bendre s.d. & ghule p.j. int. res. j. pharm. 2016, 7...

Bendre S.D. & Ghule P.J. Int. Res. J. Pharm. 2016, 7 (12)

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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY

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ISSN 2230 – 8407

Research Article ANALYTICAL METHOD DEVELOPMENT, VALIDATION, AND ASSAY OF BETAMETASONE DIPROPIONATE CREAM BY HPLC METHOD Bendre S.D. *, Ghule P.J. Mula Rural Institute of Pharmacy, Sonai, Tal. Newasa, Dist. Ahmednagar, India *Corresponding Author Email: [email protected] Article Received on: 23/10/16 Revised on: 23/11/16 Approved for publication: 03/12/16 DOI: 10.7897/2230-8407.0712151 ABSTRACT The efficient development and validation of analytical methods are critical elements in the development of pharmaceuticals. The scope of developing and validating a method is to ensure a suitable strategy for a particular analyte which is more specific, accurate, and precise. Here, the main focus is drawn to achieve improvement in conditions and standard operating procedures to be followed. The principal objective of this study was, therefore, to develop a new, simple, economical, selective, precise, reproducible, and stability-indicating high-performance liquid chromatographic (HPLC) method with a wide linear range and good sensitivity for assay of Betametasone Dipropionate and related substance study in the bulk and pharmaceutical formulations. The related substance study and assay is carried by In-house method. So, as In-house method we have selected the HPLC method. Keywords: RP-HPLC, Assay Development and validation, Betamethasone Dipropionate INTRODUCTION A survey of literature reveals that good analytical methods are not available for the drugs like Betamethasone Dipropionate. No RP-HPLC method has been reported so far for Assay and Related Substances identification by using gradient mode i.e. Mobile phase A- (Water: IPA) (70:30) and Mobile phase B-(ACN: THF) (80:20) Even though very few methods of determination of above drug is available, many of them suffer from one disadvantage or the other, such as low sensitivity, lack of selectivity and simplicity etc. The existing physicochemical methods are inadequate to meet the requirements; hence it is proposed to improve the existing methods and to develop new methods for the assay and related substance of the drugs in pharmaceutical dosage forms and different available analytical techniques like HPLC. The objective of the research work are;

1. To optimize the chromatographic condition for assay of Betametasone Dipropionate cream.

2. To optimize the chromatographic condition of estimation of related substances of Betametasone Dipropionate in Betametasone Dipropionate cream.

Method is optimized according to different parameters by taking different individual conditions to avoid error, unreliability and contamination. Moreover, it provides adequate sensitivity and specificity. The validation of this method is carried out according to the ICH (Q1A, Q2B) guidelines.4,6,7,8 This method is one of the useful methods owing to its ruggedness and robustness and found to be selective, specific, sensitive, linear, precise and economic.1,2,3,4,9,10,11

MATERIALS AND METHODS

Table 1: List of Material

Sr. No. Name Supplier 1. Betamethasone Dipropionate (BD) Sample from R and D Laboratory, Glenmark

Research Center, Mahepe, Navi Mumbai. 2. Chlorocresol

Table 2: List of Chemical and Reagent

Sr. No. Name Grade Supplier 1. Acetonitrile(ACN) HPLC Rankem Ltd,Mumbai 2 Methanol(CH3OH) HPLC Rankem Ltd,Mumbai 3. Glacial Acetic Acid

(CH3COOH) AR Merck Specialities Ltd,Mumbai.

4. IPA HPLC Rankem Ltd,Mumbai. 5. THF HPLC Rankem Ltd,Mumbai. 6. Sodium Hydroxide(NaOH) LR Rankem Ltd,Mumbai. 7. Hydro Chloric Acid (Hcl) LR Qualigens Fine Chemicals,Mumbai. 8. Milli Q Water NA In-House


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A. Preliminary Tests / Qualitative Tests2,11-15 Physical State of Drugs: Solid and white or almost white crystalline powder. Solubility · Practically insoluble in water. · Freely soluble in acetone and in methylene chloride. · Sparingly soluble in alcohol. Melting Point: 176-180 0C Identification of Drugs by IR- Spectrum

