapter 6 Result and Discussion
101
Trial 6
FIGURE 6.8 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(0), F3(1.68)
Chapter 6 RP-HPLC Method development
102
Trial 7
FIGURE 6.9 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(0), F3(-1.68)
Chapter 6 Result and Discussion
103
Trial 8
FIGURE 6.10 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(0), F3(0)
Chapter 6 RP- HPLC Method Development
104
Trial 9
FIGURE 6.11 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (1), F2(1), F3(-1)
Chapter 6 Result and Discussion
105
Trial 10
FIGURE 6.12 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (-1), F2(1), F3(1)
Chapter 6 RP-HPLC Method Development
106
Trial 11
FIGURE 6.13 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2 (0), F3(0)
Chapter 6 Result and Discussion
107
Trial 12
FIGURE 6.14 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (1), F2(-1), F3(1)
Chapter 6 RP-HPLC Method Development
108
Trial 13
FIGURE 6.15 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(0), F3(0)
Chapter 6 Result and Discussion
109
Trial 14
FIGURE 6.16 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (1.68), F2(0), F3(0)
Chapter 6 RP- HPLC Method Development
110
Trial 15
FIGURE 6. 17 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (-1), F2(-1), F3(-1)
Chapter 6 Result and Discussion
111
Trial 16
FIGURE 6.18 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (-1), F2(-1), F3(1)
Chapter 6 RP-HPLC Method Development
112
Trial 17
FIGURE 6.19 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (0), F2(-1.68), F3(0)
Chapter 6 Result and Discussion
113
Trial 18
FIGURE 6.20 Chromatogram of Gallic acid, Ellagic acid and Curcumin at F1 (1), F2(1), F3(1)
Chapter 6 RP-HPLC Method Development
114
MODEL SELECTION AND STATISTICAL PARAMETERS OBTAINED FROM
ANOVA:
The selection of model was done which have lowest PRESS value and r2 value was nearer to 1
as shown in Table 6.10- 6.14. Investigation of the associated probability revealed that for
response 1 (Asymmetry of Gallic Acid), 2 (Asymmetry of Ellagic Acid) Quadratic models
resulted in the best fitted. The cubic models were aliased, as expected, because the central
composite matrix provided very few unique design points to determine all the terms in the cubic
model. Response 4 (Resolution between Gallic Acid and Ellagic Acid), 5 (Resolution between
Ellagic Acid and Curcumin) Linear models resulted in best fitted. The cubic models were
aliased, as expected, because the central composite matrix provided very few unique design
points to determine all the terms in the cubic model. Response 3 (Asymmetry of Curcumin) both
the Model, Quadratic as well as Linear Model are best fitted and cubic model was aliased. In
this work, the following quadratic model was used to describe the response surface for response
1-5 respectively.
TABLE 6.10 Summary of results of regression analysis for models and response 1.
Source Std. Dev. R-Squared Adjusted
R-Squared
Predicted
R-Squared PRESS
Linear 0.49 0.1745 -0.0024 -0.5040 6.21
2FI 0.51 0.2986 -0.0839 -0.6723 6.90
Quadratic 0.31 0.8090 0.5941 -0.3124 5.41 Suggested
Cubic 0.25 0.9412 0.7503 -6.3376 30.27 Aliased
TABLE 6.11 Summary of results of regression analysis for models and response 2.
Source Std. Dev. R-Squared Adjusted
R-Squared
Predicted
R-Squared PRESS
Linear 0.33 0.2232 0.0567 -0.3068 2.51
2FI 0.33 0.3591 0.0095 -0.3591 2.61
Quadratic 0.22 0.8016 0.5784 -0.1456 2.20 Suggested
Cubic 0.21 0.9121 0.6266 -4.7384 11.02 Aliased
TABLE 6.12 Summary of results of regression analysis for models and response 3.
Source Std. Dev. R-Squared Adjusted
R-Squared
Predicted
R-Squared PRESS
Linear 0.17 0.6044 0.5197 0.3384 0.71 Suggested
2FI 0.17 0.7193 0.5662 0.1191 0.94
Quadratic 0.13 0.8786 0.7419 0.2937 0.75 Suggested
Cubic 0.12 0.9467 0.7733 -2.6442 3.89 Aliased
Chapter 6 Result and Discussion
115
TABLE 6.13 : Summary of results of regression analysis for models and response 4.
Source Std. Dev. R-Squared Adjusted
R-Squared
Predicted
R-Squared PRESS
Linear 9.93 0.8996 0.8780 0.8199 2473.92 Suggested
2FI 10.81 0.9064 0.8554 0.6402 4942.83
Quadratic 11.33 0.9253 0.8413 0.4766 7190.40
Cubic 6.98 0.9858 0.9397 -0.4777 20300.99 Aliased
TABLE 6.14 Summary of results of regression analysis for models and response 5.
Source Std.
Dev. R-Squared
Adjusted
R-Squared
Predicted
R-Squared PRESS
Linear 10.29 0.8000 0.7571 0.6801 2373.05 Suggested
2FI 11.34 0.8092 0.7051 0.1909 6001.95
Quadratic 9.27 0.9074 0.8032 0.3262 4998.25
Cubic 8.11 0.9645 0.8493 -5.1327 45490.90 Aliased
The model was validated by analysis of variance (ANOVA) using Design Expert 10.0.1
software that had been used to develop the experimental matrix for RSM. In ANOVA analysis,
a significant model is desired (Table 6.10 – 6.14).
TABLE 6.15 ANOVA table for response surface quadratic model for Response 1.
Source Sum of
Squares df
Mean
Square F Value
p-value
Prob > F
Model 3.34 9 0.37 3.77 0.0377 Significant
A-
composition
of aq. phase
at starting
point
0.022 1 0.022 0.22 0.6515
B-Flow rate 0.64 1 0.64 6.50 0.0342
C-pH of
M.P. (% of
F.A.)
0.058 1 0.058 0.59 0.4654
AB 0.047 1 0.047 0.48 0.5074
AC 0.18 1 0.18 1.80 0.2162
BC 0.29 1 0.29 2.92 0.1261
A2 0.90 1 0.90 9.16 0.0164
B2 0.68 1 0.68 6.92 0.0301
C2 0.11 1 0.11 1.13 0.3178
Residual 0.79 8 0.098
Lack of Fit 0.68 5 0.14 3.86 0.1479 not significant
Pure Error 0.11 3 0.035
Cor Total 4.13 17
Chapter 6 RP-HPLC Method Development
116
Ag = 0.78 + 0.040(A) – 0.22(B) - 0.065(C) + 0.077(AB) + 0.15(AC) + 0.19(BC) + 0.27(A2) –
0.23(B2) + 0.094(C2)
The statistical data obtained from ANOVA for quadratic model are given in Table 6.15. The
Model F-value of 3.77 implies the model is significant. There is only a 3.77% chance that an F-
value this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model
terms are significant. In this case B, A2, B2 are significant model terms. Values greater than
0.1000 indicate the model terms are not significant. If there are many insignificant model terms
(not counting those required to support hierarchy), model reduction may improve your model.
The "Lack of Fit F-value" of 3.86 implies the Lack of Fit is not significant relative to the pure
error. There is a 14.79% chance that a "Lack of Fit F-value" this large could occur due to noise.
TABLE 6.16 ANOVA table for response surface quadratic model for Response 2.
Source Sum of
Squares df
Mean
Square F Value
p-value
Prob > F
Model 1.54 9 0.17 3.59 0.0428 Significant
A-
composition
of aq. phase
at starting
point
0.030 1 0.030 0.62 0.4530
B-Flow rate 0.35 1 0.35 7.27 0.0272
C- pH of
M.P. .(% of
F.A.)
0.053 1 0.053 1.11 0.3234
AB 0.14 1 0.14 2.88 0.1279
AC 0.11 1 0.11 2.32 0.1663
BC 0.013 1 0.013 0.28 0.6138
A2 0.023 1 0.023 0.48 0.5075
B2 0.26 1 0.26 5.53 0.0465
C2 0.57 1 0.57 11.96 0.0086
Residual 0.38 8 0.048
Lack of Fit 0.26 5 0.052 1.31 0.4389 not significant
Pure Error 0.12 3 0.040
Cor Total 1.92 17
Ae = 0.73 + 0.047(A) – 0.16(B) + 0.062(C) + 0.13(AB) - 0.12(AC) + 0.041(BC) – 0.043(A2)
+ 0.14 (B2) + 0.21 (C2)
The statistical data obtained from ANOVA for quadratic model are given in Table 6.16. The
Model F-value of 3.59 implies the model is significant. There is only a 4.28% chance that an F-
value this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model
terms are significant. In this case B, B2, C2 are significant model terms. Values greater than
Chapter 6 Result and Discussion
117
0.1000 indicate the model terms are not significant. If there are many insignificant model terms
(not counting those required to support hierarchy), model reduction may improve your model.
The "Lack of Fit F-value" of 1.31 implies the Lack of Fit is not significant relative to the pure
error. There is a 43.89% chance that a "Lack of Fit F-value" this large could occur due to noise.
TABLE 6.17 ANOVA table for response surface quadratic model for Response 3.
Source Sum of
Squares df
Mean
Square F Value
p-value
Prob > F
Model 0.94 9 0.10 6.43 0.0077 Significant
A-
composition
of aq. phase
at starting
point
4.643E-005 1 4.643E-005 2.863E-003 0.9586
B-Flow rate 0.017 1 0.017 1.04 0.3373
C- pH of
M.P. (% of
F.A.)
0.63 1 0.63 38.77 0.0003
AB 0.027 1 0.027 1.65 0.2346
AC 0.011 1 0.011 0.67 0.4365
BC 0.085 1 0.085 5.25 0.0512
A2 0.034 1 0.034 2.07 0.1877
B2 6.554E-003 1 6.554E-003 0.40 0.5428
C2 0.16 1 0.16 9.82 0.0139
Residual 0.13 8 0.016
Lack of Fit 0.090 5 0.018 1.36 0.4250 not significant
Pure Error 0.040 3 0.013
Cor Total 1.07 17
Ac = 0.81 – 0.0018 (A) – 0.035(B) + 0.21(C) + 0.058(AB) – 0.037 (AC) - 0.10 (BC) +
0.052(A2) + 0.023(B2) + 0.11(C2)
The statistical data obtained from ANOVA for quadratic model are given in Table 6.17.The
Model F-value of 6.43 implies the model is significant. There is only a 0.77% chance that an F-
value this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model
terms are significant. In this case C, C2 are significant model terms. Values greater than 0.1000
indicate the model terms are not significant. If there are many insignificant model terms (not
counting those required to support hierarchy), model reduction may improve your model. The
"Lack of Fit F-value" of 1.36 implies the Lack of Fit is not significant relative to the pure error.
There is a 42.50% chance that a "Lack of Fit F-value" this large could occur due to noise.
Chapter 6 RP-HPLC Method Development
118
TABLE 6.18 ANOVA table for response surface quadratic model for Response 4.
Source Sum of
Squares Df
Mean
Square F Value
p-value
Prob > F
Model 12711.66 9 1412.41 11.01 0.0013 Significant
A-
composition
of aq. phase
at starting
point
6.63 1 6.63 0.052 0.8259
B-Flow rate 12288.69 1 12288.69 95.81 < 0.0001
C- pH of
M.P. (% of
F.A.)
62.62 1 62.62 0.49 0.5045
AB 16.94 1 16.94 0.13 0.7257
AC 0.31 1 0.31 2.433E-003 0.9619
BC 77.00 1 77.00 0.60 0.4607
A2 4.547E-003 1 4.547E-003 3.545E-005 0.9954
B2 234.71 1 234.71 1.83 0.2131
C2 0.99 1 0.99 7.720E-003 0.9321
Residual 1026.12 8 128.27
Lack of Fit 922.23 5 184.45 5.33 0.0996 not significant
Pure Error 103.90 3 34.63
Cor Total 13737.78 17
Rs2 = 34.69 + 0.70 (A) + 30 (B) + 2.14 (C) + 1.45(AB) +0.20 (AC) – 3.10 (BC) + 0.019 (A2)
+ 4.31 (B2) – 0.28 (C2)
The statistical data obtained from ANOVA for quadratic model are given in Table 6.18.The
Model F-value of 11.01 implies the model is significant. There is only a 0.13% chance that an
F-value this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model
terms are significant. In this case B is a significant model term. Values greater than 0.1000
indicate the model terms are not significant. If there are many insignificant model terms (not
counting those required to support hierarchy), model reduction may improve your model. The
"Lack of Fit F-value" of 5.33 implies there is a 9.96% chance that a "Lack of Fit F-value" this
large could occur due to noise.
Chapter 6 Result and Discussion
119
TABLE 6.19 ANOVA table for response surface quadratic model for Response 5.
Source Sum of
Squares Df
Mean
Square F Value
p-value
Prob > F
Model 6730.87 9 747.87 8.71 0.0028 Significant
A-
composition
of aq. phase
at starting
point
5.01 1 5.01 0.058 0.8151
B-Flow rate 591.98 1 591.98 6.89 0.0304
C- pH of
M.P. (% of
F.A.)
5337.08 1 5337.08 62.16 < 0.0001
AB 61.16 1 61.16 0.71 0.4232
AC 0.061 1 0.061 7.133E-004 0.9793
BC 7.03 1 7.03 0.082 0.7820
A2 404.89 1 404.89 4.72 0.0617
B2 181.10 1 181.10 2.11 0.1845
C2 422.54 1 422.54 4.92 0.0573
Residual 686.90 8 85.86
Lack of Fit 629.63 5 125.93 6.60 0.0757 not significant
Pure Error 57.27 3 19.09
Cor Total 7417.77 17
Rs3 = 36.54 + 0.61 (A) + 6.58 (B) + 19.77 (C) + 2.77 (AB) + 0.088 (AC) – 0.94 (BC) + 5.66
(A2) + 3.78 (B2) + 5.78 (C2)
The statistical data obtained from ANOVA for quadratic model are given in Table 6.19. The
Model F-value of 8.71 implies the model is significant. There is only a 0.28% chance that an F-
value this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model
terms are significant .In this case B, C are significant model terms.
Values greater than 0.1000 indicate the model terms are not significant. If there are many
insignificant model terms (not counting those required to support hierarchy), model reduction
may improve your model. The "Lack of Fit F-value" of 6.60 implies there is a 7.57% chance
that a "Lack of Fit F-value" this large could occur due to noise.
PERTURBATION PLOT:
The predicted models are presented in the form of perturbation plots for better understanding of
results (Figure 1.2). These graphs give the idea about how the response changes as each factor
moves from its defined reference value, with all other factors held constant. A steepest slope or
curvature indicates sensitiveness of the response to a specific factor.
Chapter 6 RP-HPLC Method Development
120
(1) (2)
(3) (4)
Chapter 6. Result and Discussion
121
(5)
FIGURE 6.21 Perturbation graph showing the effect of each factor A, B, and C on (1) Asymmetry of Gallic
Acid (2)Asymmetry of Ellagic Acid (3) Asymmetry of Curcumin (4) Resolution between Gallic Acid-
Ellagic Acid and (5) Resolution between Ellagic Acid- Curcumin.
Chapter 6. RP-HPLC Method Development
122
THREE DIMENSIONAL RESPONSE SURFACE PLOT:
Response 1:
FIGURE 6.22 (A-O) Three-dimensional plots of the RSM for five responses
(A) Variation in Asymmetry of Gallic Acid as function of A and B while fixed factor C,
(B) Variation in Asymmetry of Gallic Acid as function of A and C while fixed factor B,
(C) Variation in Asymmetry of Gallic Acid as function of C and B while fixed factor A,
Chapter 6. Result and Discussion
123
Response 2:
(D) Variation in Asymmetry of Ellagic Acid as function of A and B while fixed factor C,
(E) Variation in Asymmetry of Ellagic Acid as function of A and C while fixed factor B,
(F) Variation in Asymmetry of Ellagic Acid as function of C and B while fixed factor A,
Chapter 6. RP-HPLC Method Development
124
Response 3:
(G) Variation in Asymmetry of Curcumin as function of A and B while fixed factor C,
(H) Variation in Asymmetry of Curcumin as function of A and C while fixed factor B,
(I) Variation in Asymmetry of Curcumin as function of C and B while fixed factor A,
Chapter 6. Result and Discussion
125
Response 4:
(J) Resolution between Gallic & Ellagic Acid as function of A and B while fixed factor C,
(K) Resolution between Gallic & Ellagic Acid as function of A and C while fixed factor B,
(L) Resolution between Gallic & Ellagic Acid as function of C and B while fixed factor A,
Chapter 6. RP-HPLC Method Development
126
Response 5:
(M) Resolution between Ellagic Acid & Curcumin as function of A and B, fixed factor C,
(N) Resolution between Ellagic Acid & Curcumin as function of A and C, fixed factor B,
(O) Resolution between Ellagic Acid & Curcumin as function of C and B, fixed factor A.
Chapter 6. Result and Discussion
127
As shown in Fig. 6.22 (A–O), the analysis produces three-dimensional graphs of RSM by
plotting the response model against two of the factors, while the third is held constant at a
specified level. 3D plots represent a graphical representation of increase in the concentration of
% Formic Acid has significant impact on Asymmetry of Gallic Acid. Increase in concentration
of % Formic Acid & increase in starting composition of Aqueous mobile phase responsible for
increase in Asymmetry of Ellagic Acid. Lower starting composition of Aqueous mobile phase
& and higher % of Formic Acid responsible for increase in Asymmetry of Curcumin. Increase
in flow rate responsible for increase in Resolution between Gallic Acid & Ellagic Acid. Increase
in % of Formic Acid concentration responsible for increase in Resolution between Ellagic Acid
& Curcumin.
PREDICTED V/S ACTUAL CURVE:
FIGURE 6.23 Predicted vs. Actual Responses for Asymmetry of Gallic Acid.
Chapter 6. RP-HPLC Method Development
128
FIGURE 6.24 Predicted vs. Actual Responses for Asymmetry of Ellagic Acid.
FIGURE 6.25 Predicted vs. Actual Responses for Asymmetry of Curcumin.
Chapter 6. Result and Discussion
129
FIGURE 6.26 Predicted vs. Actual Responses for Resolution between Gallic Acid & Ellagic Acid.
FIGURE 6.27 Predicted vs. Actual Responses for Resolution between Ellagic Acid & Curcumin
Fig. 6.23 – 6.27 shows graphical representation of the predicted value versus actual value of
the responses for Asymmetry of Gallic Acid, Ellagic Acid, Curcumin & Resolution between
Gallic acid and Ellagic Acid as well as Resolution between Ellagic Acid and Curcumin.
