44 Khatri et al.

, 2014

Original Article

TJPS
The Thai Journal of Pharmaceutical Sciences
38 (1), January - March 2014: 1-56

A new RP-HPLC method for estimation of clindamycin

and adapalene in gel formulation: development and
validation consideration
Rohit. H. Khatri1 , Rashmin. B. Patel1 and Mrunali R. Patel2
1
Department of Quality Assurance, A. R. College of Pharmacy & G. H. Patel institute of Pharmacy, Vallabh Vidyanagar
388 120, Gujarat, India
2
Department of Pharmaceutics and Pharmaceutical Technology, Indukaka Ipcowala College of Pharmacy, New Vallabh
Vidyanagar – 388 121, Gujarat, India

Abstract
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. The calibration curve showed good linear relationship with
r2 = 0.9997 for clindamycin and r2 = 0.9994 for adapalene in concentration range of 100-500 µg/ml and 10-50 µg/ml,
respectively. LOD and LOQ were found to be 5.39 and 16.32 µg/ml for clindamycin and 0.89 and 2.72 µg/ml for
adapalene, respectively. Assays of clindamycin found to 99.76 ± 0.65 % and adapalene found to 99.58 ± 0.73 %.
Clindamycin undergoes thermal degradation but adapalene was found stable in this condition. The method was validated
as per ICH guideline (Q2 R1). The method was successfully applied for routine analysis of clindamycin and adapalene in
pure powder and gel formulation.

Key Words: Adapalene, Clindamycin, Gel formulation, RP-HPLC, Validation.

Introduction Clindamycin inhibits bacterial protein synthesis by

Clindamycin (CLI), chemically ((2S,4R)-N-{2-chloro-
1- [ ( 2R , 3R , 4S , 5R , 6R ) – 3 , 4 , 5 – trihydroxy – 6 -
(methylsulfanyl) oxan – 2 – yl ] propyl } - 1 – methyl – 4 -
propylpyrrolidine-2 carboxamide) is a semisynthetic
lincosamide antibiotic that has largely replaced lincomycin
due to an improved side effect profile.
Correspondence to: Rashmin. B. Patel, Department of Quality
Assurance, A. R. College of Pharmacy & G. H. Patel institute of
Pharmacy, Vallabh Vidyanagar 388 120, Gujarat, India.
Tel.: 02692230788, e-mail: rbp.arcp@gmail.com
Academic Editor: Vipaporn Panapisal
binding to bacterial 50S ribosomal subunits. It may be
bacteriostatic or bactericidal depending on the organism
and drug concentration [1]. Adapalene (ADA), chemically
(6-[3-(adamantan-1-yl)-4-methoxyphenyl] naphthalene-2-
carboxylic acid) is a topical retinoid primarily used in the
treatment of acne and is also used (off-label) to treat
keratosis pilaris as well as other skin conditions. It is
currently marketed by Galderma under the trade names
Differin  in some countries, and Adaferin  in India [1-2].
Both CLI and ADA are official in pharmacopoeia.
Combination of CLI and ADA has been shown to be
effective in the management of acne [3]. A literature
survey revealed that high-performance liquid
chromatography (HPLC) for determination of CLI in

