A Simple Gradient Rp-HPLC Method for The Quantification of Hydrochlorothiazide, Amlodipine Besylate and Nebivolol Hydrochloride in Pharmaceutical Dosage Forms

Reverse phase HPLC method has been developed for the simultaneous quantification of Hydrochlorothiazide,
Amlodipine besylate and Nebivolol in pharmaceutical dosage forms. chromatographic test was carried out by
using a Develosil C-8 UG-5, 150x3mm column with the gradient mobile phase composed of Sol-A: ammonium
acetate buffer (1.4g of ammonium acetate in to 1000ml of HPLC water) and sol-B: acetonitrile with simple
gradient program (0-4min, sol-A:86-86; 4-8min- sol-A:86-63; 8-13min- sol-A:63-63; 13-15min- sol-A:63-86
and 15-20min- sol-A:86-86) with 1.0ml per min flow rate. Column oven temperature maintained at 35°C and
UV absorbance measured at 225nm. The retention time of Hydrochlorothiazide is 2.28min, Amlodipine
besylate is 11.24 min and Nebivolol is 12.56min, respectively. Area percent RSD (relative standard deviation)
for five replicate standard injections is below 1.5percent. Method validation was performed with specificity,
precision, linearity, accuracy, ruggedness and robustness. The response was linear over the concentration range
of 10 to 60 microgram per mL for each ingredient, with correlation coefficients value is greater than 0.999.
Recovery results were satisfactory. The developed method is simple, reproducible and accurate.