15 Chapter-V BUTYLATED HYDROXY TOLUENE...

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CHAPTER V

BUTYLATED HYDROXY TOLUENE (BHT) METHOD

DEVELOPMENT AND METHOD VALIDATION

This chapter describes about development and validation of a RP-LC test procedure to

determine Butylated Hydroxytoluene in Paricalcitol Hard Gelatin Capsule formulation

dosage form. The chapter also explains about materials used and scope of the present study. It

describes the reagents, glass-ware, standards and test samples (dosage forms) used in this

present study, Brief description of equipment and methods employed in the present study.

5.1 REAGENTS AND CHEMICALS

Acetonitrile (CH3CN):

HPLC grade solvent was used for the analysis, manufactured by Rankem and Merck

India Ltd.

Methanol (CH3OH):

HPLC grade solvent was used for the analysis, manufactured by Merck India Ltd and

Rankem Ltd.

5.2 GLASSWARE:

Brand new of Borosil and Glaxo of class-A, grade glassware like pipette, volumetric

flask, conical flask, glass bottle, test tubes, measuring cylinder and glass beaker were used for

analysis.

5.3 STANDARDS:

Working standards: Butylated hydroxyl toluene (BHT)

5.4 TEST SAMPLE (DOSAGE FORMS)

Finished products: Paricalcitol Hard gelatin capsule

5.5 EQUIPMENTS AND INSTRUMENTS

HPLC:

Waters HPLC, equipped with 2489 UV/Visible detector are used in the present study.

The signals were recorded and processed by utilizing Empower2 software.

Analytical balance:

Mettler Toledo make AB265-5/fact model is used for this research work.

Centrifuge:

Remi limited make centrifuge instrument is used for samples preparation.

Sonicator:

Partech scientific suppliers manufactured 25 Liter sonicator and Life care

manufactured 10Litre was used for analysis.

Degasser:

Borosil manufactured glass degasser apparatus is used and Millipore 0.45micron filter

paper is used for degassing.

Milli-Q water purification system:

Milli pore manufactured water purification system which is having 0.22µm membrane

filter is used for analysis.

5.6 SCOPE OF THE PRESENT STUDY:

Keeping in view of the importance of antioxidant presence in drud product to be

monitored the scope of the current study is designed. The present research work focuses on

the method development and validation of Quantification methods for antioxidant BHT

present in drug products by RP

in Table: 5.6.a. Butylated hydroxytoluene (BHT)

fat soluble compound. A regulatory agency permits small percentages use of BHT in food

additives and some of pharmaceutical dosage form. Main use of Butylated

as an antioxidant is to prevent drug degradation during the stability.

The author selected the BHT antioxidant for the present study. The Literature review

reveals that no test procedure as available for separation and Quantification of th

present in paricalcitol hard gelatin capsule pharmaceutical dosage forms by the proposed

techniques.

Table- 5.6.a: Butylated Hydroxy toluene structure and chemical name

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manufactured 10Litre was used for analysis.


er purification system:


SCOPE OF THE PRESENT STUDY:


d the scope of the current study is designed. The present research work focuses on


present in drug products by RP-HPLC which are sophisticated analytical techniques as give

in Table: 5.6.a. Butylated hydroxytoluene (BHT) is useful for its antioxidant properties and is


additives and some of pharmaceutical dosage form. Main use of Butylated

as an antioxidant is to prevent drug degradation during the stability.


reveals that no test procedure as available for separation and Quantification of th


5.6.a: Butylated Hydroxy toluene structure and chemical name





d the scope of the current study is designed. The present research work focuses on


HPLC which are sophisticated analytical techniques as given

is useful for its antioxidant properties and is


additives and some of pharmaceutical dosage form. Main use of Butylated Hydroxy toluene


reveals that no test procedure as available for separation and Quantification of the BHT


5.6.a: Butylated Hydroxy toluene structure and chemical name

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5.7 INTRODUCTION:

Paricalcitol hard gelatin capsules contain the active ingredient paricalcitol. Paricalcitol

is analog of calcitriol and it made of synthetic process. It is a active metabolite of Vitamin D

and prescribe for the avoidance and curing of secondary hyperparathyroidism in chronic

kidney disease. Paricalcitol HG capsule oral administration contains 1, 2 or 4 μg of

paricalcitol.