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Khatri et al., 2014
tablet [4], plasma [5, 6], topical formulation [7] and
capsule [8]. Further, the residual determination of CLI
along with tretinoin on surface of manufacturing
equipment using HPLC method was reported [9]. A few
HPLC methods for estimation of ADA in transdermal
formulation [10] and gel [11-14] were reported. In view of
the fact that no analytical method is available for the
quantitative analysis of ADA and CLI in combined gel
formulation, the authors were motivated to develop a new
HPLC method for the estimation of ADA and CLI in pure
powder and combined gel formulation.
The present study describes the development and
validation of a RP-HPLC method for the simultaneous
quantitative estimation of ADA and CLI in pure powder
and a marketed gel formulation. The developed method
was successfully applied for the routine analysis of ADA
and CLI in the gel formulation.
Materials and Methods
Materials. The pure CLI and ADA were obtained from
Apex laboratory (Hyderabad, India). HPLC grade
acetonitrile, water, tetrahydrafuran and disodium hydrogen
phosphate were purchased from E. Merck Ltd. (Mumbai,
India).
Apparatus. The method was developed using a Shimadzu
LC-2010HT instrument equipped with SPD 20A detector,
isocratic pump system, auto injector, and Luna C-18(2)
(phenomenex) column (250 × 4.6 mm id, 5µ particle size).
Chromatographic condition. The mobile phase consisted
of acetonitrile and phasphate buffer pH 3.0 (60:40, v/v)
was pumped at a flow rate of 1.0 ml/min. The mobile
phase was filtered through a 0.45 µm nylon membrane and
degassed before use. The elution was monitored at 210 nm,
and the sample injection loop volume was 20 µL. Standard
and sample solutions were filtered through a 0.45 μm
nylon membrane prior to HPLC injection.
Preparation of a clindamycin standard stock solution. A
100 mg of clindamycin was weighed and transferred to a
100 ml volumetric flask and dissolved with 75 ml mobile
phase. The flask was sonicated for 10 min and the final
volume was made up to the mark with mobile phase to
give 1000 µg/ml of CLI (Stock solution A). One ml of the
stock solution A was transferred into a 10 ml volumetric
flask and diluted upto the mark using mobile phase to give
100 µg/ml of CLI.
Preparation of an adapalene standard stock solution. A 10
mg of adapalene was weighed and transferred to a 100 ml
volumetric flask and dissolved in 30 ml of tetrahydrofuran.
The flask was sonicated for 10 min and the final volume
was made up to mark with mobile phase to give a solution
containing 100 µg/ml ADA (Stock solution B). One ml of
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45
the prepared solution was transferred into a 10 ml
volumetric flask and make up to volume with mobile phase
to obtain a final solution containing 10 µg/ml of ADA.
Preparation of mixed standard solutions. Series of
solutions were prepared by transferring and mixing 1 ml,
2 ml, 3 ml, 4 ml and 5 ml of stock solution A and stock
solution B, respectively, in a separate 10 ml volumetric
flask and volume was made up to the mark using mobile
phase to give mixed standard solutions.
Sample preparation for determination of CLI and ADA in
gel formulation. One gm of gel equivalent to 10 mg CLI
and 1 mg ADA was transferred to a 100 ml volumetric
flask and dissolved in 30 ml tetrahydrofuran. The flask
was sonicated for 10 min and the final volume was made
up to the mark with mobile phase to give a solution
containing 100 µg/ml of CLI and 10 µg/ml of ADA.
Method validation
Specificity: Placebo was prepared from commonly
used formulation excipients (i.e., propylene glycol-7.0 gm,
hydroxyethylcellulose-0.2 gm, ethanol-1.8 gm) and was
spiked into a preweighed quantity of drugs (100 mg CLI
and 10 mg ADA). One gm content (mixture of placebo and
drugs) equivalent to 10 mg CLI and 1 mg ADA was
transferred to a 100 ml volumetric flask and dissolved in
30 ml tetrahydrofuran. The flask was sonicated for 10 min
and the final volume was made up to the mark with mobile
phase (sonicated for further 5 mins) to give a solution
containing 100 µg/ml CLI and 10 µg/ml ADA.
Linearity: Calibration curves were constructed by
plotting peak area vs. concentrations of CLI and ADA and
regression equations were calculated. The calibration
curves were plotted over five different concentrations of
CLI (100, 200, 300, 400, and 500 µg/ml) and ADA (10, 20,
30, 40, and 50 µg/ml).
Precision: Precision (repeatability) of the instruments
was checked by repeatedly injecting (n = 6) the mixed
standard solution of CLI (200 µg/ml) and ADA (20 µg/ml)
for the HPLC method. The interday and intraday precisions
(reproducibility) of proposed methods were determined by
analyzing mixed standard solutions of CLI (100, 200, 300,
µg/ml) and ADA (10, 20, 30 µg/ml) at 3 different
concentrations.
Limit of detection (LOD) and limit of quantitation
(LOQ) : LOD and LOQ were calculated according to the
following equations: LOD= 3.3 × σ/S and LOQ = 10 × σ/S;
where σ the standard deviation of the response and the y -
intercept of the regression line; S = slope of the regression
line.
Accuracy: The accuracy of the method was
determined by calculating recovery of CLI and ADA by
the standard addition method. Known amounts of the
standard solutions of CLI (80, 100 and 120 μg/ml) and
ADA (8, 10 and 12 μg/ml) were added to the prequantified
TJPS 2014, 38 (1): 44-48
46 Khatri et al., 2014

