Enantioselective metabolism of the chiral herbicide diclofop-methyl and diclofop by HPLC in loach (Misgurnus anguillicaudatus) liver microsomes in vitro

• Diclofop-methyl and diclofop were simultaneously enantioseparation on Chiralpak IC.
• Diclofop-methyl was rapidly degraded to diclofop by esterases from liver microsomes.
• The biotransformation was configurationally stable by single-enantiomer incubation.
• The degradation of diclofop-methyl in loach liver microsomes was enantioselective.
• The (S)-diclofop-methyl degraded markedly faster than the (R)-enantiomer did.

An efficient and reliable enantioseparation method by high-performance liquid chromatography (HPLC) for the chiral herbicide diclofop-methyl (DM) and its primary metabolite diclofop (DC) was developed and validated, and the biological process of the enantiomers in loach liver microsomes (LLM) in vitro was investigated. The enantiomers of DM and DC were first separated on an immobilized-type stationary phase of Chiralpak IC. The best resolutions were achieved under the chromatographic condition of n-hexane/IPA/TFA 96:4:0.1(v/v/v) at 20 °C with each Rs > 2 and the two pairs of enantiomers could be eluted in about 10 min. The extraction recoveries of the analytes from LLM were 79.6–108.9% with RSD ≤ 11.5%. The enzyme kinetics of DM enantiomers were different, with the Km value 320.7 for (S)-DM and 392.1 for (R)-DM. The metabolism experiment in vitro showed DM underwent a rapid phase-I metabolism with or without NADPH, indicating the esterases in liver played a dominant role. No interconversion between the two enantiomers was observed by single-enantiomer incubation. The preferential degradation of (S)-DM was confirmed with the half-time (t1/2) of (S)-DM 5.27 min and 21.2 min for (R)-DM. DC enantiomer was generated by its corresponding ester form and could not be further degraded during the incubation. It was the first study on biotransformation of DM and DC enantiomers in LLM in vitro. The results may help to evaluate the ecological risks of chiral pesticides.