Azoles affect the steroid balance in all biological systems and may therefore be called endocrine disrupters. Lanosterol 14␣-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-5␣-cholesta-8,14,24-triene-3␤-ol (FF-MAS) (product of CYP51) with d 6 -2,2,3,4,4,6-cholesterol (d 6 -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 Ͻ20% (n ϭ 5) for FF-MAS, lanosterol and d 6 -cholesterol. The external calibration curve was linear from 1 to 10,000 ng/ml with R 2 Ն 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. IC 50 values of epoxiconazole and miconazole-two widely used azole fungicides used in agriculture and in human medicine, respectively-were 1.95 M and 0.057 M. (J Am Soc Mass Spectrom 2004, 15, 1216 -1221

The only relevant source for human exposure to dinitropyrenes is diesel engine emissions. Due to this specificity, dinitropyrenes may be used as biomarkers for monitoring human exposure to diesel engine emissions. Only few analytical methods have been described for the quantitation of dinitropyrenes and their metabolites, aminonitropyrenes, and diaminopyrenes. Therefore, for dinitropyrenes, aminonitropyrenes, and diaminopyrenes were selected as model compounds for the development of a sensitive HPLC-MS/MS method (high performance liquid chromatography coupled to triple quadrupole mass spectrometry) was to quantify polyaromatic amines and nitroarenes in biological matrices was developed optimal methods by comparing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) sources. Dinitropyrene was not effectively ionized and diaminopyrene yielded mainly [M(.)](+) ions by electrospray ionization. With APCI and APPI, precursor ions of diaminopyrene and aminonitropyrene were [M + H](+) and [M(.)](-) for dinitropyrene. Precursor ions with [M - 30(.)](-) for dinitropyrene and [M - 30 + H](+) for aminonitropyrene were observed. Reversed and normal phase HPLC-MS/MS with ESI, APCI and APPI were optimized separately with respect to unequivocal analyte identification and sensitivity. Normal phase HPLC coupled to APPI-MS/MS gave the highest precision and sensitivity for aminonitropyrene (6%/0.2 pg on column) and dinitropyrene (9%/0.5 pg on column). The limit of detection in spiked rat plasma was 5 pg/100 microL for aminonitropyrene (accuracy 82%) and 10 pg/100 microL for dinitropyrene (accuracy 105%). In plasma of rats treated with dinitropyrene by oral administration, no detectable levels of dinitropyrene but higher aminonitropyrene levels compared with intratracheal instillation were observed. These findings clearly demonstrate that dinitropyrene was absorbed after oral and intratracheal application and that a reduction of nitro groups occurs to a high extent in the reductive environment of the intestine. To our knowledge, this is the first time that aminonitropyrene was observed in plasma after intratracheal or oral administration directly demonstrating the reductive metabolism of dinitropyrene in vivo.

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Diesel engine emissions have been classified as a potential human carcinogen and may cause a variety of other health effects. Human exposure to diesel engine emissions is highly variable within the population. Therefore, specific methods for the biomonitoring of human exposure to diesel engine emissions are required for exposure assessment within epidemiological studies. Haemoglobin adducts of dinitropyrenes may serve as biomarkers for human exposures to diesel engine emissions.To characterize structures of dinitropyrene reaction products with sulfhydryl groups, glutathione was used to trap electrophilic nitroso intermediates formed from dinitropyrenes and glutathione S-conjugates were identified and characterized by Qtrap techniques using (HPLC-MS/MS) high-performance liquid chromatography coupled to triple quadrupole mass spectrometry. Nitrosonitropyrene-derived sulfinamides, sulfenamides and glutathione thioethers bound to carbon atoms in the aromatic ring, presumably formed by a rearrangement of intermediate sulfenamide cations, were formed in low yields. In haemoglobin from rats orally administered dinitropyrenes, mild alkaline hydrolysis of haemoglobin released aminonitropyrenes, which were identified by HPLC-MS/MS. The results demonstrate that dinitropyrenes undergo nitroreduction in rats and that the intermediate nitrosonitropyrenes bind to heamoglobin. The haemoglobin adducts formed from dinitropyrenes seem, in contrast to previous studies, to be hydrolysable and thus represent sulfen- and sulfinamides derived from the intermediate nitrosonitropyrenes. The developed Qtrap methods to detect and characterize glutathione S-conjugates rapidly may have wide applications in attempts to characterize reactive intermediates formed in complex mixtures in low concentrations.