title: Use of isotope differential derivatization for simultaneous determination of thiols and oxidized thiols by liquid chromatography tandem mass spectrometry.
abstract: Here we report a new isotopic pair of derivatization reagents, ω-bromoacetonylquinolinium bromide (BQB) and d(7)-ω-bromoacetonylquinolinium bromide (d(7)-BQB). BQB and d(7)-BQB both rapidly and selectively reacted with thiols in acidic medium within 3min with the aid of a microwave. Reduced thiols and total thiols in urine were labeled with BQB and d(7)-BQB, respectively. The BQB- and d(7)-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 3.27nmol/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.
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China.
authors.forename: Yun-Qing
authors.initials: YQ
authors.lastname: Huang
authors.forename: Ge-Deng
authors.initials: GD
authors.lastname: Ruan
authors.forename: Jia-Qi
authors.initials: JQ
authors.lastname: Liu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Gao
authors.forename: Yu-Qi
authors.initials: YQ
authors.lastname: Feng
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003404:Creatinine / Q000652:urine; D003545:Cysteine / Q000652:urine; D005978:Glutathione / Q000652:urine; D006710:Homocysteine / Q000652:urine; D006801:Humans; D006842:Hydrocarbons, Brominated / Q000138:chemical synthesis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D008872:Microwaves; D010084:Oxidation-Reduction; D011806:Quinolinium Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000652:urine*; D053719:Tandem Mass Spectrometry
chemical_list: D006842:Hydrocarbons, Brominated; D011806:Quinolinium Compounds; D013438:Sulfhydryl Compounds; C562166:bromoacetonylquinolinium bromide; D006710:Homocysteine; D003404:Creatinine; D005978:Glutathione; D003545:Cysteine