RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF CLINDAMYCIN PHOSPHATE AND NICOTINAMIDE IN PHARMACEUTICAL DOSAGE FORM ABRAR M. CHAUDHARY 1, JIGNASA MODI 2, MAZHARUDDIN SHAIKH 3 ABSTRACT The present work describes a simple, rapid, and reproducible reverse phase high performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Clindamycin phosphate (CLINDA) and Nicotinamide (NICO). The separation was achieved on a Hypersil BDS C18 (250mm X 4.6 mm i.d., 5 μm particle size) with an isocratic system of Phosphate buffer (ph-3.5): Methanol in the ratio of 60:40 v/v. The mobile phase at a flow rate of 1.0 ml/min, Injection volume 20μl and wavelength of detection used was 210nm. The retention time for Nicotinamide and Clindamycin phosphate were obtained as 4.053±0.1min and 5.730±0.1min, respectively. The linearity of the proposed method was investigated in the range of 5-15µg/ml and 20-60 µg/ml for Clindamycin phosphate and Nicotinamide, respectively. Correlation coefficients were 0.998 and 0.999 for Clindamycin phosphate and Nicotinamide, respectively. The developed method was validated as per ICH guideline, for its accuracy, precision, LOD & LOQ and the results were found to be satisfactory, thus the method is specific, rapid and simple with good sensitivity for estimation of Clindamycin phosphate and Nicotinamide. These analytical methods are also applicable in ordinary laboratories. It can also be adopted for quality control tests for these drugs in cream formulation. KEYWORDS Clindamycin phosphate, Nicotinamide, RP-HPLC method, Validation. AUTHORS AFFILIATION 1,2&3. Department of Quality Assurance, Parul Institute of Pharmacy and research, Vadodara, India-391760 *Address for correspondence E-mail: abrarchaudhary91@gmail.com [160]
INTRODUCTION Clindamycin phosphate, Methyl 7-chloro-6,7,8-trideoxy-6-(1-methyl-trans-4-propyl-L-2- pyrrolidinecarboxamido)-1-thio-l-threo-α-d-galacto-octopy-ranoside2-(dihydrogen phosphate) is a semi synthetic derivative of lincomycin, a natural antibiotic produced by the actinobacterium Streptomyces lincolnensis. It is obtained by 7(S)-chloro-substitution of the 7(R) hydroxyl group of lincomycin. Clindamycin is used primarily to treat infections caused by susceptible anaerobic bacteria, including infections of the respiratory tract, skin and soft tissue infections, and peritonitis. Clindamycin binds to the 50S ribosomal subunits of susceptible bacteria and prevents elongation of peptide chains by interfering with peptidyl transfer, thereby suppressing bacterial protein synthesis. Clindamycin have been shown to have in vitro activity against Propionibacterium acnes, an organism which has been associated with acne vulgaris. Clindamycin has a bacteriostatic effect. It is a bacterial protein synthesis inhibitor by inhibiting ribosomal translocation, in a similar way to macrolides. It does so by binding to the 50SrRNA of the large bacterial ribosome subunit. O Cl N N H O S HO O HO O P OH OH Fig. 1: Structure of Clindamycin phosphate. Nicotinamide, pyridine-3-carboxamide also known as Niacinamide and Nicotinic acid amide, is the amide of nicotinic acid (vitamin B3 / niacin). Nicotinamide is a water-soluble vitamin and is part of the vitamin B group. Nicotinamide has demonstrated anti-inflammatory actions that may be of benefit to patients with inflammatory skin conditions. These conditions include acne vulgaris, and the compound can suppress antigen-induced, lymphocytic transformation and inhibit 3'-5' cyclic AMP phosphodiesterase. N NH 2 Fig. 2: Structure of Nicotinamide. Combination of Clindamycin phosphate and Nicotinamide is available as marketed formulation and prescribed by physician and dermatologist for the treatment of mild to moderate acne vulgaris and to reduce inflammatory acne lesions. A few of the brands available in the market are CLIN-3 (UNIMARK), ACNESTAR, FACECLIN and CLINKA N GEL. O [161]