Figure 1: Infrared spectrum of Betamethasone Dipropionate API UV –Spectrum

Figure 2: UV –Spectrum of Betamethasone Dipropionate API in Methnol (10ppm)

B. Assay method development and optimization2,11-15 Selection of chromatographic method Important information’s about the sample composition and properties that should be collected are as follows; · Number of compounds present · Chemical structure (functionality) of the compounds · Molecular weight of the compounds · pKa values of the compounds · UV-spectra of the compounds · Concentration range of the compounds in the sample. Sample solubility Method development was optimised by considering the various system suitable parameter such as · Number of theoretical plates (efficiency) · Capacity factor, · Separation (relative retention) · Resolution, · Tailing factor

Table 3: Optimised Chromatographic Condition for Assay Method

Parameter Condition Stationary Phase Discovery HS, C18, (150 x 4.6mm), 3µ or

equivalent. Mobile Phase Mobile phase A-Water: IPA(80:20)

Mobile phase B-ACN: THF(70:30) Flow Rate 1.0 ml/min Detection 240nm

Pump Mode Gradient Injection Volume 20 µl

Run Time 35min Column

Temperature 500C

Retention Time About 8.5 minutes for Chlorocresol About 21.0 minutes for Betamethasone.

Needle wash Methanol Gradient programme

Table 4: Composition of Mobile Phase

Time (min) Mobile phase A Mobile phase B 0 80 20 22 80 20 25 40 60 30 80 20

C. Assay method validation The optimized HPLC method has been developed for the determination of the assay of Betametasone Dipropionate in Betametasone Dipropionate cream (0.05%) w/w. This protocol is intended for the validation of assay of Betametasone Dipropionate in Betametasone Dipropionate cream (0.05%) w/w.2,7,9-15 Methodology followed Reagents Methanol (HPLC grade) Glacial acetic acid (AR Grade) Acetonitrile (HPLC grade) Isopropyl Alcohol (HPLC grade, Rankem) Tetrahydrofuran (HPLC grade, Rankem) Water (Milli Q or equivalent) Preparation of Mobile phase Mobile phase A: Mixture of Water and Isopropyl Alcohol in the ratio 80:20 was prepared. Mobile phase B: Mixture of Acetonitrile and Tetrahydrofuran in the ratio 70:30 was prepared. Preparation of diluent: (0.1 % Glacial Acetic acid in Methanol) 1 ml of Glacial acetic acid was diluted in 1000 ml of Methanol. Chromatographic Conditions Column :Discovery HS, C18, (150 x 4.6mm), 3µ or equivalent. Flow Rate:1.0 ml / min. Detection :240 nm. Column Temp:50°C. Injection Volume:20µL.

Run Time:35 minutes. Retention Times: About 8.5 minutes for Chlorocresol About 21.0 minutes for Betamethasone.


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Gradient programme

Table 5: Composition of Mobile Phase

Time (min) Mobile phase A Mobile phase B 0 80 20 22 80 20 25 40 60 30 80 20 35 80 20

Preparation of standard solution 40 mg of Betamethasone Dipropionate (equivalent to 31.08 mg of Betamethasone) and 60 mg of Chlorocresol working standard was accurately weighed and transferred into a 100 ml volumetric flask and diluted with 60 ml of diluent and sonicated to dissolve, then diluted the above solution to volume with diluent and mix. 5.0 ml of the above solution was pipetted out into a 100 ml volumetric flask and diluted to volume with diluents to obtained the standard concentration of Betamethasone Dipropionate 20ppm and Standard concentration of Chlorocresol 30ppm. Preparation of sample solution: (For 0.05% w/w Cream) 3.11g of sample (equivalent to 2mg of Betamethasone Dipropionate) was accurately weighed and transferred into 100ml volumetric flask. 60 ml of diluent was added and warmed on a water bath at 60°C for 30 minutes with intermittent vigorous shaking and diluted up the volume with diluent. Then filtered through teflon membrane 0.45µ filter to obtained the sample preparation –20ppm. Evaluation of system suitability Standard solution was injected five times into the HPLC and the chromatograms were recorded. The area counts of Betamethasone and Chlorocresol peaks were measured. The relative standard deviation of five replicate injections for both the peaks should not be more than 2.0%. Procedure Equal volumes of the blank (diluents) and sample solution (in duplicate) were injected into the HPLC and the chromatograms

