Development And Validation Of A Liquid Chromatographic Method For The Simultaneous Analysis Of Amlodipine, Valsartan And Hydrochlorothiazide
Treatment of hypertension using one drug is desirable because compliance is likely to be better, lower overall cost and fewer adverse effects. However, most patients with hypertension require two or more drugs, preferably acting by different mechanisms to give the desired effect. A simple, specific, accurate, precise and affordable high performance liquid chromatographic method for the simultaneous determination of antihypertensive drug combination consisting of a calcium channel blocker-amlodipine, an angiotensin II receptor antagonist-valsartan and a thiazide diuretic-hydrochlorothiazide was developed and validated. The mobile phase systems consisted of varying mixtures of acetonitrile, distilled water and phosphate buffer adjusted to the required pH with orthophosphoric acid. The mobile phases were degassed by ultrasonication before use. The optimized conditions for the separation of the 3 analytes amlodipine, valsartan and hydrochlorothiazide consisted of Hypersil C-18 (250 mm × 4.6 mm i.d. 5 μm) column, mobile phase: acetonitrile-potassium dihydrogen phosphate pH 3.0- water, (75:6:19, % v/v/v). Column temperature was maintained at 40 oC; Flow rate: 1 ml/min; detection: 229 nm; and injection of 20 μl. The samples were dissolved in mobile phase for optimal chromatographic parameters. The precision of the method was shown through adequate repeatability or intraday precision (CV ≤ 2) and interday precision (CV ≤ 2). The method demonstrated adequate linearity of detector response over the range of 25-150%. The linearity equations were y = 4537 x + 26628, R2 = 0.991 for hydrochlorothiazide, y = 5344 x + 124106, R2 = 0.997 for valsartan and y = 4227x + 9893, R2 = 0.995 for amlodipine. The limit of detection for hydrochlorothiazide, valsartan and amlodipine were 10.72, 21.20 and14.45 ng, while the limits of quantification were 35.76, 71.23 and 48.16 ng respectively. The method also showed adequate robustness to small variations in mobile phase pH, column temperature and acetonitrile concentrations. The full recoveries of each working standard for all compounds were within ICH specifications of 98-103% which showed that the method was accurate. The developed method is rapid (run time 6 min), selective, requires simple sample preparation procedures and simple mobile phase combinations. It is also cost effective and represents a good procedure for determination of hydrochlorothiazide, valsartan and amlodipine in bulk raw materials and pharmaceutical dosage forms. The method was applied in the assay of 6 commercial products containing all the three, combination of any two or any one of the three API obtained from randomly selected retail pharmacies located within the Thika town. Three batches of each product were analyzed. The assay results indicated that there was no significant inter-batch variation in the content of active ingredients in the products tested except in two. The most noticeable feature was the high content in most of samples of the amlodipine component whose assay value was found to be more than 110% with content as high as 129.6%. For all the commercial samples tested only 2 batches complied with the assay limits defined in the B.P. and U.S.P.