Effect of different anticoagulants in determining metformin hydrochloride levels in human plasma using high-performance liquid chromatography

Objective: This study aimed to develop and validate an analytical method and to determine the effect of different anticoagulants for analyzing metformin HCl level in human plasma. Methods: Analysis was performed with high-performance liquid chromatography method using a photodiode array detector set at 234 nm, a C₁₈ SunFire™ column (5 µm, 250 mm×4.6 mm), a temperature of 40°C, a mobile phase comprising 10 mM sodium dodecyl sulfate and 10 mM phosphate buffer in water-acetonitrile (60:40 v/v) at pH 7.0, with a flow rate of 1.0 mL/min, and atorvastatin calcium as the internal standard. Plasma extraction was performed through protein precipitation using human plasma (300 µL) and acetonitrile (600 µL; 1:2 v/v). The method was linear at a concentration range of 20.0-5000.0 ng/mL, with r>0.9998. Results: The stability and recovery of metformin HCl in human plasma were not different between citrate with ethylenediaminetetraacetic acid (EDTA) and heparin with EDTA as anticoagulants. However, a significant difference in the peak area response ratio (p<0.05) was observed between the three anticoagulants. Conclusion: The validation results for the three types of plasma anticoagulants met validation requirements based on the European Medicines Agency bioanalytical guidelines of 2011 for accuracy and precision, selectivity, calibration curve linearity, dilution integrity, carry-over, and stability.