Cyclodextrin modified micellar electrokinetic chromatography for the chiral direct resolution of (+), (−)‐trans‐1,2‐dihydrodiol metabolite of chrysene in vitro activated by rat liver microsome S9 fraction

Abstract

A γ‐cyclodextrin (γ‐CD) modified electrokinetic micellar capillary chromatography (MEKC) method was used for the enantiomer separation of a racemic trans‐1,2‐dihydro‐1,2‐dihydroxy‐chrysene (chry‐trans‐1,2‐diOH) mixture. The chiral resolution was strongly influenced by several important parameters: surfactant concentration and addition of organic modifier to the background electrolyte (BGE). An optimized electrophoretic system was used, consisting of the following conditions: 25 mM phosphate buffer, pH 7.8, 50 mM sodium dodecyl sulfate, 20 mM γ‐CD, 7.4% V/V 2‐propanol as BGE; the applied voltage, 18 kV, corresponded to 37 μA at a constant temperature of 25°C. This electrophoretic method was applied for monitoring the chry‐trans‐1,2‐diOH enantiomer formation in a real sample, obtained from in vitro metabolic activation of chrysene by phenobarbital‐ß‐naphthoflavone‐induced rat microsomes. The (+) and (‐) enantiomers were identified by the racemate and the single enantiomer standard addition method and by spectra comparison with the synthetic compound. Under the experimental conditions used for chrysene activation, the (+) optical isomer was the prevailing form. The CD‐MEKC system showed high reproducibility and selectivity, allowing a fast and interference‐free analysis even of the in vitro metabolic sample extract, without any pretreatment.