CHROMATOGRAPHIC METHOD DEVELOPMENT AND …

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Vineeta et al. World Journal of Pharmacy and Pharmaceutical Sciences

CHROMATOGRAPHIC METHOD DEVELOPMENT AND

VALIDATION FOR SIMULTANEOUS ESTIMATION OF

STIGMASTEROL AND EMBELIN

Vineeta V. Khanvilkar*, Preeti N. Gunjal, Dr. Vilasrao J. Kadam

Bharati Vidyapeeth’s College of Pharmacy, C.B.D Belapur, Navi Mumbai-400614,

Maharashtra, India.

ABSTRACT

Standardization of herbal formulation is essential in order to assess the

quality, purity, safety and efficacy of drugs. The present work

describes a chromatographic method for simultaneous estimation of

stigmasterol and embelin which can be further applied for

standardization of chopchinyadi churna. Chopchinyadi churna is a

polyherbal formulation prescribed for treatment of skin diseases. This

preparation also works well in rheumatoid arthritis and gout. It

contains Smilax china and Embelia ribes as major components in

formulation from which stigmasterol and embelin respectively were

used as marker compounds. A simple, rapid, precise, accurate, and

reproducible High Performance Thin Layer Chromatographic

(HPTLC) method was developed. The separation was performed on TLC aluminium plates

precoated with silica gel 60 F254, wherein good separation was achieved in the mobile phase

of Toluene: ethyl acetate: methanol: formic acid (6:2:1:1 v/v/v/v). Densitometric scanning for

stigmasterol and embelin was done using single wavelength i.e 212nm. The retardation

factor(Rf) value of stigmasterol and embelin was found to be 0.63+0.02 and 0.45+0.02,

respectively. The developed method was validated as per ICH guidelines and applied for

standardization of chopchinyadi churna from in-house and marketed formulations.

KEYWORDS: Chopchinyadi churna, stigmasterol, embelin, HPTLC, polyherbal

formulations, ICH.

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Article Received on

19 June 2015,

Revised on 10 July 2015,

Accepted on 29 July 2015

*Correspondence for

Author

Vineeta V. Khanvilkar

Bharati Vidyapeeth’s

College of Pharmacy,

C.B.D Belapur, Navi

Mumbai-400614,

Maharashtra, India.


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INTRODUCTION

Herbal formulations have been used by the majority of Indians since ancient times. In recent

years, there has been an increased inclination towards the herbal formulations.[1]

It is known

that herbal formulations are prepared in a number of dosage forms, in which mostly all of

them are Polyherbal formulations.[2]

Standardization of herbal formulation is essential in

order to assess the quality, purity, safety and efficacy of drugs.

Chopchinyadi churna is a polyherbal formulation comprising of 9 ingredients i.e.

madhusnuhi(smilax china), vidanga (embelia ribes), asvattaha (ficus religiosa),

tvak pippali (piper longum), lavanga(clove), akarakarabha (Anacyclus pyrethrum), marica

(piper nigrum), yavani(Trachyspermum ammi), sunthi(Zinziber officinale).

Smilax china L. is known as chobchini in Hindi, madhusnuhi in Sanskrit and china root in

english. It possesses anti-inflammatory, diuretic, anti-diabetic, anti-psoriatic, digestive

properties.[3-6]

Embelia ribes Burm F., a medicinal woody climber, belongs to the

Myrsinaceae family. It is also commonly known as false black pepper or vidanga. Embelia

ribes is a highly valuable medicinal plant with anthelmintic, carminative, antibacterial,

antibiotic, hypoglycemic, and antifertility properties.[7-8]

Literature survey shows that there are analytical methods like HPLC, HPTLC, UV

Spectroscopy for estimation of stigmasterol and embelin individually and in combination

with other drugs. However, no modern analytical method like HPLC, HPTLC have been

developed for estimating stigmasterol and embelin simultaneously.

The present research work deals with development of HPTLC method for standardization of

chopchinyadi churna by detection and quantification of markers stigmasterol and embelin

simultaneously from in-house and marketed formulations. The proposed method was

validated on the basis of its linearity, accuracy, precision, limit of detection (LOD), limit of

quantification (LOQ), and robustness according to ICH guidelines.

Structure of stigmasterol and embelin

Fig 1. Structure of stigmasterol Fig 2. Structure of embelin


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MATERIALS AND METHODS

Materials

Raw materials used for the preparation of chopchinyadi churna and two different marketed

brands (M-01, M-02) of chopchinyadi churna were procured from ayurvedic medical shop,

Mumbai. The raw materials were stored in air tight containers at room temperature. The

stationary phase used was precoated with silica gel 60 F254 (20×20 cm) TLC plates of 0.2 mm

thickness obtained from E. Merck Ltd. Mumbai, India.

