title: Separation, isolation and stereochemical assignment of imazalil enantiomers and their quantitation in an in vitro toxicity test.
abstract: 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.
authors.affiliation: Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark.
authors.forename: Mònica Escolà
authors.initials: ME
authors.lastname: Casas
authors.affiliation: Section of Analytical Biosciences, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 København Ø, Denmark.
authors.forename: Andreas Christopher
authors.initials: AC
authors.lastname: Kretschmann
authors.affiliation: Institute of Organic Chemistry, Johannes Gutenberg-University, Duesbergweg 10-14, 55128 Mainz, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Andernach
authors.affiliation: Institute of Organic Chemistry, Johannes Gutenberg-University, Duesbergweg 10-14, 55128 Mainz, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Opatz
authors.affiliation: Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark. Electronic address: kb@envs.au.dk.
authors.forename: Kai
authors.initials: K
authors.lastname: Bester
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005659:Fungicides, Industrial / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity; D006801:Humans; D007093:Imidazoles / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification* / Q000633:toxicity; D066298:In Vitro Techniques; D009904:Optical Rotation; D013237:Stereoisomerism; D013256:Steroids / Q000096:biosynthesis; D053719:Tandem Mass Spectrometry; D018675:Toxicity Tests; D014732:Vibration
chemical_list: D005659:Fungicides, Industrial; D007093:Imidazoles; D013256:Steroids; C017435:enilconazole
keywords: Enantiomers; HPLC-UV; HPLC–MS/MS; Imazalil; Isolation; Vibrational circular dichroism