Butylated hydroxytoluene (BHT) which is used in paricalcitol HG capsule is a

phenolic compound and chemically described as 2, 6-bis (1, 1-dimethylethyl)-4-

methylphenol (Fig.5.7.a). Its molecular formula C15H24O, and MW is 220.34. It’s available in

solid form that is often added as antioxidant in pharmaceutical dosage products [1, 2].

[2,6-bis (1,1-dimethylethyl)-4-methylphenol]

Fig-5.7.a :It shows Structure and Chemical name of Butylated Hydroxy toluene

BHT is essentially used to prevent oxidative rancidity in pharmaceutical dosage form

[3]. The concentration of BHT based on the compounds and it makes compound stable [4, 5].

BHT is capable for reaction inhibition which endorsed by oxygen [5]. To avoid drug product

degradation from peroxide radicals we should add antioxidant compound [6, 7].

Reversed phase liquid chromatography (RP-LC) with UV/Vis detector is useful

technique [9]. In literature review research has been done for determination of BHT by RP-

HPLC, GC and by LC-MS [5, 8, 10-16].Beside the reported method, as per our current

knowledge no method is reported by RP-LC at very low concentration of BHT as 0.0039

mcg/mL in paricalcitol capsule .This paper described procedure is a stability indicating

method that can separate BHT from oil based excipients. The proposed chromatographic

method validation was performed. The parameter covers for method validation was

establishment of LOD and LOQ, selectivity, precision, recovery, linearity, ruggedness and

robustness. Placebo interference study was conducted in selectivity parameter to demonstrate

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a specificity of proposed test procedure. These studies were conducted as per ICH guidelines [17].

5.8 OPTIMIZED TEST PROCEDURE

Chromatographic Conditions:

Column : ACE C18 column 4.6 x 250 millimeter, 5 micron

Mobile phase :

Solvent-A : Water

Solvent-B : Methanol

Isocratic : 50:950(Solvent A: Solvent B).

Flow rate : 0.8 mL per minute

UV detector Wavelength: 277nm

Injection volume : 500μL

Column Temperature : 45°C

Run time : 25 min (sample) and 12 min(standard)

Diluent:

90% Acetonitrile and 10% water well mixed combination was used as diluent-1, and 70%

Acetonitrile and 30% water well mixed combination was utilized as diluent-2.

Standard solution:

Standard solution (200μg/mL) of Butylated hydroxyl toluene was prepared in diluent-

1. Transferred about 40 mg of butylated hydroxyl toluene WS in 200mL of flask. The BHT

was dissolved by adding diluent-1 and sonicate the solution till clear solution appears.

Diluted this clear solution up to 200 mL mark with diluent-1. Further it is diluted 125 times

with diluent-1. Finally above solution of BHT was diluted 5 times with diluent-2.This diluted

BHT standard has 0.32 ppm concentration in solution. The SST was checked with 5 replicate

injection of standard.

Test Solution Preparation:

Five capsules of paricalcitol were taken and cut the each capsule shell and transferred

accurately the whole capsules with content in to a 50 milliliter flask. 25 milliliter of diluent-1

poured, sonicate it for about 20 minutes in the sonicator with vigorous intermediate shaking.

Allow the volumetric flask to bench top to set at RT and make the dilution with diluent-1 and

mixed. Further, it was diluted 5 times with diluent-2 to get the concentration of BHT as 0.32

μg per mL.

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Procedure:

Separately injected equal 500μL volume of blank, standard preparation (Five times),

test solution to HPLC instrument. The chromatograms were recorded and measured the areas

of peaks in the chromatograms and calculated the results using below formula.

