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www.setscholars.org October – 2012 IJASETR

Volume – 1, Issue – 5 Research Paper
Knowledge is Power ISSN: 1839-7239
Article #01

Development and validation of RP-HPLC method for the simultaneous

determination of Adapalene-Benzoyl Peroxide combination gel
Md. Raihan Chowdhury1*, Mahedy Hasan Chowdhury2, Partha Saha1, Md. Hafizur Rahman1, Md. Fuadh-Al-
Kabir1, S.M. Estiar Haque1
1ACI Pharmaceuticals Limited, Narayanganj-1400, Bangladesh.
2Sunbeams School, Plot# 6-12, Road#13/A, Sector#10, Uttara, Dhaka, Bangladesh.
E-mail: mcraihan@gmail.com

Abstract

A reliable and sensitive RP-HPLC method has been developed and validated for simultaneous assay of
Adapalene & Benzoyl peroxide. An isocratic separation of Adapalene & Benzoyl peroxide was achieved on C18,
250Χ4.6 mm i.d., 5 μm particle size columns with a flow rate of 1.0 ml/min and using a UV detector to monitor
the elute at 270 nm. The mobile phase consisted of a mixture of water, acetonitrile, tetrahydrofuran and
trifluroacetic acid at a ratio of 29:33:38:0.2 by volume. Application of the suggested procedures laid on ICH
,
guidlines was successfully applied to the determination of these compounds in active pharmaceutical
ingredients and in pharmaceutical preparations, with high percentage of recovery, good accuracy and
precision. Response was a linear function of drug concentration in the range of (0.79-1.20) mg/mL range with
an R2 of 0.999 for Benzoyl peroxide & (39.68-59.90) µg/mL range with an R2 of 0.999 for adapalene, accuracy
with percent RSD of 100.65±0.23 (Benzoic peroxide) & 100.48±0.45 (Adapalene) and with a limit of detection
and quantification of 24.74PPM, 92.53 PPM and 0.032 PPM, 0.126 PPM for Benzoyl peroxide & Adapalene
respectively. No major (Not more than 10%) degradation products was obtained from the stress studies which
indicate the validity of the method for the analysis of Benzoyl Peroxide and Adapalene combination gel in the
pharmaceutical formulations.

Keywords: Adapalene, Benzoyl Peroxide, Gel, RP-HPLC.

Citation: Chowdhury R. et al. (2012), Development and validation of RP-HPLC method for the simultaneous
determination of Adapalene-Benzoyl Peroxide combination gel. IJASETR 1(5): p. 1 - 9.

Copyright: @ 2012 Chowdhury R. et al. This is an open access article distributed under the terms of the
Creative Common Attribution 3.0 License.

Introduction

Adapalene is a synthetic topical retinoid and a naptholic acid derivative.[1, 2] The chemical name for Adapalene is (6-[3-
(1-adamantyl)-4-methoxyphenyl]-2- naphthoic acid). The structure formula for this drug is following:

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 Figure 1: Adapalene, Molecular formula: C28H28O3 Molecular weight: 412.5

Adapalene is used to treat mild to moderate acne. Benzoyl Peroxide is one type of Lipohilic oxidizing agent used as
antimicrobial, exfoliative and keratolytic drug.[1] The chemical name of Benzoyl Peroxide is dibenzoyl peroxide. The
structure formula for this drug is following:

Figure 2: Benzoyl Peroxide, Molecular formula: C14H10O4 Molecular weight: 242.23

The combination of Adapalene and Benzoyl Peroxide gel provided a greater net benefit in patients with all grades of acne
than the individual constituents given separately.[3] The mechanism of synergistic action is not clear. The
pharmacokinetics studies[4] are available for the Adapalene-Benzoyl Peroxide combination gel and various Chemical
stability of Adapalene combined with Benzoyl are experimented[5] but the method validation study was not developed for
this combination pharmaceutical dosage formulation. The purpose of this study was to validate a simultaneous estimation
method by reverse-phase HPLC for the quantitative analysis of Adapalene and Benzoyl Peroxide according to the
guideline of ICH and USP.[6, 7]

