Ion-pair HPLC Method for the Quantification of Metformin in Human Plasma and Its Application to a Pharmacokinetic Study

Aims: To develop and validate a rapid, selective and sensitive ion-pairing HPLC-UV method for the determination of metformin in human plasma, using a conventional reverse phase column.

Study Design: Experimental study.

Place and Duration of Study: Department of Department of Biomedical Sciences, Faculty of Medicine, University of Medicine, “Mother Tereza” hospital center, between November 2014 and February 2015.

Methodology: Ion-pair separation followed by UV detection performed on deproteinised and dichloromethane washed plasma samples was chosen for the determination of metformin. The separation was performed on an analytical LiChrocart® 100 RP 18 (125 × 4.0 mm i.d., 5 µm particle size) C18 column. A mobile phase consisting of acetonitrile and 10 mM sodium phosphate buffer (pH=6.0; 32.5:67.5, v/v) and sodium dodecyl sulfate (0.3%) was pumped at an isocratic flow rate of 1.25 mL/minute, and quantification was achieved at 236 nm using a UV/Vis DAD.

Results: The calibration curves were linear (r > 0.9998) in the concentration ranges of 50-1600 ng/mL for metformin in plasma. The assay enables the measurement of metformin for therapeutic drug monitoring with a minimum detectable limit of 18 ng/ml. The coefficients of variation for inter-day and intra-day assay were within the range of clinical usefulness. Absolute recovery was found to be > 90% for all three concentrations of plasma quality controls studied.

Conclusion: The proposed method was found to be rapid, precise and accurate for quantification of metformin in human plasma. This method was successfully applied to a pharmacokinetic study at humans through oral administration.