System Suitability:

The Butylated Hydroxy toluene peak asymmetry factor in standard solution was NMT

2.0 , Theoretical plate count was NLT 3000 for BHT peak from standard preparation and %

Relatives standard deviation for butylated Hydroxy toluene from five injection was NMT 2%.

Calculation:

BHT Content in mg/Capsule =

At * WS * 2 * 5 * 50 * 25 * P As * 200 * 250 * 25 * 5 * 5 * 100

Where, “As” is the area of Butylated hydroxyl toluene in standard preparation. “At” is

the area of Butylated hydroxyl toluene in test preparation. “WS” is weight of the Butylated

hydroxyl toluene standard took for STD solution in milligram and “P” is the Butylated

hydroxyl toluene potency as such basis.

5.9 METHOD DEVELOPMENT:

IR Spectra:

Butylated hydroxytoluene was scanned in Fourier Transform-Infrared range from

4000 to 450cm-1 and the individual infrared Spectra’s are represented as Spectrum 5.9.a

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Spectrum 5.9.a: Butylated Hydroxy toluene (BHT)

UV-Spectra:

BHT was scanned across the UV range from 200-400nm using 2996 PDA Detector of

Waters HPLC system equipped with 2695 separation module. Butylated Hydroxy toluene has

UV maximum absorbance. Spectrum 5.9.b represents the absorption spectra of BHT.

Spectrum 5.9.b: UV spectrum of BHT.

The above UV spectra indicate that, BHT has maximum Absorbance at 277 nm

wavelength was selected and the results were found satisfactory and reproducible at this

wavelength.

Selection of HPLC column:

Preliminary phase of method development experiments was conducted with isocratic

method and Alltima C18 250mm x 4.6 millimeter five micron column. The mixtur of 20%

watar and 80 % methnol as mobil phase but BHT peak was merged with excipient peak. To

resolve the merging issue HPLC column was changed to differant brande as Ace

octadecylsilane, 250 x 4.6 millimeter, five micron colum. BHT peak was slightly resolved but

peak shape was not symmetrical. Hence using same column mobile phase was changed.

Finally resolution was obtained in octadecylsilane, 250 x 4.6 millimeter, five micron column

and column has proved for Reproducibility, and Ruggedness.

Selection of Diluent:

Selection of diluent for Butylated Hydroxy toluene was a major challenge in the method

development as it is Insolubl in watar; frely solubl in CH3CH2OH and acetonitrile. Initially

acetonitrile was used for dilution but the peak shape was not symmetric for BHT peak.

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Different types of solvent like methanol, THF, acetonitrile and water in different ratio,

experiment were conducted for improvement of BHT peak shape and recovery problem.

Further experiment were conducted by different diluent ratio of water and acetonitrile and

decided to keep two diluent, first diluent for extraction of BHT from paricalcitol capsule

where organic solvent ratio is more and second diluent to get symmetrical peak shape.

Finally, 90% Acetonitrile and 10% water mixture was used as diluent-1, and 70%

Acetonitrile and 30% water mixture was used as diluent-2.

Selection of run time and flow rate:

With this developed method the retention times for BHT was found within 7 minutes

with a mobile phase FR was 0.8 milliliter per minute. Above 0.8 milliliter per minute flow

rates leads to early retention and merging of excipient peak with BHT, it causes

quantification problems for BHT.

Selection of standard and sample solution concentration:

BHT content per paricalcitol capsule is extremely low. The standard concentration

was based on concentration of test concentration. The sample concentration selected as 0.32

µg/mL which can be maximum achieved by sample preparation.

Selection of other chromatographic parameters:

As Butylated Hydroxy toluene (BHT) concentration is very low, the First main

objective was to develop a method for detection of BHT and the test procedure should able to

resolve close eluting molecule from BHT. The test procedure should have chromatographic

run very short. The solution having 0.32 mcg per milliliter of BHT was used for method

optimization. An isocratic method employed using 20% Milli-Q water and 80% methanol

mixture as mobile phase, Alltima octadecylsilane (4.6 X 250 millimeter) five micron column.