The present study was designed to develop a simple, precise, and rapid analytical RP-HPLC procedure, which can be used for the analysis of assay method for simultaneous estimation of Clindamycin phosphate and Nicotinamide as there was only individual methods reported for both drugs. The combination of these two drugs is not official in any pharmacopoeia; hence no official method is available for the simultaneous estimation of these two drugs in their combined dosage forms. Literature survey of Clindamycin phosphate and Nicotinamide revealed several methods for detecting these drugs individually but there is no method for their simultaneous estimation using RP-HPLC. The developed method was validated as per ICH guidelines and its updated international convention. The linearity of response, precision, ruggedness and robustness of the described method has been checked. Chemicals and Reagents EXPERIMENTAL Clindamycin phosphate API was gifted by Parry Pharma PVT Ltd., Ahmedabad, India. Nicotinamide was provided from Parul Institute of Pharmacy and Research, Limda, Waghodia, Vadodara, India. The combination of these drugs is manufactured by Abbott Healthcare Pvt. Ltd. under the name of FACECLIN. All the reagents including potassium dihydrogen phosphate and ortho phosphoric acid were purchased from Merck and Rankem Ltd. New Delhi, India. HPLC Instrumentation and Conditions The analysis was carried out on a HPLC system (Shimadzu-LC 20AT) equipped with UV detector, pressure controlled by prominence pump and operated by LC Solution. C18 column (250 mm 4.6 mm i.d., particle size 5 μm) was used for separation. Mobile phase used for separation was containing Phosphate buffer: Methanol in the ratio of 60:40 v/v adjusted to ph 3.5 with orthophosphoric acid. The flow rate was kept at 1.0 ml/min, column temperature was ambient (25 C), eluents were detected by UV detector at 210 nm, and the injection volume was 20 μl. Preparation of Phosphate Buffer Phosphate buffer was used for the estimation of both the drugs in their combined dosage form. 6.72 gm of potassium dihydrogen phosphate (KH2PO4) was weighed accurately and transferred in to 1000 ml beaker. 800 ml HPLC grade water was added to dissolve it. The ph 3.5 was adjusted with diluted ortho phosphoric acid (1%) and made up the volume up to 1000 ml with HPLC grade water to give 0.05 M Phosphate buffer. Preparation of Mobile Phase Mobile phase was prepared by mixing 60 volumes of 0.05 M Phosphate buffer (adjusted to ph 3.5 with orthophosphoric acid) and 40 volumes of Methanol. The mobile phase was ultrasonicated, filtered through 0.45 μm membrane filter, and degassed. Preparation of mixed standard stock solutions of Clindamycin phosphate and Nicotinamide 10 mg of Clindamycin phosphate and 40 mg of Nicotinamide were weighed accurately and [162]
transferred to a 100 ml volumetric flask and dissolved in 50 ml mobile phase. The flask was shaken and volume was made up to the mark with mobile phase to give a solution containing Clindamycin phosphate (100µg/ml) and Nicotinamide (400µg/ml). From this stock solution different aliquots were prepared. Preparation of working stock solution of Clindamycin phosphate (10 μg/ml) and Nicotinamide (40 μg/ml) From above prepared standard solution, 10ml of standard solution of Clindamycin phosphate and Nicotinamide were taken in 100ml volumetric flask individually and volume was made up to the mark with diluent (Mobile phase) to give solution containing 10μg/ml of Clindamycin phosphate and 40μg/ml of Nicotinamide. Selection of detection wavelength In the present study individual drug solutions of 10μg/ml were prepared in different solvent mixtures of HPLC grade organic and inorganic solvents at different ratio. These drug solutions were then scanned in the UV region of 200-400 nm and the spectrums were recorded to get λmax of analyte in Mobile Phase. 