Materials and Methods

Adapalene API & Benzoyl Peroxide API were obtained as a gift sample from ACI Pharmaceuticals Ltd, Narayanganj,
Bangladesh, having purities of 98.99% and 70.00% respectively. Each gram of Gel contains 1.25 mg of Adapalene & 25
mg of Benzyl Peroxide as active ingredient. Acetonitrile (RCI Lab Scan), Tetrahydrofuran (RCI Lab Scan),
Trifluoroacetic acid (Scharlau) & Water (Milli-Q) used were of HPLC grade to prepare the mobile phase & diluents.

Instrument and Chromatographic condition

Chromatographic separation was performed by set up of High Performance Liquid Chromatography (HPLC) system, LC
2010 HT, (Shimazdu) equipped with LC -20AT pump, two analytical columns, variable wavelength programmable
UV/VIS detector, SPD-20A, CTO-10ASVP column oven and universal injector (Rheodyne). A Waters NovaPak C 18
◦
column [250 x 4.6 mm, 5 µm bead size] was used as column 1 and maintained at 37 C temperature in a thermostated
column compartment and Hichrom, C18 [250 x 4.6 mm, 5 µm bead size] was used as column 2. The separation was
performed by isocratic elution with a mixture of water, acetonitrile, Tetrahydrofuran and Trifluroacetic acid (29:33:38:0.2

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by volumn) at a flow rate of 1.0 ml/min & detector wavelength of 270 nm. The injection volumn was 20 µL and the run
time was 14 min for each injection.

Preparation of mobile phase & diluting solution

A Mixture of water, acetonitrile(ACN), Tetrahydrofuran(THF) and Trifluroacetic(TFA) acid at a volume ratio of
29:33:38:0.2 was prepared and the resulting solution was sonicated for 5 min using ultrasonic bath and finally the mixture
was filtered using 0.2 µm nylon membrane filters. The obtained solution was used as mobile phase and diluting solution.

Preparation of standard solution

Stock Standard Solution
50 mg of Adapalene working standard (WS) was accurately weighed and was transferred into a clean and dry 100-mL
volumetric flask. Then it was dissolved by adding 5 mL of THF and diluted to volumn with diluting solution.

Analytical Standard Solution (Nominal concentration)

50 mg of Benzoyl Peroxide WS was accurately weighed and was transferred into a clean and dry 50-mL volumetric flask.
5 mL of stock standard solution was added into the volumetric flask and finally add mobile phase q.s to 50 mL. It was
filtered through a filter having a nominal pore size not greater than 0.45 µm, discarding the first 10 mL of filtrate.

Results
Chromatographic separation and detection
System suitability
System suitability of the analytical method was performed by estimating the repeatability, theoretical plates, tailing factor
and retention time of six replicate injections of standard solution at nominal concentration. The % RSD was calculated in
each cases (Table 1) which indicate the good performance of the system.
Table 1: Chromatographic Characteristics of System Suitbility of Study

Parameters Value (Mean ± % RSD)*

Benzoyl Peroxide Adapalene
Peak Area 11235787±0.070 3760243± 0.33
Tailing Factors 1.187±0.043 0.973± 0.053
Retention Time 4.346±0.031 10.066±0.039
Theoretical Plates 2872±1.016 3943±0.671
*Mean and % Relative Standard Deviation of six replicates.

