Box-Behnken optimized salting-out assisted liquid-liquid extraction coupled with LC-MS/MS for sustainable amisulpride...

A sustainable salting-out assisted liquid-liquid extraction coupled with liquid chromatography-tandem mass spectrometry (SALLE-LC-MS/MS) method was developed and validated for amisulpride quantification in human plasma according to ICH M10 guidelines. Box-Behnken experimental design systematically optimized SALLE parameters through evaluation of sample pH (5.0-10.0), acetonitrile volume (500-1500 μL), salt concentration (2.0-6.0 mol/L), and centrifugation time (3.0-10.0 min). The developed polynomial model demonstrated strong predictive capability with subsequent numerical optimization identifying optimal conditions of pH 8.5, acetonitrile 1200 μL, ammonium acetate 4.5 mol/L, and 3-minute centrifugation. These conditions achieved 97.6% extraction recovery for amisulpride with minimal deviation from predicted values. Following optimization, chromatographic separation was achieved using a C18 column with isocratic elution (acetonitrile:water with 0.1% formic acid, 70:30 v/v) and 4-minute analysis time, with multiple reaction monitoring employing transitions m/z370.1→242.1 for amisulpride and m/z376.2→165.1 for haloperidol internal standard. Comprehensive validation subsequently demonstrated linearity across 2-1500 ng/mL with acceptable accuracy, precision, and stability under clinical storage conditions. Matrix effects ranged from 88-105% with low variability, while extraction recovery exceeded 98% across all quality control levels. Clinical application in healthy volunteers (n = 5) following 200 mg oral amisulpride administration successfully characterized key pharmacokinetic parameters including C_max (506 ng/mL), t_max (3.8 h), and t_1/2 (13.3 h) over 48 h. Finally, multi-metric sustainability assessment using CaFRI (75/100), BAGI (77.5/100), and RGB12 (83.1/100 whiteness) frameworks confirmed achievement of white analytical chemistry through balanced analytical performance, environmental friendliness, and practical implementation feasibility. The method offers significant advantages including minimal waste generation, reduced solvent consumption, and enhanced throughput while maintaining regulatory compliance for amisulpride therapeutic drug monitoring applications.