A new RP-HPLC method for metformin determination in human serum
Nasir Ibrahim , Amina J. Yusuf , Abdullahi M. Ismail , Musa Aminu , Awwalu Salisu
1Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria.
2Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria.
Corresponding author: Nasir Ibrahim
Email: thenasir25@gmail.com, nasir.ibrahim@udusok.edu.ng;
Phone: +2348035169018
ABSTRACT
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.
Keywords: Therapeutic metformin monitoring, RP-HPLC, serum, isocratic elution.