Thiol radical-based chemical isotope labelling for sterols quantitation through high performance liquid...

Enlightened by the high specificity and reactivity of thiol radical toward allyl, here, we first established a rapid thiol radical-based chemical isotope-labelling (CIL) strategy coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis for the quantitative profiling of sterols. In this strategy, N-(4-(carbazole-9-yl)-phenyl)-N-maleimide labelled derivative of ethylenedithiol (NCPM-d₀-SH) and its deuterated analogue NCPM-d₂-SH were employed as a novel pair of CIL reagents to efficiently label sterols. Under lighting condition, the thiol radical obtained from NCPM-d₀/d₂-SH attacks one allyl hydrogen in the B-ring of sterols to produce a reactive radical intermediate which can quickly react with another thiol radical to form the last labelled derivatives. This labelling reaction can rapidly complete only within 1.5 min. Absorbingly, the NCPM-d₀-SH and NCPM-d₂-SH labelled derivatives of sterols can produce two specific product ions (PIs) containing different isotope tags at m/z of 431.6 and 433.6 via collision induced dissociation, which were employed to develop the multiple reaction monitoring (MRM) mode-based analysis. According to the specific mass differences with a fixed value, the peak pairs with similar retention times can be easily extracted from the two PIs spectrums and designated as the candidates for the identification of sterols. NCPM-d₀-SH and NCPM-d₂-SH labelled derivatives of sterols can be readily distinguished from their several ion chromatograms. Thus, sterols from two samples labelled by different isotope tags were ionized at the same conditions and measured respectively, providing excellent identification and precise quantitation by compensating the matrix effect and instrument fluctuation during MS-based analysis. The detection sensitivities of thiol-containing drugs improved by 53.5-560.3-fold due to NCPM-labelling. The limits of detection (LODs) and the limits of quantitation (LOQs) were in the range of 0.15-0.40 μg kg^-1 and 0.50-1.30 μg kg^-1, respectively. Using the developed method, we quantitatively profiled five sterols in vegetable oils with good applicability. As promising, the proposed thiol radical-based CIL strategy is a potential platform for the quantitation of sterols.