Standards and reagents

The organic solvents and chemicals of analytical grade were procured from S.D Fine

chemicals Pvt. Ltd. Mumbai, India. Standard stigmasterol and embelin were procured from

Sigma Aldrich Pvt. Ltd. Mumbai, India, Total herbal solution Pvt.ltd., Mumbai, India,

respectively.

Instrumentation

Camag Linomat 5 semiautomatic sample applicator equipped with a 100μl Hamilton syringe

(Camag, Switzerland) and winCATS software (CAMAG Ver.1.4.1), Camag TLC Scanner 3,

Twin trough chamber.

EXPERIMENTAL

Preliminary studies

The quality of raw materials used in the preparation of chopchinyadi churna was assessed by

determining the proximate parameters like ash value, extractive value and loss on drying

using standard pharmacopoeial methods. Powder extracts were qualitatively evaluated by

chemical tests for the presence of various phytoconstituents like alkaloids, glycosides,

saponins, phenolic compounds tannins and phytosterols.

HPTLC Method Development

Preparation of standard solution

Stock solutions of stigmasterol and embelin (1000μg/ml) were prepared separately by

dissolving 10 mg of accurately weighed standard in 10 ml of methanol. From this stock

solution 100μg/ml was prepared by transferring 1 ml stock solution to 10 ml volumetric flask

and volume was then adjusted with methanol.


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Preparation of In-house formulation

All the ingredients were collected, dried and powdered separately, passed through 100 # sieve

and then mixed together in specified proportions in a geometrical manner to get uniform

mixture and packed in air tight containers for further analysis.

Extraction of stigmasterol and embelin from marketed and in-house formulations

5g of the formulation was refluxed using 50ml of methanol for 2 hrs and then was filtered

using Whatman filter paper no.41. The extract was further filtered using the syringe filter.

The filtered extract was diluted to 100ml.

Chromatographic conditions

Chromatographic separation was achieved on HPTLC plates (10×10 cm) pre-coated with

silica gel 60 F254 of 0.2 mm thickness with aluminium sheet support. Standard solutions of

markers and extracts were applied to the plates as bands 6.0 mm wide, 10.0 mm from the

bottom edge of the same chromatographic plate by use of a Camag (Muttenz, Switzerland)

Linomat 5 sample applicator equipped with a 100μl Hamilton syringe. Ascending

development to a distance of 80 mm was performed at room temperature (24 ± 2°C) with

mobile phase, in a Camag glass twin-trough chamber previously saturated with mobile phase

vapour for 30 min. After development, the plates were dried and then scanned at 212 nm with

a Camag TLC Scanner 3 using the deuterium lamp with winCATS software.

Optimization of Mobile phase

Mobile phase composition was optimized to provide accurate, precise and reproducible

results for the determination of stigmasterol and embelin. The standard stock solution

containing 100μg/ml of stigmasterol and embelin was spotted on to TLC plate and developed

in different solvent systems. Many preliminary trials were carried out for selection of mobile

phase.

Calibration curves of stigmasterol and embelin

Serial dilutions were made in the concentration range of 300-900ng/spot and 30-90ng/spot for

stigmasterol and embelin respectively. Appropriate volume of above solutions were applied

with the band width of 6 mm, in triplicate on TLC plate (10×10 cm) to obtain a concentration

range of 300-900 ng/spot for stigmasterol and 30-90 ng/spot for embelin. Peak area for each

band was recorded. Separate calibration curves were obtained by plotting a graph of peak

area vs. concentration of stigmasterol and embelin.


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Assay

For assay purpose standard and sample (extract) solutions were applied on HPTLC plate in

triplicates. Standard solutions of stigmasterol and embelin 100μg/ml were applied. Extracted

solution was directly used for quantification of stigmasterol. The amount of stigmasterol and

embelin present per gram of formulation was calculated by comparison of the areas

measured for the sample with the calibration curves constructed from peak areas obtained

from standard solutions of stigmasterol and embelin.

Method validation

In accordance with ICH guidelines Q2 (R1) the optimized HPTLC method was validated

with respect to following parameters.[9]

Linearity

The linearity of an analytical procedure is its ability (within a given range) to obtain test

results which are directly proportional to the concentration of analyte in the sample. It was

determined by plotting a graph of peak area v/s concentration of standards to obtain

correlation coefficient (r2) and equation of the line.

