Fenotipizzazione del CYP 2E1 in vivo come biomarker di esposizione, effetto e suscettibilità a xenobiotici: una nuova metodica

Abstract A modification of a high-performance liquid Chromatographic (HPLC) assay was developed for the quantitation of chlorzoxazone (CHZ) and its major metabolite 6-hydroxychlorzoxazone(6-OH-CHZ). These compounds along with phenacetin, the internal standard, were extracted from human serum using ter-butyl-methyl ether extraction. The extracts were analyzed by HPLC on a C18 ODS 5 μm column with a mobile-phase of acetonitrile-0.5% acetic acid (30:70, v/v). The assay utilized UV detection at 287 nm. The mean correlation coefficient of the standard curves for 6-OH-CHZ and CHZ was 0.9994 and 0.9996, respectively, over the range of 0.05-5 μg/ml and 0.25-5 μg/ml, respectively. The inter-day and intra-day coefficients of variation were <12 and <9%, respectively. The advantages of the new method towards the original technique are: an increased accuracy due to the introduction of an internal standard, a decreased preparation time of the sample due to the volatility of ter-butyl methyl ether, a sensible reduction of the retention time by means of a new column, a larger number of samples measurable in the same time and safer conditions for the operator due to the use of the vacuum system.