Chapter 6. RP-HPLC Method Development
130
It will help to detect a value, or group of values, that are not easily predicted by the model.
From the scattered diagram for response it is conclude that the developed models are adequate
because the residuals for the prediction of each response are minimum, since the residuals tend
to be close to the diagonal line.
CONTOUR PLOT:
Contour plots between one independent variable versus another holding magnitude of response
and other variables constant. Fig. 6.28 (A-I) depicts the graphical representation of the Contour
Plot for Asymmetry of Gallic Acid, Ellagic Acid & Curcumin respectively. Figure 6.28 (J-O)
depicts the graphical representation of the counter plots for Resolution between Gallic Acid,
Ellagic Acid & Curcumin.
Chapter 6. Result and Discussion
131
Response 1:
(A) (B)
(C)
FIGURE 6.28 (A-O) Contour Plots for five responses
(A) Variation in Asymmetry of Gallic Acid as function of A and B while fixed factor C,
(B) Variation in Asymmetry of Gallic Acid as function of A and C while fixed factor B,
(C) Variation in Asymmetry of Gallic Acid as function of C and B while fixed factor A,
Chapter 6. RP-HPLC Method Development
132
Response 2:
(D) (E)
(F)
(D) Variation in Asymmetry of Ellagic Acid as function of A and B while fixed factor C,
(E) Variation in Asymmetry of Ellagic Acid as function of A and C while fixed factor B,
(F) Variation in Asymmetry of Ellagic Acid as function of C and B while fixed factor A,
Chapter 6. Result and Discussion
133
Response 3:
(G) (H)
(I)
(G) Variation in Asymmetry of Curcumin as function of A and B while fixed factor C,
(H) Variation in Asymmetry of Curcumin as function of A and C while fixed factor B,
(I) Variation in Asymmetry of Curcumin as function of C and B while fixed factor A,
Chapter 6. RP-HPLC Method Development
134
Response 4:
(J) (K)
(L)
(J) Resolution between Gallic & Ellagic Acid as function of A and B while fixed factor C,
(K) Resolution between Gallic & Ellagic Acid as function of A and C while fixed factor B,
(L) Resolution between Gallic & Ellagic Acid as function of C and B while fixed factor A,
Chapter 6. Result and Discussion
135
Response 5:
(M) (N)
(O)
(M) Resolution between Ellagic Acid & Curcumin as function of A and B, fixed factor C,
(N) Resolution between Ellagic Acid & Curcumin as function of A and C, fixed factor B,
(O) Resolution between Ellagic Acid & Curcumin as function of C and B, fixed factor A.
Chapter 6. RP-HPLC Method Development
136
NUMERICAL OPTIMIZATION:
After completion of analysis of model, optimization of different factor was done by derringer’s
desirability function. In optimization first the target of individual factors and responses were
fixed as shown in Table 6.20.
TABLE 6.20 Criteria for optimization of individual responses and factors with Targeted values.
Lower Upper Lower Upper Importance
Name Goal Limit Limit Wt. Wt.
A:composition of aq. phase at
starting point is in range -1 0 1 1 3
B:Flow rate is in range -0.5 1 1 1 3
C:pH of M.P.(% of F.A.) is in range -1 1 1 1 3
Ag none 0.18 1.754 1 1 3
Ae none 0.423 1.632 1 1 3
Ac none 0.582 1.595 1 1 3
Rs2 none 3.83 83.89 1 1 3
RS3 none 12.75 83.97 1 1 3
The goal of optimization is to find a good set of conditions that will meet all the goals, not to
get to a desirability value of 1.0. After defining the target, software had provided 1 solutions of
optimization as depicts in Table 6.20.
Chapter 6. Result and Discussion
137
FIGURE 6.29 Optimized Graphical representation for selection of Factors.
TABLE 6.21 Suggested best solutions having desirability scores nearer to 1.00 for the optimization
Coded Value Actual Value
Factor 1 % of Aq. Phase at starting point 0.00 70
Factor 2 Flow rate -0.50 0.95
Factor 3 pH of Mobile Phase (% of F.A) 0 2.55 (0.1%)
Response 1 Asymmetry of Gallic Acid 0.825 -
Response 2 Asymmetry of Ellagic Acid 0.849 -
Response 3 Asymmetry of Curcumin 0.835 -
Response 4 Resolution between Gallic Acid & Ellagic
Acid 20.72 -
Response 5 Resolution between Ellagic Acid &
Curcumin 34.18 -
After optimization experimental runs were carried out according to the best optimized
conditions. In order to investigate the predictability of the proposed model, the agreement
between experimental and predicted responses for the predicted optimums.
Design-Expert® SoftwareFactor Coding: ActualOverlay Plot
AgAeAcRs2RS3
Design Points
X1 = A: composition of aq. phase at starting pointX2 = B: Flow rate
Actual FactorC: % of F.A. = 0
-1 -0.5 0 0.5 1
-1
-0.5
0
0.5
1Overlay Plot
A: composition of aq. phase at starting point
B:
Flo
w r
ate
Ag: 0.4
Ag: 0.85
Ag: 0.85
Ae: 0.9
Ac: 0.85
Rs2: 25
RS3: 40
4Ag: 0.825285 Ae: 0.848086 Ac: 0.835671 Rs2: 20.8573 RS3: 34.2028 X1 0.003125 X2 -0.496591
Ag: 0.825206 Ae: 0.849634 Ac: 0.835966 Rs2: 20.7259 RS3: 34.1884 X1 0.003125 X2 -0.501705
Chapter 6. RP-HPLC Method Development
138
Then predicted errors were calculated using formula:
Predicted Error = Experimental-Predicted/Predicted × 100
TABLE 6.22 Comparison of experimental and predictive value of different experimental runs under
optimum conditions
Optimum
Conditions Response
Responses
(predicted)
Responses
(observed)
Predicted
error %
1
Asymmetry of Gallic Acid 0.825 0.803 2.66
Asymmetry of Ellagic Acid 0.849 0.864 1.76
Asymmetry of Curcumin 0.835 0.849 1.67
Resolution between Gallic Acid & Ellagic
Acid
20.72 21.36 3.08
Resolution between Ellagic Acid &
Curcumin
34.18 35.21 3.01
From the Table 6.22, %predicted error, it is concluded that a set of coordinates producing high
desirability value (D = 1) at condition 1. Hence, Condition 1 had been proposed for selecting an
optimum experimental condition for analyzing. The graphical representation of the response-
surface plot corresponding to this D value is depicted in Fig. 6.30, where the best compromise
is obtained at the top of the graph, D=1.000.
FIGURE 6.30 Bar graph showing individual values of various responses and their Association as a
geometric mean (D Combine)
1
1
1
1
1
1
1
1
1
Desirability
0.000 0.250 0.500 0.750 1.000
A:composition of aq. phase at starting point
B:Flow rate
C:% of F.A.
Ag
Ae
Ac
Rs2
RS3
Combined
Chapter 6. Result and Discussion
139
6.2.5 Optimized Chromatographic Condition of HPLC Method : Refer to Table 4.5
FIGURE 6.31 Final Optimized Chromatogram
In the final optimized chromatogram, Curcumin showed retention time at 12.527 min. Around
the Retention time of 12 min., two other peaks were seen. All three peaks of Curcuminoids were
separated and further confirmed by LC-MS/MS Study [7-9].
Chapter 6. RP-HPLC Method Development
140
FIGURE 6.32 Synthetic Pathway of Curcumin [10]
From the above synthetic pathway of curcumin, It was concluded that, when Curcuminoids are
synthesized from Phenyl alanine, cinnamic acid and p-coumaric acid; bisdesmethoxycurcumin
and desmethoxy curcumin are present in small quantity.
Approximately 2%–6% (w/w) of turmeric is curcuminoids. The curcuminoids contains 80%
curcumin, 18% desmethoxycurcumin, and 2% bisdesmethoxycurcumin. The United States Food
and Drug Administration has approved curcumin as being GRAS (generally recognized as
safe).So, from this, we can say that, from all three peaks, highest peak area has curcumin. It can
be also prove by following chromatogram.
Chapter 6. Result and Discussion
141
FIGURE 6.33 Separation of Bisdesmethoxycurcumin, desmethoxycurcumin and Curcumin by UPLC [11].
LCMSMS Analysis was done to confirm the presence of Curcuminoids.
6.2.6 LC-MS/MS Analysis for Curcuminoids.
OPTIMIZED CONDITION FOR LCMSMS: Please refer Table 4.6.
Chapter 6. RP-HPLC Method Development
142
LC-MS/MS Spectra are as follow. For the First peak out of three peaks.
FIGURE 6.34 LC-MS/MS Chromatogram for Mass confirmation (1st Peak)
Chapter 6. Result and Discussion
143
FIGURE 6.35 Fragmentation Pattern in LC-MS/MS for a peak on Rt 13.68 min
So, the first peak having molecular ion peak at 307.08 m/z ratio. So it is of bisdesmethoxy
curcumin (molecular weight: 308.333 g/mol)
Chapter 6. RP-HPLC Method Development
144
For the second peak out of three peaks.
FIGURE 6.36 LC-MS/MS Chromatogram for Mass confirmation (2nd Peak)
Chapter 6. Result and Discussion
145
FIGURE 6. 37 Fragmentation Pattern in LC-MS/MS for a peak on Rt 13.80 min
So, the second peak having molecular ion peak at 337.08 m/z ratio. So it is of desmethoxy
curcumin (molecular weight: 338.333 g/mol).
Chapter 6. RP-HPLC Method Development
146
For the third peak out of three peaks:
FIGURE 6.38 LC-MS/MS Chromatogram for Mass confirmation (3rd Peak)
Chapter 6. Result and Discussion
147
FIGURE 6.39 Fragmentation Pattern in LC-MS/MS for a peak on Rt 13.97 min
So, the third peak having molecular ion peak at 367.08 m/z ratio. So it is of curcumin
(molecular weight: 368.333 g/mol).
Chapter 6. RP-HPLC Method Development
148
6.2.7 APPLICABILITY OF THE METHOD
Analysis of Formulations
Applicability of the proposed method was tested on Different formulation. Results are shown in
below Fig. 6.40-6.42 and Table 6.23.
FIGURE 6.40 Chromatograph of formulation (Glysikot)
FIGURE 6.41 Chromatogram of formulation (Diasol)
FIGURE 6.42 Chromatogram of Formulation (Diabeta plus)
Chapter 6. Result and Discussion
149
TABLE 6.23 Assay for formulations by RP-HPLC Method.
Formulation Constituents Assay
(%w/w)
Assay
(mg) %RSD
Glysikot
Gallic acid 1.3216 0.1620 0.4517
Ellagic acid 2.4913 0.2772 0.0743
Curcumin 0.5079 0.0562 0.7011
Diasol
Gallic acid 2.3447 0.2978 0.5929
Ellagic acid 0.1568 0.0181 1.5022
Curcumin 0.2872 0.0314 1.0072
Diabeta Plus
Gallic acid 0.3296 0.0303 0.2790
Ellagic acid 0.0162 0.0025 1.3765
Curcumin 0.0884 0.0091 0.4504
6.2.8 Validation Parameters:
SYSTEM SUITABILITY PARAMETERS:
TABLE 6.24 Observed values for system suitability test *(n=6)
Parameter
Constituent Mean (n=6) ± S.D. % R.S.D.
Retention time
Gallic Acid 3.287 ± 0 0.0000
Ellagic Acid 4.6332 ± 0.0041 0.0889
Curcumin 12.5306 ± 0.0029 0.0235
Tailing factor
Gallic Acid 0.7308 ± 0.0121 1.6588
Ellagic Acid 1.0294 ± 0.015 1.4611
Curcumin 0.8276 ± 0.0103 1.2407
Theoretical Plate
Gallic Acid 14220.8 ± 279.2185 1.9634
Ellagic Acid 15282 ± 260.0169 1.7014
Curcumin 166176.2 ± 3249.6887 1.9555
Resolution
Gallic Acid - -
Ellagic Acid 10.2724 ± 0.1548 1.5073
Curcumin 58.8466 ± 0.4829 0.8206
SPECIFICITY:
The specificity of the method was ascertained by analysing standard drugs and sample of Gallic
acid, Ellagic Acid and Curcumin. The results suggested that proposed method is specific, the
other constituents present in the formulation does not affect the result. The chromatogram taken
by running only with the mobile phase and after injection the sample overlain with standard are
given in Fig. 6.43-6.47.
Chapter 6. RP-HPLC Method Development
150
FIGURE 6 43 Chromatogram of placebo
FIGURE 6.44 Chromatogram for formulation (Glysikot)
FIGURE 6.45 Chromatogram of Gallic acid (4 µg/ml)
FIGURE 6.46 Chromatogram of Ellagic acid (10 µg/ml)
Chapter 6. Result and Discussion
151
FIGURE 6.47 Chromatogram of Curcumin (2 µg/ml)
LINEARITY:
The linearity study was carried out for three markers at seven different concentration levels. The
linearity of Gallic Acid, Ellagic Acid and Curcumin was in the range of 2-14 μg/ml, 5-35 μg/ml
and 1-7 μg/ml of Gallic acid, Ellagic acid and Curcumin respectively depicted in table.
FIGURE 6.48 Overlain Chromatogram of Gallic Acid (2-14 μg/ml), Ellagic Acid (5-35 μg/ml) and
Curcumin (1-7 μg/ml)
Chapter 6. RP-HPLC Method Development
152
TABLE 6.25 Linearity of Gallic Acid (2-14 μg/ml) by RP-HPLC
Conc. (µg/ml) Mean Area ± SD (n=5) % RSD
2 21839.33 ± 141.76 0.6491
4 42452.33 ± 250.71 0.5906
6 59304.67 ± 482.38 0.8134
8 75129.33 ± 87.3 0.1162
10 86323.67 ± 345.15 0.3998
12 105448.33 ± 2729.88 0.4000
14 118140 ± 1856.65 1.3454
FIGURE 6.49 Calibration curve of Gallic Acid (2-14 μg/ml)
TABLE 6.26 Linearity of Ellagic Acid (5-35 μg/ml) by RP-HPLC
Conc. (µg/ml) Mean Area ± SD (n=5) % RSD
5 162900.33 ± 1690.83 1.0380
10 293026 ± 2040.37 0.6963
15 441507.67 ± 234.41 0.0531
20 581998.67 ± 162.24 0.0279
25 756475.67 ± 89.89 0.0119
30 896566.67 ± 632.65 0.0706
35 1054884.67 ± 440.8 0.0418
FIGURE 6.50 Calibration curve of Ellagic Acid (5-35 μg/ml)
y = 7798.4x + 9927.3
R² = 0.9950
0
50000
100000
150000
0 5 10 15
Are
a
Conc. (g/ml)
Gallic Acid at 257 nm
y = 29986x - 1520.4
R² = 0.9989
0
200000
400000
600000
800000
1000000
1200000
0 10 20 30 40
Are
a
Conc. (µg/ml)
Ellagic Acid at 257 nm
Chapter 6. Result and Discussion
153
TABLE 6.27 Linearity of Curcumin (1-7 μg/ml) by RP-HPLC
Conc. (µg/ml) Mean Area ± SD (n=5) % RSD
1 5067 ± 32.69 0.6452
2 12736 ± 86.84 0.6818
3 18456 ± 212.76 1.1528
4 22761.33 ± 234.41 1.0298
5 28464 ± 84.93 0.2984
6 34385 ± 189.49 0.5511
7 40294 ± 101.12 0.2510
FIGURE 6.51 Calibration curve of Curcumin (1-7 μg/ml)
PRECISION:
Repeatability: - The data of repeatability for Gallic Acid, Ellagic Acid and
Curcumin are shown in table. % RSD was found to be 0.0983, 0.0239 and 0.8038
for Gallic acid, Ellagic acid and Curcumin respectively.
TABLE 6.28 Repeatability of Gallic Acid, Ellagic Acid and Curcumin by RP-HPLC
Constituent Conc. (µg/ml) Mean Area ± S.D. (n=6) %R.S.D.
Gallic Acid 8 75164.3333 ± 73.9136 0.0983
Ellagic Acid 20 581956.6667 ± 139.0068 0.0239
Curcumin 4 22829.6667 ± 183. 5135 0.8038
y = 5678.1x + 453.76
R² = 0.9964
0
10000
20000
30000
40000
50000
0 2 4 6 8
Are
a
Conc. (µg/ml)
Curcumin at 257 nm
Chapter 6. RP- HPLC Method Development
154
Intraday precision
The data for intraday precision for Gallic Acid, Ellagic Acid and Curcumin are shown in Table
6.29.
TABLE 6.29 Intraday precision of Gallic Acid, Ellagic Acid & Curcumin by RP-HPLC
Constituent
Conc.(µg/ml)
Area (Mean) (n=3)
± SD
%RSD
Gallic acid
6 59304.67 ± 482.3804 0.8134
8 75129.33 ± 87.3015 0.1162
10 86323.67 ± 345.1544 0.3998 0.4431
Ellagic acid
15 441507.67 ± 234.4075 0.0531
20 581998.67 ± 162.2433 0.0279
25 756475.67 ± 89.8901 0.0119 0.0310
Curcumin
3 18456 ± 212.7596 1.1528
4 22761.33 ± 234.4075 1.0298
5 28464 ± 84.9274 0.2984 0.8270
Interday precision
The data for intraday precision for Gallic Acid, Ellagic Acid and Curcumin are shown in Table
6.30.
TABLE 6.30 Interday precision of Gallic Acid, Ellagic Acid & Curcumin by RP-HPLC
Constituent
Conc.(µg/ml)
Area (Mean) (n=3)
± SD
%RSD
Gallic acid
6 59610.33 ± 551.0041 0.9243
8 75147.33 ± 96.4446 0.1283
10 86133.67 ± 581.6054 0.6752 0.5760
Ellagic acid
15 441719 ± 85.3971 0.0193
20 582517 ± 373.364 0.0641
25 756316.33 ± 138.3578 0.0183 0.0339
Curcumin
3 18535.33 ± 280.6295 1.5140
4 22721 ± 290.7313 1.2796
5 28654.33 ± 201.1406 0.7020 1.1652
Chapter 6. RP-HPLC Method Development
155
ACCURACY:
The % Recovery experiment was performed by the Standard Addition Method. Known amounts
of standard solutions of Gallic Acid, Ellagic Acid and curcumin were added at 80%, 100% and
120 % level to pre-quantified sample solutions of Gallic Acid, Ellagic Acid and Curcumin. The
amounts of Gallic Acid, Ellagic Acid and Curcumin were estimated by using the regression
equation of the calibration curve. The low value of standard deviation indicates that the proposed
method is accurate. Results of recovery studies are shown in Table 6.31.