Specificity

Specificity is the ability to assess the analyte in the presence of components that may be

expected to be present in the sample matrix. The specificity of the method was ascertained by

comparing the Rf value and the peak purity was assessed by comparing the spectrum of

standard stigmasterol and embelin with sample.

Precision

Precision is the measure of the degree of repeatability of an analytical method under normal

operation and is normally expressed as the percent relative standard deviation (%RSD) for a

statistically significant number of samples. As per the ICH guidelines precision should be

performed at three different levels i.e. low quality control (LQC), medium quality control

(MQC) and high quality control (HQC). Repeatability expresses the precision under the same

operating conditions over a short interval of time. Repeatability is also termed as intra-assay

precision. It is assessed by using minimum of 9 determinations covering the specified range

for the procedure. The intra-day assay precision was performed 3 times on same day, while

inter-assay precision was performed on 3 different days.


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Limit of Detection (LOD) and Limit of Quantification (LOQ)

Limit of detection (LOD) is the lowest amount of an analyte in a sample that can be detected,

but not necessarily quantitated, under the stated experimental conditions. Limit of

Quantification (LOQ) is the lowest amount analyte in a sample that can be determined with

acceptable precision and accuracy under the stated experimental conditions. LOD and LOQ

were determined by k x SD/s where k is a constant (3.3 for LOD and 10 for LOQ), SD is the

standard deviation of the analytical signal and s is the slope of the calibration curve.

Accuracy

Accuracy should be reported as percent recovery by the assay of known added amount of

analyte in the sample or as the difference between the mean and the accepted true value

together with the confidence intervals. Accuracy should be assessed using a minimum of 9

determinations over a minimum of 3 concentration levels covering the specified range (e.g. 3

concentrations /3 replicates each of the total analytical procedure). The percent recovery was

calculated by performing recovery studies in triplicates of three concentration levels viz.

80%, 100%, 120% by adding known amount of standard mixture of stigmasterol and

embelin. These samples were then analyzed and the results obtained were compared with

expected results.

Robustness

The robustness of an analytical procedure 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.

It was studied in triplicate at 400 ng/spot and 600 ng/spot for stigmasterol and 40ng/spot and

60ng/spot for embelin by making small changes in mobile phase composition, the mobile

phase saturation time. The final results were examined by calculation of %RSD of

concentration.

RESULTS AND DISCUSSION

Preliminary studies

Evaluation means confirmation of identity and determination of quality and purity of the

herbal drug. Preliminary evaluation of herbal formulation was carried out for marketed

formulations of three different brands (M1, M2) and In-house formulation (I) in terms of

following parameters.


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The results of preliminary studies are given in Table 1.

Table 1. Results of Preliminary studies.

Parameters Marketed

formulation (M-01)

Marketed

formulation (M-02)

In-house

formulation

Alcohol soluble extractive

value (% w/w) 7.2 4.5 5.7

Water soluble extractive

value (% w/w) 30.42 36.73 28.6

Loss on drying (% w/w) 5.52 4.5 6.34

Ash value (% w/w) 12.5 17.2 19.5

Fig. 3: HPTLC in situ overlain spectra of stigmasterol and embelin

Good resolution and sharp peaks with minimum tailing were obtained with mobile phase

consisting of Toluene: ethyl acetate: methanol: formic acid(6:2:1:1) (v/v/v/v). embelin and

stigmasterol were satisfactorily resolved with Rf values at 0.45+ 0.02 and 0.63+ 0.02,

respectively.

Fig. 4: Chromatogram of standard embelin [Rf: 0.45 ± 0.02] and stigmasterol [Rf: 0.63

± 0.02] [peak1: Embelin, peak 2: Stigmasterol]


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HPTLC Method Validation

Linearity

Linear relationship was observed by plotting drug concentration against peak area for each

compound. stigmasterol and embelin showed linear response in the concentration range of

300-900 ng/spot and 30-90 ng/spot, respectively (Fig 5A and 5B). The linearity was validated

by the high value of the correlation coefficients. The results are tabulated in Table 2.

Fig 5A. Calibration curve of stigmasterol

Fig 5B. Calibration curve of embelin


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Table 2: Linear regression data for calibration plot for stigmasterol and embelin

Parameters stigmasterol embelin

Linearity range 300-900 ng/spot 30-90 ng/spot

Regression

equation y = 0.266x + 804.1 y = 4.782x + 1136

Correlation

coefficient 0.991 0.990

Slope 0.266 4.782

Intercept 804.1 1136

Specificity

When the spectra of standard stigmasterol and embelin were overlayed or compared with

extracts of chopchinyadi churna it was observed that constituents present in the extract did

not interfere with the peaks of stigmasterol and embelin. Thus the proposed method was

proved to be specific. The spectra of the standard stigmasterol and embelin corresponded

with the extract of chopchinyadi churna. ( Fig 6Aand 6B)

Fig 6 (A) Overlay spectra of stigmasterol and stigmasterol from formulation extracts

Fig 6 (B) Overlay spectra of embelin and embelin from formulation extracts.


