Analysis of hydroxylated and N-dealkylated metabolites of terfenadine in microsomal incubates by liquid chromatography--mass spectrometry.

This report describes an assay for the H(1)-receptor antagonist, terfenadine, and its two primary metabolites, terfenadine alcohol (TOH) and azacyclonol (AZ), using positive-ion, electrospray ionization-liquid chromatography-mass spectrometry. The assay was developed in support of kinetic studies of terfenadine oxidative metabolism in human liver and intestinal microsomes, which required quantification of incubate metabolites at low nanomolar concentrations. Terfenadine metabolites were extracted from basified microsomal incubates into methylene chloride. Reconstituted extracts were subject to liquid chromatographic separation on a cyano-reverse phase column. The [M+H]+ ions of terfenadine, terfenadine metabolites, and internal standard were monitored in the effluent by quadrupole mass spectrometry. The assay demonstrated linearity over an incubate concentration range of 5-250 and 12.5-1250 ng/ml for the metabolites and the parent drug, respectively. The respective limits of detection and quantitation for all three analytes were 1.5 and 5 ng/ml of microsomal incubate. Replicate analysis of quality control samples exhibited intra-day coefficients of variation ranging from 3.3% to 7.8% for the three analytes. The corresponding inter-day coefficients of variation ranged from 4.2% to 8.6%. The reproducibility and sensitivity of the assay, combined with the selectivity of mass spectrometric detection, should allow an accurate kinetic characterization of terfenadine oxidation mediated by the high affinity CYP3A enzymes in human liver and intestinal microsomes.