Isocratic HPLC Method for Simultaneous Determination of Amlodipine and Xipamide in Human Plasma

Abstract

An HPLC method had been developed and validated for rapid simultaneous separation and determination of the two antihypertensive drugs, amlodipine and xipamide in human plasma within 5 minutes. Drugs are extracted from plasma using methanol, the environmentally benign solvent, for protein precipitation technique. Separation was carried out on a Thermo Scientifi c ® BDS Hypersil C 8 column (5μm, 2.50x4.60 mm) using a mobile phase of methanol: 0.025M KH 2 PO 4 (70: 30, v/v) adjusted to pH 3.49 with ortho -phosphoric acid at ambient temperature. The fl ow rate was 1 ml/min and maximum absorption was measured using DAD detector at 235nm. The retention times of amlodipine and xipamide were recorded to be 4.51 and 3.38 minutes, respectively, indicating a shorter analysis time. Limits of detection were reported to be 0.17 and 0.25 μg/ml for amlodipine and xipamide, respectively, showing a high degree of the method sensitivity. The method was then validated according to FDA guidelines for the determination of the drugs clinically in human plasma specially regarding pharmacokinetic and bioequivalence studies.