The FR was set as 2.0 milliliter per minute on HPLC equipped with PDA detector. 60%

acetonitrile was used as diluent. BHT Peak was merged with Excipient peak. To resolve the

peak an attempt were done by different ratio of water and MeOH in MP by changing

octadecylsilane (4.6 X 250 millimeter) five micron column. BHT peak was very well

resolved but the recovery found in lower side. Therefore to achieve satisfactory recovery,

different experiments were conducted in various diluents. Recovery was increased by

changing the diluent and sonication time but peak shape was not found symmetrical. Further

experiment were conducted by different diluent ratio of water and acetonitrile and decided to

keep two diluent, first diluent for extraction of BHT from paricalcitol capsule where organic

solvent ratio is more and second diluent to get symmetrical peak shape. Based above trials the

method final conditions were proposed as mentioned below.

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The method was concluded with ACE octadecylsilane (4.6 X 250 millimeter) five

micron column .The MP having a composition of 5% sol-A (water) and 95% Sol-B

(MeOH).Isocratic method was used with runtime of 25 minute for sample and 12minute for

standard. 10% H2O and 90% ACN mixture was utilized as diluent-1and 30% H2O and 70%

ACN mixture as diluent-2. The FR was 0.8 milliliter per minute. The column oven temp was

set at 45 degree centigrade. The outcome signal was detected at 277 nanometer wavelength.

The sample inj. volume was 500 micro liter.

5.10. METHOD VALIDATION:

The method validation optimized test procedure was performed par ICH guidelines.

The method was validated by executing the studies for the parameter are, selectivity,

linearity, recovery, precision, and robustness.

System suitability

This parameter was verified by test procedure characteristic criteria before initiate the

analysis which determination has been done base on specification. The SST was assessed

based on tailiing factore and theoritical plats of BHT and relative standrd deviation (RSD) of

five inj. of STD. SST was checked previous to analysis with 5 replicate injections of the

standard preparation having 0.32µg/mL BHT.(Fig.5.10.a) The specification for % RSD

should be NMT 2.0%, USP tailing factor should be NMT 2.0 and theoretical plate should be

NLT 3000 for BHT peak from standard preparation. All SST parameters are found to be

complying to the specification (Table 5.10.a).

Tab. 5.10.a. System suitability data

Parameters Specification Observed values

Theoretical plates for BHT peak from standard injection

>3000 13030

The% RSD for peak areas in five standard injections

<2.0% 0.3

The USP Tailing factor for BHT peak <2.0 1.1

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Fig. 5.10.a: Representative chromatograms of standard solution

Specificity

A specificity study to determine placebo interference was performed. Study was

performed on Placebo (Placebo contains without Butylated hydroxytoluene, with Paricalcitol

and other excipients) in duplicate. Placebo preparation was made by taking equal weight

placebo composition in test preparation as per proposed procedure. Placebo chromatogram

had shown no interference at the RT of Butylated hydroxytoluene, this indicats that

excipientes use in the formuletion do not have any interference in estimasion of Butylated

hydroxytoluene in Paricalcitol capsules (Fig.5.10.b).

Fig. 5.10.b: Representative chromatograms of Placebo. Precision

This parameter was studied by repeatability and intermediate precision. The proposed

test procedure evaluation has been done at target concentration level (0.32µg/mL).

Repeatability was determined with 6 sample preparations of BHT in paricalcitol Hard Gelatin

capsule as per test method (Fig.5.10.c). %RSD of result for BHTs was calculated. The test

procedure intermediate precision study was assessed by different analyst, instrument and day.

Based on outcome of the study it is found that proposed test procedure is very precise.

