Development of validated HPLC-UV method for simultaneous determination of Metformin, Amlodipine, Glibenclamide and Atorvastatin in human plasma and application to protein binding studies

A simple, sensitive, fast, and economical HPLC method was developed and validated for simultaneous estimation of two fixed dose combinations frequently prescribed in diabetes (Metformin plus Glibenclamide) and hypertension with dyslipidemia (Amlodipine plus Atorvastatin) in Human plasma for the first time. The validated HPLC method was used to quantify the concentration of selected actives in ultrafiltrate. Optimum separation conditions were obtained with Water's Novapack Phenyl (150 mm × 4.6 mm, i.d., 5.0 μm) column with mobile phase consisting of 0.1% Phosphoric acid (pH 3.0) and acetonitrile (ACN) in gradient mode with column oven temperature maintained at 30 °C and elution monitored by a UV detector at 227 nm. Protein precipitation was employed to extract the selected analyte form human plasma. The recoveries were more than 90% for all analytes in cold aqueous 10% trichloroacetic acid (TCA) and acetonitrile. The optimized HPLC-UV was validated in the calibration range of 10-10,000 ng mL−1 for Metformin, 25-5000 ng mL−1 for amlodipine, 50-10,000 ng mL−1 for glibenclamide and 10-5000 ng mL−1 for atorvastatin. The mean relative error was least when weighing of 1/×2 was applied for calibration curve. The accuracy of samples for six replicate measurements at LLOQ level was within limit. The precision and accuracy of samples for six replicate measurements at LLOQ level was within limit. The validated method was applied for quantitation of selected analytes in ultrafiltrate from protein binding experiments. A four to five fold increase in unbound fraction was observed when spiked to human serum albumin. Further the unbound fraction of highly albumin bound drugs was increased nearly to double when incubated with Gly-HSA as compare to HSA.