TABLE 6.31 Accuracy data for Gallic Acid, Ellagic Acid and Curcumin by RP-HPLC.
Constituent
Amt.
Taken
(mg)
Amt.
added
(mg)
Amt. Found (n=3)
± SD
(mg)
Recovery (%)
(n=3) %RSD
Gallic
Acid
0% 1.32 0 1.31 ± 0.0082 99.2424 0.6233
80% 1.32 1.05 2.3433 ± 0.0249 98.8748 1.0645
100% 1.32 1.32 2.6133 ± 0.034 98.9899 1.3008
120% 1.32 1.58 2.86 ± 0.0408 98.6207 1.4274
Ellagic Acid
0% 2.49 0 2.4733 ± 0.0125 99.3307 0.5043
80% 2.49 1.99 4.4633 ± 0.017 99.6280 0.3808
100% 2.49 2.49 4.9833 ± 0.0249 100.0669 0.5006
120% 2.49 2.98 5.49 ± 0.0216 100.3656 0.3935
Curcumin
0% 0.51 0 0.5033 ± 0.0047 98.6928 0.9366
80% 0.51 0.41 0.9133 ± 0.0047 99.2754 0.5161
100% 0.51 0.51 1.0267 ± 0.0094 100.6536 0.9183
120% 0.51 0.61 1.13 ± 0.0082 100.8929 0.7226
LIMIT OF DETECTION (LOD):
The LOD for Gallic Acid, Ellagic Acid and curcumin were 0.0168, 0.0074 and 0.0123 μg/ml
respectively.
LIMIT OF QUANTITATION (LOQ):
The LOQ for Gallic Acid, Ellagic Acid and curcumin were 0.0510, 0.0223 and 0.0374μg/ml
respectively.
ROBUSTNESS:
The robustness of the method was established by making deliberate minor variations in the
following method parameters.
Change in Flow Rate: ± 0.05 units
Change in pH: ± 0.1 unit
Change in wavelength: ± 2 unit
Chapter 6. RP-HPLC Method Development
156
TABLE 6.32 Robustness data for change in flow rate by RP-HPLC.
CHANGE IN FLOW RATE
Drugs Gallic Acid (8 μg/mL) Ellagic Acid (20 μg/mL) Curcumin (4 μg/mL)
Level Peak Area Retention time Peak Area Retention time Peak Area Retention time
0.9
mL/min
73789.00 3.2860 594321.00 4.6320 22874.00 12.5330
75158.00 3.2870 598721.00 4.6330 22963.00 12.5340
73879.00 3.2860 592111.00 4.6330 22789.00 12.5290
Avg 74275.33 3.2863 595051.00 4.6327 22875.33 12.5320
SD 625.22 0.0005 2747.45 0.0005 71.04 0.0022
%RSD 0.8418 0.0143 0.4617 0.0102 0.3106 0.0172
0.95
mL/min
75219.00 3.2870 581791.00 4.6400 22874.00 12.5270
75158.00 3.2870 582018.00 4.6330 22435.00 12.5330
75011.00 3.2870 582187.00 4.6330 22975.00 12.5330
Avg 75129.33 3.2870 581998.67 4.6353 22761.33 12.5310
SD 87.30 0.0000 162.24 0.0033 234.41 0.0028
%RSD 0.1162 0.0000 0.0279 0.0712 1.0298 0.0226
1.0
mL/min
75154.00 3.2870 598721.00 4.6330 22435.00 12.5290
75158.00 3.2860 581791.00 4.6350 22576.00 12.5310
75123.00 3.2880 582018.00 4.6330 22967.00 12.5320
Avg 75145.00 3.2870 587510.00 4.6337 22659.33 12.5307
SD 15.64 0.0008 7927.92 0.0009 225.04 0.0012
%RSD 0.02082 0.0248 1.3494 0.0203 0.9931 0.0100
TABLE 6.33 Robustness data for change in pH by RP-HPLC.
CHANGE IN PH
Drugs Gallic Acid (8 μg/mL) Ellagic Acid (20 μg/mL) Curcumin (4 μg/mL)
Level Peak Area Retention
time Peak Area Retention time Peak Area Retention time
2.45
75154.00 3.2870 598721.00 4.6330 22823.00 12.5290
75258.00 3.2880 581791.00 4.6350 22576.00 12.5310
75123.00 3.2880 581918.00 4.6340 22967.00 12.6240
Avg 75178.33 3.2877 587476.67 4.6340 22788.67 12.5613
SD 57.74 0.0005 7951.11 0.0008 161.46 0.0443
%RSD 0.0768 0.0143 1.3534 0.0176 0.7085 0.3528
2.55
75219.00 3.2870 581791.00 4.6400 22874.00 12.5270
75158.00 3.2870 582018.00 4.6330 22435.00 12.5330
75011.00 3.2870 582187.00 4.6330 22975.00 12.5330
Avg 75129.33 3.2870 581998.67 4.6353 22761.33 12.5310
SD 87.30 0.0000 162.24 0.0033 234.41 0.0028
%RSD 0.1162 0.0000 0.0279 0.0712 1.0298 0.0226
2.65
73789.00 3.2850 594321.00 4.6320 22874.00 12.5330
75184.00 3.2870 598721.00 4.6330 22963.00 12.5980
73879.00 3.2870 592725.00 4.6360 22824.00 12.5290
Avg 74284.00 3.2863 595255.67 4.6337 22887.00 12.5533
SD 637.46 0.0009 2535.51 0.0017 57.49 0.0316
%RSD 0.8581 0.0287 0.4260 0.0367 0.2512 0.2519
Chapter 6. Result and Discussion
157
TABLE 6.34 Robustness data for change in Wavelength by RP-HPLC.
CHANGE IN WAVELENGTH
Drugs Gallic Acid (8 μg/mL) Ellagic Acid (20 μg/mL) Curcumin (4 μg/mL)
Level Peak Area Retention
time Peak Area
Retention
time Peak Area
Retention
time
255 nm
73527.00 3.2850 597536.00 4.6540 22465.00 12.5290
75158.00 3.2870 598721.00 4.6330 22963.00 12.5340
73879.00 3.2860 592111.00 4.6330 22789.00 12.5290
Avg 74188.00 3.2860 596122.67 4.6400 22739.00 12.5307
SD 700.79 0.0008 2877.63 0.0099 206.36 0.0024
%RSD 0.9446 0.0248 0.4827 0.2134 0.9075 0.0188
257 nm
75219.00 3.2870 581791.00 4.6400 22874.00 12.5270
75158.00 3.2870 582018.00 4.6330 22435.00 12.5330
75011.00 3.2870 582187.00 4.6330 22975.00 12.5330
Avg 75129.33 3.2870 581998.67 4.6353 22761.33 12.5310
SD 87.30 0.0000 162.24 0.0033 234.41 0.0028
%RSD 0.1162 0.0000 0.0279 0.0712 1.0298 0.0226
259 nm
75834.00 3.2850 599534.00 4.6450 22834.00 12.5350
75158.00 3.2860 581791.00 4.6350 22576.00 12.5310
75123.00 3.2880 582018.00 4.6330 22967.00 12.5320
Avg 75371.67 3.2863 587781.00 4.6377 22792.33 12.5327
SD 327.23 0.0012 8311.14 0.0052 162.32 0.0017
%RSD 0.4342 0.0380 1.4140 0.1132 0.7122 0.0136
6.3 SUMMARY OF THE DEVELOPED RP-HPLC METHOD
TABLE 6.35 Summary of Validation Parameters for RP-HPLC Method.
Parameters Gallic Acid Ellagic Acid Curcumin
Concentration Range 2-14 µg/ml 5-35 µg/ml 1-7 µg/ml
Regression equation y = 7798.4x + 9927.3 y = 29986x - 1520.4 y = 5678.1x + 453.76
Regression co-efficient 0.9950 0.9989 0.9964
Correlation co-efficient 0.9974 0.9994 0.9981
LOD (n = 5) (µg/ml) 0.0168 0.0074 0.0123
LOQ (n = 5) (µg/ml) 0.0510 0.0223 0.0374
Repeatability ( n = 6) 0.0983 0.0239 0.8038
Intraday precision (n = 3) 0.1162 – 0.8134 0.0119 – 0.0531 0.2984 – 1.1528
Interday precision (n = 3) 0.1283 – 0.9243 0.0183 – 0.0641 0.7020 – 1.5140
% Recovery
0% 99.2424 99.3307 98.6928
80% 98.8748 99.6280 99.2754
100% 98.9899 100.0669 100.6536
120% 98.6207 100.3656 100.8929
Chapter 6. RP-HPLC Method Development
158
References:
1. Adnan M, Mustafa K et al, 2010, “Simultaneous HPLC analysis of pseudophedrine hydrochloride,
codeinephosphate, and triprolidine hydrochloride in liquid dosage forms”, Journal of pharmaceutical and
Biomedical Analysis, 51, 991–993, ISSN No. 0731-7085.
2. Patel R, Patel V, 2012, “Development and validation of a RP-HPLC method for the simultaneous determination of
Embelin, Rottlerin and Ellagic acid in Vidangadi churna”, Journal of Pharmaceutical Analysis 2(5), 366–371, ISSN
No. 2095-1779.
3. Yadav N, Raghuvanshi A et al, 2016, “QbD-Based Development and Validation of a Stability-Indicating HPLC
Method for Estimating Ketoprofen in Bulk Drug and Proniosomal Vesicular System”, Journal of Chromatographic
Science 54(3), 377–389, ISSN No. 0021-9665
4. Manikandan K, Lakshmi K et al, “QbD Approach in RP-HPLC Method development for the Assay of Benzocaine
and Diclofenac in dosage forms”, The 11th National Conference on Mathematical Techniques and Applications
AIP Conf. Proc. 2112, 020083-1–020083-9.
5. Gundala A, Bharathi K et al, 2018, “Analytical Quality by design approach in RP-HPLC method development for
the assay of pitavastatin in tablet dosage form”, IJPSR , 9(11), 4992-5001, ISSN No. 0975-8232.
6. Bondea S, Bondea C etal, 2019, “Quality by design based development and validation of HPLC method for
simultaneous estimation of paclitaxel and vinorelbine tartrate in dual drug loaded liposomes”, Microchemical
Journal, 149, 103982, ISSN No. 0026-265X.
7. Barth C, De Souza G et al, “RP-HPLC and LC–MS–MS determination of a bioactive artefact from Ipomoea pes-
caprae extract”, Revista Brasileira de Farmacognosia xxx (2019) xxx–xxx, ISSN No. 0102-695X.
8. Haneefa J, Mohommad S et al, 2013, “Application of LC–MS/MS for quantitative analysis of glucocorticoids and
stimulants in biological fluids”, Journal of Pharmaceutical Analysis, (5) 341–348, ISSN No. 2095-1779.
9. Yun Zeng, Yi-Ling Quek et al, 2015,“Analysis of 32 toxic natural substances in herbal products by liquid
chromatography quadrupole linear ion trap mass spectrometry”, Journal of Pharmaceutical and Biomedical
Analysis 115, 169–173, ISSN No. 0731-7085.
10. Tomoko K, Shinsuke I et al, 2008, “The Biosynthetic Pathway of Curcuminoids in Turmeric (Curcuma longa) as
Revealed by 13C-Labeled Precursors”, Bioscience, Biotechnology and Biochemistry 72(7), 1789-98, ISSN No.
0916-8451.
11. Adam Horkey, Application note, “Rapid analysis of Curcuminoids in Turmeric extract using the Agilent 1290
infinity LC and STM Columns” by Agilent Technology, 1-4.
Chapter 7. Experimental Work
159
Chapter 7
HPTLC Method Development
7.1 Experimental work [1]
7.1.1 Materials and Instruments: Refer Table 4.1, 4.2 and 4.3
7.1.2 Solvents and Reagents: Refer Table 4.4
7.1.3 Preparation of Stock solution and working standard solution
PREPARATION OF STANDARD STOCK SOLUTION OF GALLIC ACID:
Accurately weighed 4 mg of Gallic Acid was transferred into 10 mL volumetric flask,
Dissolved and diluted up to mark with methanol to get 400 ng/μL solution of Gallic acid.
PREPARATION OF WORKING STOCK SOLUTION OF GALLIC ACID:
40ng/μL of Gallic Acid solution was prepared by diluting 1 mL of stock solution in 10
mL with Methanol.
PREPARATION OF STANDARD STOCK SOLUTION OF ELLAGIC ACID:
Accurately weighed 10 mg of Ellagic Acid was transferred into 10 mL volumetric flask,
dissolved and diluted up to mark with methanol to get 1000 ng/µL solution of Ellagic
acid.
PREPARATION OF WORKING STOCK SOLUTION OF ELLAGIC ACID:
100 ng/μL of Ellagic Acid solution was prepared by diluting 1 mL of stock solution in
10 mL with Methanol.
PREPARATION OF STANDARD STOCK SOLUTION OF CURCUMIN:
Accurately weighed 2 mg of Curcumin was transferred into 10 mL volumetric flask,
dissolved and diluted up to mark with methanol to get 200 ng/μL solution of Curcumin.
Chapter 7. HPTLC Method Development
160
PREPARATION OF WORKING STOCK SOLUTION OF CURCUMIN:
20ng/μL of Curcumin solution was prepared by diluting 1mL of stock solution in 10 mL
with Methanol
7.1.4 Preparation of Calibration curve
One mL working stock solutions from Gallic acid, Ellagic acid and Curcumin were
combined together (Mixture solution) and 0.5, 1, 2, 4, 6, 8 and 10 μL from that solution
were injected in HPTLC Instrument to get 20- 400 ng/band of Gallic Acid, 50- 1000
ng/band of Ellagic Acid and 10- 200 ng/band of Curcumin.
7.1.5 Determination of wavelength
Solution of Gallic acid (4 g/mL), Ellagic acid (10 g/mL) and Curcumin (2 g/mL)
were scanned between 200-800 nm in UV Spectrophotometry.
At wavelength of 300 nm, all three markers show absorbance. So, it is selected for further
determination of markers.
7.1.6 Preparation of Mobile phase
Twin Trough Chamber 20x10cm was taken and to that 6 mL of Toluene, 7 mL of Ethyl
acetate and 2 mL of Formic acid were added.
Chamber was kept aside for 15 minutes preconditioning time.
7.1.7 Determination of Formulation: Refer 6.1.6.
7.1.8 METHOD VALIDATION [2-3]
Parameters to be considered for the validation of method are
SPECIFICITY:
Specificity is a procedure to detect quantitatively the analytes in the presence of components
that may be expected to be present in the sample matrix. While Selectivity is the procedure to
detect qualitatively the analytes in the presence of components that may expect to be present in
Chapter 7. Experimental work
161
the sample matrix. Specificity of Developed method was established by spiking of Gallic Acid,
Ellagic Acid & Curcumin in Polyherbal formulation and expressing that analytes peak were not
interfered from other constituents.
LINEARITY:
The linearity response was determined by analyzing different concentration for calibration curve
in the range of 20- 400 ng/band, 50- 1000 ng/band and 10- 200 ng/band for Gallic Acid, Ellagic
Acid and Curcumin respectively. Plot the calibration curve of Peak Area vs concentration and
determine correlation coefficient and regression line for Gallic Acid, Ellagic Acid & Curcumin.
PRECISION:
Repeatability
6 μL solution from the mixture solution were injected 6 times in HPTLC Instrument. The Area
of the each substance was measured at selected wavelength and % RSD was calculated.
Intraday Precision
2, 4 and 6 μL from the mixture solution were injected 3 times in HPTLC Instrument. The area
of each substance were analyzed three times on the same day and % RSD was calculated.
Interday Precision
2, 4 and 6 μL from the mixture solution were injected 3 times in HPTLC Instrument. The area
of each substance were analyzed on three different days and % RSD was calculated.
ACCURACY:
Accuracy is the closeness of the test results obtained by the method to the true value. Recovery
studies were carried out by addition of standard drug to the sample at 3 different concentration
levels (80%, 100%, 120%) taking into consideration percentage recovery of added bulk drug
samples.
Formulation (A): 0.1 gm of Formulation ( It contains 1.3172 mg of Gallic Acid, 2.4998 mg of
Ellagic acid and 0.5115 mg of Curcumin)
Standard samples: Gallic acid, Ellagic acid and Curcumin
Chapter 7. HPTLC Method Development
162
TABLE 7. 1 Steps for Accuracy study for Gallic Acid by HPTLC Method.
Sr.
No. Step 1 Step 2
Total Gallic Acid
(mg)
1 0.1 gm of Formulation Make up to 10 mL with mobile phase and
filter 1.32
2 0.1 gm of formulation
+1.05 mg of Gallic acid
Make up to 10 mL with mobile phase and
filter 2.37
3 0.1 gm of formulation
+1.32 mg of Gallic acid Make up to 10 mL with mobile phase and
filter 2.64
4 0.1 gm of formulation
+1.58 mg of Gallic acid Make up to 10 mL with mobile phase and
filter 2.9
TABLE 7. 2 Steps for Accuracy study for Ellagic Acid by HPTLC Method.
Sr.
No. Step 1 Step 2
Total Ellagic Acid
(mg)
1 0.1 gm of Formulation Make up to 10 mL with mobile phase and
filter 2.5
2 0.1 gm of formulation
+ 2 mg of Ellagic acid
Make up to 10 mL with mobile phase and
filter 4.5
3 0.1 gm of formulation
+ 2.5 mg of Ellagic acid Make up to 10 mL with mobile phase and
filter 5
4 0.1 gm of formulation
+ 3 mg of Ellagic acid Make up to 10 mL with mobile phase and
filter 5.5
TABLE 7. 3 Steps for Accuracy study for Curcumin by HPTLC Method.
Sr.
No. Step 1 Step 2 Total Curcumin (mg)
1 0.1 gm of Formulation Make up to 10 mL with mobile phase and
filter 0.51
2 0.1 gm of formulation
+ 0.41 mg of Curcumin
Make up to 10 mL with mobile phase and
filter 0.92
3 0.1 gm of formulation
+ 0.51 mg of Curcumin Make up to 10 mL with mobile phase and
filter 1.02
4 0.1 gm of formulation
+ 0.61 mg of Curcumin
Make up to 10 mL with mobile phase and
filter 1.12
LIMIT OF DETECTION (LOD):
The LOD was estimated from the set of six calibration curves used to determine method
linearity. The LOD may be calculated as
LOD = 3.3 x (SD / Slope)
Where, SD = the standard deviation of Y- intercept of six calibration curves.
