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

Abstract

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

Quantitative determination of cholesterol, sitosterol, and sitostanol in cultured Caco-2 cells by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

“…Because sterols are highly lipophilic with few polar functional groups, they are difficult to ionize through conventional electrospray methods [16,21]. APCI ionization is usable but is not the most sensitive method.…”

“…APCI ionization is usable but is not the most sensitive method. Electron impact (EI) ionization in particular and the recently developed atmospheric pressure photoionization (APPI) are effective alternatives for lipophilic compounds [19][20][21]. However, APCI ionization is most widely used for the sterols analysis, APCI instruments are commonly available, and APCI ionization can be easily coupled with HPLC system.…”

Dopant-assisted atmospheric pressure photoionization mass spectrometry of polyisobutylene derivatives initiated by mono- and bifunctional initiators

“…This method has been successfully applied to the analysis of various classes of nonpolar compounds, such as polyaromatic hydrocarbons [20 -22], lipids [23,24], and steroids [25][26][27][28]. Very recently, we have shown that the negative ion mode of APPI-MS is also capable of the analysis of low molecular weight polyisobutylene [29] and polyethylene derivatives [30] by means of the formation of adducts of these polymers with chloride ions formed "in situ" from chlorinated solvents, e.g., CCl 4 , CH 2 Cl 2 , in the presence of toluene dopant.…”

APPI-MS: Effects of mobile phases and VUV lamps on the detection of PAH compounds

“…Recent work has focused on the direct comparison of different sources (APPI, APCI, and ESI) with specific target compounds, e.g., polyaromatic hydrocarbons [6,7], hydrophobic peptides [8], pesticides [9,10], as well as fatty acids and lipids [4,5]. In many cases atmospheric pressure photoionization (APPI) [1,7] has demonstrated extended linear dynamic range [11], enhanced sensitivity and thus lower detection limits [6,9,[12][13][14][15], and reduced or no off-line sample cleanups [6,9] in comparison with direct APCI or ESI. Adding a dopant (dopant-assisted, DA) to the mobile phase in many cases can further increase sensitivity [2].…”

“…(J Am Soc Mass Spectrom 2007, 18, 589 -599) © 2007 American Society for Mass Spectrometry M ethods to detect nonpolar compounds by liquid chromatography-mass spectrometry (LC-MS) have advanced significantly over the last few years, owing to development of newer source technologies [1][2][3] and improved choices of mobile phase(s) for chromatographic separation [4,5]. Recent work has focused on the direct comparison of different sources (APPI, APCI, and ESI) with specific target compounds, e.g., polyaromatic hydrocarbons [6,7], hydrophobic peptides [8], pesticides [9,10], as well as fatty acids and lipids [4,5]. In many cases atmospheric pressure photoionization (APPI) [1,7] has demonstrated extended linear dynamic range [11], enhanced sensitivity and thus lower detection limits [6,9,[12][13][14][15], and reduced or no off-line sample cleanups [6,9] in comparison with direct APCI or ESI.…”