210nm wavelength was selected for estimation of this combination. Method Validation Linearity and Range The linearity response was determined by analyzing independent levels of concentrations in the range of 5-15μg/ml and 20-60μg/ml for Clindamycin phosphate and Nicotinamide respectively. Peak area of each solution was measured using developed method. Calibration curve of peak area Vs concentration was plotted. The correlation coefficient and regression line equations for Clindamycin phosphate and Nicotinamide were determined. Accuracy To study the accuracy powdered tablet analysis of the same was carried out. Recovery studies were carried out by addition of standard drug to the sample at 3 different concentration levels (80, 100 and 120 %) taking into consideration percentage purity of added bulk drug samples. It was determined by calculating the recovery of Clindamycin phosphate and Nicotinamide by standard addition method. Accuracy is the closeness of the test results obtained by the method to the true value. Preparation of sample solution for % recovery: Cream equivalent to 10 mg Clindamycin phosphate and 40 mg Nicotinamide was accurately weighed and transferred to volumetric flask of and transferred to a 125 ml conical flask, 100 ml of mobile phase was added in it. The stopper was inserted in the flask securely and shaken for 1 hour. The flask was cooled in ice bath for 20 min and centrifuged. The cloudy lower layer was filtered with 2µ filter paper & filtrate was used for % recovery. From this solution 1 ml was transferred to volumetric flask of 10 ml capacity. Volume was made up to the mark to give a solution containing 5 µg/ml CLINDA and 20 µg/ml NICO. The chromatogram of sample solutions was taken at selected wavelength for Clindamycin phosphate and Nicotinamide and concentration is calculated which is known as pre-analyzed sample. In pre- [163]
analyzed sample 80, 100 and 120 % of Clindamycin phosphate and Nicotinamide was spiked. Chromatogram of spiked samples was measured and total amount of drug was calculated and from which % recovery was calculated. Precision Repeatability 6 replicates of standard mixture solution having 10μg/ml Clindamycin phosphate and 40μg/ml Nicotinamide were prepared and chromatograms were recorded and RSD was calculated. Intraday precision Standard solutions containing 5, 10 and 15μg/ml of Clindamycin phosphate and 20, 40, and 60 μg/ml of Nicotinamide were analyzed 3 times on the same day as per the procedure. Chromatogram of each sample was taken. SD and RSD were calculated Interday precision Standard solutions containing 5, 10 and 15μg/ml of Clindamycin phosphate and 20, 40, and 60 μg/ml of Nicotinamide were analyzed on three different days as per the procedure. Chromatogram of each sample was taken. SD and RSD were calculated. Limit of Detection Calibration curve was repeated for 6 times and the standard deviation (SD) of the intercepts was calculated than LOD was calculated as follow from the formula LOD= (3.3*SD)/Slope Where, SD = the standard deviation of Y- intercept of 6 calibration curves. Slope = the mean slope of the 6 calibration curves. Limit of Quantitation Calibration curve was repeated for 6 times and the standard deviation (SD) of the intercepts was calculated than LOQ was calculated as follow from the formula LOQ= (10*SD)/Slope Robustness Study Change in Flow Rate One concentration of mixture Clindamycin phosphate(5μg/ml) and Nicotinamide (66.5μg/ml) was analyzed 3 times at 3 different flow rate and record the peak area, resolution and tailing factor were recorded and percentage was R.S.D. calculated. Acceptance criteria % RSD should be less than 2 and SST (system suitability test parameters) should be in range. Change in Mobile Phase Ratio [164]
