Development and validation of a green DLLME-LC-MS/MS method for apixaban quantification in human plasma using...

A novel dispersive liquid-liquid microextraction coupled with liquid chromatography-tandem mass spectrometry method (DLLME-LC-MS/MS) was developed and validated for apixaban quantification in human plasma. Box-Behnken experimental design was employed to systematically optimize DLLME parameters including disperser solvent volume (acetonitrile, 500-1500 μL), extraction solvent volume (ethyl acetate, 100-250 μL), sample pH (3-9), and centrifugation time (3-9 min). The optimized DLLME conditions achieved 97.97% extraction recovery with excellent precision, consuming only 1.5 mL total organic solvents per sample and representing significant solvent reduction compared to conventional extraction methods. Chromatographic separation was achieved on a Poroshell 120 EC-C18 column with 5-min isocratic elution using positive electrospray ionization tandem mass spectrometry detection. The method demonstrated excellent analytical performance with lower limit of quantification of 1 ng/mL, wide linear range of 1-1000 ng/mL, and comprehensive validation meeting ICH M10 guidelines for selectivity, accuracy, precision, recovery, matrix effects, and stability. Clinical applicability was confirmed through pharmacokinetic studies in healthy volunteers following single 5 mg apixaban administration, with pharmacokinetic parameters (C_max 107.12 ng/mL, t_max 4 h, t_½ 11.16 h, AUC_0→∞ 1706.82 ng·h/mL) demonstrating excellent agreement with published literature values. Multi-metric greenness assessment yielded favorable scores: MoGAPI 70%, CaFRI 72/100, BAGI 75.0/100, and WAC RGB 12 whiteness 80.1%, confirming superior environmental sustainability and practical applicability. The developed method provides a robust, sensitive and green analytical platform suitable for therapeutic drug monitoring, bioequivalence studies, and pharmacokinetic investigations of apixaban.