The % RSD for the area of BHT was maximum 1.5% and 0.2% in repeatability and

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intermediate precision correspondingly. The results for each impurity are tabulated in Table

5.10.b.

Tab. 5.10.b.: Repeatability and intermediate precision test results

Sample No.

% Butylated hydroxytoluene (BHT) Repeatability Intermediate precision

1 96.4 97.6 2 100.7 97.2 3 98.5 97.6 4 99.7 97.9 5 98.0 97.4 6 98.2 97.7

Mean 98.6 97.6 %RSD 1.5 0.2

Fig. 5.10.c. Representative chromatograms of Sample solution

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

A S/N data for proposed test procedure was employed to establish LOD/LOQ value

for Butylated Hydroxy toluene. For LOD and LOQ 3:1 and 10:1 respectively S/N was

achieved by different concentration BHT known solution. LOQ precision also studied for

BHT by 6 replicate injections as per test method.RSD was calculated for BHT precision at

LOQ level which is reported in Table 5.10.c and 5.10.d.

TAB 5.10.C: Precision at limit of Quantification

Sample No. % BHT content 1 104.8 2 101.1 3 98.8 4 93.1 5 112.1 6 101.7

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Mean 101.9 % RSD 6.2

Linearity

The different concentration solutions were made to the necessary concentrations by

covering the range between 0.0039µg/mL and 0.64 µg/mL of BHT (Table 5.10.e). The

preparations were made at 6 different levels from LOQ to 200 percent of target level (LOQ,

25percent. 50percent, 100percent, 150percent and 200percent). Calibration graph were

designed where in ‘X’ axis concentration of the analyte in µg/mL and in ‘Y’ axis repose of

the analyte. (Fig.5.10.d) The R2 was found more than 0.999. and % bis at target leval are

with-in ± 2%. The slope, y-intercept, coefficient correlation, and bias at 100% response were

summarized in Tab. 5.10.d.

Tab. 5.10.d. Linearity and LOD-LOQ data

Parameter BHT LOD (microgram per mL) 0.0013

LOQ (microgram per mL) 0.0039

Correlation coefficient 0.9999

Intercept (a) 38.3016

Slope (b) 306579.0448

Bias at 100% response 0.04

Precision at LOQ (%RSD) 6.2

Tab. 5.10.e. Linearity study concentration of BHT and Peak Area

concentration of BHT in µg/mL Area 0.0039 968 0.0798 24534 0.1596 48964 0.3191 98217 0.4787 147062 0.6382 195323

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Fig.5.10.d: Linearity graph

Accuracy

Recovery for the proposed method for BHT were assessed by three preparation like

LOQ (0.0039), 0.08,0.16,0.32,0.48 and 0.64 µg/mL. The % Accuracy for Butylated Hydroxy

toluene was varied from 98.8% to 104.8%. The recovery values are tabulated below in Table

5.10.f.

Tab. 5.10.f. Recovery data

Sample No.

%Spike level “microgram”

added “microgram”

found %

Recovery Mean % Recovery

1 LOQ 0.0040 0.0042 104.8

101.6 2 LOQ 0.0040 0.0040 101.1

3 LOQ 0.0040 0.0040 98.8

1 25% 0.0803 0.0827 103.0

102.3 2 25% 0.0803 0.0820 102.1

3 25% 0.0803 0.0817 101.7

1 50% 0.1607 0.1623 101.0

102.1 2 50% 0.1607 0.1644 102.3

3 50% 0.1607 0.1654 102.9

1 100% 0.3214 0.3255 101.3 101.7

2 100% 0.3214 0.3284 102.2

968

24534

48964

98217

147062

195323

y = 30657x + 38.30

R² = 0.999

0

50000

100000

150000

200000

250000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Are

a

Concentration (µg/mL)

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3 100% 0.3214 0.3261 101.5

1 150% 0.4820 0.4959 102.9

102.8 2 150% 0.4820 0.4937 102.4

3 150% 0.4820 0.4965 103.0

1 200% 0.6427 0.6448 100.3

100.7 2 200% 0.6427 0.6494 101.0

3 200% 0.6427 0.6469 100.7

Robustness

To establish the proposed procedure robustness, optimized chromatographic

conditions deliberate change was made. The outcome or response was measured is SST

parameters for BHT standard. The MP composition, FR and column oven tem was altered in

this study to check test procedure performance.The MP flow rate was 0.8 milliliter per min.