One concentration of mixture Clindamycin phosphate(10μg/ml) and Nicotinamide (40 μg/ml) was analyzed 3 times at 3 different mobile phase ratio and record the peak area, resolution and tailing factor were recorded and percentage R.S.D. was calculated Acceptance Criteria % RSD should be less than 2 SST (system suitability test parameters) should be in range. Change in ph One concentration of mixture Clindamycin phosphate (10 μg/ml) and Nicotinamide (40 μg/ml) was analyzed 3 times at 3 different ph and record the peak area, resolution and tailing factor were recorded and percentage was R.S.D. calculated. Acceptance Criteria % RSD should be less than 2 SST (system suitability test parameters) should be in range. System suitability 10 μg/ml Clindamycin phosphate and 40 μg/ml Nicotinamide in mixture was injected and the chromatograms were recorded. The resolution, number of theoretical plates, and peak asymmetry were calculated to determine whether the result complies with the recommended limit. Analysis of Pharmaceutical Preparations %Assay of Clindamycin phosphate and Nicotinamide in their Cream Dosage Form (Formulation: FACECLIN Cream) Sample preparation 10 mg CLINDA and 40 mg NICO were equivalently weighed from their dosage form and transferred to a 125 ml conical flask; 100 ml of mobile phase was added in it. The stopper was inserted in the flask securely and shaken for 1 hour. The flask was cooled in ice bath for 20 min and centrifuged. The cloudy lower layer was filtered with 2µ filter paper & filtrate was used as the assay preparation. From this solution 1 ml was transferred to volumetric flask of 10 ml capacity. Volume was made up to the mark to give a solution containing 10 µg/ml CLINDA and 40 µg/ml NICO. This solution was used for the estimation of Clindamycin phosphate and Nicotinamide. The amount of Clindamycin phosphate and Nicotinamide present in the sample solution were determined by fitting peak area into the equation of the straight line representing the calibration curves for Clindamycin phosphate and Nicotinamide [165]
RESULTS AND DISCUSSION Optimization of Chromatographic Conditions To develop suitable RP-HPLC method for simultaneous estimation of Clindamycin phosphate and Nicotinamide, different chromatographic conditions were applied and optimized chromatographic conditions were developed. (See figure 3) Fig. 3: Optimized chromatogram of Clindamycin phosphate and Nicotinamide. Table 1. Optimized chromatographic conditions. Parameters Stationary phase Mobile phase Pump mode Condition Hypersil BDS C18 column (250mm X 4.6 mm i.d., 5 μm particle size) Buffer (0.05 M Potassium dihydrogen phosphate), ph:3.5 adjusted with orthophosphoric acid : Methanol (60:40) Isocratic Flow rate (ml/min) 1.0 Run time (min) 10.0 Volume of injection (μl) 20 Detection wavelength (nm) 210 Validation Linearity Linearity was performed on synthetic mixture of Clindamycin phosphate and Nicotinamide as per marketed formulation composition.(see figure 4) [166]
Fig. 4: Chromatograms of Clindamycin phosphate and Nicotinamide for Linearity. Calibration curve for the Clindamycin phosphate (5-15 μg/ml) Chromatogram for following concentrations 5, 7.5, 10, 12.5 and 15 μg/ml for Clindamycin phosphate at 210 nm at flow rate of 1 ml/min was obtained. Peak areas were obtained as tabulated in Table 2 and the graph of calibration curve was obtained as shown in Figure 5. Table 2: Calibration curve for Clindamycin phosphate. s (µg/ml) Peak area of Clindamycin phosphate (Mean ± S.D.) Coefficient of Variation 5 477.218 ± 4.52 0.94 7.5 706.303 ± 2.82 0.40 10 967.476 ± 6.83 0.70 12.5 1175.678 ± 10.62 0.90 15 1450.452 ± 15.09 1.04 Fig. 5: Graph of Calibration curve for Clindamycin phosphate. [167]
Discussion: Linearity range for Clindamycin phosphate was found to be 5-15 μg/ml in Mobile Phase. Regression Equation for Clindamycin phosphate at 210 nm: Y= 96.63x- 10.91 r 2 value: 0.998 Calibration curve for the Nicotinamide (20-60 μg/ml) Chromatogram for following concentrations 20, 30, 40, 50 and 60 μg/ml for Nicotinamide at 210 nm at flow rate of 1 ml/min. Peak area were obtained as tabulated in Table 3 and the graph of calibration curve was obtained as shown in Figure 6. Table 3: Calibration curve for Nicotinamide. s (µg/ml) Peak area of Nicotinamide (Mean ± S.D.) Coefficient of Variation 20 2102.164 ± 5.45 0.25 30 3103.89 ± 20.08 0.64 40 4243.219 ± 34.88 0.82 50 5241.081 ± 58.14 1.10 60 6357.941 ± 39.30 0.61 Fig. 6: Graph of Calibration curve for Nicotinamide. Discussion: Linearity range for Nicotinamide was found to be 20-60 μg/ml in Mobile Phase. Regression Equation for Clindamycin phosphate at 210 nm: Y= 106.4x 49.83, r 2 value: 0.999 Accuracy (% Recovery study) Clindamycin phosphate: (Target concentration 5 μg/ml) [168]