were recorded. The area counts of Betamethasone and Chlorocresol peaks were measured. A. Specificity Preparation of standard test solution 40 mg of Betamethasone Dipropionate (equivalent to 31.08 mg of Betamethasone) and 60 mg of Chlorocresol working standard was accurately weighed and transferred into a 100 ml volumetric flask and diluted with 60 ml of diluent and sonicated to dissolve, then diluted the above solution to volume with diluent and mix. 5.0 ml of the above solution was pipetted out into a 100 ml volumetric flask and diluted to volume with diluent. Preparation of sample solution 3.11g of sample (equivalent to 2mg of Betamethasone Dipropionate) was accurately weighed and transferred into 100ml volumetric flask. 60 ml of diluent was added and warmed on a water bath at 60°C for 30 minutes with intermittent vigorous shaking. Allowed to attain room temperature and diluted up the volume with diluent. Then filtered through Teflon membrane 0.45µ filter to obtained the sample preparation 20ppm. Preparation of placebo solution 3.11g of placebo was accurately weighed into 100ml volumetric flask. 60 ml of diluent was added and warmed on a water bath at 60°C for 30 minutes with intermittent vigorous shaking. Allowed to attain room temperature and diluted upto the volume with diluent. Centrifuge at 2000 rpm for 15 minutes. Then filtered through teflon membrane 0.45µ filter. Preparation of spike solution To the sample solution 1ml of impurity solution (1ppm) was spiked. Experimental approach Blank solution, Standard, Sample, spike sample and placebo of Betamethasone Dipropionate were injected as per the sequence details given in Table-5.2. Percent interference will be determined by comparing the response for any peak detected at the retention time for Betamethasone Dipropionate.

Table 6: Sequence for Selectivity

Sample Name No. of Inj. Inj. volume (µl) Run time (minutes)

Blank 1 20 35 Placebo Solution 1 20 35

Betamethasone standard 1 20 35 Betamethasone sample 1 20 35

Spiked Solution 1 20 35 Acceptance Criterion There should not any interference at the retention time of main peak i.e. the main peak should be pure. B. Linearity Experimental approach For establishing the linearity for Betamethasone Dipropionate andChlorocresol, a series of standard preparation of Betamethasone Dipropionate andChlorocresol were prepared to cover a range of 50 % to 150 % of sample concentration i.e 20ppm for Betamethasone Dipropionate, based on this the range proposed for Linearity determination is 50% to 150% test concentration (i.e. 10 ppm to 30 ppm) for Betamethasone

Dipropionate and 30ppm for Chlorocresol, based on this the range proposed for Linearity determination is 50% to 150% test concentration (ie 15 ppm to 45 ppm) for Chlorocresol. A detail of dilutions is given in Table-5.5 and sequence for injection is given in Table-5.6. The Linearity graph should be plotted from50% to 150%. Preparation of linearity stock solution 40 mg of Betamethasone Dipropionate 60mg Chlorocresol working standard was accurately weighed and transferred into two different 100mL volumetric flask. Add 70mL of diluent to each and sonicate to dissolve, cool and make up volume with diluent and mix properly to get the stock solution of both.


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Table 7: Dilutions for Linearity Betamethasone Dipropionate

% Conc of Sample Amount of stock solution to be transferred (ml)

Final volume with diluent (ml) Concentration (ppm)

50% 2.5 100 10 80% 4.0 100 16 90% 4.5 100 18

100% 5.0 100 20 110% 5.5 100 22 120% 6.0 100 24 150% 7.5 100 30

Table 8: Dilutions for Linearity Chlorocresol

% Conc of Sample Amount of stock solution to be

transferred (ml) Final volume with diluent

(ml) Concentration (ppm)

50% 2.5 100 15 80% 4.0 100 24 90% 4.5 100 27 100% 5.0 100 30 110% 5.5 100 33 120% 6.0 100 36 150% 7.5 100 45