To check the method performance with respect to flow rate optimized flow was altered ± 0.2

unit (0.6 to 1.0 milliliter per minute) and evaluated SST parameter. The effect of composition

of mobile was checked in between 90% to 110% of the method organic phase composition.

The column temp change on SST was checked on 40 degree centigrade and 50 degree

centigrade in place of 45° C. In all the deliberate changes of chromatographic conditions,

highest tailing of BHT peak was 1.1, minimum theoretical plate was 12059 and the %RSD of

peak areas was maximum 1.9. The system suitability parameters evaluated are shown in

Table 5.10.g.

Tab. 5.10.g: Method Robustness results

Variation in chromatographic condition

Observed system suitability parameters

Theoretical plate

> 3000

USP Tailing for BHT peak

<2.0

The %RSD of peak areasa

<2.0%

Column Temp

40°C 13652 1.1 0.4

Column Temp

50°C 12329 1.1 1.9

FR 0.6 mL/min

13580 1.1 0.8

FR 1.0 mL/min

12538 1.1 0.7

90% organic 12059 1.1 0.7

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composition

110% organic composition

13400 1.1 0.4

aThe relative standard deviation of peak response of five standard inj. Stability of the Solutions and Mobile Phase

The study was determned by Keping sample and standad solusions in closed container

at RT for 5 days. Every day BHT content level and STD preparation similarity factor was

evaluated with new STD preparation. The same process was followed to established mobile

phase stability for 5 days where every day sample was evaluated against old mobile phase.

The MP was not changed throughout the experiment. The result shows that mobil phas is

stabl up to 5 days. The sampl result is not varying more than 10%. Hence, samples are stable

up to 5 days. The similrity factore of new and old prepared standard is within the

specification limit. Therefore, STD is also stable up to 5 days on room temperature. The

bench top stability of standard and test are tabulated in table 5.10.h.

Table 5.10.h: Ruggedness –Bench top Stability Of Standard and Test Preparation

Time in days

Similarity Factor of Standard

preparation

% content of BHT

% Difference

Test-1 Test-2 Test-1 Test-2

Initial NA 97.6 97.2 NA NA

1

1.0 98.0 97.6 0.4 0.4

2

1.1 102.5 104.5 4.9 7.3

5

1.1 97.4 104.6 0.2 7.4

Assay Result and Method Application

To check the Application of the method three different types of paricalcitol HG

capsules formulations were selected where BHT concentration were different. These each

sample were analyzed three times as per above mentioned method and results are found

satisfactory which is summarized in below mentioned table 5.10.h.

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Tab. 5.10.i: Assay results of RP-LC method

Sample

Amount of BHT in mg/cap %labeled claim

+ %RSD Labeled Estimated

Paricalcitol HG capsule Formulation-1

0.0080 0.0081 101.2+ 2.3


0.0160 0.0160 100.0+ 0.9


0.0240 0.0242 100.8+ 1.3

The results (precision, specifecity, linerity, acuracy, robustnes and ruggednes) of the

present study indicate that the developed and validated methods are reproducible, rugged and

stability indicating.

An uncomplicated and reliable RP-HPLC test procedure development and method

validation was performed for analysis of Butylated Hydroxy toluene in paricalcitol capsule

drug product. The validation confirmed that propose procedure is precice, liner, acurate,

ruged and robst. The method validation results are complies the acceptance criteria. The

developed method is useful for commercial batch routine analysis for checking the stability

of the BHT in paricalcitol capsules.

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