Eco-friendly microextraction technique using SFO-DLLME coupled with HPTLC for plasma analysis of metformin and...

This study aimed to develop and validate a green, sensitive, and high-throughput analytical method for the simultaneous determination of metformin and imeglimin in plasma samples using gliclazide as an internal standard. A novel microextraction technique was proposed, combining salt-induced dispersive liquid-liquid microextraction based on solidified floating organic droplet (SI-DLLME-SFOD) with high-performance thin-layer chromatography HPTLC-densitometric detection. This approach significantly improves greenness, sensitivity, and simplicity compared to conventional extraction and chromatographic techniques. The method was optimized for maximum resolution using silica gel 60 F254 pre-coated TLC plates as the stationary phase and a mobile phase comprising methanol: chloroform: 0.5 % w/v ammonium acetate (8:1:1, v/v/v). Detection was performed at 254 nm. The method was validated following US FDA bioanalytical guidelines. The R_f values were observed at 0.45 ± 0.04 for imeglimin, 0.63 ± 0.04 for metformin, and 0.84 ± 0.04 for gliclazide. The method exhibited excellent linearity in the range of 100-1000 ng/band, with correlation coefficients (r²) exceeding 0.998. The average recoveries were 90.91 % for imeglimin and 92.61 % for metformin, with RSD values of 0.42 and 0.39, respectively, confirming high precision and accuracy. The SI-DLLME-SFOD protocol enables efficient extraction, lower solvent consumption, and improved analyte enrichment. The developed method was evaluated using green analytical chemistry metrics and its eco-friendliness was confirmed. It demonstrated reduced solvent usage, minimal waste generation, and a lower environmental burden, establishing its superiority over previously reported sample preparation techniques. Furthermore, the validated method was successfully applied to real samples (human plasma) to assess the concentration, thereby demonstrating the potential value of the method for routine use. This method provides a green, efficient, and validated strategy for the bioanalysis of antidiabetic drugs in human plasma, supporting its potential application in routine clinical diagnostics and therapeutic drug monitoring.