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ISSN: 2637-4609

Archives of Organic and Inorganic Chemical Sciences

Research Article(ISSN: 2637-4609)

Novel Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Clindamycin and Adapalene in Pharmaceutical Dosage Forms Volume 1 - Issue 4

P Nagaraju*, V Mounika and G Indira Priyadarshini

  • Department of Pharmaceutical Analysis, Hindu College of Pharmacy, India

Received: February 02, 2018;   Published: February 16, 2018

Corresponding author: P Nagaraju, Department of Pharmaceutical Analysis, Hindu College of Pharmacy, India

DOI: 10.32474/AOICS.2018.03.000120

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A simple, accurate, rapid and precise isocratic stability indicating reversed-phase high-performance liquid chromatographic method has been developed and validated for simultaneous determination of Clindamycin and Adapalene in tablets. The chromatographic separation was carried out on C18 BDS Hypersil (150 x 4.6mm, 5μ) with a mixture of mixed phosphate buffer: acetonitrile (55:45%v/v) as a mobile phase at a flow rate of 1.0mL/min. UV detection was performed at 230nm. The retention times were 2.84 and 3.999 min for Clindamycin and Adapalene respectively. Calibration plots were linear (r2=0.999) over the concentration range of 25-150μg/mL for Clindamycin and 2.5-15μg/mL for Adapalene. The method was validated for accuracy, precisio006E, specificity, linearity and sensitivity. The proposed method was successfully used for quantitative analysis of tablets. No interference from any component of pharmaceutical dosage form was observed. Validation studies revealed that method is specific, rapid, reliable, and reproducible. The high recovery and low relative standard deviation confirm the suitability of the method for routine determination of Clindamycin and Adapalene in bulk and tablet dosage form.

Keywords: Clindamycin; Adapalene; RP-HPLC; Tablets

Abbrevations: CLIN: Clindamycin; ADA: Adapalene; ICH: International Conference on Harmonization; LOD: Limit of Detection; LOQ: Limit of Quantification


Figure 1: Molecular structure of Clindamycin


Clindamycin (CLIN) (Figure 1) is a semisynthetic lincosamide antibiotic that has largely replaced lincomycin due to an improved side effect profile. Clindamycin is an antibiotic, similar to and a derivative of lincomycin. Clindamycin can be used in topical or systemic treatment. It is effective as an anti-anaerobic antibiotic and antiprotozoal. It is chemically Methyl (5R)-5-[(1S)-2-chloro- 1- {[(4R)-1-methyl-4-propyl-L-prolyl]amino}propyl]-1-thio-p-L- arabinopyranoside Adapalene (ADA) (Figure 2) is a chemically stable retinoid-like compound. Biochemical and pharmacological profile studies have demonstrated that adapalene is a modulator of cellular differentiation, keratinization, and inflammatory processes all of which represent important features in the pathology of acne vulgaris. Mechanistically, adapalene binds to specific retinoic acid nuclear receptors (gamma and beta) and retinoid X receptors but does not bind to the cytosolic receptor protein [1-4]. It is chemically 6-[3-(adamantan-1-yl)-4-methoxyphenyl] naphthalene- 2- Carboxylic acid. Literature survey [5-10] reveals that few spectrophotometric and chromatographic methods were reported for estimation of CLIN and ADA in single and combination with other drugs. In this study, an attempt has been made to develop an accurate, rapid and reproducible reverse phase HPLC method for simultaneous determination of CLIN and ADA in tablet dosage form and validate it, in accordance with International Conference on Harmonization (ICH) [11,12] guidelines.

Figure 2: Molecular structure of Adapalene.


Material and Methods

Chemicals and Reagents

The reference samples of CLIN (API) and ADA (API) were obtained from Pulse Pharmaceuticals, Hyderabad. The branded formulations LACNE gel was procured from the local market. Gel claimed to contain 1% CLIN and 0.1% ADA have been utilized in the present work. All chemicals and reagents used were HPLC grade and purchased from Merck chemicals, India.

