Development and Validation of Two Chromatographic Methods for Simultaneous Determination and Quantification of Amiloride Hydrochloride, Hydrochlorothiazide, and Their Related Substances, in Pure and Tablet Forms.

Abstract

Amiloride hydrochloride (AM) is a potassium sparing diuretic, while hydrochlorothiazide (HCZ) is the protype of thiazide diuretics. The combining of the studied drugs exhibits a synergistic effect. Moreover, HCZ prevents the potassium depletion side effect caused by AM. Two accurate and precise simultaneous chromatographic separation methods were promoted and investigated to quantify AM, HCZ, official impurities of HCZ (cholorothiazide and salamide), and the official impurities of AM (methyl 3, 5-diamino-6-chloropyrazine-2-carboxylate). The components of the quintuple mixture were quantified by two methods. The first method was high-performance thin layer chromatography (HPTLC), where exemplary separation was achieved on silica gel HPTLC F254 plates at the stationary phase using ethyl acetate-ethanol-ammonia solution (8 + 2 + 0.2, v/v) as a developing system. Scanning of bands at 273 nm was done. The second method was a reversed-phase chromatography (RP-HPLC) method using C18 (4.6 × 100 mm) column and mobile phase comprising 0.1% phosphoric acid solution-acetonitrile (90 + 10, v/v) with UV determination at 273 nm. Adjustment of the flow rate at 1 mL/min and pH at 3.6 was performed. Regarding RP-HPLC, optimum separation of the quintuple mixture was achieved within just five minutes. According to HPTLC, symmetric and sharp peaks were separated on the resulted chromatogram. Validity of the introduced methods was investigated by applying international conference on harmonization (ICH) guidelines. The methods were successfully applied for assays of the studied drugs in their pure and tablet forms. No significant difference was revealed through application of statistical comparison between results of the suggested methods and those of the reported method regarding both accuracy and precision.