Use of isotope differential derivatization for simultaneous determination of thiols and oxidized thiols by liquid chromatography tandem mass spectrometry

Here we report a new isotopic pair of derivatization reagents, ω-bromoacetonylquinolinium bromide (BQB) and d⁷-ω-bromoacetonylquinolinium bromide (d⁷-BQB). BQB and d⁷-BQB both rapidly and selectively reacted with thiols in acidic medium within 3 min with the aid of a microwave. Reduced thiols and total thiols in urine were labeled with BQB and d⁷-BQB, respectively. The BQB- and d⁷-BQB-labeled urine samples were then mixed and separated on a HILIC (hydrophilic interaction chromatography) column followed by electrospray ionization tandem mass spectrometry (ESI–MS/MS) detection. The new strategy, which we have named isotope differential derivatization, allows us to simultaneously determine thiols and oxidized thiols in a single run. Compared with positive mode ESI detection of unlabeled thiols, the positive mode ESI–MS signal intensities of BQB-labeled thiols were found to increase by 10-, 20-, and 40-fold for cysteine (Cys), homocysteine (HCys), and glutathione (GSH), respectively (unlabeled N-acetylcysteine (Nac) is difficult to detect by ESI–MS in positive mode due to its low ionization efficiency). The detection limits calculated at a signal-to-noise ratio of 3 were found to be 8.02, 1.56, 0.833, and 3.27 nmol/L for Cys, HCys, Nac, and GSH, respectively. Recoveries of thiols and disulfides from spiked urine samples were between 80% and 105%. The method was successfully used to determine thiols and oxidized thiols in urine samples of 25 healthy volunteers.