Linearity and Range

The linearity of the analytical method was evaluated by linear regression analysis obtaining a calibration curve by plotting
the response (Peak Area) versus a series of concentration, 80%,90%,100%(nominal concentration),110%,& 120% of

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analytical standard solution. Significant regression correlation coefficient (R2) value demonstrate the linearity of the
method (Table 2).
Table 2: Parameter of regression analysis

Parameters Benzoyl Peroxide Adapalene

Linearity range (0.79-1.20)mg/ml (39.68-59.90)µg/ml
Correlation coefficient (r2) 0.999 0.999
Slop 31262 42374
% Y-Intercept 0.278 1.126

Specificity
Placebo interference and degradation studies were carried out to investigate the specificity of the active analytes
(Adapalene & Benzoylperoxide) in pharmaceutical preparation (Adaben Duo Gel). The chromatogram of the blank,
placebo, standard (nominal concentration) and test sample (nominal concentration) justify the specificity of the analytical
method (Fig. 3) since no placebo interference (extra peak for excipients) was observed.

Figure 3: Comparative chromatograms for specificity indication.

Test sample was treated under acid hydrolysis, alkali hydrolysis, oxidation, reduction and elevated temperature (80 ◦C) and
finally compared with freshly prepared sample to evaluate the degradation study. No detectable degradation peak was
observed in the chromatogram of treated test samples (Table3).

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 Table 3: Results showing different degradative outcome

Parameters Label Claim (mg/gm) Assay at Force Condn Degradation (%)

(mg/gm)**
Adapalene Benzoyl* Adapalene Benzoyl* Adapalene Benzoyl*
1.25 25.0
◦
Heat (60 C) 1.21 21.72 3.2 13.12
Acidic 1.17 23.24 6.4 7.04
Alkaline 1.19 22.10 4.8 11.6
Oxidation 1.23 21.23 1.6 15.08
Reduction 1.16 23.14 7.2 7.44
Purified H2O 1.24 24.82 0.8 0.72
Benzoyl* =Benzoyl peroxide

**Mean of three replicates.

Accuracy
Accuracy was carried out for the drug matrix solution that was perform by the recovery test which was consisted of
adding known amounts of active ingredients (Adapalene & Benzoylperoxide) into the placebo sample solution. The test
was conducted by three different concentrations (80%, 100% and 120%) of the solution in three replicate sample
preparation and percent recoveries (mean±%RSD) of the drug matrix solutions indicate the excellent accuracy of the
analytical method (Table4).

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 Table 4: Accuracy studies of Benzoylperoxide & Adapalene in drug-matrix solution

Amount Recovery
Amount added % Overall
Peak Area recovered (Mean ±
(mg of API*) Recovery (Mean±%RSD)
(mg) %RSD)
54.8 8805134 55.48 101.25
54.8 8703222 54.84 100.08 100.85±0.66
54.8 8804321 55.48 101.24
Benzoyl peroxide

72.2 11500225 72.47 100.37

72.2 11617261 73.20 101.39 100.71±0.58 100.65±0.23
72.2 11498926 72.46 100.36
83.8 13421478 84.57 100.92
83.8 13209964 83.24 99.33 100.39±0.91
83.8 13419824 84.56 100.91
39.6 2942174 39.80 100.51
39.6 2920557 39.51 99.77 100.25±0.41
39.6 2941337 39.79 100.48
49.2 3634581 49.17 99.93
Adapalene

49.2 3647080 49.34 100.28 100.19±0.22 100.48±0.45

49.2 3650129 49.38 100.36
59.5 4430339 59.93 100.73
59.5 4469968 60.47 101.63 101.01±0.53
59.5 4427645 59.90 100.67

Precision
Precision of the analytical method was studied by analysis of three replicate of standard solution in three different
concentrations (80%, 100% & 120%). It was demonstrated by repeatability (intra-day precision) and intermediate
precision (inter-day precision) of the solutions. Table5 shows the obtained results in both cases (intra-day precision &
inter-day precision) indicating the performance of the analytical method.