Table 9: Sequence for Linearity Betamethasone Dipropionate and Chlorocresol

Sample Name No. of Inj. Inj. Volume (µl) Run time (minutes)

Blank 1 20 35 50% 2 20 35 80% 2 20 35 90% 2 20 35 100% 2 20 35 110% 2 20 35 120% 2 20 35 150% 2 20 35

Acceptance Criterion Correlation Coefficient should not less than 0.999 C. Accuracy (Recovery) Experimental approach Recovery samples were prepared by spiking placebo preparations with known amounts of Betamethasone Dipropionate and Chlorocresol standard in triplicate at three levels (total nine determinations). Each test sample will be prepared as described below.

Betamethasone Dipropionate placebo solution Placebo equivalent to 3110 mg of Betamethasone Dipropionate cream was accurately weighed and transferred into nine 100mL volumetric flasks. Betamethasone Dipropionate spiked solution Transferred accurate amount of API of both to above flasks to make the spiked solution in three levels i.e. 80%, 100% and 120%. About 60ml of diluents was added and sonicated for 30 min. Then cool and dilute up the volume with diluent to get the spiked solution of Betamethasone Dipropionate and Chlorocresol at different levels as described below in the Table 10.

Table 10: Sample preparation for Accuracy

Accuracy Level Conc. Of spiked sample of

Betamethasone Dipropionate Conc. Of spiked sample of

Chlorocresol 80%_sample_1 16 ppm 24 ppm 80%_ sample _2 16 ppm 24 ppm 80%_ sample _3 16 ppm 24 ppm

100%_ sample _1 20 ppm 30 ppm 100%_ sample _2 20 ppm 30 ppm 100%_ sample _3 20 ppm 30 ppm 120%_ sample _1 24 ppm 36 ppm 120%_ sample _2 24 ppm 36 ppm 120%_ sample _3 24 ppm 36 ppm


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Table 11: Sequence for Accuracy

Sample Name No. Of Injections Injection Volume (µl) Run time (minutes) Blank 1 20 35

Standard-1 5 20 35 Standard-2 2 20 35

Accuracy-80%/1 2 20 35 Accuracy-80%/2 2 20 35 Accuracy-80%/3 2 20 35

Standard-1 1 20 35 Accuracy-100%/1 2 20 35 Accuracy-100%/2 2 20 35 Accuracy-100%/3 2 20 35

Standard-1 1 20 35 Accuracy-120%/1 2 20 35 Accuracy-120%/2 2 20 35 Accuracy-120%/3 2 20 35

Standard-1 1 20 35 D. Precision System Precision Experimental approach Six replicate of standard solution were injected and the response was recorded.

Acceptance criterion The Relative standard deviations (%RSD) of the six replicate injections of standard solution will NMT 2.0%. Method precision Experimental approach One analyst was independently prepared six sample preparations of Betamethasone Dipropionate and analyze as per the method. Details of injection sequence are given Table 12.

Table 12: Sequence for System and Method Precision

Sample Name No. of Injections Injection Volume

(µl) Run time (minutes)

Blank 1 20 35 Standard solution-1 1 20 35 Standard solution-2 1 20 35 Standard solution-3 1 20 35 Standard solution-4 1 20 35 Standard solution-5 1 20 35 Standard solution-6 1 20 35

Sample-1 2 20 35 Sample-2 2 20 35 Sample-3 2 20 35 Sample-4 2 20 35 Sample-5 2 20 35 Sample-6 2 20 35

Intermediate precision (Ruggedness) Six standard solutions and six sample solutions of Betamethasone Dipropionate of the same lot using a different HPLC system, a different column on a different day will be analyzed. The mean and percent RSD values for area will be calculated. Acceptance criterion The Relative standard deviation (%RSD) of the twelve replicate injections ie the fresh six sample preparation and that of method precesion of sample solution will NMT 2.0%.