Figure 1 A chromatogram of a standard solution containing 100 μg/ml of Clindamycine and 10 μg/ml of Adapalene.

sample solution (100 μg/ml CLI and 10 μg/ml ADA) of gel
dosage form. The amounts of CLI and ADA were
estimated by applying values of peak area to the regression
equations of each calibration curve, respectively.
Robustness: Robustness of the method was studied by
changing the flow rate, composition of organic phase and
pH of mobile phase, and column oven temperature using
the solution containing 100 µg/ml CLI and 10 µg/ml ADA
as the test solution.
Solution Stability: Stability of the sample solution
containing 100 µg/ml CLI and 10 µg/ml ADA was studied
at ambient temperature for 24 h.
System suitability test: The system suitability test was
carried out to evaluate the resolution and reproducibility of
the system for the analysis to be performed, using five
replicate injections of the mixed standard solution
containing 100 µg/ml CLI and 10 µg/ml ADA. The
measured parameters were retention time, theoretical
plates, tailing factor and peak purity.
Results and Discussion
HPLC method development and optimization. To obtain
the best chromatographic conditions, the mobile phase was
optimized to provide sufficient selectivity and sensitivity in
a short separation time. Phosphate buffer resulted in high
sensitivity compared with ammonium acetate buffer and
phosphoric acid solution. The use of acetonitrile resulted in
better sensitivity and short analysis time, improving the
peak symmetry. Luna C-18(2) analytical column was
evaluated and selected, as it provided the best
chromatographic performance and acceptable peak
characteristics, including tailing factor, number of
theoretical plates and acceptable resolution of CLI and
ADA, confirming the capability of the proposed method.
For selection of the best wavelength of detection, a PDA
detector was used.
A satisfactory separation with good peak symmetry and
steady baseline was achieved with Luna C-18(2) column

Table 1 Regression analysis of calibration graphs for clindamycine and adapalene for proposed HPLC method

Parameter CLI ADA

Concentration range 100-500 µg/mL 10-50 µg/mL
Slope 5488.99 21042.73
Residual SD 12198.96 7822.74
intercept 41379.56 203995.40
SD of the intercept 5721.82 3669.19
Correlation coefficient 0.9997 0.9994
LOD 5.39 µg/mL 0.89 µg/mL
LOQ 16.32 µg/mL 2.72 µg/mL

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Khatri et al., 2014 47

Table 2 Summary of validated parameters of the proposed RP-HPLC method for clindamycine and adapalene

Parameter CLI ADA

Accuracy, % 99.6-100.1 99.3-100.1

Repeatability(RSD, %, n=6) 0.054 0.109
Precision (RSD, %)
Interday (n=3) 0.19-0.82 0.29-0.69
Intraday (n=3) 0.21-0.25 0.31-0.77
Specificity Specific Specific
Solvent suitability Suitable Suitable

