Stability-Indicating Method Development and Validation for the Assay of Hydrochlorothiazide and Determination of Impurities/Degradants in Hydrochlorothiazide Raw Material and Tablets using Reverse-Phase Liquid Chromatography

Hydrochlorothiazide is a drug that belongs to the class of diuretics used to treat hypertension and edema by inhibiting the ability of kidneys to retain water. A simple, accurate and precise reversed-phase liquid chromatography method was developed and validated to analyze Hydrochlorothiazide in raw material and determine impurities and degradants. A method was developed on a 15mm Luna Column C8, Particle size: 3μ, I.D. 4mm under ambient temperature. Mobile phase used: Monobasic Potassium Phosphate at pH 2.9 and Acetonitrile at a flow rate of 1.0 ml/min with UV-Vis detection at 273nm. Separation Mode: Isocratic elution 7/93% ACN/Buffer in 20min. Hydrochlorothiazide was subjected to the following stress conditions: acid hydrolysis, base hydrolysis, hydrogen peroxide oxidation, heat and UV light. The method was validated for specificity, robustness, linearity, accuracy, and precision, limit of detection and limit of quantitation. Linearity for active ingredient was investigated in the range from 250 to 1200 μg/mL with correlation coefficient found to be 0.9995. Linearity for impurities was studied in 0.001 to 1μg/mL range and gave a correlation coefficient of 0.9999. Calculated %Recovery for the active ingredient ranged from 95.0 to 98.0 percent. The method was then applied to quantify Hydrochlorothiazide in the Lisinopril and Hydrochlorothiazide tablets. Percent recovery of Hydrochlorothiazide in the finished product was calculated to be 98.7%. The molecular formula for degradant was established to be C6H8ClN3O4S2 corresponding to 4,6- sulfonylamido-3-chloroaniline, an ingredient used to prepare Hydrochlorothiazide raw material.