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Precision

Intraday precision is used to describe the variation of the method, at three different

concentration levels within the same day while interday precision is for variation between

different days. The % RSD values for both intraday and interday precision were found within

acceptable limit as shown in Table 3.

Table.3 Results of Interday and Intraday precision

Concentratio

n

(µg/ml)

Intraday Interday

Mean

Area SD %RSD

Mean

Area SD %RSD

stigmasterol

400 1905.178 18.789 0.986 1913.12 28.071 1.467

600 2311.156 31.038 1.342 2345.83 43.422 1.851

800 2922.878 51.291 1.754 2929.19 10.602 0.361

embelin

40 2798.233 55.120 1.969 2828.72 59.004 2.08

60 3907.322 49.640 1.270 3914.02 33.505 0.856

80 5161.544 38.462 0.745 5199.05 83.803 1.611

Limit of detection (LOD) and Limit of quantification (LOQ): The LOD and LOQ values

were tabulated in table 4.

Table 4. results of LOD and LOQ values for stigmasterol and embelin.

stigmasterol (ng/spot) embelin (ng/spot)

Limit of Detection (LOD) 30.53 29.046

Limit of Quantification(LOQ) 92.52 88.018

Accuracy

Accuracy of the method is reported as percent recovery of known added amount of analyte in

the sample. The percent recovery was calculated by performing recovery studies in triplicates

of three concentration levels viz. 80%, 100%, 120% by adding known amount of standard

mixture of stigmasterol and embelin.


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Table 5A: Results of accuracy of stigmasterol

stigmasterol Level of

% recovery

Total amount

of marker

(ng)

Amount of

marker

found (ng)

Recovery

%

%

RSD

Mean

recovery

Marketed

Formulation M1

80 504.8 502.67 99.57 0.94

99.07 100 631 620.92 98.40 1.23

120 757.2 751.6 99.26 1.65

Marketed

Formulation M2

80 540.8 534.41 98.81 1.47

98.96 100 676 660.43 97.69 0.87

120 811.2 790.14 98.75 0.92

In house

formulation

80 544 538.79 99.04 1.75

98.57 100 680 668.43 98.29 1.93

120 816 802.68 98.38 1.68

Table 5B: Results of accuracy of embelin

embelin

Level of

%

recovery

Total

amount of

marker

(ng)

Amount of

marker

found (ng)

%

Recovery

%

RSD

Mean

recovery

(%)

Marketed

Formulation

M1

80 111 109.124 98.19 1.28

98.63 100 124 122.603 98.87 1.79

120 136 134.428 98.84 0.45

Marketed

Formulation

M2

80 102.6 106.763 103.9 0.71

99.01 100 114 111.921 98.17 1.37

120 125.4 122.144 98.625 0.91

In house formulation

80 117 115.23 99.14 1.43

98.89 100 130 128.46 98.46 1.86

120 143 141.68 99.07 2.05

Robustness

The % RSD of the peak area was calculated in triplicate for changes in mobile phase

composition, duration of saturation time and volume of mobile phase for 400 and 600 ng/spot

for stigmasterol and 40 and 60ng/spot for embelin. The values of % RSD were less than 2%

which indicated that the developed method is robust as shown in Table 6

Table. 6 Results of Robustness

Parameters

stigmasterol embelin

400ng/spot 600ng/spot 40ng/spot 60ng/spot

%RSD %RSD %RSD %RSD

Mobile phase composition

Toluene:

ethyl acetate:

0.69

0.72

0.93

0.97


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methanol:

formic acid

6:2:0.8:1

Toluene:

ethyl acetate:

methanol:

formic acid

6:2:1:0.8

0.52

0.55

0.63

0.75

Saturation time

28 minutes 1.23 1.33 0.85 1.27

32 minutes 0.46 1.52 0.67 0.51

Estimation of stigmasterol and embelin in marketed and In-house formulations

The developed method was applied for the detection and quantification of stigmasterol and

embelin from marketed and in-house formulations of chopchinyadi churna. The peaks for

stigmasterol and embelin were observed at Rf 0.63+ 0.02 and 0.45+ 0.02 respectively in the

densitogram of extracts( Fig 7,9and 11). There was no interference from other compounds

present in the churna. The total stigmasterol and embelin content in different marketed and

in-house formulations of Chopchinyadi churna was found to be satisfactory.