and acetonitrile-phosphate buffer pH 3.0 (60 : 40, v/v) as
mobile phase at flow rate of 1.0 ml/min. The quantitations
of CLI and ADA were achieved at 210 nm.
The optimized conditions of the HPLC method were
validated for the analysis of CLI and ADA in gel
formulations and application for QC. Figure 1 shows a
typical chromatogram obtained by the proposed HPLC
method, demonstrating the resolution of the symmetrical
peak corresponding to CLI and ADA. The retention times
were observed for CLI (3.03 ± 0.01 min) and ADA
(4.92 ± 0.01 min) which allows a fast determination of
both drugs, and is suitable for QC laboratories.
Method validation. In present study, the ability of the
method to separate the drug from each other and the non-
interference of the excipients indicates the specificity of
the method. Values of peak purity index were 0.9999 for
both drugs. These results indicated that the proposed
method is specific, and can be applied for stability studies
and QC analysis of CLI and ADA in pharmaceutical
products. The linearity data described in the present study
demonstrated acceptable linearity for CLI and ADA over
the range of 80 to 120 % of the target concentration,
respectively. Linear correlation was obtained between peak
areas and concentrations of CLI and ADA in the range of
100 - 500 µg/ml and 10 - 50 µg/ml, respectively. Data of
regression analysis were summarized in (Table 1). The
obtained RSD values from the precision study were
0.054 % for CLI and 0.109 % for ADA, respectively.
The RSD values for the repeatability study was found
to be <1 %, which could be concluded that the proposed
method is repeatable. The intermediate precision was
assessed by analyzing two gel formulations on 3 different
days (interday); the RSD values obtained for CLI and
ADA were 0.19 - 0.82 % and 0.29 - 0.69 %, respectively.
The between-analysts precision was determined by
calculating the RSD for the analysis of two samples of the
pharmaceutical formulation by three analysts; the values
were found to be 0.21 - 0.25 % for CLI and 0.31 - 0.77 %
for ADA, respectively. The RSD for intermediate precision
was found to be <2 %, which could be concluded that the
proposed method is reproducible. The recoveries from the
accuracy study were obtained in a range of 99.6 to 100.1 %
for CLI and 99.3 and 100.1 % for ADA (Table 2). Nearly
100 % recovery indicates that the proposed HPLC method
is accurate. The LOD and LOQ for CLI and ADA were
found to be 5.39 and 16.32 µg/ml and 0.89 and 2.72 µg/ml,
respectively (Table 1).
Results and the experimental range of the evaluated
variables in the robustness assessment are given in Table 3,
together with the optimized values. There were no
significant changes in the chromatographic pattern when
the modifications were made in the experimental
conditions, thus could be represented the robustness.
Solvent suitability study was carried out to establish the
stability of the sample in an analytical solution (diluent)
over a period of time during a routine analysis. The
stability of sample solutions was tested at intervals of 12
hours, 24 hours, and up to 48 hours. The method was found
to be rugged as there was no change in the areas of CLI
and ADA. Results of this solvent suitability study showed
non-significant change (< 2 %) relative to freshly prepared
samples. The RSD values calculated in the system
suitability test for the studied parameters were within the

Table 3 System suitability test parameters for clindamycine and adapalene for the proposed HPLC method

Parameter CLI ± %RSD(n=6) ADA ± %RSD(n=6)

Retention time (min.) 3.03 ± 0.01 4.92 ± 0.01
Tailing factor 1.62 ± 0.01 1.69 ± 0.02
Theoretical plate No. 3278 ± 0.32 2843 ± 0.20
Peak purity 0.9999 0.9999
Resolution - 4.90 ± 0.03

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48 Khatri et al., 2014

Table 4 Chromatographic conditions and range investigated during robustness study for proposed RP-HPLC method

Variables Range CLI % RSD % ADA % RSD % Optimized

Value
Flow rate (ml/min) 0.8 99.25 0.26 99.17 0.28 1.0
1.0 99.94 0.13 99.89 0.11
1.2 99.66 0.20 99.55 0.21
pH of Mobile phase 2.8 99.67 0.29 99.39 0.38 3.0
3.0 99.92 0.14 100.1 0.17
3.2 99.40 0.31 99.51 0.29
Temperature (°C) 28 99.65 0.27 99.46 0.28 30
30 99.89 0.13 99.94 0.12
Table 5 Chromatographic conditions
32 and range investigated0.32
99.52 during robustness
99.61 study for proposed
0.24 RP-HPLC method
Percentage of
Parameter 50
CLI ± SD (n99.41
= 5), % 0.32 ADA ±99.51
SD (n = 5), % 0.31 60
Acetonitrile in
Marketed Gel formulation 60
99.76 ± 0.6599.97 0.14 99.92
99.58 ± 0.73 0.19
Mobile Phase
70 99.64 0.26 99.47 0.43

acceptable range (RSD < 2 %), as shown in Table 4,
indicating that the system is suitable for the analysis
intended.
Method application. The proposed HPLC method was
applied for the simultaneously determinations of CLI and
ADA in the gel dosage form, without prior separation of
the excipients of the formulation. The results in Table 5
demonstrate the quality of the analyzed pharmaceutical
samples and the applicability of the method for QC
analysis.
Conclusion
This study is a typical example of the development of
an assay method following ICH guidelines. A new
isocratic RP-HPLC method has been developed and
validated for determination of CLI and ADA in the gel
dosage form. The results of the validation studies showed
that the RP-HPLC method possesses significant linearity,
precision, accuracy, specificity, sensitivity, high efficiency
and resolution, and no interference from the excipients, as
were demonstrated. The proposed method was successfully
applied and is suggested for the quantitative analysis of
CLI and ADA in combined pharmaceutical formulations
for QC, where economy and time are essential and to
assure therapeutic efficacy.
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