Swami Vivekananda Pharmacy College

1. A NEW ANALYTICAL DEVELOPMENT AND
VALIDATION OF A HPLC METHOD FOR
ADAPALENE AND BENZOYL PEROXIDE IN BULK
AND PHARMACEUTICAL DOSAGE FORMS
BY
SWAMI VIVEKANANDA INSTITUTE OF PHARMACEUTICAL SCIENCES,
VANGAPALLY, YADADRI BHUVANAGIRI, TELANGANA.
1

2. CONTENTS
 INTRODUCTION
 LITERATURE SURVEY
 AIM AND OBJECTIVE
 DRUG PROFILE
 EXPERIMENTAL WAORK
 RESULTS
 SUMMURY & CONCLUSION
 BIBLIOGRAPHY
2

3. INTRODUCTION
• 1.1 Analytical Chemistry
• Analytical chemistry is a scientific discipline used to study the chemical composition,
structure and behaviour of matter. The purposes of chemical analysis are together and
interpret chemical information that will be of value to society in a wide range of contexts.
• Qualitative analysis is the identification of elements, species and or compounds present in
sample.
• Quantitative analysis is the determination of the absolute or relative amounts of elements,
species or compounds present in sample.
• 1.2 Chromatography
• 1.2.1 Introduction
• The chromatography was discovered by Russian Chemist and botanist
Micheal Tswett (1872-1919) who first used the term chromatography (colour writing
derived from Greek for colour – Chroma , and write – graphein) to describe his work on the
separation of coloured plant pigments into bands on a column of chalk and other material
such as polysaccharides, sucrose and insulin.
• “] Chromatography is a method in which the components of a mixture are separated on an
adsorbent column in a flowing system".
3

4. LITERATURE SURVEY
• .Yi-Cheng Chen et al ., The aim of this study was to develop a simple and reliable
high-performance chromatographic (HPLC) method for simultaneous analysis of
adapalene and benzoyl peroxide in pharmaceutical formulation by response surface
methodology (RSM). An optimized mobile phase composed of acetonitrile,
tetrahydrofuran and water containing 0.1% acetic acid at a ratio of 25:50:25 by
volume was successfully predicted by using RSM.
• Md. Raihan Chowdhury et al .,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
• Laura A. Martins et al .,A simple stability indicating high-performance liquid
chromatography method for the analysis of adapalene in pharmaceutical gel
formulation is developed and validated. An isocratic separation is performed using a
Merck RP-8 (150 mm × 4.6 mm i.d., particle size 5 m) column and a mixture of
acetonitrile–water (67:33, v/v, pH adjusted to 2.5 with phosphoric acid) as the
mobile phase.
4

5. • S.S.DEO et al., A simple, reliable and reproducible method applicable for
topical creams is developed to monitor the in vitro performance of adapalene
0.1% cream formulations using manual diffusion cell with suitable synthetic
membrane and receptor medium. The membranes which have permeability
similar to skin and receptor media that can maintain the sink conditions have
been selected. For solid oral dosage forms such as tablets, extensive study and
analytical methods were developed to evaluate the in vitro performance of
the drug.
• Rohit. H. Khatri et al ., This paper describes a validated high-performance
liquid chromatographic (HPLC) method for simultaneous estimation of
clindamycin (CLI) and adapalene (ADA) in pure powder and gel formulation.
The HPLC separation was achieved on a Luna C18 (2) (250 × 4.6 mm i.d., 5 µm
particle size) using acetonitrile : phosphate buffer pH 3.0 (60:40, v/v) as a
mobile phase delivered at a flow rate of 1.0 ml/min.
5

6. AIM AND OBJECTIVE
•AIM:
• The literature review reveals the few HPLC methods for the
estimation of adapalene and benzoyl peroxidealone and in
combination with other drugs. Few methods are also reported for
estimation of both drugs from formulation .we intend to develop a
Stability indicating RP-HPLC method by simultaneous determination
with simple, rapid, greater sensitivity and faster elution.
•OBJECTIVE:
• Development of a HPLC method for analysis of both the drugs.
• Validation of the method using formulations.
6

