A new RP-HPLC method for metformin determination in human serum

Background: Anticoagulant in the sample bottles could form complex with metformin thereby affecting its concentration. This necessitates the need to develop HPLC method for determination of metformin in serum

Objectives: This study was aimed at developing a simple, less tedious RP-HPLC method for the determination of metformin in human serum.

Methods: Blank serum (2 mL) was spiked into solution containing 2 mL metformin (2-65 µg mL-1) and 1 mL caffeine (5.0 µg mL-1) as internal standard (IS) centrifuged at 3000 rpm for 5 minutes. Portion (0.5 mL) of the resultant solution was injected into the HPLC auto sample machine (Agilent 1260 infinity). The optimized conditions included a mobile phase of methanol-water (80:20 v/v) containing 0.1 % orthophosphoric acid, isocratic elusion mode, an injection volume of 10 µL, flow rate of 1.2 mL min-1, at 35 °C and detection wavelength of 255 nm. Calibration curve (0.40 to 12.2 µg mL-1) was constructed by plotting the peak area ratios against their corresponding concentrations. The method was validated according to ICH guidelines.

Results: Metformin and caffeine eluted at 1.29 and 1.42 minutes respectively. The method was precise (4.77 % RSD), accurate (% Er of 0.22 and % recovery of 99.78 %) with a linear calibration curve (r = 0.988). LOD and LOQ of the developed method are 1.93 and 5.87 µg/mL respectively. All the parameters were within the acceptable limits.

Conclusion: The developed method was found to be simple, precise, accurate and rapid for the routine therapeutic metformin monitoring in human serum.