Slope = the Mean slope of the six calibration curves.
Chapter 7. Experimental work
163
LIMIT OF QUANTITATION (LOQ):
The LOQ was estimated from the set of six calibration curves used to determine method
linearity. The LOQ may be calculated as
LOQ = 10 x (SD / Slope)
Where, SD = the standard deviation of Y- intercept of six calibration curves.
Slope = the Mean slope of the six calibration curves.
ROBUSTNESS:
Robustness of the method was determined by subjecting the method to slight change in the
method condition, individually, i.e. Change in wavelength and change in preconditioning time.
Three replicates were made for the same concentration (240 ng/band of Gallic Acid, 600
ng/band of Ellagic Acid and 120 ng/band of Curcumin). % RSD was calculated.
Chapter 7. HPTLC Method Development
164
7.2 RESULTS AND DISCUSSION
7.2.1 Trials for HPTLC Method Development
TABLE 7. 4 HPTLC TRIALS
Sr.
No. Mobile Phase
Wave
length
(nm)
Densitogram Remarks
1
Toluene: Ethyl
Acetate: Formic
acid
(4.8 :5 : 0.2)
v/v/v
Rf of Gallic acid - 0.41
Rf of Curcumin – 0.75
Ellagic acid merged with Gallic acid
(From literature review of Gallic acid and Curcumin)
2
Toluene: Ethyl
Acetate : Formic
Acid: Methanol
(9 : 9 : 3 : 0.6)
v/v/v
Rf of Gallic acid - 0.45
Rf of Gallic acid - 0.43
Rf of Curcumin – 0.68
Spot is little clear but, Ellagic acid merged with Gallic acid
(From literature review of Gallic acid and Ellagic acid)
3
Toluene: Ethyl
acetate: Formic
acid
(4: 3: 0.8) v/v/v
300
Gallic acid and
Ellagic acid
merged
4
Toluene: Ethyl
acetate: Formic
acid
(3: 4: 0.8) v/v/v
350
Rf of
Curcumin is
above 0.8
Chapter 7. Result and Discussion
165
5
Toluene: Ethyl
acetate: Formic
acid
(3.5: 4: 0.8)
v/v/v
350
Peak of Ellagic
acid is
Asymmetric
6
Toluene: Ethyl
acetate: Formic
acid
(3.5: 4.5: 0.8)
v/v/v
300
Curcumin peak
is not proper
and Rf is
above 0.8
7
Toluene: Ethyl
acetate: Formic
acid
(3: 4: 1.2) v/v/v
250
Splitting of
Curcumin peak
and Rf is
above 0.8
8
Toluene: Ethyl
acetate: Formic
acid
(3: 4: 1.2) v/v/v
300
Splitting of
Curcumin peak
and Rf is
above 0.8
Chapter 7. HPTLC Method Development
166
9
Optimized
Toluene: Ethyl
acetate: Formic
acid
(3: 3.5: 1) v/v/v
300
Well separated
all peaks with
Rf G- 0.59
Rf E - 0.51
Rf C – 0.78
7.2.2 Selection of Wavelength: At wavelength of 300 nm, all three markers show good
absorbance. So, it is selected for further determination of markers.
FIGURE 7. 1 Selection of Wavelength for HPTLC Method, Overlain spectrum of Gallic acid (4 µg/ml),
Ellagic acid (10 µg/ml) and Curcumin (2 µg/ml).
Chapter 7. Result and Discussion
167
7.2.3 Optimized Chromatographic Condition of HPTLC Method
Optimized Chromatographic Condition of HPTLC Method: please refer Table 4.7.
Optimized Densitogram:
FIGURE 7. 2 Final Optimized Densitogram
TABLE 7. 5 Optimized condition for Densitogram.
7.2.4 Applicability of the Method [4-5]
Analysis of Formulations:
Applicability of the proposed method was tested on Different formulations. Results are shown
in following Fig 7.3, 7.4 & 7.5 and Table 7.6.
Peak Max. Rf ± SD
(n = 5)
Area %Area %RSD Assigned Substance
1 0.51 ± 0.0048 10208.47 56.62 0.9531 Ellagic Acid
2 0.59 ± 0.0066 4908.833 27.22 1.1261 Gallic Acid
3 0.78 ± 0.0063 2912.3 16.15 0.8108 Curcumin
Chapter 7. HPTLC Method Development
168
FIGURE 7. 3 Densitogram of Glysikot granules
FIGURE 7. 4 Densitogram of Diasol Capsule
FIGURE 7. 5 Densitogram of Diabeta plus Capsule
Chapter 7. Result and Discussion
169
TABLE 7. 6 Assay of Formulations by HPTLC method.
Formulation Constituents Assay
(%w/w)
Assay
(mg) %RSD
Glysikot
Gallic acid 1.3172 0.1485 0.6343
Ellagic acid 2.4998 0.3160 0.0521
Curcumin 0.5115 0.0663 0.2358
Diasol
Gallic acid 2.3648 0.2775 0.9750
Ellagic acid 0.1592 0.0395 1.1998
Curcumin 0.2923 0.0332 1.3827
Diabeta plus
Gallic acid 0.3313 0.0272 0.4106
Ellagic acid 0.0150 0.0023 0.6721
Curcumin 0.0874 0.1661 0.3517
7.2.5 Validation Parameters
SPECIFICITY:
The specificity of the method was ascertained by analysing standard drugs and sample of Gallic
acid, Ellagic Acid and Curcumin. The results suggested that proposed method is specific, the
other constituents present in the formulation does not affect the result.
FIGURE 7. 6 Chromatogram for Specificity.
FIGURE 7. 7 Overlay Densitogram to confirm markers from mixture
Chapter 7. HPTLC Method Development
170
LINEARITY:
The linearity study was carried out for three markers at seven different concentration levels. The
linearity of Gallic Acid, Ellagic Acid and Curcumin was in the range of 20-400 ng/μL, 50-1000
ng/μL and 10-200 ng/μL of Gallic acid, Ellagic acid and Curcumin respectively
Depicted in Table 7.7, 7.8 and 7.9 respectively.
FIGURE 7. 8 Overlain spectra for Linearity of Gallic Acid (20-400 ng/band), Ellagic Acid (50-1000
ng/band) and Curcumin (10-200 ng/band).
TABLE 7. 7 Linearity of Gallic Acid (20-400 ng/band) by HPTLC Method.
Conc. (ng/band) Mean Area ± SD (n=5) %RSD
20 476.6 ± 9.3 1.9513
40 871.83 ± 6.01 0.6894
80 1825.6 ± 25.97 1.4225
160 3483.67 ± 28.71 0.8241
240 4908.83 ± 51.17 1.0424
320 6467.1 ± 84.14 1.3010
400 7475.57 ± 37.91 0.5071
FIGURE 7. 9 Calibration curve and Peak Purity data of Gallic Acid (20-400 ng/band)
y = 18.821x + 256.43
R² = 0.9946
0
2000
4000
6000
8000
10000
0 100 200 300 400 500
Are
a
Conc. (ng/band)
Gallic Acid
Chapter 7. Result and Discussion
171
TABLE 7. 8 Linearity of Ellagic Acid (50-1000 ng/band) by HPTLC Method.
Conc. (ng/band) Mean Area ± SD (n=5) %RSD
50 890.03 ± 11.77 1.3224
100 2337.51 ± 30.42 1.3014
200 4417.37 ± 58.18 1.3171
400 7488.9 ± 124.29 1.6597
600 10208.47 ± 11.48 0.1125
800 12796.53 ± 190.19 1.4863
1000 15431.84 ± 239.44 1.5516
FIGURE 7. 10 Calibration curve and Peak purity data of Ellagic Acid (50-1000 ng/band)
TABLE 7. 9 Linearity of Curcumin (10 - 200 ng/ band) by HPTLC Method
Conc. (ng/band) Mean Area ± SD (n=5) %RSD
10 480.77 ± 8.48 1.7638
20 644.99 ± 8.16 1.2651
40 1064.03 ± 8.52 0.8007
80 2033.57 ± 26.63 1.3095
120 2912.3 ± 30.06 1.0322
160 3898.43 ± 16.46 0.4222
200 4515.73 ± 66.23 1.4667
FIGURE 7. 11 Calibration curve and Peak purity data of Curcumin (10- 200 ng/band)
y = 14.899x + 948.44
R² = 0.9915
0
5000
10000
15000
20000
0 200 400 600 800 1000 1200
Are
a
Conc. (ng/band)
Ellagic Acid
y = 5678.1x + 453.76
R² = 0.9964
0
10000
20000
30000
40000
50000
0 2 4 6 8
Are
a
Conc. (µg/ml)
Curcumin at 257 nm
Chapter 7. HPTLC Method Development
172
PRECISION:
Repeatability: The data of repeatability for Gallic Acid, Ellagic Acid and Curcumin are
shown in Table 7.10.
TABLE 7. 10 Repeatability of Gallic Acid, Ellagic Acid and Curcumin by HPTLC Method
Constituent Concentration
(ng/band) Mean Area ± SD (n=6) %RSD
Gallic Acid 240 4404.88 ± 45.7015 1.0375
Ellagic Acid 600 9035.93 ± 1.9093 0.0211
Curcumin 120 2925.75 ± 27.7014 0.9468
Intraday precision: The data for intraday precision for Gallic Acid, Ellagic Acid and
Curcumin are shown in Table 7.11.
TABLE 7. 11 Intraday precision of Gallic Acid, Ellagic Acid & Curcumin by HPTLC Method
Constituent Conc.
(ng/band) Area (Mean) (n=3) SD %RSD
Gallic acid
80 1825.60 25.9742 1.4228
160 3483.67 28.7143 0.8243
240 4908.83 51.1729 1.0425 1.0965
Ellagic acid
200 4417.37 58.1835 1.3172
400 7488.90 124.2889 1.6596
600 10208.47 11.4837 0.1125 1.0298
Curcumin
40 1064.03 8.5203 0.8008
80 2033.57 26.6315 1.3096
120 2912.30 30.0647 1.0323 1.0476
Chapter 7. Result and Discussion
173
Interday precision: The data for intraday precision for Gallic Acid, Ellagic Acid
and Curcumin are shown in Table 7.12.
TABLE 7. 12 Interday precision of Gallic Acid, Ellagic Acid & Curcumin by HPTLC Method
Constituent Conc.
(ng/band) Area (Mean) (n=3) SD %RSD
Gallic acid
80 1887.67 29.7461 1.5758
160 3501.93 53.7461 1.5348
240 4949.90 54.5397 1.1018 1.4041
Ellagic acid
200 4398.20 82.1531 1.8679
400 7478.20 136.9063 1.8307
600 10250.57 67.8033 0.6615 1.4534
Curcumin
40 1069.77 16.3563 1.5290
80 2026.50 33.3543 1.6459
120 2925.77 37.1082 1.2683 1.4811
ACCURACY:
The % Recovery experiment was performed by the Standard Addition Method. Known amounts
of standard substance of Gallic Acid, Ellagic Acid and curcumin were added at 80%, 100% and
120 % level to Glysikot granules. The amounts of Gallic Acid, Ellagic Acid and Curcumin were
estimated by using the regression equation of the calibration curve. The low value of standard
deviation indicates that the proposed method is accurate. Results of recovery studies are shown
in Table 7.13.
Chapter 7. HPTLC Method Development
174
TABLE 7. 13 Accuracy data for Gallic Acid, Ellagic Acid and Curcumin by HPTLC Method
Constituent
Amt. Taken
(mg)
Amt. added
(mg)
Amt. Found
(n=3)
± S.D. (mg)
Recovery
(%) (n=3) % RSD
Gallic Acid
0% 1.32 0 1.3367 ± 0.0125 101.2626 0.9331
80% 1.32 1.05 2.3667 ± 0.0125 99.8594 0.5270
100% 1.32 1.32 2.61 ± 0.0356 98.8636 1.3636
120% 1.32 1.58 2.8867 ± 0.0556 99.5402 1.9253
Ellagic Acid
0% 2.5 0 2.4833 ± 0.0287 99.3333 1.1547
80% 2.5 2 4.5167 ± 0.0492 100.3704 1.0897
100% 2.5 2.5 4.97 ± 0.0572 99.4000 1.1500
120% 2.5 3 5.52 ± 0.0497 100.3636 0.8997
Curcumin
0% 0.51 0 0.5133 ± 0.0047 100.6536 0.9183
80% 0.51 0.41 0.9133 ± 0.0047 99.2754 0.5161
100% 0.51 0.51 1.0267 ± 0.0047 100.6536 0.4592
120% 0.51 0.61 1.1333 ± 0.0125 101.1905 1.1005
LIMIT OF DETECTION(LOD) AND LIMIT OF QUANTITATION (LOQ):
The LOD for Gallic Acid, Ellagic Acid and Curcumin were 2.4356, 1.6257 and 2.2471 ng/band
respectively. The LOQ for Gallic Acid, Ellagic Acid and curcumin were7.3805, 4.9264 and
6.8093 ng/band respectively.
ROBUSTNESS:
The robustness of the method was established by making deliberate minor variations in the
following method parameters and shown in Table 7.14 and 7.15
Change in wavelength: ± 2 nm.
Change in preconditioning time: ± 2 min.
Chapter 7. Result and Discussion
175
TABLE 7. 14 Robustness data for change in Wavelength by HPTLC Method
CHANGE IN WAVELENGTH
Drugs Gallic Acid (240 ng/band) Ellagic Acid (600 ng/band) Curcumin (120 ng/band)
Level Peak Area Rf value Peak Area Rf value Peak Area Rf value
298 nm
4339.00 0.5700 10752.60 0.4800 3473.40 0.7700
4419.50 0.5800 10454.70 0.4800 3567.50 0.7600
4254.34 0.5800 10280.20 0.4900 3528.90 0.7800
Avg 4337.61 0.5767 10495.83 0.4833 3523.27 0.7700
SD 67.43 0.0047 195.04 0.0047 38.62 0.0082
%RSD 1.5546 0.8175 1.8582 0.9753 1.0962 1.0604
300 nm
4981.2 0.5800 10213.4 0.5100 2946.9 0.7900
4873.2 0.6000 10192.6 0.5200 2873.6 0.7800
4872.1 0.5900 10219.4 0.5200 2916.4 0.7800
Avg 4908.83 0.5900 10208.47 0.5167 2912.30 0.7833
SD 51.17 0.0082 11.48 0.0047 30.06 0.0047
%RSD 1.0425 1.3839 0.1125 0.9124 1.0323 0.6018
302 nm
3666.00 0.5600 9409.60 0.4900 3363.00 0.7700
3664.90 0.5600 9450.60 0.4800 3348.10 0.7600
3534.20 0.5600 9502.90 0.4800 3491.80 0.7600
Avg 3621.70 0.5600 9454.37 0.4833 3400.97 0.7633
SD 61.87 0.0000 38.18 0.0047 64.52 0.0047
%RSD 1.7084 0.0000 0.4039 0.9753 1.8970 0.6176
TABLE 7. 15 Robustness data for Change in Preconditioning Time by HPTLC Method
PRECONDITIONING TIME
Drugs Gallic Acid (240 ng/band) Ellagic Acid (600 ng/band) Curcumin (120 ng/band)
Level Peak Area Rf value Peak Area Rf value Peak Area Rf value
13 min
2463.57 0.6000 6231.40 0.5500 2171.70 0.7900
2519.50 0.5900 6160.30 0.5400 2212.40 0.7800
2537.70 0.5900 6382.10 0.5400 2151.30 0.7700
Avg 2506.92 0.5933 6257.93 0.5433 2178.47 0.7800
SD 31.54 0.0047 92.47 0.0047 25.40 0.0082
%RSD 1.2582 0.7945 1.4777 0.8676 1.1659 1.0468
15 min
4981.2 0.5800 10213.4 0.5100 2946.9 0.7900
4873.2 0.6000 10192.6 0.5200 2873.6 0.7800
4872.1 0.5900 10219.4 0.5200 2916.4 0.7800
Avg 4908.83 0.5900 10208.47 0.5167 2912.30 0.7833
SD 51.17 0.0082 11.48 0.0047 30.06 0.0047
%RSD 1.0425 1.3839 0.1125 0.9124 1.0323 0.6018
17 min
5072.80 0.5700 9265.67 0.5000 2606.10 0.7800
4889.80 0.5600 9291.80 0.5000 2657.32 0.7800
4992.00 0.5600 9008.20 0.5000 2654.32 0.7800
Avg 4984.87 0.5633 9188.56 0.5000 2639.25 0.7800
SD 74.88 0.0047 127.98 0.0000 23.47 0.0000
%RSD 1.5021 0.8368 1.3928 0.0000 0.8893 0.0000
Chapter 7. HPTLC Method Development
176
7.3 Summary of the developed HPTLC method
TABLE 7. 16 Summary of Validation parameters for HPTLC Method.
Parameters Gallic Acid Ellagic Acid Curcumin
Concentration Range 20 - 400 ng/band 50 - 1000 ng/band 10 - 200 ng/band
Regression equation y = 18.821x + 256.43 y = 14.899x + 948.44 y = 22.008x + 240.72
Regression co-efficient 0.9946 0.9915 0.9973
Correlation co-efficient 0.9972 0.9957 0.9986
LOD (n = 5)(ng/band)) 2.4356 1.6257 2.2471
LOQ (n = 5) (ng/band)) 7.3805 4.9264 6.8093
Repeatability (n = 6) (%RSD) 1.0375 0.0211 0.9468
Intraday precision (n=3) (%
RSD) 0.8243 – 1.4228 0.1125 – 1.6596 0.8008 – 1.3096
Interday precision (n=3) (%
RSD) 1.1018 – 1.5758 0.6615 – 1.8679 1.2683 – 1.6459
% Recovery
0% 101.2626 99.3333 100.6536
80% 99.8594 100.3704 99.2754
100% 98.8636 99.4000 100.6536
120% 99.5402 100.3636 101.1905
References:
1. Bhole, 2015, “A High-Performance Thin Layer Chromatography (HPTLC) Method for Simultaneous
Determination of Diphenhydramine Hydrochloride and Naproxen Sodium in Tablets”, Analytical Chemistry
Insights. 10, 47-51, ISSN NO. 1177-3901
2. Dhalwal K, Shinde V, Mahadik K, 2007, “Rapid Densitometric method for Simultaneous Analysis of
Umbelliferone, Psoralen, and Eugenol in Herbal raw materials using HPTLC”, Journal of Separation Science, 30,
2053-2058, ISSN No. 1615-9314.
