Int. J. Pharm. Sci. Rev. Res., 28(2), September – October 2014; Article No. 14, Pages: 64-68 ISSN 0976 – 044X International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net © Copyright protected. Unauthorised republication, reproduction, distribution, dissemination and copying of this document in whole or in part is strictly prohibited. © Copyright pro 64 Tuani YT* 1 , Kissi FA 2 , Sackey J 1,3 , Gordon AJ 4 , Akanji O 5 1 Letap Pharmaceuticals Limited, Accra, Ghana. 2 Entrance Industries and Research Centre, Ghana. 3 Graduate School of Nuclear and Allied Sciences, Ghana. 4 Accra Polytechnic, Ghana. 5 U.S. Pharmacopoeia (Centers for Pharmaceutical Advancement and Training), Accra, Ghana. * Corresponding author’s E-mail: yayratuani@hotmail.com Accepted on: 20-07-2014; Finalized on: 30-09-2014. ABSTRACT A simple, precise, accurate and rapid reverse phase high performance liquid chromatographic isocratic method has been developed for the quantification of Ampicillin and Cloxacillin in Oral suspension dosage forms. The chromatographic separation was attained on P. Hypersorb ODS (250 x 4.6) mm with precolumn as the stationary phase at ambient temperature. The mobile phase was composed of Acetonitrile and 0.02 M Phosphate buffer pH 3.0, 35:65 with a flow rate of 1.8 mL/ min, and UV detection at 225 nm. The run time was 9 minutes. The injection volume was 20 µL. The retention times of Ampicillin and Cloxacillin were 1.943 ± 0.001 minutes and 6.058 ± 0.002 minutes respectively. Flucloxacillin was used as the internal standard and its retention time was 7.057± 0.004 minutes. The linearity of Ampicillin and Cloxacillin were investigated with the concentration ranges of 0.0185 %w/ v – 0.0277 %w/ v and 0.0175 %w/ v – 0.0263 % w/ v respectively. The correlation coefficients were greater than 0.99. The mean percentage recovery of Ampicillin at 80 %, 100 % and 120 % concentration levels were 101.5 ± 0.5, 99.6 ± 0.9 and 99.4 ± 0.3 respectively. That of Cloxacillin were 99.7 ± 0.6, 100.2 ± 0.1 and 101.4 ± 0.3 for 80 %, 100 % and 120 % concentration levels respectively. The results obtained show a method that is simple, rapid and precise. It can be used for the estimation of Ampicillin, Cloxacillin or Flucloxacillin in single as well as combination pharmaceutical dosage forms. Keywords: Ampicillin, Cloxacillin, Flucloxacillin, RP-HPLC, Validation. INTRODUCTION mpicillin is a beta-lactam antibiotic. It is bactericidal and has a similar mode of action to that of benzylpenicillin, but as an aminopenicillin with an amino group side-chain attached to the basic penicillin structure, Ampicillin is better able to penetrate the outer membrane of some gram-negative bacteria and has a broader spectrum of activity. The chemical formula is C 16 H 19 N 3 O 4 S·3H 2 O and its molecular weight is 403.45 g/mol. Cloxacillin is isoxazolyl penicillin used similarly to flucloxacillin in the treatment of infections due to staphylococci resistant to benzylpenicillin. It may be used with other antibacterials, including ampicillin, to produce a wider spectrum of activity. The molecular formula is C 19 H 18 ClN 3 NaO 5 S H 2 O and the molecular weight is 475.55 g/mol. 1 A number of methods for the simultaneous determination of Ampicillin and Cloxacillin have been reported. 2-4 The aim of this research is to develop a cost- effective method that can be used to analyze pharmaceutical dosage forms containing either single or combinations of Ampicillin, Cloxacillin and / or Flucloxacillin. This study was done in accordance with the International Conference on Harmonization (ICH) guidelines. 5 M ATERIALS AND M ETHODS M aterials and Reagents Knauer HPLC system with autosampler and UV detection, Measuring cylinder, P. Hypersorb ODS (250 x 4.6) mm column; particle size 5 µm (Make: Knauer (Germany)), Analytical balance (Make: Kern and Sohn GmbH, Model: ABJ 120-4M), pH Meter (Make: Labindia Model: SAB 5000), Potassium dihydrogen orthophosphate, Phosphoric acid, Acetonitrile (HPLC Grade) (Make: Merck, India). Distilled water, volumetric pipettes (100 mL, 50 mL), Column oven (Make: Knauer (Germany)), Syringe- driven filter unit (Make: Millipore Millex, Lot Number: ROAAB2861), Ampicillin trihydrate working standard (potency: 98.95 % on dried basis), Cloxacillin sodium working standard; Potency: 100.79 on dried basis; Flucloxacillin working standard; Potency: 98.79 % on dried basis, Millipore vacuum pump (Make: Millipore, Model: WP6122050), 0.2 µm Mobile phase filter (Make: Millipore M odel: GNWP). Instrumentation and Chromatographic conditions A Reverse phase - High Performance Liquid Chromatographic (HPLC) system, controlled by Knauer Advanced Scientific Instrument data handling system 6 and coupled with an auto sampler was used for the analysis. The data was recorded using Euro Chrome software. A stainless steel column 250 mm long, 4.6 mm internal Development and Validation of RP-HPLC M ethod for the Simultaneous Determination of Ampicillin and Cloxacillin in Oral Suspension Dosage Form A Research Article