7. PLAN OF WORK
• PLAN OF WORK:
• To develop a new analytical method for the simultaneous estimation
of Adapalene and Benzoyl peroxide by RP-HPLC.
• The dissertation work has been carried out in the following steps.
SELECTION OF DRUGS
LITERATURE REVIEW
SELECTION OF DRUGS
ANALYTICAL METHOD DEVELOPMENT
ANALYTICAL METHOD VALIDATION
SELECTION OF λmax
SELECTION OF COLUMN
SELECTION OF MOBILE PHASE
SELECTION OF FLOW RATE
OPTIMIZED CHROMATOGRAPHIC
CONDITION
7

8. DRUG PROFILE
ADAPALENE:
IUPAC Name : 6-[3-(1-adamantyl)-4-methoxy-phenyl] naphthalene-2-carboxylic acid
Chemical formula : C28H28O3
Molecular weight : 412.52 g/mo
Solubility : Sparingly soluble in water , completly soluble in methanol
Category : Topical retinoid
Generic Name : ADAPALENE
Brand Name : Differin gel
8

9. Chemical Data
IUPAC Name : 1-carbamimidamido-N,N-
dimethylmethanimidamide
Chemical formula : C14H10O4
Molecular weight : 242.23 g·mol−1
• BENZOYL PEROXIDE
9

10. MECHANISM OF ACTION
• ADAPALENE:
• Unlike tretinoin,adapalene inhibits keratinocyte differentiation.
• This inhibition of keratinocyte differentiation and proliferation is responsible
for adapalene’s comedolytic effect.
• It has both exfoliating and anti-inflammatory effects. In an in vivo study,
adapalene’s ability to reduce comedo formation was demonstrated by a 50–
60% reduction in comedo counts compared with vehicle.
• BENZOYL PEROXIDE:
• Benzoyl peroxide (BPO) is effective for reducing the number and severity
of acne lesions.
10

11. • Benzoyl peroxide has a bactericidal effect on Propionibacterium acnes bacteria
associated with acne and does not induce antibiotic resistance.
• It may be combined with salicylic
acid, sulfur, erythromycin or clindamycin (antibiotics),or adapalene (a
synthetic retinoid).
• Two common combination drugsinclude benzoyl
peroxide/clindamycin and adapalene/benzoyl peroxide, an unusual
formulation considering most retinoids are deactivated by peroxides.
• Benzoyl peroxide for acne treatment is typically applied to the affected areas
in gel or cream form, in concentrations of 2.5% increasing through 5.0%, and
up to 10%. No strong evidence supports the idea that higher concentrations of
benzoyl peroxide are more effective than lower concentrations.
• Irritation can also be reduced by avoiding harsh facial cleansers and
wearing sunscreen prior to sun exposure.
11

12. EXPERIMENTAL WORK
MATERIALS & METHODS
Equipment / Instrument details
Chemicals and Reagents
Environmental health and safety
ANALYTICAL METHOD DEVELOPMENT
Selection of wavelength
Selection of Chromatographic condition
Intial separation
Preparation of standard solution
Preparation of sample solution
Preparation of placebo
12

13. TRIALS
 TRIAL 1:
 Method development for the drugs was initiated based on the
individual chemical characteristics’ and their methods given in
individual journals.
 Mobile phase: Methanol: Phosphate buffer PH3 (70:30)
 Diluent: Methanol
 Chromatographic conditions:
 Flow rate : 0.8ml/min
 Column : Symmetry C18 (4.6 x 150mm, 5m)
 Detector wavelength : 280 nm
 Column oven : Ambient
 Injection volume: 10l
13

14. TRAIL 2:
In order to improve resolution and remove fronting of the peak and avoid
unwanted peaks interfering, mobile phase was changed and again the
same experiment was performed.
Mobile phase: Methanol: Sodium acetate PH 4 (60:40)
Diluent: methanol
Chromatographic conditions:
Flow rate : 0.8ml per min
Column : Symmetry C18 (4.6 x 150mm, 5m)
Detector wavelength : 280 nm
Column oven : Ambient
Injection volume : 10l
14

