Development and Validation of a Simple Micromethod for the Determination of Metformin in Plasma by HPLC-UV for Application in Pharmacokinetic Studies

Background: 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. Objective: 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. Methods: 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 (95: 5, v,v). Results: 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 <20% 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. Conclusion: 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.