3. Singh M, Younus K,Thajudeen K et al, 2011, “Development and Validation of a Stability-Indicating HPTLC
Method for Analysis of Arjunolic Acid in a Herbal Formulation”, Journal of Planar Chromatography, 24(2), 172–
175, ISSN No. 1789-0993.
4. Anjoo Kamboj, Ajay Kumar Saluja, 2013, “Development of Validated HPTLC method for Quantification of
Stigmasterol from leaf and stem of Bryophyllum Pinnatum”, Arabian Journal of Chemistry. xxx, xxx–xxx, ISSN
No. 1878-5352.
5. Sheikh ZA, Shakeel S et al, 2015, “A Novel HPTLC method for Quantitative Estimation of Biomarkers in Poly-
herbal Formulation”, Asian Pacific Journal of Tropical Biomedicine, ISSN No. 2221-1691.
Chapter 8 Experimental work
177
CHAPTER - 8
Chemometric Methods Development
8.1 Experimental Work
8.1.1 Materials and Instruments: Refer Table 4.1, 4.2 and 4.3
8.1.2 Solvents and Reagents: Refer Table 4.4
8.1.3 Preparation of Stock solution and working standard solution: Refer 6.1.3
8.1.4 Preparation of Calibration and Validation set[1-2]
CONSTRUCTION OF THE CALIBRATION (TRAINING) SET :
A training set consisting of 20 mixture solutions in the possible combinations containing 2 -
20g/ml of Gallic Acid 5-50g/ml of Ellagic Acid & 1-10g/ml of Curcumin was used for
Chemometric Calibrations. Randomly take the mixture of all three markers and zero order
absorbance spectra were measured & stored in the computer. To estimate the CLS and ILS
models for the training set, the computer was fed with absorbance & concentration matrices,
then calculations were carried out with the use of proposed software (MATLAB R2015a)
CONSTRUCTION OF THE VALIDATION SET:
Different mixtures of the three drugs were prepared by diluting different volumes of Gallic Acid,
Ellagic Acid and Curcumin standard solutions in 10 ml measuring flask & diluting to volume
with methanol.
8.1.5 Determination of Wavelength range for Measurement
0.4 mL of working standard solution of Gallic Acid (4 μg/mL), 1 mL of working
standard solution of Ellagic Acid (10 μg/mL) and 0.2 mL of working standard solution
of Curcumin were diluted to 10 mL with mobile Phase individually to get 4 μg/mL of
Gallic Acid, 10 μg/mL of Ellagic Acid and 2 μg/mL of Curcumin.
Chapter 8. Chemometric Methods
178
Each solution was scanned between 200-800 nm in UV Spectrophotometry.
Wavelength range from 241-279 nm were selected as in this range all three markers
show variation in absorbance.
8.1.6 Determination of Formulation: Refer 6.1.6
8.1.7 Validation Parameters
ACCURACY:
The accuracy of the method was obtained by the recovery studies on validation sets.
Concentration obtained from the software and compare with Actual data to calculate %
Recovery of the developed Method.
PRECISION:
The precision was determined by means of a one way ANOVA including 10 replicates carried
out on three successive days for Formulation. F values below the tabulated levels were obtained
and there was no significant difference between the results obtained in the determination of each
drug in the presence of other on different days.
LIMIT OF DETECTION (LOD):
LOD was measured as follows
LOD=3 * SD of mean of analytical signal /slope of calibration curve
LIMIT OF QUANTITATION (LOQ):
LOQ was measured as follows
LOQ=10 * SD of mean of analytical signal /slope of calibration curve
Chapter 8. Result and Discussion
179
8.2 Results and Discussion
In the spectral work, the following steps can explain the fundamental concepts of CLS and ILS.
8.2.1 Determination of Wavelength range for Measurement
Although CLS and ILS are the full spectrum method, 20 wavelengths were selected from 241nm
to 279nm with the interval of = 2 nm in the zero order spectra.
8.2.2 Measurement of the Absorbance
The absorbance matrices were produced by measuring absorbance at 20 wavelengths. In this
calibration was obtained by measuring absorbance data matrix & concentration data matrix to
predict the concentration of Gallic Acid, Ellagic Acid & Curcumin in their tertiary mixtures &
Formulations. The numerical calculations were performed using MATLAB R2015a
SOFTWARE & EXCEL.
FIGURE 8.1 Overlay spectra of Gallic Acid, Ellagic Acid and Curcumin showing spectral region
241nm- 279 nm (20 wavelengths range)
Chapter 8. Chemometric Method
180
Calibration set was prepared from the working standard solutions of Gallic acid, Ellagic acid
and Curcumin combinely in the same volumetric flask. For example, 0.2 mL from working stock
solution of Gallic acid; 0.5 mL from working stock solution of Ellagic acid and 0.1 mL from
working stock solution of Curcumin and dilute up to 10 mL with methanol to obtain 2, 5 and 1
µg/mL solution of Gallic acid, Ellagic acid and Curcumin respectively. (Mixture No. 1).
TABLE 8. 1 Composition of Calibration set for three constituents used in CLS Techniques
Mix. No. Gallic Acid (µg/ml) Ellagic Acid (µg/ml) Curcumin (µg/ml)
1 2 5 1
2 4 10 2
3 6 15 3
4 8 20 4
5 10 25 5
6 12 30 6
7 14 35 7
8 16 40 8
9 18 45 9
10 20 50 10
11 2 50 10
12 4 45 9
13 6 40 8
14 8 35 7
15 10 30 6
16 12 25 5
17 14 20 4
18 16 15 3
19 18 10 2
20 20 5 1
Absorbance of all mixtures were determined on each 20 wavelength at the interval of 2 nm
(241 to 279 nm).
The following Table 8.2 showing Absorbance data for all mixtures at each wavelength
181
TABLE 8. 2 Absorbance data for the Calibration set at wavelength range (241-279 nm).
Mix. No. ABSORBANCE
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
WA
VE
LE
NG
TH
241 0.0600 0.1155 0.2305 0.3233 0.3762 0.4288 0.6089 0.7256 0.8097 0.9200 0.6351 0.7531 0.3185 0.5538 0.4486 0.2661 0.2389 0.1966 0.1236 0.1077
243 0.0633 0.1243 0.2486 0.3493 0.4020 0.4601 0.6437 0.7877 0.8796 0.9992 0.6848 0.8155 0.3368 0.5979 0.4844 0.2852 0.2575 0.2124 0.1352 0.1182
245 0.0662 0.1320 0.2652 0.3732 0.4247 0.4885 0.6745 0.8471 0.9451 1.0755 0.7302 0.8724 0.3536 0.6383 0.5157 0.3026 0.2744 0.2270 0.1461 0.1297
247 0.0685 0.1382 0.2791 0.3933 0.4432 0.5114 0.6977 0.8996 1.0024 1.1426 0.7681 0.9200 0.3677 0.6720 0.5410 0.3173 0.2894 0.2412 0.1574 0.1411
249 0.0705 0.1438 0.2921 0.4119 0.4601 0.5332 0.7180 0.9488 1.0566 1.2065 0.8022 0.9622 0.3797 0.7024 0.5634 0.3319 0.3040 0.2547 0.1687 0.1534
251 0.0734 0.1501 0.3045 0.4301 0.4778 0.5543 0.7411 0.9951 1.1075 1.2664 0.8332 1.0030 0.3922 0.7321 0.5866 0.3467 0.3184 0.2679 0.1800 0.1663
253 0.0756 0.1549 0.3152 0.4444 0.4926 0.5728 0.7597 1.0281 1.1447 1.3090 0.8566 1.0311 0.4029 0.7535 0.6048 0.3596 0.3316 0.2798 0.1896 0.1782
255 0.0773 0.1578 0.3191 0.4491 0.5008 0.5822 0.7669 1.0338 1.1506 1.3133 0.8584 1.0316 0.4094 0.7585 0.6128 0.3681 0.3397 0.2888 0.1969 0.1884
257 0.0780 0.1586 0.3163 0.4432 0.5003 0.5809 0.7591 1.0065 1.1208 1.2803 0.8382 1.0033 0.4117 0.7449 0.6079 0.3714 0.3437 0.2937 0.2017 0.1956
259 0.0783 0.1589 0.3094 0.4311 0.4953 0.5737 0.7425 0.9591 1.0723 1.2192 0.8036 0.9595 0.4123 0.7196 0.5965 0.3732 0.3460 0.2972 0.2056 0.2013
261 0.0787 0.1592 0.3026 0.4185 0.4901 0.5668 0.7256 0.9105 1.0205 1.1564 0.7692 0.9116 0.4125 0.6937 0.5851 0.3756 0.3481 0.3005 0.2086 0.2055
263 0.0796 0.1608 0.2966 0.4067 0.4861 0.5617 0.7109 0.8650 0.9718 1.0982 0.7377 0.8680 0.4142 0.6690 0.5748 0.3788 0.3509 0.3045 0.2122 0.2093
265 0.0804 0.1621 0.2891 0.3933 0.4801 0.5526 0.6934 0.8190 0.9230 1.0377 0.7034 0.8233 0.4145 0.6430 0.5623 0.3806 0.3524 0.3064 0.2146 0.2110
267 0.0808 0.1626 0.2791 0.3769 0.4703 0.5398 0.6711 0.7670 0.8661 0.9697 0.6654 0.7715 0.4130 0.6112 0.5459 0.3804 0.3515 0.3087 0.2160 0.2106
269 0.0814 0.1624 0.2679 0.3582 0.4582 0.5249 0.6453 0.7101 0.8044 0.8976 0.6238 0.7173 0.4107 0.5759 0.5265 0.3787 0.3492 0.3071 0.2164 0.2081
271 0.0812 0.1613 0.2545 0.3368 0.4438 0.5049 0.6158 0.6490 0.7383 0.8198 0.5780 0.6591 0.4071 0.5365 0.5029 0.3747 0.3445 0.3053 0.2157 0.2033
273 0.0807 0.1595 0.2399 0.3144 0.4267 0.4824 0.5846 0.5884 0.6709 0.7422 0.5325 0.6008 0.4014 0.4959 0.4778 0.3684 0.3384 0.3015 0.2138 0.1967
275 0.0804 0.1563 0.2253 0.2913 0.4074 0.4566 0.5530 0.5312 0.6074 0.6696 0.4877 0.5446 0.3933 0.4557 0.4499 0.3596 0.3287 0.2954 0.2103 0.1885
277 0.0787 0.1516 0.2097 0.2683 0.3860 0.4288 0.5213 0.4794 0.5500 0.6044 0.4465 0.4928 0.3815 0.4170 0.4209 0.3470 0.3160 0.2863 0.2054 0.1791
279 0.0768 0.1454 0.1941 0.2464 0.3624 0.4003 0.4905 0.4352 0.4992 0.5473 0.4080 0.4452 0.3680 0.3807 0.3903 0.3307 0.3002 0.2739 0.1979 0.1688
Chapter 8 Chemometric Methods
182
Data obtained from Calibration set will be used to prepare one matrix with the help of
which validation of the method will be possible. We can compare with validation set data
to validate developed Method.
TABLE 8. 3 Composition of Validation set for all three constituents used in CLS Techniques.
Mixture No. Gallic acid (g/mL) Ellagic acid (g/mL) Curcumin (g/mL)
1 20 5 10
2 18 10 9
3 16 15 8
4 14 20 7
5 12 25 6
6 10 30 5
7 8 35 4
8 6 40 3
9 4 45 2
10 2 50 1
Absorbance of prepared validation set were determined from 241 to 279 nm. The
following Table 8.4 shows the data of Absorbance.
TABLE 8. 4 Absorbance data for the above Validation set at wavelength range (241-279 nm).
ABSORBANCE
1 2 3 4 5 6 7 8 9 10
WA
VE
LE
NG
TH
241 0.1404 0.189 0.2614 0.321 0.4349 0.5026 0.5151 0.7456 0.7981 0.731
243 0.1504 0.2021 0.2803 0.3448 0.4706 0.5455 0.5585 0.8117 0.8691 0.794
245 0.1611 0.2149 0.2988 0.3673 0.5042 0.584 0.5965 0.8726 0.933 0.8516
247 0.1721 0.2275 0.3154 0.3864 0.5335 0.6185 0.6297 0.9261 0.9887 0.8993
249 0.184 0.2398 0.3317 0.4048 0.5609 0.6499 0.6586 0.9753 1.0404 0.9414
251 0.1963 0.2534 0.3482 0.423 0.5876 0.6801 0.6865 1.0204 1.0865 0.9811
253 0.2078 0.2656 0.3631 0.4392 0.6082 0.7017 0.7084 1.0513 1.1171 1.0085
255 0.2174 0.2749 0.3721 0.4476 0.6137 0.7065 0.7125 1.0504 1.1155 1.0083
257 0.2246 0.2804 0.3741 0.4489 0.604 0.6917 0.6995 1.0184 1.0797 0.9802
259 0.2298 0.284 0.3725 0.4455 0.5849 0.6656 0.6759 0.9643 1.0231 0.9335
261 0.2336 0.286 0.3693 0.441 0.5642 0.6375 0.6511 0.9082 0.964 0.8856
263 0.2361 0.2876 0.3662 0.4367 0.5452 0.6117 0.629 0.8574 0.9101 0.8434
265 0.2364 0.2869 0.3607 0.4308 0.524 0.5852 0.6046 0.8071 0.8562 0.7997
267 0.2345 0.2834 0.3523 0.4213 0.499 0.5534 0.576 0.7491 0.7955 0.7502
269 0.2306 0.2777 0.3409 0.409 0.4707 0.5188 0.5439 0.6898 0.7312 0.6964
271 0.2243 0.2693 0.327 0.3937 0.4387 0.4807 0.5084 0.6253 0.6631 0.6395
273 0.2169 0.2596 0.3111 0.3754 0.4055 0.4417 0.4719 0.5627 0.5965 0.5828
275 0.2079 0.2479 0.2933 0.3555 0.373 0.4035 0.4351 0.5031 0.5334 0.528
277 0.1982 0.235 0.275 0.3335 0.3413 0.3681 0.3995 0.4505 0.4777 0.4779
279 0.1873 0.2211 0.257 0.31 0.3123 0.3361 0.366 0.4057 0.4293 0.4334
Chapter 8 Result and Discussion
183
The absorbance values of the samples, at the 20 wavelengths in the spectral region from 241 to
279 nm were placed in the above equation and the amounts of Gallic Acid, Ellagic Acid &
Curcumin in the synthetic mixture and formulations were found.
8.2.3 Equation for CLS (Classical Least Square) Method [3-4]
The mathematical expression A=K C in the matrix is given as
A1 =K11A1 + K12C2 + ………K1cCc
A2 =K21A1 + K22C2 + ………K2cCc
A3 =K31A1 + K32C2 + ………K3cCc
: : : :
Aw =Kw1A1 + Kw2C2 + ………KcwCc
Where,
Aw = Absorbance at the wth wavelength.
Kcw = Calibration coefficient for the cth component at the wth wavelength.
Cc = Concentration of the cth component.
In this method the calibration coefficient (K) was obtained from the linear equation system using
the absorbance data & the training set.
The absorbance values of the samples at 20 wavelegths were placed in the above equation & the
amounts of Gallic Acid, Ellagic Acid & Curcumin in the synthetic mixtures & formulation were
found.
Introducing (K) into linear equation system, the calibration for CLS was obtained as:
Introducing (K) in to the linear equation system with an absorbance matrix of sample
gives the concentration of Gallic Acid, Ellagic Acid and Curcumin in the sample mixture.
Chapter 8 Chemometric Methods
184
8.2.4 Equation for ILS (Inverse Least Square) Method:
The mathematical expression C=P×A in the matrix is given as
C1 =P11A1 + P12A2 + ………P1wAw
C2 =P21A1 + P22A2 + ………P2wAw
C3 =P31A1 + P32A2 + ………P3wAw
: : : :
Cc =Pc1A1 + Pc2A2 + ………PcwAw
Where,
Aw = Absorbance at the wth wavelength.
Pcw = Calibration coefficient for the cth component at the wth wavelength.
Cc = Concentration of the cth component
In this method the calibration coefficient (P) was obtained from the linear equation system using
the absorbance data & the training set.
Chapter 8 Result and Discussion
185
The absorbance values of the samples at 20 wavelengths were placed in the above equation & the
amounts of Gallic acid, Ellagic Acid & Curcumin in the synthetic mixtures & formulation were
found.
Introducing (P) into linear equation system, the calibration for ILS was obtained as follows.
Chapter 8 Chemometric Method
186
8.2.5 Validation Parameter
ACCURACY:
Recovery study from CLS and ILS were found in Table 8.5 and 8.6.
TABLE 8. 5 Recovery results obtained for the determination of Gallic Acid, Ellagic Acid &
Curcumin by CLS Method.
GALLIC ACID ELLAGIC ACID CURCUMIN
Added
(µg/ml)
Found
(µg/ml)
%
Recovery
Added
(µg/ml)
Found
(µg/ml)
%
Recovery
Added
(µg/ml)
Found
(µg/ml)
%
Recovery
20 20.03 100.1500 5 4.94 98.8000 10 10.21 102.1000
18 18.19 101.0556 10 10.13 101.3000 9 9.04 100.4444
16 15.98 99.8750 15 15.11 100.7333 8 8.12 101.5000
14 14.12 100.8571 20 19.93 99.6500 7 6.94 99.1429
12 12.05 100.4167 25 25.30 101.2000 6 6.07 101.1667
10 9.97 99.7000 30 30.04 100.1333 5 5.10 102.0000
8 7.96 99.5000 35 35.17 100.4857 4 3.96 99.0000
6 6.05 100.8333 40 40.14 100.3500 3 3.03 101.0000
4 3.96 99.0000 45 44.95 99.8889 2 2.01 100.5000
2 2.03 101.5000 50 50.18 100.3600 1 0.98 98.000
Mean Recovery 100.2888 Mean Recovery 100.2901 Mean Recovery 100.4854
%RSD 0.7402 %RSD 0.6985 %RS 1.2917
TABLE 8. 6 Recovery results obtained for the determination of Gallic Acid, Ellagic Acid & Curcumin by ILS
Method.