15. • TRIAL-3 & 4
• In order to avoid poor response and tailing mobile phase was changed i.e
organic phase was changed
• Mobile phase: Methanol: Ammonium acetate PH3 (70:30)
• Diluent: methanol.
• Chromatographic conditions:
• Flow rate : 0.8 ml /min
• Column : Symmetry C18 (4.6 x 150mm, 5m)
• Detector wavelength : 280 nm
• Column oven : Ambient
• Injection volume : 20l.
15

16. OPTIMIZED METHOD
• Preparation of mobile phase:
• Take 6.8 gm of KH2PO4 into 1000ml volumetric flask
• Dissolved in HPLC grated water and adjust the PH up to 3 with ortho
phosphoric acid
• Test procedure:
• 20 µl of the Standard, Sample and Blank preparations in duplicate
were injected separately into HPLC system and the peak responses for
Adapalene and Benzoyl peroxide were measured.
• The developed RP-HPLC method for the simultaneous estimation of
Adapalene and Benzoyl peroxide were carried out On symmetry C18
(4.6 x 250mm), 5m column in gradient mode using mobile phase
composition of Methanol: Phosphate buffer PH 3 [70 : 30, v / v] with
flow rate of 0.8 ml / min at 280nm.
16

17. METHOD VALIDATION
The objective of validation of an analytical procedure is to demonstrate that
it is suitable for its intended purpose. According to ICH guidelines, typical
analytical performance characteristics that should be considered in the
validation of the type of methods are
System suitability
Linearity
Precision
Accuracy
Specificity
Ruggedness
Robustness
Limit of detection
Limit of quantification
17

18. System suitability
• Sample solution of Adapalene and Benzoyl peroxide were injected three
times into HPLC system as per test procedure. The system suitability
parameters were evaluated from standard Chromatograms obtained, by
calculating the % RSD of retention times, tailing factor, theoretical plates
and peak areas from three replicate injections.
• Acceptance criteria
• The % RSD for the retention times of principal peak from 3 replicate
injections of each Standard solution should be not more than 2.0 %
• The number of theoretical plates (N) for the Adapalene and Benzoyl
peroxide peaks should be not less than 2000.
• The Tailing factor (T) for the Adapalene and Benzoyl peroxide peaks should
be not more than 2.0.
• From the system suitability studies it was observed that all the parameters
were within limit. Hence it was concluded that the Instrument, Reagents and
Column were suitable to perform the Assay.
18

19. Tailing factor Obtained from the standard injection is 1.6
Theoretical Plates Obtained from the standard injection is 2496
• ADAPALENE
Injection Rt Peak Area
USP
Plate count
USP
Tailing
1 2.799 1225763 2887 1.72
2 2.799 1274867 2889 1.78
3 2.813 1259849 2896 1.74
Mean 1278360
SD 3890.679
% RSD 0.257
19

20. BENZOYL PERIOXIDE
Injection Rt Peak Area
USP
Plate count
USP
Tailing
USP
Resolution
1 3.861 950747 2281 1.51 3.04
2 3.863 932591 2244 1.47 3.09
3 3.886 948856 2261 1.47 3.05
Mean 937329
SD 5974.93
% RSD 0.473
Tailing factor Obtained from the standard injection is 1.51
Theoretical Plates Obtained from the standard injection is 2281 20

21. RESULTS
•From the above chromatogram it was
observed that the adapalene and benzoyl
peroxide peak was splitted
•Chromatogram of Trial 1
From the above chromatogram it was observed that
the adapalene and benzoyl peroxide peaks are
splitted
Chromatogram of Trial 2

22. From the above chromatogram it was observed
that there are no interferences
Chromatogram of blank
From the above chromatogram it was observed
that there are no interferences
Chromatogram for Placebo

23. METHOD VALIDATION
System suitability:
Chromtograms for system suitability

24. LINEARITY
Chromatogram for Linearity level 1 Chromatogram for Linearity level 2
Chromatogram for Linearity level 3 Chromatogram for Linearity level 4

