High-performance liquid chromatographic method for the determination of (−)-verbenone 10-hydroxylation catalyzed by rat liver microsomes

A sensitive assay for the determination of (−)-verbenone 10-hydroxylation catalyzed by rat liver microsomes was developed using high-performance liquid chromatography. Verbenone was incubated in vitro with liver microsomes of untreated rats and rats treated with phenobarbital and the products thus formed were extracted with CH2Cl2 and the extracts were separated by HPLC with a C18 5-μm analytical column. Elution was conducted with 40% methanol containing 20 mM NaClO4 and the detection of UV absorbance was done at 251 nm. Product formation was dependent on the incubation time at least up to 30 min and the microsomal protein concentration between 0.01 and 0.1 mg protein/ml. The limit of detection of (−)-10-hydroxyverbenone with the HPLC was found to be about 40 pg, indicating that this method is about 100-fold sensitive than the GC–MS method. Optimized pH for the reaction was at 7.4 when examined with 100 mM potassium phosphate buffer in different pHs. Kinetic analysis showed that Km values for liver microsomes of untreated and phenobarbital-treated rats were 206 and 41 μM and Vmax values were 5.8 and 44 nmol/min/mg protein, respectively. Thus the present results provided a sensitive and useful method for the determination of verbenone 10-hydroxylation catalyzed by rat liver microsomes.