Separation, isolation and stereochemical assignment of imazalil enantiomers and their quantitation in an in vitro toxicity test

•Enantiomers of imazalil were isolated using HPLC with an enantioselective column.•Assignment of the absolute configuration by different chiroptical methods.•An enantioselective HPLC–MS/MS method was developed to quantify imazalil enantiomers in aqueous samples.•HPLC–MS/MS analyses validated enantiomer exposure in toxicological in vitro-tests. A simple method for the separation of the enantiomers of the fungicide imazalil was developed. Racemic imazalil was separated into its enantiomers with an enantiomeric purity of 99% using HPLC-UV with an enantioselective column (permethylated cyclodextrin) operated in reversed phase mode (water with 0.2% trimethylamine and 0.08% acetic acid and methanol). The absolute configuration of the separated enantiomers was assigned and unequivocally confirmed by optical rotation as well as by vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) combined with ab-initio calculations. The same enantioselective column was also used to develop an HPLC–MS/MS method for the quantification of imazalil enantiomers. The HPLC–MS/MS method reached limits of quantification (LOQs) of 0.025mg/mL with 5μL injections. This method was used to verify imazalil concentrations and enantiomeric fractions in samples from an in vitro test on effects on human steroidogenesis (H295R steroidogenesis assay). The quantification verified the stability of the enantiomers of imazalil during the in vitro tests.