Development and Validation of a Simple Micromethod for the Determination of Metformin in Plasma by HPLC-UV for application in pharmacokinetics studies

Abstract

Metformin is the first-line drug to enhance glycemic control of type 2 diabetes mellitus (DM2) patients. Some reported methods to determine plasma metformin by HPLC-UV are not sensitive enough. Other methods require long extraction processes. The objective of this study was to develop and validate a simple and rapid analytical method to determine plasma metformin by HPLC-UV for application in a population pharmacokinetic study. Analyte was extracted from plasma by a simple protein precipitation technique using trichloroacetic acid (15%, w/v) as the precipitating agent. Plasma samples were analyzed using a C18 column (3.0 x 150 mm, 3.5 µm) under isocratic elution with 30 mM sodium hexansulfonate (pH 5) and acetonitrile (97: 3, v/v). The limit of quantification (LOQ) was 0.1 µg mL-1 and the calibration curve was linear up to 4 µg mL-1 with a correlation coefficient >0.99. The mean recovery for metformin using this extraction procedure was 84.4 - 86.6%. The intra- and inter-day coefficients of variation and percent error values of the assayed method were <0% and <15% for LOQ and QCs, respectively. Metformin was stable in plasma samples by subjecting it to three freeze-thaw cycles and storing it up to 60 days at -80°C. This method was applied to determine plasma metformin concentrations in patients with type 2 diabetes mellitus treated with this drug. The HPLC-UV method developed is selective, accurate and precise for the quantification of metformin in plasma samples. Since sample, processing is fast and simple, in addition to being applicable in pharmacokinetic studies.