White analytical chemistry-driven, blue-guided LC-MS/MS method for simultaneous quantification of metformin and sitagliptin in human plasma

Abstract

Abstract A liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of metformin (MET) and sitagliptin (SIT) in human plasma. Chromatographic separation was achieved on an Endurus C18 pro-XR 4.6 × 100 mm column, using methanol and 2 mM ammonium formate (0.1% formic acid, pH 3) (50:50, v/v) under isocratic conditions at a flow rate of 0.6 mL/min with a total run time of 5 min. Conversely, sequential protein precipitation was employed for both drugs. The operational settings for MET, SIT, and their internal standards were optimized using Multiple Reaction Monitoring (MRM) with ESI operated in positive ion mode. The protonated precursor ions $$\:\left[M+H{]}^{+}\right.$$ were monitored at m/z 130.06 for MET, m/z 136.1 for MET-d6, m/z 408.11 for SIT, and m/z 412.11 for SIT-d4. The retention times of MET and SIT were 2.40 and 2.98 min, respectively. The method exhibited linearity over concentration ranges of 2.983–2983.2 ng/mL for MET and 1.986–794.4 ng/mL for SIT, with corresponding LLOQ values of 2.983 and 1.986 ng/mL (r 2 − 0.9995 and 0.999). Intra- and inter-day precision (%CV) ranged from 0.94 to 11.12 and 1.21–7.08, respectively, while accuracy ranged from 96.24 to 103.02% and 98.45–105.18%. Mean recoveries of MET and SIT were 86.12% and 94.61%, respectively, with negligible matrix effects. Furthermore, stability studies confirmed acceptable analyte stability under benchtop, autosampler, freeze-thaw, and whole blood conditions. Notably, the green-blue-white analytical chemistry assessment of the developed method for simultaneous determination of MET and SIT in human plasma was evaluated using AGREE, AGREE Prep, MoGAPI, BAGI, CACI, and RGB (12 algorithms), yielding scores of 0.73, 0.70, 76, 67.5, 67, and 77.3%, respectively, indicating balanced environmental, analytical, and practical performance for routine pharmaceutical and clinical bioanalysis approaches.