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 Table 5: Intra-day & inter-day precision of HPLC method

Sample Peak Area Peak Area Peak Area Peak Area Over all
Concentration (Day 1) (Mean±%RSD) (Day 2) (Mean±%RSD) (Mean±%RSD)
(%) Day1 & Day2
8841376 8838152
80 8810387 8836221±0.27 8910346 8891542±0.53 8863882±0.51
8856901 8926129
Benzoy lperoxide

11502518 11495672
100 11523787 11524655±0.20 11482763 11493962±0.09 11509309±0.20
11547659 11503452
13478715 13128974
120 13510123 13490541±0.13 13013432 13118550±0.76 13304546±01.61
13482785 13213245
2916763 2934532
80 2931587 2946037±0.27 2915134 2933685±0.62 2939861±0.95
2989761 2951389
3598781 3678563
Adapalene

100 3609125 3599224±0.27 3600259 3630578±1.16 3614901±0.89

3589765 3612912
4512997 4401776
120 4498715 4504363±0.17 4561991 4497880±1.88 4501121±1,20
4501376 4529873

Sensitivity
Both limit of detection (LOD) & limit of quantitation (LOQ) were estimated by determining of signal to noise ratio. The
obtained LOD & LOQ for Adapalene were 0.032 µg/ml & 0.126 µg/ml as well as for Benzoylperoxide were 24.74 µg/ml
& 92.53 µg/ml respectively.

Ruggedness & Robustness

Ruggedness of the current method was evaluated by analysis six assay sample solution of Adaben Duo Gel (contains
combined 25 mg Adapalene & 0.5mg Benzoylperoxide) by different instrument, column and analyst.
Table6 shows the results obtained from robustness study which was determined by doing small changes or variations in
the method’s parameter such as flow rate, column temperature, wavelength and mobile phase composition.

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 Table 6: Robustness of method

Actual
Parameters Retention Time Peak Area % Deviation
Variance
Benzoic* Adapalene Benzoic* Adapalene Benzoic* Adapalene
Unaffected
Normal 4.346 10.066 11369483 3819212 NA NA
method
0.98ml/min 4.703 11.214 12049817 3915932 -5.98 -2.53
Flow Rate
1.02ml/min 4.012 9.761 10892512 3722516 4.20 2.53
Elution Solvent 31 4.778 11.342 11419924 3956542 -0.44 -3.60
ratios 35 3.927 9.568 10728761 3658916 5.64 4.20
Detector 268nm 4.401 10.113 10925875 3800984 3.90 0.48
Wavelength
272nm 4.382 10.253 11671269 3799829 -2.65 0.51
(λ max)
35◦C 4.569 10.981 11453282 3900156 -0.74 -2.12
Temperature
37◦C 4.201 9.423 11217786 3581762 1.33 6.22

Conclusions
The study of RP-HPLC method validation showed that the System Suitability, Linearity and Range, Accuracy, Precision
(Repeatability, Intermediate), Limit of Quantitation and Specificity were within the required range for the simultaneous
assay of Adapalene- Benzoyl Peroxide combination Gel. This simple method is proposed for the use of sample analysis.

Acknowledgement:
The entire program was supported by ACI Pharmaceuticals Ltd., Narayanganj, Bangladesh, by providing their
laboratories & suggestions.

References

1. JK., T., Adapalene 0.1% and benzoyl peroxide 2.5%: a novel combination for treatment of acne vulgaris. Skin Therapy Lett.,
2009. 14(6): p. 4-5.
2. Gollnick H, C.W., Berson D, Dreno B, Finlay A, Leyden JJ, Shalita AR, Thiboutot D, Management of acne: a report from a
Global Alliance to Improve Outcomes in Acne. J Am Acad Dermatol., 2003. 49(1 Suppl): p. S1-37
3. Gollnick H, C.W., Berson D, et al, Global Alliance to Improve Outcomes in Acne. Management of acne: a report from a
Global Alliance to Improve Outcomes in Acne. J Am Acad Dermatol., 2003. 49(suppl 1): p. S1-S37
4. Abi Adebowale, L.V. Clinical Pharmacology Review. 2008

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 5. Martin B, M.C., Montels D, Watts O., Chemical stability of adapalene and tretinoin when combined with benzoyl peroxide in
presence and in absence of visible light and ultraviolet radiation. Br J Dermatol., 1998. 139( Suppl 52): p. 8-11.
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