Chromatographic Conditions

Separation was performed on an isocratic waters HPLC 2965 system instrument equipped with a with binary pump and variable wavelength PDA detector with auto injector. Data was analysed by using Empower2 software. Degassing of the mobile phase was done by using bath sonicator. A Shimadzu balance was used for weighing the materials. The separation was achieved on a BDS C18 (150 x 4.6mm, 5m) analytical column. The mobile phase consisted of mixed phosphate buffer: acetonitrile (55:45%v/v). The flow rate was 1.0mL/min and UV detection was performed at 230nm. The mobile phase was shaken on an ultrasonic bath for 30min. The resulting transparent mobile phase was filtered through a 0.45μ membrane filter (Millipore, Ireland). The injection volume was 10μL and all the experiments were performed at ambient temperature.

Preparation of standard solution

Accurately Weighed and transferred 100mg of CLIN and 10mg of ADA working Standards into 100ml clean dry volumetric flasks, add 3/4th volume of diluent, sonicated for 5 minutes and make up to the final volume with diluents.

Preparation of sample preparation

Lacne Gel with label claim of 1% CLIN and 0.1% ADA in 10g was extracted with 50ml of 0.1% glacial acetic acid in methanol in centrifuge tube. The sample mixture was heated at 500C for 10 min and mixed occasionally during the heating process. After heating, the sample was allowed to cool for 10 min and add 2ml of water into the centrifuge tube and then cooled in an ice bath for 20min and centrifuged at 3000RPM for 10min. After centrifugation, the supernatant liquid layer, which contains the analytes of interest, was separated.

Method Validation

The developed method was validated according to ICH guidelines. The system suitability was evaluated by five replicate analysis of CLIN and ADA mixture at concentrations of 1000μ/mL and 100μ/mL. The acceptance criteria are number of theoretical plates (N) at least 2000 per each peak and tailing factor is not more than 2.0.


Standard calibration curves were plotted against the concentration ranging from 25-150μ/mL for CLIN and 2.5-15μ/ mL for ADA. Different linearity levels were prepared and injected into the HPLC system keeping the injection volume constant.


To study the reliability and suitability of developed method, recovery experiments were carried out at three levels 50%, 100% and 150%. Known concentration of sample was spiked with known amount of standard. At each level, three determinations were performed with expected results. The %RSD of individual measurements was also determined.


Precision of assay was determined by System and Method Precision. Every sample was injected six times. The repeatability of sample application and measurements for peak area were expressed in terms of %RSD.


All chromatograms were examined to determine whether compound of interest coeluted with each other or with any additional excipient peaks. Marketed formulation was analysed to determine the specificity of the optimized method in presence of common excipients.

Limit of Detection and Limit of Quantification

Limit of detection (LOD) and limit of quantification (LOQ) were estimated from signal-to-noise ratio. LOD and LOQ were calculated using 3.3 σ/s and 10 σ/s formulae, respectively. Where, a is the standard deviation of the peak areas and S is the slope of the corresponding calibration curve.


To evaluate robustness of HPLC method a few parameters were deliberately varied. The parameters included are variation of flow rate and Detection Wavelength.

Force Degradation studies

    i. Oxidation: To 1 ml of stock solution, 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 600c. For HPLC study, the resultant solution was diluted to obtain 100μg/ml&10μg/ml solution and 10 μl were injected into the system and the chromatograms were recorded to assess the stability of sample.

    ii. Acid degradation studies: To 1ml of stock solution of Adapalene and Clindamycin, 1ml of 2N Hydrochloric acid was added and refluxed for 30mins at 600c .The resultant solution was diluted to obtain 100μg/ml&10μg/ml solution and 10 μl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

    iii. Alkali degradation studies: To 1ml of stock solution of Adapalene and Clindamycin, 1ml of 2N sodium hydroxide was added and refluxed for 30mins at 600c. The resultant solution was diluted to obtain 100μg/ml&10μg/ml solution and 10 μl were injected into the system and the chromatograms were recorded to assess the stability of sample.