E. Robustness Experimental approach Prepare Three sample preparations of the same lot of Betamethasone Dipropionate were prepared. The following modifications to the Chromatographic conditions will be evaluated: · Change in column Temperature (±5°C) · Change in wavelength (±5 nm) · Change in Flow rate (± 0.1 ml\min)10% change For each set of change in the experimental condition, following sequence given in Table 13 will be followed.

Table 13: Sequence for Change in Experimental Condition

Sample Name No. of Injections Injection Volume (µl) Run time (minutes)

Blank 1 20 35 Standard-1 6 20 35 Standard-2 1 20 35 Sample-1 1 20 35 Sample-2 1 20 35 Sample-3 1 20 35


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F. Change in column temperature Normal experimental condition for column temperature is 50°C. Change in column temperature was studied for actual ±5°C. G. Change in wavelength Normal experimental condition for detection is 240 nm. Change in wavelength was studied for actual ±5 nm. H. Change in flow rate Normal experimental condition for flow rate is 1.0 ml/minute. Change in flow rate was studied for actual ±0.1ml/minute. RESULT AND DISCUSSION1,7,8,9

A. Linearity and Range

Table 14: Linearity for Betamethasone Dipropionate

% Concentration Concentration (µg per ml)

Response (Area) Statistical analysis

50% 10.034 360695 Slope 36321.6 80% 16.055 577523 90% 18.062 649383 Intercept -7305.6 100% 20.069 720764 110% 22.075 792247 Correlation

Coefficient 0.99956

120% 24.082 854761 150% 30.103 1095931

Table 15: Linearity for Chlorocresol

% Concentration Concentration

(µg per ml) Response (Area) Statistical analysis

50% 15.179 163386 Slope 10653.6 80% 24.286 260865 90% 27.322 293332 Intercept 966.2 100% 30.358 322393 110% 33.394 354744 Correlation

Coefficient 0.99984

120% 36.430 387424 150% 45.537 488589

Figure 3: Linearity Assay Graph of Betamethasone dipropionate

Figure 4: Linearity Assay Graph of Chlorocresol

Acceptance criteria: Correlation coefficient should not be less than 0.999. Conclusion: Correlation coefficient for Betamethasone dipropionate is 0.9991 and for chlorocresol is 0.9997. Therefore, the HPLC method for the determination of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream is linear. B. Precision System Precision: Six replicate injections of the Standard preparation were injected into the HPLC system using the method as described under Methodology.

Table 16: Data sheet for system precision

Injection Betamethasone Area

Chlorocresol Area

1 730368 344416 2 732315 344484 3 730726 344654 4 736851 344857 5 731718 344623 6 730179 344976

Mean 731968 344705 SD 2793.263 217.595

%RSD 0.38 0.06


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Acceptance criteria: RSD should not be more than 2.0%. Conclusion: The RSD of system precision is 0.38% for Betamethasone dipropionate and 0.06% for Chlorocresol. Therefore, the HPLC method for the determination of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream is precise. Method precision: Six replicate injections of the Sample preparation were injected into the HPLC system using the method as described under Methodology.

Table 17: Data sheet for method precision

Sample % Label claim Betamethasone

% Label claim Chlorocresol

1 100.5 100.6 2 100.9 101.1 3 101.0 101.1 4 100.4 100.5 5 100.7 101.4 6 100.2 101.0

Mean 100.6 101.0 SD 0.306 0.339

%RSD 0.30 0.34 Acceptance criteria: RSD should not be more than 2.0%. Conclusion: The RSD of method precision is 0.30% for Betamethasone dipropionate and 0.34% for Chlorocresol. Therefore, the HPLC method for the determination of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream is reproducible. C. Ruggedness (Intermediate Precession) Different HPLC system chosen for ruggedness study. Experiment: Six sample preparations of the same lot of Betamethasone dipropionate augmented cream 0.05% were made by a different analyst, using different column on a different day and injected in duplicate into a different HPLC using the method as described under Methodology, along with Standard preparation.

