Development and Validation of a Simple and Sensitive LC-MS/MS Method for Quantification of Metformin in Dried Blood...

Metformin (MET), an oral biguanide agent, can improve insulin resistance and decrease hepatic glucose production, leading to a reduction in blood-sugar levels. The objective of the present study was to develop and validate simple and rapid LC-MS/MS method for analysis of MET in dried blood spot (DBS) sample for patient monitoring studies purposes (drug adherence).

The chromatographic separation was achieved with Waters HSS-T3 column using gradient elution of mobile phases of two solvents: 1) solvent A, consisted of 10mM ammonium formate, 0.2% formic acid 1%; and 2) acetonitrile solvent B, contained 0.2% formic acid in acetonitrile at a flow rate of 0.2 mL/min. The total run time was 3.0 min. The effectiveness of chromatographic conditions was optimized, and afatinib was used as the internal standard. The assay method was validated using USP 26 and the ICH guidelines.

The method showed good linearity in the range 8-48 ng/mL for MET with correlation coefficient (r) >0.9907. The intra- and inter‑day precision values for MET met the acceptance criteria as per regulatory guidelines. MET was stable during the stability studies at ambient temperature 25 °C, at refrigerator 4 °C, at 10 °C autosampler, freeze/thaw cycles and 30 days storage in a freezer at -30 ± 0.5 °C.

This method has successfully fulfilled all validation requirements referring to EMA and FDA guidelines, and successfully can be applied for MET adherence study. All the six studied patients were approved to metformin adherence.