    iv. Dry heat degradation studies: The standard drug solution was placed in oven at 1050c for 6 h to study dry heat degradation. For HPLC study, the resultant solution was diluted to 100μg/ml & 10μg/ml solution and 10μl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

    v. Photo Stability Studies: The photochemical stability of the drug was also studied by exposing the 300μg/ml & 10μg/ ml & 25μg/ml solution to UV Light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m2 in photo stability chamber. For HPLC study, the resultant solution was diluted toobtain 100μg/ml & 10μg/ml solutions and 10μl were injected into the system and the chromatograms were recorded to assess the stability of sample.

    vi. Neutral degradation studies: Stress testing under neutral conditions was studied by refluxing the drug in water for 6hrs at a temperature of 60°. For HPLC study, the resultant solution was diluted to 100μg/ml & 10μg/ml solution and 10μl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Results and Discussion

During the optimization of HPLC method, two columns symmetry C-18 and C-8 analytical column (4.6x250mm; 5μm) and (4.6x150 mm; 5μm), organic solvent (acetonitrile), one buffer (phosphate) were tested. Initially Water: Acetonitrile and Phosphate buffer were tried in different ratios. Finally mobile phase consisting of mixture of acetonitrile: Phosphate buffer in ratio 45:55 (v/v) was selected as mobile phase to achieve clear separation and sensitivity. Flow rates between 0.8 to 1.2mL/min were studied. A flow rate of 1.0 mL/min gave an optimum signal to noise ratio with reasonable separation time using a C18 analytical column (4.6x250mm; 5μm), the retention times for CLIN & ADA were observed to be 2.84 and 3.999min respectively. Total run time was less than 7min. The chromatogram at 230nm showed a complete resolution for all peaks (Figure 3). Validity of the analytical procedure as well as the resolution between different peaks of interest is ensured by the system suitability tests. All critical parameters tested meet the acceptance criteria on all days. As shown in chromatogram, two analytes are eluted by forming symmetrical peaks.

Figure 3: Typical chromatogram of standard for CLIN & ADA.


Linearity was obtained for CLIN & ADA in the range of 25-150μg/ mL and 2.5-15μg/mL. The correlation coefficient (r2) was found to be greater than 0.999 in all instances. The results of calibration studies are summarized in Table 1. The proposed method afforded high recoveries for CLIN and ADA in dosage form. Results obtained from recovery studies presented in Table 2 indicate that this assay procedure can be used for routine quality control analysis of binary mixture in sample. Precision of the analytical method was found to be reliable based on %RSD (<2%) corresponding to peak areas and retention times. As can be seen in Table 3 the %RSD values were less than 2 for System & Method precision. Hence, the method was found to be precise for these two drugs.

The chromatograms were checked for appearance of any extra peaks under optimized conditions, showing no interference from common excipients and impurities. Also the peak areas were compared with standard and percentage purity calculated was found to be within limits. LOD and LOQ were found to be 0.13μg/ mL and 0.4μg/mL for CLIN, 0.08μg/mL and 0.24μg/mL for ADA. In all deliberately varied conditions, the %RSD for replicate injections of CLIN & ADA were found to be within the acceptable limit. The tailing factors for two peaks were found to be less than 1.5 and assay showed good agreement with label claims and the results are the results are shown in Table 4. The validate method was used in shown in Table 5. Degradation studies results were shown in Tables analysis of marketed tablet dosage form. The results for the drugs 6 & 7.

Table 1: System suitability parameters of proposed method.


Table 2: Accuracy data for proposed method.


Table 3: Precesion data of proposed method.


Table 4: Robustness for flow rate variation of CLIN & ADA.


Table 5: Analysis of marketed formulation by proposed method.


Table 6: Degradation Data of Clindamycin.


Table 7: Degradation Data of Adapalene.



The developed stability indicating RP-HPLC method is simple, specific, accurate and precise for the simultaneous determination of CLIN & ADA in dosage form. The developed method provides good resolution between CLIN & ADA. It was successfully validated in terms of system suitability, linearity, precision, accuracy, specificity, LOD, LOQ and robustness in accordance with ICH guidelines. Thus the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing these drugs either as such or in combination.


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