Quantitation of lanosterol and its major metabolite FF-MAS in an inhibition assay of CYP51 by azoles with atmospheric pressure photoionization based LC-MS/MS

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Abstract
Azoles affect the steroid balance in all biological systems and may therefore be called endocrine disrupters. Lanosterol 14α-demethylase (CYP51) is an enzyme inhibited by azoles. Only few data have been reported showing their inhibitory potency since an assay in an in vitro system is not available so far. In the present work an inhibition assay using human recombinant CYP51, coexpressed with human P450 oxido-reductase by the baculovirus/insect cell expression system, and LC-MS/MS as analytical method is described. Atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI) sources were used with a triple quadrupole mass spectrometer to compare quantitation of lanosterol (substrate) and 4,4-dimethyl-5α-cholesta-8,14,24-triene-3β-ol (FF-MAS) (product of CYP51) with d6-2,2,3,4,4,6-cholesterol (d6-cholesterol) as internal standard. Optimization of analytical parameters resulted in a LC-APPI-MS/MS method with a LOQ of 10 pg on column for FF-MAS. The sensitivity of the method (LOD 0.5 ng/ml) makes it possible to analyze supernatants of inhibition experiments after precipitation of proteins by isopropanol without any sample enrichment. The coefficient of variation of the analytical method was n=5) for FF-MAS, lanosterol and d6-cholesterol. The external calibration curve was linear from 1 to 10,000 ng/ml with R2≥0.999 and an accuracy of 94–115%. Compared with APCI, APPI provides a ten- to 500-fold increase in sensitivity for the analytes in this study. IC50 values of epoxiconazole and miconazole—two widely used azole fungicides used in agriculture and in human medicine, respectively—were 1.95 µM and 0.057 µM.

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