25. S.No
Linearity
Level
Concentratio
n
Area
1 I 20 ppm 839286
2 II 40 ppm 1067774
3 III 60 ppm 1246474
4 IV 80 ppm 1439994
5 V 100 ppm 1639065
Correlation Coefficient 0.999
Linearity results for Adapalene
S.No
Linearity
Level
Concentration Area
1 I 10 ppm 626221
2 II 15 ppm 778750
3 III 20 ppm 931447
4 IV 25 ppm 1070162
5 V 30 ppm 1196060
Correlation Coefficient 0.999
Linearity results for Benzoyl peroxide
Calibration curve of Adapalene
Calibration curve of Benzoyl peroxide

26. PRECISION
Sample Chromatograms for Repeatability

27. ACCURACY
Standard Chromatogram for Accuracy Chromatograms for Accuray 50%
Chromatograms for Accuracy 100% Chromatograms for Accuray 150%

28. LIMIT OF DETECTION (LOD)
Chromatogram for Adapalene and Benzoyl peroxide
LIMIT OF QUANTIFICATION (LOQ)
Chromatogram For Adapalene and Benzoyl peroxide

29. SUMMARY AND CONCLUSION
• The proposed HPLC method was found to be simple, specific, precise,
accurate, rapid and economical for simultaneous estimation of
Adapalene and Benzyol peroxide in tablet dosage form.
• The Sample recoveries in all formulations were in good agreement
with their respective label claims.
• From literature review and solubility analysis initial chromatographic
conditions Mobile phase ortho phosphoric acid buffer:Methanol
65:35 were set (Buffer PH 3 adjusted with opa ), symmetry C 18
(250×4.6mm, 5µ) Column, Flow rate 0.8 ml/min and temperature was
ambient, eluent was scanned with PDA detector in system and it
showed maximum absorbance at 280 nm.
29

30. As the methanol content was increased Adapalene and Benzyol peroxide
got eluted with good peak symmetric properties. The retention times for
Adapalene and Benzyol peroxide was found to be 2.972 min and 3.548 min
respectively.
 System suitability parameters were studied by injecting the standard five
times and results were well under the acceptance criteria.
Linearity study was carried out between 50% to150 % levels, R2 value was
found to be as 0.999.
By using above method assay of marketed formulation was carried out,
100.8% was present.
Full length method was not performed; if it is done this method can be
used for routine analysis of Adapalene and Benzyol peroxide.
30

31. BIBLIOGRAPHY
Yi-Cheng Chen, Pi-Ju Tsai, Yaw-Bin Huang, and Pao-Chu Wu, Optimization and
Validation of High-Performance Chromatographic Condition for Simultaneous
Determination of Adapalene and Benzoyl Peroxide by Response Surface
Methodology Journal List v.10(3); 2015
Md. Raihan Chowdhury , Mahedy Hasan Chowdhury, Partha Saha, Md. Hafizur
Rahman, Md. Fuadh-AlKabir, S.M. Estiar Haque , Development and validation of
RP-HPLC method for the simultaneous determination of Adapalene-Benzoyl
Peroxide combination gel IJASETR Research Paper ISSN: 1839-7239 , October –
2012 Volume – 1, Issue – 5
Laura A. Martins, Leonardo Z. Meneghini, César A. Junqueira, Danieli C. Ceni, and
Ana M. Bergold A Simple HPLC-DAD Method for Determination of Adapalene in
Topical Gel Formulation , Journal of Chromatographic Science, Vol. 49,
November/December 2011
31

32. S.S.DEO, F. INAM and N. P. KARMARKAR , Analytical Method Development for
Determination of Performance of Adapalene in Adapalene 0.1% Gel Formulation Using
Manual Diffusion Cell Chem Sci Trans., 2013, 2(1), pp 251-257
Rohit. H. Khatri , Rashmin. B. Patel and Mrunali R. Patel A new RP-HPLC method for
estimation of clindamycin and adapalene in gel formulation: development and validation
consideration The Thai Journal of Pharmaceutical Sciences 38 (1), January - March 2014: 1-
56
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