DLLME and LC-MS for stereoselective hydrolysis of acephate and methamidophos in human liver microsomes: Enzyme characterization and in vitro-in vivo extrapolation

Acephate (ACP) and its metabolite methamidophos (MTD) are chiral organophosphate pesticides with recognized neurotoxic potential. However, due to analytical challenges in separating their enantiomers, the stereoselective metabolism of these compounds in humans remains poorly understood. The present work reports the first tandem chiral-chiral LC-MS approach under the polar organic mode, combined with a chemometrically optimized dispersive liquid-liquid microextraction (DLLME) method, for the simultaneous enantioseparation and extraction of ACP and MTD from human liver microsomes. Using a serial arrangement of Chiralpak IG-3 and Chiralpak AS-H columns with an acetonitrile-based mobile phase containing acetic acid, stereoselective metabolism was characterized, key hydrolytic enzymes involved were identified, and in vitro findings were extrapolated to predict in vivo hepatic clearance. This analytical setup enabled accurate and precise quantification of the enantiomers, revealing negligible metabolism for ACP, whereas (S)-(−)-MTD was metabolized 2.5-fold faster than its (R)-(+)-enantiomer, resulting in total hepatic clearance values of 5 and 2 mL min−1 kg−1, respectively. Carboxylesterase 1 was identified as the primary enzyme responsible for MTD hydrolysis, with contributions from other hydrolases yet to be determined.