Figure 7: Densitogram of Methanolic Extract of Chopchinyadi churna (Formulation

M1) The stigmasterol and embelin content in marketed formulation(M-1) was found to

be 0.1262% and 0.0124% w/w respectively.


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Figure 8(a): HPTLC Fingerprinting Profile of Extract of chopchinyadi churna

(Marketed Formulation M1) at 254nm.

T1, T5- Standard stigmasterol and embelin, T2 to T4- Extract used for quantification of

stigmasterol and embelin

Figure 8(b): HPTLC Fingerprinting Profile of Extract of chopchinyadi churna

(Marketed Formulation M1) at 366nm.


stigmasterol and Embelin


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Figure8(c): HPTLC Fingerprinting Profile of Extract of chopchinyadi churna

(Marketed formulation M1) after derivatization.



Figure 9: Densitogram of Methanolic Extract of Chopchinyadi churna (Formulation

M2)

The stigmasterol and embelin content in marketed formulation(M-2)was found to be

0.1352%w/w and 0.0114%w/w respectively.


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Figure 10(a) HPTLC Fingerprinting Profile of Extract of chopchinyadi churna

(Marketed Formulation M2) at 254 nm



Figure 10(b): HPTLC Fingerprinting Profile of Extract of chopchinyadi churna

(Marketed Formulation M2) at 366 nm


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Figure10(c): HPTLC Fingerprinting Profile of Extract of chopchinyadi churna

(Marketed Formulation M2) after derivatization



Figure 11: Densitogram of Methanolic Extract of Chopchinyadi churna (Inhouse

ormulation)

The stigmasterol and embelin content in In-house formulation was found to be

0.1360%w/w and 0.0130%w/w respectively.


1629


Figure 12(a): HPTLC Fingerprinting Profile of Extract of chopchinyadi churna

(Inhouse Formulation) at 254 nm

T1, T5- Standard stigmasterol and embelin, T2 to T4- Extract used for quantification

of stigmasterol and Embelin

Figure12(b) HPTLC Fingerprinting Profile of Extract of chopchinyadi churna (Inhouse

Formulation) at 366 nm


stigmasterol and Embelin


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Figure 12(c): HPTLC Fingerprinting Profile of Extract of chopchinyadi churna

(Inhouse Formulation) after derivatization


stigmasterol and Embelin respectively

CONCLUSION

Chopchinyadi churna contains Smilax china and Embelia ribes as major ingredients

containing marker compounds stigmasterol and embelin respectively. The HPTLC method

was developed for simultaneous estimation stigmasterol and embelin. This method was

further applied for standardization of chopchinyadi churna. The developed HPTLC method

was found to be rapid, simple, linear, precise and accurate for quantitative estimation of

stigmasterol and embelin from chopchinyadi churna. The proposed method was found to be

useful, to evaluate various formulations available in the market containing both the drugs.

ACKNOWLEDGEMENT

Authors are thankful to Bharati Vidyapeeth's College of Pharmacy for providing facilities to

complete this work successfully.

REFERENCES

1. Rasheed A, Reddy S, Roja C. A review on standardisation of herbal formulation. Inter. J.

Phytotherapy. 2012; 2: 74-88.


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2. Subramani Parasuraman, Gan Siaw Thing, and Sokkalingam Arumugam Dhanaraj,

Polyherbal formulation: Concept of ayurveda, Pharmacogn Rev. 2014 Jul-Dec; 8(16): 73–

80.

3. Jayprakasam R, Ravit K,Athira Kuryakoise, Asha Jyothi B, Quantitative determination of

Diosgenin in polyherbal formulation and various extracts of smilax china linn using

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4. C.P Khare, Indian herbal Remedies- rational western therapy,ayurvedic and other

traditional uses, botany, published by-springer, 2004; 428.

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6. Rajesh Bhati, Anupama Singh, Vikas Anand Saharan, Veerma Ram, Anil Bhandari,

Pharmacognostical standardization, extraction and anti-diabetic activity of Smilax china

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7. Syed asadulla, Ramangdang, Rajashekaran, Pharmacognosy of Embelia ribes Burm F,

IJRPC, 2011; 1(4): 1236-1250.

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http://www.ncbi.nlm.nih.gov/pubmed/?term=Parasuraman%20S%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Thing%20GS%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Dhanaraj%20SA%5Bauth%5D

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