Table 4: %Recovery data for Clindamycin phosphate. Level of recovery Amt of Sample added Amt of std spiked Total amt of sample Peak area of sample Amt of std found Amount of std recovered % recovery mean ± SD (n=3) 80%(n=3) 5 4 9 925.87 9.02 4.02 100.60±1.35 100%(n=3) 5 5 10 1028.836 9.98 4.98 99.74±0.57 120%(n=3) 5 6 11 1136.326 10.99 5.99 99.92±0.44 (n=3 Determination) Nicotinamide: (Target concentration 20 μg/ml) Table 5: %Recovery data for Nicotinamide. Level of recovery Amt of Sample added Amt of std spiked Total amt of sample Peak area of sample Amt of std found Amount of std recovered % recovery mean ± SD (n=3) 80%(n=3) 20 16 36 3944.546 35.97 15.97 99.88±1.02 100%(n=3) 20 20 40 4382.322 39.92 19.92 99.67±0.64 120%(n=3) 20 24 44 4823.764 43.91 23.91 99.68±0.49 (n=3 Determination) Precision Repeatability Table 6: Repeatability data for Clindamycin phosphate and Nicotinamide Standard Drug Target Peak Area of Sample Found Mean SD % RSD Clindamycin phosphate 10 963.272 10.08 10 965.207 10.10 10 926.806 9.70 10 969.089 10.14 10 064.239 10.09 10 966.173 10.11 10.03 0.16 1.65 [169]
40 4226.792 40.19 40 4216.701 40.09 Nicotinamide 40 4243.722 40.35 40 4252.255 40.43 40 4231.032 40.23 40 4239.449 40.31 40.26 0.12 0.30 Discussion: The % RSD for Repeatability of both the drugs was found to be less than 2. So, it was concluded that proposed method for estimation of Clindamycin phosphate and Nicotinamide is precised in nature. Intraday precision Table 7: Intraday precision data for Clindamycin phosphate and Nicotinamide. Standard Drug Clindamycin phosphate Nicotinamide Target Peak Area of Sample Found 5 474.065 5.01 5 459.747 4.87 5 474.457 5.02 10 960.4 10.05 10 950.084 9.94 10 965.232 10.10 15 1440.816 15.02 15 1407.993 14.68 15 1447.947 15.09 20 2065.165 19.87 20 2091.591 20.12 20 2097.861 20.18 40 4186.175 39.81 40 4244.526 40.15 40 4235.233 40.27 60 6261.391 59.31 60 6326.546 59.92 60 6345.662 60.10 Mean SD % RSD 4.96 0.083 1.68 10.03 0.081 0.81 14.93 0.21 1.46 20.05 0.16 0.81 40.07 0.23 0.59 59.77 0.41 0.69 [170]
Discussion: % RSD for Intraday precision of both the drugs were found less than 2. So, it was concluded that proposed method for estimation of Clindamycin phosphate and Nicotinamide is precised in nature. Interday precision Table 8: Interday precision data for Clindamycin phosphate and Nicotinamide. Standard Drug Target Peak Area of Sample Found Mean SD % RSD 5 474.541 5.02 5 461.706 4.89 4.97 0.075 1.50 Clindamycin phosphate Nicotinamide 5 474.55 5.02 10 961.362 10.06 10 943.278 9.87 10 961.295 10.06 15 1442.25 15.03 15 1417.302 14.78 15 1442.161 15.03 20 2070.678 19.92 20 2093.684 20.14 20 2089.47 20.10 40 4185.841 39.80 40 4226.751 40.19 40 4210.293 40.03 60 6267.66 59.37 60 6332.922 59.89 60 6320.279 59.86 9.96 0.10 1.09 14.94 0.144 0.96 20.05 0.11 0.58 40.0 0.19 0.49 59.70 0.29 0.48 Discussion: The % RSD for Interday precision of both the drugs were found to be less than 2. So, it was concluded that proposed method for estimation of Clindamycin phosphate and Nicotinamide is precised in nature. [171]
Limit of Detection (LOD) and Limit of Quantitation (LOQ) Table 9: LOD and LOQ data for Clindamycin phosphate and Nicotinamide. Parameters Clindamycin phosphate Nicotinamide Mean Slope (n=6) 96.62 106.46 SD of Y intercept (n=6) 5.928 24.24 LOD 0.20 0.75 LOQ 0.61 2.27 Discussion: The proposed method can detect Clindamycin phosphate and Nicotinamide at very low level. So, it was concluded that the proposed method is very sensitive in nature. Robustness Study Table 10: Robustness data. Parameter Changed Variation % RSD Theoretical Plates Tailing factor CLIN NIC CLIN NIC CLIN NIC Resolution Flow Rate (ml/min) Mobile Phase (Ratio) ph +0.2ml 1.48 0.87 4474.33 4489.66 1.65 1.30 5.736-0.2ml 1.92 0.60 4422.66 4536 1.63 1.29 5.73 62:38 1.07 0.98 4476 4418.33 1.62 1.31 5.736 58:42 1.56 0.65 4430.66 4519 1.66 1.29 5.725 +0.2 1.60 0.66 4442 4450 1.65 1.27 5.71-0.2 1.67 0.74 4457 4519 1.7 1.29 5.725 CLIN=Clindamycin phosphate, NIC=Nicotinamide System suitability Table 11: System suitability Parameters. Name Rt (min) Area Tf Resolution Theoretical Plate# Nicotinamide 4.053 4260.768 1.273 4430 Clindamycin 5.692 5.73 971.018 1.659 4399 phosphate [172]