Table 18: Ruggedness of Betamethasone dipropionate

Sample Analyst -1 % Label claim

Analyst -2 % Label claim

1 100.5 98.8 2 100.9 100.5 3 101.0 99.9 4 100.4 102.7 5 100.7 100.9 6 100.2 100.8

Mean 100.6 100.6 SD 0.306 1.287

%RSD 0.30 1.28 Overall Mean 100.6

Overall SD 0.892 Overall %RSD 0.89

Table 19: Ruggedness of Chlorocresol

Sample Analyst -1

% Label claim Analyst -2

% Label claim 1 100.6 101.2 2 101.1 101.9 3 101.1 101.1 4 100.5 101.9 5 101.4 101.6 6 101.0 101.4

Mean 101.0 101.5 SD 0.339 0.343

%RSD 0.34 0.34 Overall Mean 101.2

Overall SD 0.440 Overall %RSD

0.43

Acceptance criteria: Overall RSD for twelve results should not be more than 2.0% Conclusion: The RSD of intermediate precision is 0.89% for Betamethasone dipropionate and 0.43% for Chlorocresol. Therefore, the HPLC method for the determination of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream is rugged. D. Accuracy (Recovery) Placebo of Betamethasone dipropionate augmented cream was spiked with Chlorocresol and Betamethasone dipropionate Drug Substance at three different levels: 80%, 100% and 120% of the label claim in triplicate (in total nine determinations) and then proceeded with Sample solution as described under Methodology. Each of the Sample solution was injected in duplicate and the average area count was taken for calculation.

Table 20: Accuracy for Betamethasone dipropionate

Sample No. Amount added (mg) Amount recovered (mg) % Recovery Accuracy 80% -1 1.605 1.609 100.2 Accuracy 80% -2 1.605 1.608 100.2 Accuracy 80% -3 1.605 1.611 100.4

Accuracy 100% -1 2.007 2.018 100.5 Accuracy 100% -2 2.007 2.000 99.7 Accuracy 100% -3 2.007 2.018 100.5 Accuracy 120% -1 2.408 2.422 100.6 Accuracy 120% -2 2.408 2.410 100.1 Accuracy 120% -3 2.408 2.419 100.5

Mean SD

% RSD

100.3 0.283 0.28


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Table 21: Accuracy for Chlorocresol

Sample No. Amount added (mg) Amount recovered (mg) % Recovery Accuracy. 80% -1 2.429 2.427 99.9 Accuracy 80% -2 2.429 2.444 100.6 Accuracy 80% -3 2.429 2.436 100.3 Accuracy 100% -1 3.036 3.031 99.8 Accuracy 100% -2 3.036 3.064 100.9 Accuracy 100% -3 3.036 3.054 100.6 Accuracy 120% -1 3.643 3.671 100.8 Accuracy 120% -2 3.643 3.632 99.7 Accuracy 120% -3 3.643 3.649 100.2

Mean SD

% RSD

100.3 0.443 0.44

Acceptance criteria: Mean recovery should be in the range of 98.0% to 102.0%. The RSD should not be more than 2.0%. Conclusion: Mean recovery for Betamethasone dipropionate is 100.3% and RSD is 0.28%. Mean recovery for Chlorocresol is 100.3% and RSD is 0.44%. Therefore, the HPLC method for the determination of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream is accurate.

F. Robustness Experiment: Three Sample preparations of the same lot (as used in precision study) of Betamethasone dipropionate augmented cream 0.05% were prepared as described under Methodology. The samples along with standard were injected in duplicate under different chromatographic condition as shown below.

G. Change in Temperature (+ 5°C)

Table 22: Change in Temperature for Betamethasone (+ 5°C)

Control (+5°C) Control (-5°C) 101.8 102.6 100.8 101.6 101.2 102.1 100.9 101.5 102.2 103.1 100.8 101.4

Cumulative Mean 102.2 Cumulative Mean 101.2 Cumulative SD 0.653 Cumulative SD 0.372

Cumulative %RSD 0.64 Cumulative %RSD 0.37

Table 23: Change in Temperature for Chlorocresol (+ 5°C)

Control (+5°C) Control (-5°C) 100.9 99.4 100.5 100.9 99.7 100.0 100.2 100.5

100.3 100.7 100.0 100.8 Cumulative Mean 100.2 Cumulative Mean 100.5

Cumulative SD 0.579 Cumulative SD 0.343 Cumulative %RSD 0.58 Cumulative %RSD 0.34

