Novel Stability-Indicating RP-HPLC Method for Determination of Hydrochlorothiazide, Amiloride Hydrochloride and Timolol Maleate in Tablet Dosage Form

Abstract

A novel stability indicating reversed phase high performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous determination of hydrochlorothiazide (HCT), amiloride hydrochloride (AMD) and timolol maleate (TIM) in a tablet dosage form. The chromatographic separation was performed on a Shimadzu Nexera LC-30AD system using Inertsil ® C8-3 column (4.6 mm x 150 mm, 5µ) and isocratic elution of methanol and a solution containing ion-pairing agent (1.5 g of 1-hexane sulfonic acid sodium salt, 30 mL glacial acetic acid,1 mL triethylamine dissolved in 1800 mL purified water and pH adjusted to 2.4 ± 0.05 with phosphoric acid then volume completed to 2000 mL by purified water) (40: 60, v/v) at a flow rate of 1.0 mL/min. UV detection was performed at 286 nm. Total run time was 7 min within which main compounds and degradants were separated. Stability indicating capability was established by forced degradation experiments. The method was validated for linearity, repeatability, trueness, specificity and robustness. Limit of detection (LOD) and limit of quantification (LOQ) were also established.