GALLIC ACID ELLAGIC ACID CURCUMIN
Added
(µg/ml)
Found
(µg/ml)
%
Recovery
Added
(µg/ml)
Found
(µg/ml)
%
Recovery
Added
(µg/ml)
Found
(µg/ml)
%
Recovery
20 20.21 101.0500 5 5.03 100.6000 10 9.92 99.2000
18 18.19 101.0556 10 10.02 100.2000 9 8.95 99.4444
16 15.96 99.7500 15 14.95 99.6667 8 8.13 101.6250
14 14.22 101.5714 20 20.01 100.0500 7 7.03 100.4286
12 11.93 99.4167 25 25.25 101.0000 6 5.98 99.6667
10 10.07 100.7000 30 30.16 100.5333 5 4.94 98.8000
8 7.96 99.5000 35 35.42 101.2000 4 4.08 102.0000
6 5.89 98.1667 40 40.18 100.4500 3 3.02 100.6667
4 4.02 100.5000 45 45.49 101.0889 2 2.04 102.0000
2 2.03 101.5000 50 49.37 98.7400 1 0.99 99.0000
Mean Recovery 100.3210 Mean Recovery 100.3529 Mean Recovery 100.2831
%RSD 1.0291 %RSD 0.7024 %RSD 1.1785
Chapter 8 Result and Discussion
187
PRECISION:
F ration in below table was < 0.005 in both methods for all markers (Gallic acid, Ellagic acid and
Curcumin) so that variation observed between obtained result and actual result. So from above
data, Results were very much precise.
TABLE 8. 7 Data for precision studies for Gallic Acid, Ellagic Acid and Curcumin by one way ANOVA
Parameters CLS ILS
Gallic Acid Ellagic Acid Curcumin Gallic Acid Ellagic Acid Curcumin
Between days
variance 36.6666 229.1666 9.1666 36.6666 229.1667 9.1666
Within days
variance 36.7858 229.2531 9.2899 37.2650 228.8556 9.3260
F ratio 0.0002424 0.000002 0.000576 0.0004091 0.000011 0.000678
TSS 661.081963 4125.779 166.1149 665.4010 4122.203 166.4408
LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTITATION (LOQ):
LOD and LOQ values were determined to check whether Linearity starting points for all
constituents can be measured accurately and precisely or not. Here, LOQ for Gallic acid, Ellagic
acid and Curcumin were 0.5118, 0.7900 and 0.2646 µg/mL from CLS Method and LOQ for Gallic
acid, Ellagic acid and Curcumin were 0.5470, 0.6133 and 0.3662 µg/mL from ILS Method which
are quite less as compare to starting point of range.
TABLE 8. 8 LOD and LOQ for Gallic Acid, Ellagic Acid & Curcumin by CLS & ILS method
Parameters
CLS ILS
Gallic Acid Ellagic
Acid Curcumin Gallic Acid
Ellagic
Acid Curcumin
LOD (µg/ml) 0.1689 0.2607 0.0873 0.1805 0.2024 0.1208
LOQ (µg/ml) 0.5118 0.7900 0.2646 0.5470 0.6133 0.3662
PREDICTED VERSUS KNOWN CONCENTRATION PLOT:
The predicted concentrations of the validation samples were plotted against the known
concentration values. This tool is used to determine whether the model accounts for the
concentration variation in the validation set or not. Plots were expected to fall on a straight line
with a slope of 1 and 0 intercept. The predicted versus known concentration plots of the prepared
concentration plot of the prepared validation samples.
Chapter 8. Chemometric Methods
188
TABLE 8. 9 Actual, Predicted and Residual values by CLS method.
Sr.
No.
Actual Conc.
(µg/ml)
Predicted Conc.
(µg/ml) Residual Percentage
Gall
ic a
cid
Ell
agic
aci
d
Cu
rcu
min
Gall
ic a
cid
Ell
agic
aci
d
Cu
rcu
min
Gall
ic a
cid
Ell
agic
aci
d
Cu
rcu
min
Gall
ic a
cid
Ell
agic
aci
d
Cu
rcu
min
1 20 5 10 20.11 5.03 10.05 -0.11 -0.026 -0.046 100.55 100.52 100.46
2 18 10 9 18.09 10.10 9.03 -0.094 -0.096 -0.028 100.52 100.96 100.31
3 16 15 8 15.95 14.96 8.05 0.044 0.04 -0.048 99.73 99.73 100.60
4 14 20 7 14.02 20.05 7.04 -0.016 -0.048 -0.04 100.11 100.24 100.57
5 12 25 6 12.06 24.63 6.14 -0.056 0.366 -0.136 100.47 98.54 102.27
6 10 30 5 10.02 30.21 5.02 -0.022 -0.208 -0.022 100.22 100.69 100.44
7 8 35 4 8.04 35.05 4.01 -0.04 -0.05 -0.008 100.50 100.14 100.20
8 6 40 3 6.02 40.03 3.01 -0.022 -0.034 -0.012 100.37 100.09 100.40
9 4 45 2 4.1 45.14 1.98 -0.1 -0.144 0.016 102.50 100.32 99.20
10 2 50 1 2.01 49.89 1.01 -0.006 0.1046 -0.002 100.30 99.79 100.20
TABLE 8. 10 Actual, Predicted and Residual values by ILS method.
Sr.
No.
Actual Conc.
(µg/ml)
Predicted Conc.
(µg/ml) Residual Percentage
Gall
ic a
cid
Ell
agic
aci
d
Cu
rcu
min
Gall
ic a
cid
Ell
agic
aci
d
Cu
rcu
min
Gall
ic a
cid
Ell
agic
aci
d
Cu
rcu
min
Gall
ic a
cid
Ell
agic
aci
d
Cu
rcu
min
1 20 5 10 20.05 5.06 10.06 -0.05 -0.06 -0.068 100.25 101.20 100.68
2 18 10 9 18.32 10.02 9.04 -0.326 -0.022 -0.04 101.81 100.22 100.44
3 16 15 8 16.04 15.04 8.06 -0.048 -0.048 -0.066 100.30 100.32 100.83
4 14 20 7 14.03 20.04 7.04 -0.03 -0.042 -0.048 100.21 100.21 100.69
5 12 25 6 12.02 25.01 6.10 -0.026 -0.01 -0.106 100.22 100.04 101.77
6 10 30 5 10.02 29.88 4.99 -0.024 0.12 0.002 100.24 99.60 99.96
7 8 35 4 7.94 35.08 4.03 0.06 -0.086 -0.03 99.25 100.25 100.75
8 6 40 3 6.08 40.05 3.01 -0.084 -0.05 -0.006 101.40 100.13 100.20
9 4 45 2 4.01 45.05 1.99 -0.01 -0.05 0.002 100.25 100.11 99.90
10 2 50 1 2.01 49.97 0.99 -0.012 0.026 0.006 100.60 99.95 99.40
Here, we obtained Calibration curve for predicted versus known concentration. It showed
similarity between obtained data and predicted data. For Gallic acid r2 value was found to
be 0.9999 and 0.9998 for CLS and ILS methods respectively. For Ellagic acid r2 Value was
found to be 0.9999 and 1 for CLS and ILS methods respectively. For Curcumin r2 Value was
found to be 0.9997 and 0.9999 for CLS and ILS methods respectively. So, form the
Calibration curve; we obtained Good correlation between Actual and predicted
Chapter 8. Result and Discussion
189
concentration. This indicates that the prediction ability of the validation set is very much
better in terms of recovery.
FIGURE 8. 2 Linearity plots for Gallic Acid, Ellagic acid and Curcumin by CLS & ILS method
y = 1.0016x + 0.0247
R² = 0.9999
0.00
5.00
10.00
15.00
20.00
25.00
0 10 20 30
Con
c.
(g/m
l)
Conc. (g/ml)
Actual Value Vs. Predicted
Value
Gallic Acid CLS
y = 1.008x - 0.0332
R² = 0.9998
0.00
5.00
10.00
15.00
20.00
25.00
0 10 20 30
Con
c.
(g/m
l)
Conc. (g/ml)
Actual Value Vs. Predicted
Value
Gallic Acid ILS
y = 1.0001x + 0.0071
R² = 0.9999
0
20
40
60
0 20 40 60
Con
c.
(g/m
l)
Conc. (g/ml)
Actual Conc. Vs. Predicted
Conc.
Ellagic Acid CLS
y = 0.9993x + 0.0411
R² = 1
0
20
40
60
0 20 40 60Con
c.
(g/m
l)
Conc. (g/ml)
Actual Conc. Vs. Predicted
Conc.
Ellagic Acid ILS
y = 1.0103x - 0.0325
R² = 0.99970
2
4
6
8
10
12
0 5 10 15
Con
c.
(g/m
l)
Conc. (g/ml)
Actual conc. Vs. Predicted
conc.
Curcumin CLS
y = 1.0086x - 0.012
R² = 0.9999
0
2
4
6
8
10
12
0 5 10 15
Con
c.
(g/m
l)
Conc. (g/ml)
Actual conc. Vs. Predicted
conc.
Curcumin ILS
Chapter 8. Chemometric methods
190
CONCENTRATION RESIDUALS VERSUS PREDICTED
CONCENTRATION PLOT:
The difference the known and predicted concentration (residuals) were plotted against the
actual concentrations for the validation samples. It is used to determine whether the model is
valid for the concentration variation in the validation set and it also provides information
about how well the method will predict the future samples. For the validation set, it can be
found that the residual values are more close to zero and are more randomly distributed.
-0.12
-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0 5 10 15 20 25
Resi
du
al
valu
e (
g/m
l)
Predicted Value (g/ml)
Residual Vs. Predicted concentration plot for
Gallic acid (CLS)
-0.35
-0.3
-0.25
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0 5 10 15 20 25
Resi
du
al
valu
e (
g/m
l)
Predicted Value (g/ml)
Residual Vs. Predicted concentration plot for
Gallic acid (ILS)
Chapter 8. Result and Discussion
191
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0 10 20 30 40 50 60
Resi
du
al
Valu
e (
g/m
l)
Predicted Value (g/ml)
Residual Vs. Predicted concentration plot for
Ellagic Acid (CLS)
-0.1
-0.05
0
0.05
0.1
0.15
0 10 20 30 40 50 60
Resi
du
al
Valu
e (
g/m
l)
Predicted Value (g/ml)
Residual Vs. Predicted concentration plot for
Ellagic Acid (ILS)
Chapter 8. Chemometric Methods
192
FIGURE 8. 3 Residual vs. predicted concentration plot for Gallic Acid, Ellagic Acid & Curcumin.
From the above plots, we can observe that all the residual points are very much near
to the zero line so the developed method is quite correlate with predicted system.
ROOT MEAN SQUARE ERROR OF PREDICTION:
The predictive ability of a model can be defined as RMSEP (Root Mean Square Error of
Prediction). RMSEP summarizes both precision & accuracy. It is used for examining the
errors in the predicted concentrations. The use of RMSEP means we are producing the
statistics on some new things or on which were left out during Calibration. It is consider as
separate Test set or it can be use along with Cross validation scheme.
-0.16
-0.14
-0.12
-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0 2 4 6 8 10 12
Resi
du
al
Valu
e (
g/m
l)
Predicted Value (g/ml)
Residual Vs. Predicted concentration
plot for Curcumin (CLS)
-0.12
-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0 2 4 6 8 10 12
Resi
du
al
Valu
e (
g/m
l)
Predicted Value (g/ml)
Residual Vs. Predicted concentration
plot for Curcumin (ILS)
Chapter 8. Result and Discussion
193
It is calculated from the following formula.
Where,
Ciadded = added concentration of the drug.
Cifound = predicted concentration of the drug.
n =total number of synthetic mixture.
The full range of concentration residual should correspond to approximately 2-3 RMSEP
units if there is no bias.
TABLE 8.11 RMSEP values for Gallic Acid, Ellagic Acid and Curcumin for CLS & ILS method
COMPONENT RMSEP (CLS) RMSEP (ILS)
Gallic Acid 0.0623 0.1114
Ellagic Acid 0.1504 0.0598
Curcumin 0.0512 0.0501
8.2.6 APPLICABILITY OF THE METHOD
ANALYSIS OF FORMULATIONS:
Applicability of the proposed method was tested on Different formulation. Results are shown
in below Fig. 8.4 and Table 8.12.
%w/w obtained from both Methods (ILS and CLS) were quite similar and further we will
check the same using statistical comparison.
n
CC
RMSEP
N
i
found
i
added
i
1
2)(
Chapter 8. Chemometric Methods
194
FIGURE 8. 4 Overlain spectra of formulations for Assay calculation (Glysikot, Diasol and Diabeta
Plus)
TABLE 8.12 Assay Result of Formulations
Formulation Constituents Assay (%w/w)
(CLS method) %RSD
Assay (%w/w)
(ILS method) %RSD
Glysikot
Gallic acid 1.3120 0.9034 1.3064 0.8850
Ellagic acid 2.4820 0.1658 2.4908 0.1985
Curcumin 0.5006 1.1297 0.5018 1.5869
Diasol
Gallic acid 2.3335 0.5211 2.3319 0.7122
Ellagic acid 0.1596 0.4974 0.1614 1.2507
Curcumin 0.2848 1.4336 0.2816 0.7484
Diabeta plus
Gallic acid 0.3327 1.8095 0.3370 0.6475
Ellagic acid 0.0152 0.4762 0.0155 1.4619
Curcumin 0.0872 0.1123 0.0896 0.9826
8.2.7 Summary of Validation parameters
The summary of validation parameters were reported in Table 8.13.
TABLE 8.13 Summary of Validation Parameters for CLS and ILS Methods
Sr.
No.
Parameters CLS ILS
Gallic
Acid
Ellagic
Acid
Curcumin Gallic
Acid
Ellagic
Acid
Curcumin
1 Calibration design 20 Mixture Spectra
2 Validation design 10 Mixture Spectra
3 Spectral region 241-279 nm
4 Linearity range
(µg/ml) 2-20 5-50 1 - 10 2-20 5-50 1 - 10
5 RMSEP 0.0623 0.1504 0.0512 0.1114 0.0598 0.0501
6 Assay(n = 5)
(Glysikot)
1.3120 2.4820 0.5006 1.3064 2.4908 0.5018
(Diasol) 2.3335 0.1596 0.2848 2.3319 0.1614 0.2816
(Diabeta) 0.3327 0.0152 0.0872 0.3370 0.0155 0.0896
7 LOD (n = 5)(μg/ml) 0.1689 0.2607 0.0873 0.1805 0.2024 0.1208
8 LOQ (n = 5)(μg/ml) 0.5118 0.7900 0.2646 0.5470 0.6133 0.3662
Chapter 8. References
195
References:
1. Pathak A, 2012, “RP-HPLC and Chemometric Assisted UV-Spectrophotometric methods for Simultaneous in
vitro Analysis of Atrovastatin Calcium, Ezetimibe and Fenofibrate in their Pharmaceutical Formulation”, Indo
American Journal of Pharm Research, 2(9), 1178-1193, ISSN No. 2231-6876.
2. Dinc E¸ Baleanu D, 2002, “Spectrophotometric Quantitative Determination of Cilazapril and
Hydrochlorothiazide in tablets by Chemometric methods”, Journal of Pharmaceutical and Biomedical Analysis,
30, 715-723, ISSN No. 0731-7085.
3. Kumar N, Bansal A et al, 2014, “Chemometrics Assisted Quantitative Estimation of Synthetic and Marketed
Formulations”, Asian Journal of Biomedical and Pharmaceutical Sciences, 04(34), 21-26, ISSN No. 2249-
622X.
4. Xuan Zhonga, Jun Yana et al, 2014, “A Novel strategy for Quantitative Analysis of the Formulated Complex
system using Chromatographic Fingerprints combined with some Chemometric Techniques”, Journal of
Chromatography A, 13(70), 179-186, ISSN No. 0021-9673.
196
Chapter 9. Experimental Work
197
CHAPTER 9
UV Spectrophotometric Method Development
9.1 Experimental work
9.1.1 Materials and Instruments: Refer Table 4.1, 4.2 and 4.3
9.1.2 Solvents and Reagents: Refer Table 4.4
9.1.3 Preparation of Stock solution and working standard solution: Refer 6.1.3
9.1.4 Preparation of Calibration curve
CALIBRATION CURVE FOR GALLIC ACID:
The solutions of Gallic Acid ranging from 2-20 μg/mL were prepared by pipetting
out 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2 mL of the working standard solution
of Gallic Acid (100 μg/mL) into series of 10 mL volumetric flasks and the volume
was adjusted to mark with methanol to get concentration of 2, 4, 6, 8, 10, 12, 14, 16,
18 & 20 μg/mL of Gallic Acid.
CALIBRATION CURVE FOR ELLAGIC ACID:
Solutions of Ellagic Acid ranging from 5-50 μg/mL were prepared by pipetting out
0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mL of the working standard solution
of Ellagic Acid (100 μg/mL) into series of 10 mL volumetric flasks and the volume
was adjusted to mark with methanol to get concentration of 5, 10, 15, 20, 25, 30, 35,
40, 45 and 50 μg/mL of Ellagic Acid.
CALIBRATION CURVE FOR CURCUMIN:
Solutions of Curcumin ranging from 1-10 μg/mL were prepared by pipetting out 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mL of the working standard solution of
Curcumin (100 μg/mL) into series of 10 mL volumetric flasks and the volume was
adjusted to mark with methanol to get concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10
μg/mL of Curcumin.
Chapter 9. UV Spectrophotometry
198
9.1.5 Determination of Wavelength for Measurement
0.4 mL of working standard solution of Gallic Acid (4 μg/mL), 1 mL of working
standard solution of Ellagic Acid (10 μg/mL) and 0.2 mL of working standard
solution of Curcumin (2 μg/mL) were diluted to 10 mL with Methanol individually
to get 4 μg/mL of Gallic Acid, 10 μg/mL of Ellagic Acid and 2 μg/mL of Curcumin.
Each solution was scanned between 200-800 nm.
9.1.6 Determination of Formulation: Refer 6.1.6
9.1.7 Validation Parameters
LINEARITY:
The linearity response was determined by analyzing different concentration for calibration
curve in the range of 2-20 μg/mL, 5-50 μg/mL and 1-10 μg/mL for Gallic Acid, Ellagic Acid
and Curcumin respectively. Plot the calibration curve of absorbance vs concentration and
determine correlation coefficient and regression line equations for Gallic Acid, Ellagic Acid
and Curcumin.
PRECISION:
Repeatability
1.0mL of working standard solutions of Gallic Acid, 2.5 mL of Ellagic Acid and 0.5mL of
Curcumin were transferred into separate 10 mL volumetric flasks and diluted up to mark with
methanol to get 10 μg/mL, 25 μg/mL and 5 μg/mL for Gallic Acid, Ellagic Acid and
Curcumin respectively. The absorbance of the each solution was measured at selected
wavelengths 6 times and % RSD was calculated.