Analysis of Pharmaceutical Preparations: International Bulletin of Drug Research., 4(6): 160-174, 2014 %Assay of Clindamycin phosphate (10μg/ml) and Nicotinamide (40μg/ml) in their cream dosage form Fig. 7: Chromatograph of 10 μg/ml of Clindamycin phosphate and 40 μg/ml of Nicotinamide prepared from cream (FACECLIN). Table 12: % Assay of Clindamycin phosphate and Nicotinamide. Drug in marketed formulation (mg) Conc. taken for %Assay Peak area of Sample solution* found from Mixture* % Assay* ±S.D. Clindamycin phosphate 10 10 μg/ml 973.007 10.03 μg/ml 100.70 ±1.28 Nicotinamide 40 40 μg/ml 4277.14 40.35 μg/ml 100.88 ±0.39 *Mean of 3 determinations Discussion: % Assay of Clindamycin phosphate and Nicotinamide was found in an acceptance limit so this method can be used for analysis of this combination. CONCLUSION The proposed RP-HPLC method was used for the simultaneous estimation of Clindamycin phosphate and Nicotinamide, hence found to be sensitive, accurate, precise, simple, and rapid. Hence the present RP-HPLC method may be used for routine analysis of the raw materials, in combinational dosage formulations containing Clindamycin phosphate and Nicotinamide ACKNOWLEDGEMENTS The authors are thankful to the Principal Dr. T.Y.Pasha, Parul institute of pharmacy & research, Vadodara, India, for providing necessary facilities and Parry Pharma Pvt. LTD., Ahmedabad for providing the gift sample of Clindamycin phosphate. [173]
REFERENCES 1. Barar F. S. K., Essentials of Therapeutics; Interprint New Delhi, pp 378,430-31. 2. Rang M.P., and Dale M. Pharmacology; 6th Edn; Churchill Liningstone, pp 328, 672-73. 3. United State Pharmacopoeia-2009, The Official Compendia of Std, Vol-II, pp 1970, 3178. 4. Shethi P.D., HPLC-Quantitative analysis of pharmaceutical formulations; 1st Edn; CBS publishers & Distributors, New Delhi, 2001, pp 3-141. 5. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use. Validation of Analytical: Text and Methodology ICH Q2 (R1). 2005. 6. Rajameena R and Rama K, RP-HPLC method development and validation for estimation of Clindamycin phosphate and Clotrimazole. International research journal of pharmacy. July 2013, 4(7). 7. Navkhare MS and Gaidhane HK, Validated stability indicating analytical method for the determination of clindamycin phosphate and adapalene in topical formulation. Trade Science Inc. March 2013, Volume 13, Issue 6. 8. Darji RB and Patel BH, Development and validation of hplc method for simultaneous estimation of clindamycin phosphate, clotrimazole and tinidazole in pharmaceutical dosage form. Inventi Rapid: Pharm Analysis & Quality Assurance. May 2012, 374. 9. Platzer Daniel J and Brent A, Development and validation of a gradient HPLC method for the determination of clindamycin and related compounds in a novel tablet formulation. J Pharm Biomed Anal. April 2006, 84-88. 10. Yuerong Rose Y and Edin Bektic, Simultaneous determination of tretinoin and clindamycin phosphate and their degradation products in topical formulations by reverse phase HPLC. Journal of Separation Science.Jan 2004, 71-77. 11. Orwa JA and Vandenbempt K. Liquid chromatography method for separation of clindamycin from related substances. J Pharm Biomed Anal. Sep 1999, 745-52. 12. Patel Prashant and Javed Akhtar, Validated liquid chromatography method for simultaneous estimation of salicylic acid in semi-solid dosage form. Indian journal of chemical technology.may 2013, Vol. 20, 185-188. 13. Chimezie Anyakora and Ibukun Afolami, HPLC analysis of nicotinamide, pyridoxin, riboflavin and thiamin in some selected food products in Nigeria. African Journal of Pharmacy and Pharmacology, April, 2008, Vol. 2(2), 29-36. [174]