Development and Validation of Isocratic High Performance Liquid Chromatography Methods for Simultaneous Determination of Antihypertensive Combination Drugs

Abstract

Simple, precise and rapid isocratic HPLC-UV method for simultaneous determination of five multi component drugs was developed and validated. The first drug contained valsartan and hydrochlorothiazide, the separation was achieved using phenyl hexyl column (150 × 4.6 mm, 3 μm particle size), both components were determined at 275nm, simple isocratic elution was selected, the optimized mobile phase was composed of methanol and 1% formic acid solution at 75: 25 ratio, with flow rate of 0.8 ml/min, injection volume was 20μl, and the separation was performed at ambient temperature. Linearity of this method was checked using concentration range of 2.5μg/ml –10μg/ml for hydrochlorothiazide and 5μg/ml –20μg/ml for valsartan; very good linearity correlation, (R2 =1), for both components. The second, contained amlodipine and losartan, the separation was achieved using phenyl hexyl column (150 × 4.6 mm, 3μm), both components were determined at 260nm, simple isocratic elution was selected, the optimized mobile phase was composed of acetonitrile and 1% formic acid solution at 60: 40 ratio, with flow rate of 0.8ml/min, injection volume was 20μl, and the separation was performed at ambient temperature. Linearity was checked using the concentration range 8μg/ml–32μg/ml for amlodipine and 80μg/ml–320μg/ml for losartan potassium. The linearity correlation, (R2 =1), for both components. The third, contained amlodipine and atorvastatin calcium, the separation was achieved using Neucleodur polaratic colum (50mmx2mm, 1.8 μm), both components were determined at 240nm, simple isocratic elution was selected, the optimized mobile phase was composed of methanol and 1% formic acid solution at 60: 40 ratio, with flow rate of 0.3 ml/min, injection volume was 20μl, and the separation performed at ambient temperature. Linearity was checked using the concentration range of 8ng–32ng for both amlodipine and atorvastatin, R2 =0.999 and 1.000 for Amlodipine besylate and atorvastatin calcium, respectively. The fourth, contained amlodipine besylate, hydrochlorothiazide and losartan potassium, The separation was achieved using Phenyl hexyle column (150mm×4.6mm,3μm), the components were determined at 260nm, simple isocratic elution was selected, the optimized mobile phase was composed of acetonitril and 1% formic acid solution at 1:1 ratio, with flow rate of 0.8ml/min, injection volume was 20μl, and the separation was performed at ambient temperature. Linearity was checked using concentrations range 4μg/ml-16μg/ml, 10μg/ml–40μg/ml and 40μg/ml -160μg/ml, for amlodipine besylate, hydrochlorothiazide and losartan potassium, respectively. Linearity III correlation (R2) was found to be 1.000 for each of the three components. The fifth, contained amlodipine besylate, hydrochlorothiazide and valsartan, The separation was achieved using Phenyl hexyl column (150mm×4.6mm,3μm), the three components were determined at 254nm, simple isocratic elution was selected, the optimized mobile phase was composed of acetonitril and 1% formic acid solution at 1:1 ratio, with flow rate of 0.8ml/min, injection volume was 20μl, and the separation performed at ambient temperature. Linearity was checked using concentration range of 4μg/ml-14μg/ml , 5μg/ml–20μg/ml and 64μg/ml -256μg/ml, for amlodipine besylate, hydrochlorothiazide and valsartan, respectively, the linearity correlation (R2) was found to be 1.000 for each of the three components.