H. Change in Flow rate (+ 10%)

Table 24: Change in Flow rate for Betamethasone (+ 10 %)

Control (+10%) (-10%) 100.8 101.2 101.2 100.9 101.3 101.2 100.8 100.5 100.8

Cumulative Mean 100.9 101.0 Cumulative SD 0.293 0.197

Cumulative %RSD 0.29 0.20

Table 25: Change in Flow rate for Chlorocresol (+ 10 %)

Control (+10%) (-10%) 100.5 100.8 100.6 100.2 100.7 99.7 100.0 100.4 99.0



I. Change in wavelength (+ 5nm)

Table 26: Change in wavelength for Betamethasone (+ 5 nm)

Control (+ 5nm) (-5nm) 100.7 100.2 100.5 100.3 99.4 99.5 101.1 100.7 100.5



Table 27: Change in wavelength for Chlorocresol (+ 5 nm)

Control (+ 5nm) (-5nm) 99.9 100.0 100.2 98.9 98.8 99.4 100.2 99.7 100.6




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Acceptance criteria: System suitability should meet as per the test method at each variable condition. Overall RSD should not be more than 2.0% for the results obtained at control and variable conditions.

J. System suitability %RSD of five replicate injections, Retention time, USP Tailing and USP Tangent for Chlorocresol and Betamethasone dipropionate in Standard solution were maintained as per method on every day.

Table 28: System Suitability for Betamethasone

%RSD of Standard preparation Retention Time USP Tailing USP Tangent

Specificity 0.54 20.288 1.01 15834 Forced Degradation 0.84 20.318 1.05 11061

Method precision 0.38 20.595 1.04 15571 Ruggedness 0.54 21.524 0.98 11715

Solution stability 0.49 20.621 1.05 14643 Robustness

Control 0.54 20.288 1.01 15834 Flow +10% 0.21 18.678 1.18 13475 Flow -10% 0.16 22.309 1.23 13251

Wavelength +5nm 0.07 20.288 1.01 15750 Wavelength -5nm 0.64 20.287 1.01 15862

Temp +5°C 1.40 18.419 1.11 10292 Temp -5°C 0.20 22.670 1.20 14149

Table 29: System Suitability for Chlorocresol

%RSD of Standard preparation Retention Time USP Tailing USP Tangent

Specificity 0.79 8.497 1.11 10030 Forced Degradation 1.82 8.461 1.11 7822

Method precision 0.06 8.562 1.10 9995 Ruggedness 0.30 8.954 1.06 9241

Solution stability 1.61 8.711 1.12 9829 Robustness

Control 0.79 8.497 1.11 10030 Flow +10% 0.13 7.797 1.22 8514 Flow -10% 0.12 9.396 1.19 8389

Wavelength +5nm 0.41 8.499 1.16 9529 Wavelength -5nm 0.36 8.497 1.12 10026

Temp +5°C 1.82 7.859 1.18 7176 Temp -5°C 0.23 9.319 1.22 9030

Acceptance Criteria: RSD should not be more than 2.0%. USP tangent should not be less than 5000. USP Tailing should not be more than 2.0. CONCLUSION The test method is validated for Specificity, Linearity and Range, Precision, Accuracy (Recovery), Ruggedness, Stability of Analytical solution and Robustness and found to be meeting the predetermined acceptance criteria. The validated method is Specific, Linear, Precise, Accurate, Rugged and Robust for Assay of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream. Hence this method can be introduced into routine use for the assay of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream. ACKNOWLEDGMENT The author is thankful to Glenmark Research Centre Mhape, Mumbai for providing all the necessary facility for completing the research work. REFERENCES 1. Becket AH, Stenlake JB, “Practical Pharmaceutical

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Cite this article as: Bendre S.D., Ghule P.J. Analytical method development, validation, and assay of Betametasone dipropionate cream by HPLC method. Int. Res. J. Pharm. 2016;7(12):74-83 http://dx.doi.org/10.7897/2230-8407.0712151

Source of support: Nil, Conflict of interest: None Declared

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