Intraday Precision
Mixed solutions containing 8, 10, 12 μg/mL Gallic Acid; 20, 25, 30 μg/mL Ellagic Acid and
4, 5, 6 μg/mL of Curcumin respectively were analyzed three times on the same day and %
RSD was calculated.
Interday Precision
Mixed solutions containing 8, 10, 12 μg/mL Gallic Acid; 20, 25, 30 μg/mL Ellagic Acid and
4, 5, 6 μg/mL of Curcumin were analyzed on three different days and %RSD was calculated.
Chapter 9. Experimental Work
199
ACCURACY:
Accuracy is the closeness of the test results obtained by the method to the true value.
Recovery studies were carried out by addition of standard drug to the sample at 3 different
concentration levels (80%, 100%, 120%) taking into consideration percentage recovery of
added bulk drug samples.
Formulation (A): 100 mg of Formulation ( It contains 1.3002 mg of Gallic Acid, 2.48 mg of
Ellagic acid and 0.51 mg of Curcumin)
Standard samples: Gallic acid, Ellagic acid and Curcumin
TABLE 9.1 Steps for Accuracy study for Gallic Acid by UV Spectrophotometric Methods.
(Absorbance correction Method + )
(First Order Derivative Method + ) Sr.
No. Step 1 Step 2 Step 3
Total Gallic
Acid (mg)
1 0.1 gm of Formulation Add methanol to make
up to 100 mL
Dilute 1 mL
solution from 2nd
step to 10 mL
with Methanol
1.3/1.3
2
0.1gm of formulation
+ 1.04 mg of Gallic acid/
+ 1.04 mg of Gallic acid
Add methanol to make
up to 100 mL 2.34/2.34
3
0.1 0.1gm of formulation
0.2 + 1.3 mg of Gallic acid/
+ 1.3 mg of Gallic acid
Add methanol to make
upto 100 mL 2.6/2.6
4
0.1 gm of formulation
+1.56 mg of Gallic acid/
+ 1.56 mg of Gallic acid
Add methanol to make
upto 100 mL 2.86/2.86
TABLE 9.2 Steps for Accuracy study for Ellagic Acid by UV Spectrophotometric Methods.
Sr.
No. Step 1 Step 2 Step 3
Total Ellagic
Acid (mg)
1 0.1 gm of Formulation Add methanol to make
up to 100 mL
Dilute 1 mL
solution from 2nd
step to 10 mL
with Methanol
2.48/
2.5
2
0.1 gm of formulation
+1.98 mg of Ellagic acid/ + 2 mg
of Ellagic acid
Add methanol to make
up to 100 mL
4.46/
4.5
3
0.1 gm of formulation
+2.48 mg of Ellagic acid/
+ 2.5 mg of Ellagic acid
Add methanol to make
upto 100 mL 4.96/
5
4
0.1 gm of formulation
+ 2.97 mg of Ellagic acid/
+ 3 mg of Ellagic acid
Add methanol to make
upto 100 mL 5.45/
5.5
Chapter 9. UV Spectrophotometry
200
TABLE 9.3 Steps for Accuracy study for Curcumin by UV Spectrophotometric Methods.
Sr.
No. Step 1 Step 2 Step 3
Total
Curcumin (mg)
1 0.1 gm of Formulation Add methanol to make
up to 100 mL
Dilute 1 mL
solution from 2nd
step to 10 mL
with Methanol
0.51/
0.51
2
0.1 gm of formulation
+ 0.4 mg of Curcumin/
+ 0.4 mg of Curcumin
Add methanol to make
up to 100 mL
0.91/
0.91
3
0.1 gm of formulation
+ 0.51 mg of Curcumin /
+ 0.51 mg of Curcumin
Add methanol to make
upto 100 mL 1.02/
1.02
4
0.1 gm of formulation
+ 0.61 mg of Curcumin/
+ 0.61 mg of Curcumin
Add methanol to make
upto 100 mL 1.12/
1.12
LIMIT OF DETECTION (LOD):
The LOD was estimated from the set of 6 calibration curves used to determine method
linearity. The LOD may be calculated as
LOD = 3.3 x (SD / Slope)
Where, SD = the standard deviation of Y- intercept of 6 calibration curves.
Slope = the mean slope of the 6 calibration curves.
LIMIT OF QUANTITATION (LOQ):
The LOQ was estimated from the set of 6 calibration curves used to determine method
linearity. The LOQ may be calculated as
LOQ = 10 x (SD / Slope)
Where, SD = the standard deviation of Y- intercept of 6 calibration curves.
Slope = the mean slope of the 6 calibration curves.
Chapter 9. Absorbance Correction
201
9.2 RESULTS AND DISCUSSION (ABSORBANCE CORRECTION METHOD) [1-
2]
9.2.1 Determination of Wavelength for Measurement
At wavelength of 421 nm, only Curcumin shows absorbance. So, determination of
Curcumin at this wavelength is possible.
Ellagic Acid can be determine at 364.5 nm. Absorbance of Curcumin will be deducted
from the Absorbance of Ellagic Acid at same wavelength.
For Gallic acid, Absorbance is measured at difference of 2 wavelengths, i.e. 246 and
266 nm. At the difference of these 2 wavelengths, No interference of Absorbance of
Ellagic acid & Curcumin as both shows same absorbance at these selected
Wavelength.
FIGURE 9.1 Selection of Wavelength for Absorbance Correction Method, Overlain spectrum of Gallic
acid (4 µg/ml), Ellagic acid (10 µg/ml) and Curcumin (2 µg/ml)
9.2.2 Applicability of the Method
ANALYSIS OF FORMULATIONS:
Applicability of the proposed method was tested on Different formulation. Results
are shown in following fig. 9.2 and Tables 9.4.
Chapter 9. UV Spectrophotometry
202
FIGURE 9.2 Overlain spectra of formulations for Assay calculation (Glysikot, Diasol and Diabeta Plus)
TABLE 9.4 Assay result of Formulations by Absorbance Correction Method
Formulations Constituents Assay
(% w/w)
Assay
(mg) %RSD
Glysikot
Gallic acid 1.3002 0.1374 0.8496
Ellagic acid 2.4800 0.2268 0.3718
Curcumin 0.5072 0.0660 0.9315
Diasol
Gallic acid 2.3441 0.2422 0.6203
Ellagic acid 0.1560 0.0184 1.3242
Curcumin 0.2840 0.0369 0.1961
Diabeta
Gallic acid 0.3279 0.0397 0.7128
Ellagic acid 0.0160 0.0058 0.7952
Curcumin 0.0880 0.0113 0.1079
9.2.3 Validation Parameters
LINEARITY:
Linear correlation was obtained between Absorbance and concentration in the range of 2-
20µg/mL for Gallic Acid, 5-50µg/mL for Ellagic Acid and 1-10µg/mL of Curcumin. The
linearity of the calibration curves was validated by the value of correlation coefficients of the
regression (r). The optical and regression characteristics are listed in Table 9.5.
Chapter 9. Absorbance Correction
203
FIGURE 9.3 Overlay Spectra of Gallic Acid showing Linearity (2-20 µg/mL)
FIGURE 9.4 Overlay Spectra of Ellagic Acid showing Linearity (5-50 µg/mL)
FIGURE 9.5 Overlay Spectra of Curcumin showing Linearity (1-10 µg/mL)
Chapter 9. UV Spectrophotometry
204
TABLE 9.5 Linearity data for Gallic Acid, Ellagic Acid & Curcumin by Absorbance correction
Method.
FIGURE 9. 6 Calibration Curve of Curcumin FIGURE 9.7 Calibration Curve of Ellagic Acid at
364.5 nm (5 – 50 µg/mL) at 421 nm (1-10 µg/mL)
FIGURE 9.8 Calibration Curve of Gallic Acid at (266 – 246) nm (2- 20 µg/mL)
PRECISION:
Repeatability: The % RSD of repeatability of measurement of Absorbance was found to be 0.1744, 0.2092 and 0.0363 for Gallic Acid, Ellagic Acid and Curcumin,
respectively.
y = 0.1452x + 0.0021
R² = 0.9994
0
0.5
1
1.5
2
0 5 10 15
Curcumin at 421 nm
Conc. (g/ml)
Ab
s.
y = 0.0223x - 0.0098
R² = 0.99940.0000
0.2000
0.4000
0.6000
0.8000
1.0000
1.2000
0 20 40 60
Ellagic Acid at A1(364.5
nm)- B1(364.5 nm)
Conc. (g/ml)
Abs.
y = 0.0358x - 0.0208
R² = 0.99420.0000
0.2000
0.4000
0.6000
0.8000
0 10 20 30
Gallic Acid at C1(266)-
D1(246) nm
Conc. (g/ml)
Ab
s.
Conc.
(µg/ml)
Absorbance of
Curcumin at 421
nm
Mean ± S.D. (n=5)
Conc.
(µg/ml)
Absorbance of Ellagic
Acid at A1(364.5 nm) -
B1( 364.5 nm)
Mean ± S.D. (n=5)
Conc.
(µg/ml)
Absorbance of Gallic Acid
at C1(266 nm) -D1( 246
nm)
Mean ± S.D. (n=5)
1 0.1514 ± 0.0010 5 0.0975 ± 0.0002 2 0.0606 ± 0.0001
2 0.2957 ± 0.0022 10 0.2073 ± 0.0015 4 0.1254 ± 0.0016
3 0.4251 ± 0.0028 15 0.3259 ± 0.0021 6 0.1961 ± 0.002
4 0.5887 ± 0.0016 20 0.4343 ± 0.0032 8 0.2535 ± 0.0025
5 0.7292 ± 0.0004 25 0.5657 ± 0.0027 10 0.3389 ± 0.0008
6 0.8866 ± 0.0012 30 0.6551 ± 0.0014 12 0.4178 ± 0.0005
7 0.9970 ± 0.0025 35 0.7793 ± 0.0002 14 0.4578 ± 0.0009
8 1.1643 ± 0.0027 40 0.8766 ± 0.0017 16 0.5212 ± 0.0008
9 1.3047 ± 0.0027 45 0.9821 ± 0.0007 18 0.6437 ± 0.0016
10 1.4642 ± 0.0013 50 1.1068 ± 0.0027 20 0.7115 ± 0.0014
Chapter 9. Absorbance Correction
205
TABLE 9.6 Repeatability data for Gallic acid, Ellagic acid and Curcumin by Absorbance correction
Method.
Constituent Concentration
(µg/ml) Mean Abs. ± SD (n=6) %RSD
Gallic Acid 10 0.3391 ±0.0003 0.0954
Ellagic Acid 25 0.5648 ±0.0005 0.0935
Curcumin 5 0.7293± 0.0001 0.0131
Intraday Precision: % RSD for intra-day precision was found to be 0.0903, 0.1039
and 0.1412 for Gallic Acid, Ellagic Acid and Curcumin, respectively.
TABLE 9.7 Intraday Precision data for Gallic acid, Ellagic acid and Curcumin by Absorbance
correction Method.
Constituent Conc.
(µg/ml) Absorbance (Mean) (n=3) SD %RSD
Gallic acid
8 0.2534 0.0002 0.0671
10 0.3391 0.0005 0.1411
12 0.4177 0.0003 0.0628 0.0903
Ellagic acid
20 0.4326 0.0003 0.0786
25 0.5652 0.0009 0.1591
30 0.9429 0.0007 0.0740 0.1039
Curcumin
4 0.5887 0.0016 0.2646
5 0.7295 0.0002 0.0258
6 0.8865 0.0012 0.1332 0.1412
Interday Precision: % RSD for inter-day precision was found to be 0.2025, 0.1709
and 0.2563 for Gallic Acid, Ellagic Acid and Curcumin, respectively, which indicates
the method is precise.
TABLE 9.8 Interday Precision data for Gallic acid, Ellagic acid and Curcumin by Absorbance
correction Method
Constituent Conc.
(µg/ml) Absorbance (Mean) (n=3) SD %RSD
Gallic acid
8 0.2537 0.0003 0.1034
10 0.3383 0.0014 0.4055
12 0.4174 0.0004 0.0985 0.2025
Ellagic acid
20 0.4330 0.0007 0.1701
25 0.5663 0.0013 0.2244
30 0.9436 0.0011 0.1181 0.1709
Curcumin
4 0.5863 0.0029 0.4880
5 0.7297 0.0008 0.1059
6 0.8849 0.0016 0.1749 0.2563
Chapter 9. UV Spectrophotometry
206
ACCURACY:
The accuracy study was carried out by the standard addition method. The percent recovery
was found in the range of 99.74 – 101.53 %, 99.17 -100.47 % and 99.63 – 101.30 % for
Gallic Acid, Ellagic Acid and Curcumin respectively, which indicates accuracy of the
method.
TABLE 9.9 Accuracy data of Gallic acid, Ellagic acid and Curcumin by Absorbance correction Method
Constituent
Amt. Taken
(mg)
Amt.
added
(mg)
Amt. Found
(n=3)
(mg)
Mean
recovery
(%) (n=3)
%RSD
Gallic Acid
0% 1.3 0 1.32 ± 0.0163 101.5385 1.2371
80% 1.3 1.03 2.3667 ± 0.0125 101.1396 0.5270
100% 1.3 1.3 2.5933 ± 0.0499 99.7436 1.9237
120% 1.3 1.56 2.8867 ± 0.0556 100.9324 1.9253
Ellagic
Acid
0% 2.48 0 2.4667 ± 0.0262 99.4624 1.0641
80% 2.48 1.98 4.4233 ± 0.0411 99.1779 0.9291
100% 2.48 2.48 4.9833 ± 0.0047 100.4704 0.0946
120% 2.48 2.97 5.44 ± 0.0432 99.8165 0.7942
Curcumin
0% 0.51 0 0.5167 ± 0.0047 101.3072 0.9124
80% 0.51 0.4 0.9067 ± 0.0047 99.6337 0.5199
100% 0.51 0.51 1.02 ± 0.0082 100.0000 0.8005
120% 0.51 0.61 1.1267 ± 0.0094 100.5952 0.8368
Summary of Validation Parameters
TABLE 9. 10 Summary of Validation Parameters by Absorbance correction Method.
Parameters Gallic acid Ellagic acid Curcumin
Concentration Range 2-20 µg/ml 5-50 µg/ml 1-10 µg/ml
Regression equation y = 0.0358x - 0.0208 y = 0.0223x - 0.0098 y = 0.1452x + 0.0021
Regression co-efficient 0.9942 0.9994 0.9994
Correlation co-efficient 0.9970 0.9996 0.9996
LOD (n = 5) (µg/ml) 0.0905 0.1694 0.0377
LOQ (n = 5) (µg/ml) 0.2743 0.5134 0.1142
Repeatability (n = 6) (%RSD) 0.1744 0.2092 0.0363
Intraday precision (n=3) (% RSD) 0.0628- 0.1411 0.0740 - 0.1591 0.0258 – 0.2646
Interday precision (n=3) (% RSD) 0.0985 – 0.4055 0.1181 – 0.2244 0.1059 – 0.4880
% Recovery
(Glysikot)
0 101.5385 99.4624 101.3072
80 101.1396 99.1779 99.6337
100 99.7436 100.4704 100.0000
120 100.9324 99.8165 100.5952
Chapter 9. First order derivative
207
9.3 Result and Discussion (FIRST ORDER DERIVATIVE SPECTROSCOPY) [3-5]
9.3.1 Determination of Wavelength for Measurement
At the wavelength of 255 nm, Gallic acid shows good absorbance and linearity
because it is ZCP of Ellagic acid and Curcumin. At the wavelength of 343 nm, Ellagic
acid shows good absorbance and linearity because it is ZCP of Gallic acid and
Curcumin. At the wavelength of 452 nm, Curcumin shows good absorbance and
linearity because it is ZCP of Gallic acid and Ellagic acid. So, determination of
Curcumin at this wavelength is possible.
FIGURE 9.9 First order derivative Overlain spectrum of Gallic Acid (4 µg/ml ), Ellagic Acid (10 µg/ml
) and Curcumin (2 µg/ml) For Selection of ZCP
Linear correlation was obtained between Absorbance of first order Spectra and concentration
in the range of 2-20µg/mL for Gallic Acid, 5- 50µg/mL for Ellagic Acid and 1 -10µg/mL of
Curcumin. The linearity of the calibration curves were validated by the value of correlation
coefficients of the regression (r).
Chapter 9. UV Spectrophotometry
208
9.3.2 Applicability of the Method
ANALYSIS OF FORMULATIONS:
Applicability of the proposed method was tested on Different formulation. Results
are shown in following fig. 9.10 and Table 9.11.
FIGURE 9.10 First order overlain spectra of formulations for Assay calculation (Glysikot, Diasol and
Diabeta Plus)
TABLE 9.11 Assay result of Formulations by First order derivative Method
Formulation Constituents Assay
(% w/w)
Assay
(mg) % RSD
Glysikot
Gallic acid 1.3091 0.1090 0.4867
Ellagic acid 2.4963 0.2043 0.1654
Curcumin 0.5059 0.0676 1.0708
Diasol
Gallic acid 2.3386 0.1900 0.5281
Ellagic acid 0.1502 0.0257 1.2445
Curcumin 0.2832 0.0374 1.6507
Diabeta
Gallic acid 0.3305 0.0320 1.8467
Ellagic acid 0.0131 0.0153 1.0143
Curcumin 0.0886 0.0109 0.6326
9.3.2 Validation Parameters
LINEARITY:
First order Spectra were converted to first order derivative spectra. Linear correlation was
obtained between Absorbance and concentration in the range of 2-20µg/mL for Gallic Acid,
5-50µg/mL for Ellagic Acid and 1-10µg/mL of Curcumin. The linearity of the calibration
curves was validated by the value of correlation coefficients of the regression (r).
Chapter 9. First order derivative
209
The optical and regression characteristics are listed in below Fig. 9.11- 9.13 and Table 9.12.
FIGURE 9.11 First Order overlay Spectra of Gallic Acid showing Linearity (2-20 µg/mL) (ZCP of
Gallic Acid is at 343 nm and 452 nm)
FIGURE 9.12 First Order overlay Spectra of Ellagic Acid showing Linearity (5- 50 µg/Ml (ZCP of
Ellagic Acid is at 255nm and 452 nm)
Chapter 9. UV Spectrophotometry
210
FIGURE 9.13 First Order overlay Spectra of Curcumin showing Linearity (1 -10 µg/mL) (ZCP of
Curcumin is at 255 nm and 272.5 nm)
Table 9.12 Linearity data for Gallic Acid, Ellagic Acid & Curcumin by first order derivative
spectroscopy Method.
