An Improved Rapid HPLC Method for the Separation of Five Anti-Hypertensive Agents Using C18 Column: Application to Hydrochlorothiazide Determination in Bulk and Tablet Dosage Form

Abstract

A selective, sensitive and accurate HPLC method with UV detection was developed and validated for the separation of five anti-hypertensive agents and applied for the determination of hydrochlorothiazide in bulk and pharmaceutical dosage forms. One of the key goals of HPLC technique is to achieve a consistent and reproducible separation. RP-HPLC method was developed by using Welchrom C18 Column (4.6 mm i.d. X 250 mm, 5 μm particle size), Shimadzu LC-20AT Prominence Liquid Chromatograph. The mobile phase constituted of 10 mM Phosphate buffer (pH3.0, adjusted with triethylamine): acetonitrile (50:50, v/v).The flow rate was set to 1.0 mL/min with the responses measured at 235nm using Shimadzu SPD20A Prominence UV-Visible detector. The retention times of atenolol hydrochloride, metoprolol succinate, hydrochlorothiazide, amlodipine besylate and nebivolol hydrochloride were found to be 2.303 min, 2.827 min, 3.543 min, 4.253 min and 4.957 min respectively. This method successfully separated all five antihypertensive drugs in less than 6 min. Hydrochlorothiazide was found to give linear response in the concentration range of 2-10μg/mL. Recovery studies were performed to ascertain the accuracy by standard addition method and average recovery was found to be 99.70 to 100.17 %.The limit of detection and limit of quantification were found to be 0.126616 μg/mL and 0.383686 μg/mL respectively. The developed method can be used for routine quality control analysis of hydrochlorothiazide in pharmaceutical tablet dosage form. It can also be extended for the determination of other four anti-hypertensive agents. This method provides simple and fast method with excellent peak symmetry and high resolution.