Background: Metformin is an oral antidiabetic drug from the biguanide class and it is the first-line drug chosen for the treatment of type 2 diabetes. This is a drug on the list of pharmaceutical substances required to report bioequivalence study data when registering the drug.

Objectives: (1) To develope a capillary zone electrophoresis method for determining metformin hydrochloride in human plasma. (2) To validate the method. Materials and methods: Human plasma, metformin hydrochloride, ranitidine hydrochloride. The method was developed and validated according to US-FDA 2018 and EMA 2011.

Results: The procedure was developed by using the Agilent 7100 CE electrophoresis system with ranitidine hydrochloride as an internal standard. Sample preparation was accomplished through protein precipitation with acetonitrile. The optimal electrophoresis conditions are as follows: uncoated fused-silica capillary column of a total length of 40 cm (31.5 cm effective length, inner diameter 100 μm), phosphate buffer solution 100 mM (pH = 4), the voltage applied to both capillary ends of 15 kV, the inlet end of capillary dipped in water before sample injection, sample injection mode of 50 mBar in 7 seconds, using a PDA detector at 232 nm. The analysis method was validated according to the requirements of the US-FDA 2018 and EMA 2011 with the following criteria: system suitability with RSD < 3%; good specificity; the calibration curves were linear (r2 ≥ 0.98) in the concentration range of 0.1 – 4.0 μg/ml for metformin in human plasma; the lower limit of quantification was 0.1 μg/ml; the intra-day and inter-day accuracy were 99.41 – 105.28% and 92.47 – 106.26%, respectively; the intra-day and inter-day precision were 1.99 – 4.03% and 3.11 – 6.05%, respectively; the mean recovery of ranitidine (internal standard) was 86.2%, the mean recoveries of metformin at three levels LQC, MQC, and HQC were 72.9%, 75.9%, and 77.4%, respectively; plasma samples were stable to analysis.

Conclusions: The developed method meets the requirements of US-FDA 2018 and EMA 2011 to determine metformin in plasma.