Conc.
(µg/mL)
Amplitude of Gallic
acid at 255 nm
Mean ± S.D. (n=3)
Conc.
(µg/mL)
Amplitude
of Ellagic acid at 343 nm
Mean ± S.D. (n=3)
Conc.
(µg/mL)
Amplitude of
Curcumin at 452 nm
Mean ± S.D. (n=3)
2 0.0032 ± 0.0002 5 0.0026 ± 0.0001 1 0.0038 ± 0.0001
4 0.0064 ± 0.0004 10 0.0056 ± 0.0002 2 0.0071 ± 0.0002
6 0.0106 ± 0.0002 15 0.0102 ± 0.0006 3 0.0102 ± 0.0005
8 0.0140 ± 0.0001 20 0.0132 ± 0.0001 4 0.0135 ± 0.0001
10 0.0184 ± 0.0002 25 0.0184 ± 0.0001 5 0.0177 ± 0.0002
12 0.0250 ± 0.0002 30 0.0210 ± 0.0001 6 0.0216 ± 0.0002
14 0.0256 ± 0.0001 35 0.0245 ± 0.0002 7 0.024 ± 0.0002
16 0.0284 ± 0.0004 40 0.0277 ± 0.0000 8 0.0277 ± 0.0001
18 0.0351 ± 0.0003 45 0.0304 ± 0.0001 9 0.0305 ± 0.000
20 0.0391 ± 0.0001 50 0.0363 ± 0.0001 10 0.0338 ± 0.0001
Chapter 9. First Order Derivative
211
Figure 9.14 Calibration Curve of Gallic Acid Figure 9.15 Calibration Curve of Ellagic Acid
At 255 nm (2-20 µg/mL) At 343 nm (5-50 µg/mL)
Figure 9.16 Calibration curve of Curcumin at 452 nm (1-10 µg/mL)
PRECISION:
Repeatability: The % RSD of repeatability of measurement of Absorbance was
found to be 0.5763, 0.5133 and 1.0122 for Gallic Acid, Ellagic Acid and Curcumin
respectively.
TABLE 9.13 Repeatability data for Gallic Acid, Ellagic Acid & Curcumin by first order derivative
spectroscopy Method.
Constituent Concentration
(µg/ml) Mean Amplitude ± SD (n=6) %RSD
Gallic Acid 10 0.0185 ± 0.0001 0.5763
Ellagic Acid 25 0.0184 ± 0.0001 0.5133
Curcumin 5 0.0177 ± 0.0002 1.0122
Intraday Precision
The % RSD for intra-day precision was found to be 0.7166, 0.7388 and 1.0620 for Gallic
Acid, Ellagic Acid and Curcumin, respectively.
y = 0.002x - 0.0012
R² = 0.9902
0.0000
0.0100
0.0200
0.0300
0.0400
0.0500
0 10 20 30
Gallic Acid at 255 nm
Conc.(g/ ml)
Am
pli
tud
e
y = 0.0007x - 0.001
R² = 0.9955
0.0000
0.0100
0.0200
0.0300
0.0400
0 20 40 60
Ellagic acid at 343 nm
Conc. (g/ml)
Am
pli
tud
e.
y = 0.0034x + 0.0004
R² = 0.9983
0
0.01
0.02
0.03
0.04
0 5 10 15
Curcumin at 452 nm
Conc. (g/ml)
Am
pli
tud
e
Chapter 9. UV Spectrophotometry
212
TABLE 9.14 Intraday Precision data for Gallic Acid, Ellagic Acid & Curcumin by first order
derivative spectroscopy Method
Constituent Conc.
(µg/ml Mean Amplitude (n=3) SD %RSD
Gallic acid
8 0.0140 0.0001 0.5832
10 0.0184 0.0002 0.8875
12 0.0250 0.0002 0.6790
0.7166
Ellagic acid
20 0.0132 0.0001 0.9425
25 0.0184 0.0001 0.6791
30 0.021 0.0001 0.5949
0.7388
Curcumin
4 0.0135 0.0002 1.2096
5 0.0177 0.0002 1.2204
6 0.0216 0.0002 0.7560
1.0620
Interday Precision
% RSD for inter-day precision was found to be 1.2379, 1.1981, 1.3357 for Gallic Acid,
Ellagic Acid and Curcumin, respectively, which indicates the method is precise.
TABLE 9.15 Interday Precision data for Gallic Acid, Ellagic Acid & Curcumin by first order
derivative spectroscopy Method
Constituent Conc.
(µg/ml Mean Amplitude (n=3) SD %RSD
Gallic acid
8 0.0141 0.0002 1.1582
10 0.0184 0.0003 1.5913
12 0.0257 0.0002 0.9644
1.2379
Ellagic acid
20 0.0136 0.0002 1.2528
25 0.0183 0.0002 1.1805
30 0.0211 0.0002 1.1609
1.1981
Curcumin
4 0.0135 0.0002 1.5258
5 0.0177 0.0003 1.4857
6 0.0217 0.0002 0.9955
1.3357
ACCURACY:
The accuracy study was carried out by the standard addition method. The percent recovery
was found in the range of 98.71 – 101.98 %, 99.06 -100.26 % and 99.27 – 100.65 % for
Gallic Acid, Ellagic Acid and Curcumin respectively, which indicates accuracy of the method
Chapter 9. First Order Derivative
213
TABLE 9.16 Accuracy data of Gallic acid, Ellagic acid and Curcumin by First order derivative Method.
Constituent
Amt.
Taken
(mg)
Amt. added
(mg)
Amt. Found
(n=3)
(mg)
Mean recovery
(%) (n=3) %RSD
Gallic acid
0% 1.3 0 1.2833 ± 0.0125 98.7179 0.9719
80% 1.3 1.04 2.36 ± 0.0082 100.8547 0.3460
100% 1.3 1.3 2.6233 ± 0.0309 100.8974 1.1784
120% 1.3 1.56 2.9167 ± 0.017 101.9814 0.5827
Ellagic acid
0% 2.5 0 2.5067 ± 0.0125 100.2667 0.4976
80% 2.5 2 4.5067 ± 0.0205 100.1481 0.4559
100% 2.5 2.5 4.9533 ± 0.0377 99.0667 0.7614
120% 2.5 3 5.4733 ± 0.0573 99.5152 1.0478
Curcumin
0% 0.51 0 0.5067 ± 0.0047 99.3464 0.9304
80% 0.51 0.41 0.9133 ± 0.0047 99.2754 0.5161
100% 0.51 0.51 1.0267 ± 0.0047 100.6536 0.4592
120% 0.51 0.61 1.1233 ± 0.0125 100.2976 1.1103
9.3.3 Summary of Validation Parameters:
TABLE 9.17 Summary of Validation Parameters by First order derivative Spectroscopy.
Parameters Gallic acid Ellagic acid Curcumin
Concentration Range 2-20 µg/ml 5-50 µg/ml 1-10 µg/ml
Regression equation y = 0.002x - 0.0012 y = 0.0007x - 0.001 y = 0.0034x
0.0004
Regression co-efficient 0.9902 0.9955 0.9983
Correlation co-efficient 0.9950 0.9977 0.9991
LOD (n = 5) (µg/ml) 0.2804 1.0184 0.2014
LOQ (n = 5) (µg/ml) 0.8498 3.0861 0.6103
Repeatability (n = 6) (%RSD) 0.5763 0.5133 1.0122
Intraday precision (n=3) (% RSD) 0.5832 – 0.8875 0.5949- 0.9425 0.7560 – 1.2204
Interday precision (n=3) (% RSD) 0.9644 – 1.5913 1.1609 – 1.2528 0.9955 – 1.5258
% Recovery
(Glysikot)
0 98.7179 100.2667 99.3463
80 100.8547 100.1481 99.2754
100 100.8974 99.0667 100.6536
120 101.9814 99.5152 100.2976
References: 1. Rajanit S, 2015, “Absorbance Correction Method for Simultaneous Estimation of Nifedipine and Metoprolol
Succinate in Their Synthetic Mixture Using From Spectrophotometry”, International Journal of Advances in
Scientific Research, 1(03): 151-155, ISSN No. 2395-3616.
2. Patel K, Patel A, 2012, “Absorbance correction method for estimation of telmisartan and metoprolol succinate
in combined tablet dosage forms, Pharmaceutical Methods, 3(2), 106-111, ISSN No. 2229-4708.
3. Abdullah NS, 2014, “Spectrophotometric Determination of Chlorthalidone in Pharmaceutical Formulations
using different order derivative methods”, Arabian Journal of Chemistry, XXX-XXX, ISSN No. 1878-5352.
4. Rote AR, Bhalerao SR., 2011, “First-order derivative Spectrophotometric Estimation of Nabumetone and
Paracetamol in tablet dosage form”, Pharmaceutical Methods 2, 260-263, ISSN No. 2229-4708.
5. Karljikovic K, Novovic D et al, 2003, “First-order UV-derivative Spectrophotometry in the Analysis of
Omeprazole and Pantoprazole Sodium salt and Corresponding impurities”, Journal of Pharmaceutical and
Biomedical Analysis, 32, 1019-1027, ISSN No. 0731-7085.
214
Chapter 10. ANOVA
215
CHAPTER 10
Statistical comparison
10.1 ANOVA ( Analysis of Variance) Test [1]
An ANOVA test is a way to find out if survey or experiment results are significant. In other
words, they help you to figure out if you need to reject the null hypothesis or accept
the alternate hypothesis. Basically, you are testing groups to see if there is a difference
between them.
10.1.1 Types of Tests.
There are two main types: one-way and two-way. Two-way tests can be with or without
replication.
One-way ANOVA between groups: used when you want to test two groups to see if
there’s a difference between them.
Two way ANOVA without replication: used when you have one group and
you’re double-testing that same group. For example, you’re testing one set of individuals
before and after they take a medication to see if it works or not.
Two way ANOVA with replication: Two groups, and the members of those groups
are doing more than one thing. For example, two groups of patients from different hospitals
trying two different therapies.
ONE WAY ANOVA:
A one way ANOVA is used to compare two means from two independent (unrelated) groups
using the F-distribution. The null hypothesis for the test is that the two means are equal.
Therefore, a significant result means that the two means are unequal.
Chapter 10. Statistical comparison
216
10.2 ANOVA , Statistical Comparison Between Developed Methods
In Statistical Comparison, Six different methods, i.e. Two Chromatographic Methods (HPLC
and HPTLC); Two Chemometric Methods (CLS and ILS) and Two UV Spectrophotometric
(Absorbance Correction Method and first order derivative) were taken for estimation of
Gallic Acid, Ellagic Acid and Curcumin in Formulations like Glysikot, Diasol and Diabeta
Plus.
For that, One Way ANOVA test is applied.
Level of Significance : 2%
Degree of freedom : 5
Null Hypothesis (H0): There is no significance difference between all these six Analytical
Methods.
Alternate Hypothesis (H1): There is significance difference between all these six Analytical
Methods.
The result of ANOVA is shown in Table 10.1, 10.2 and 10.3 respectively for Glysikot,
Diasol and Diabeta Plus.
TABLE 10.1 ANOVA Test for Glysikot
ANOVA: Single Factor (Glysikot)
Parameters Gallic Acid Ellagic Acid Curcumin
Observations 6 6 6
Df 5 5 5
Fcrit 3.3312
F 2.5904 3.2325 2.0523
F<Fcrit Yes Yes Yes
Null Hypothesis Pass Pass Pass
Alternate Hypothesis Fail Fail Fail
Conclusion: Null Hypothesis Passes which indicates that there is no significance difference
between Six Analytical Methods employed. All Methods can be successfully used for the
determination of Gallic Acid, Ellagic Acid and Curcumin in Glysikot.
Chapter 10. ANOVA
217
TABLE 10.2 ANOVA Test for Diasol
ANOVA: Single Factor (Diasol)
Parameters Gallic Acid Ellagic Acid Curcumin
Observations 6 6 6
Df 5 5 5
Fcrit 3.3312
F 3.0947 0.7309 2.5697
F<Fcrit Yes Yes Yes
Null Hypothesis Pass Pass Pass
Alternate Hypothesis Fail Fail Fail
Conclusion: Null Hypothesis Passes which indicates that there is no significance difference
between Six Analytical Methods employed. All Methods can be successfully used for the
determination of Gallic Acid, Ellagic Acid and Curcumin in Diasol.
TABLE 10.3 ANOVA Test for Diabeta Plus
ANOVA: Single Factor (Diabeta Plus)
Parameters Gallic Acid Ellagic Acid Curcumin
Observations 6 6 6
Df 5 5 5
Fcrit 3.3312
F 0.8074 3.0818 2.3656
F<Fcrit Yes Yes Yes
Null Hypothesis Pass Pass Pass
Alternate Hypothesis Fail Fail Fail
Conclusion: Null Hypothesis Passes which indicates that there is no significance difference
between Six Analytical Methods employed. All Methods can be successfully used for the
determination of Gallic Acid, Ellagic Acid and Curcumin in Diabeta Plus.
REFERENCE:
1. https://www.statisticshowto.datasciencecentral.com/probability-and-statistics/hypothesis-
testing/anova
218
Chapter 11. Summary
219
CHAPTER 11
Summary and Conclusion
11.1 Summary
Most sensitive RP-HPLC method for simultaneous estimation of Gallic acid, Ellagic
acid and Curcumin was developed using QbD approach (CCD as Experimental
Design) and further confirmation of Curcuminoids by LCMSMS Analysis was done.
HPTLC Method for simultaneous estimation of Gallic acid, Ellagic acid and
Curcumin in Polyherbal Antidiabetic formulations was developed which is important
tool in standardization of Herbal Medical product in initial stage of Research.
Most sturdy Chemometrics Methods (CLS and ILS) for simultaneous estimation of
Gallic acid, Ellagic acid and Curcumin in Polyherbal Antidiabetic formulations were
developed.
Cost effective UV Spectrophotometric methods (Absorbance correction and First
order Spectroscopy) for simultaneous estimation of Gallic acid, Ellagic acid and
Curcumin were developed.
All developed methods were validated for Specificity, Linearity, Accuracy, Precision,
Robustness, Limit of Detection and Limit of Quantitation and for System suitability
Parameters.
Statistical comparison of Methods was performed by using one way ANOVA.
Chapter 11. Summary and Conclusion
220
11.2 Conclusion
RP-HPLC method has been developed and validated for estimation of Gallic acid, Ellagic
acid and Curcumin using DAD as a detector and Gradient elution mode for mobile phase.
Optimization of mobile phase pH, ratio of organic and aqueous phase are the critical part of
method. For better Method development QbD approach was incorporated using CCD
(Central composite Design) approach as Experimental Design. The Optimized Solution
provided from software can be validated by applying same condition and by measuring %
predicted error of the responses. Desirability can be calculated to prove better result
assurance. The separation was achieved on Agilent C18 column (25 cm, 4.6 mm, 5m) using
gradient mobile phase. Mobile Phase A: Water with % Formic acid and Mobile Phase B:
Acetonitrile. The flow rate was 0.95 mL/min. Retention time of Gallic acid, Ellagic acid and
curcumin were found to be 3.267, 4.633 and 12.527 minutes respectively. The assay was
linear over the range of 2-14 μg/ml for Gallic Acid, 5-35μg/ml for Ellagic Acid and 1-7 μg/ml
for Curcumin. The intra-and inter-day precision were less than 2%, with accuracies between
98-102% of the true values. Along with Curcumin; desmethoxycurcumin and
bisdesmethoxycurcumin are also available. So, to confirm the Curcumin peak, LCMSMS
scan was done. Shorter retention time of markers cuts down cost of experiment. Good
resolution and specificity of Method made routine Analysis for measurement in Quality
control laboratories.
HPTLC method has been developed and validated for the estimation of Gallic acid, Ellagic
acid and Curcumin. The separation was achieved on CAMAG HPTLC system, using
winCATS Planar Chromatography manager software and Linomat V as a sample Applicator.
TCL Silica gel 60 F254 (20*20) was used as Stationary Phase and Toluene: Ethyl acetate:
Formic Acid (3: 3.5: 1 v/v) as a Mobile Phase. Detection was done at 300 nm using D2 Lamp
and 50°C temperature was maintained throughout the day. Rf value of Gallic acid, Ellagic
acid and Curcumin were found to be 0.59, 0.51 and 0.78. The linearity of Gallic Acid, Ellagic
Acid and Curcumin was in the range of 20-400 ng/band, 50-1000 ng/band and 10-200
ng/band of Gallic acid, Ellagic acid and Curcumin respectively. The advantages of Method
are low cost of reagents, Rapid Analysis and good peak Shapes. Lower values of LOD and
LOQ proved high Sensitivity method.
Chapter 11. Conclusion
221
Chemometric methods (CLS and ILS) have been developed and validated which frequently
used for complex matrix. It covers whole wavelength range and all possible concentration
from the Linearity. So it is widely used for measurement of all three constituents very
precisely when they all are available in combination. Measurement of coefficient value from
the matrix is the key part of Chemometric Methods and found using the software MATLAB
R2015a. Low cost instrument and low interference of matrix play a key role for successful
application of both methods in routine Analysis.
UV spectrophotometric methods have been developed and validated for simultaneous
estimation of Gallic acid, Ellagic acid and Curcumin in their combine Polyherbal dosage
form. Linearity was investigated in concentration range of 2-20 μg/mL for Gallic acid, 5-50
μg/mL of Ellagic acid and 1-10 μg/mL of curcumin respectively. Both methods are very
much precise as % RSD was found to be less than 2. Method is also very accurate as %
Recovery was found to be within the limit 98-102%. Low cost of Instrument and accurate
and precise results are the key factor for applying UV Spectrophotometric Methods in routine
Analysis.
222
List of Publications
223
List of Publications
1. Megha Shah, Harsha Patel, Hasumati Raj, Methods for the estimation of Ellagic Acid
and Curcumin in Antidiabetic Herbal Formulations- A review, Eurasian Journal of
Analytical Chemistry, 2017, 12(4), 295-311, ISSN: 1306-3057.
2. Megha Shah, Harsha Patel, development and validation of absorbance correction
method and first order derivative Spectrophotometric method for simultaneous
estimation of Gallic acid, Ellagic acid and curcumin in Polyherbal antidiabetic dosage
Forms, International Journal of pharmaceutical Research, 2019, 11(1), ISSN: 0975-
2366.
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