期刊
ANALYTICAL BIOCHEMISTRY
卷 416, 期 2, 页码 159-166出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.05.020
关键词
Isotope differential derivatization; Thiols; Oxidized thiols; Urine
资金
- National Science Fund for Distinguished Young Scholars [20625516]
- National Nature Science Foundation [91019013, 31070327]
- Science Fund for Creative Research Groups [20921062]
- National Natural Science Foundation of China (NSFC)
Here we report a new isotopic pair of derivatization reagents, omega-bromoacetonylquinolinium bromide (BQB) and d(7)-omega-bromoacetonylquinolinium bromide (d(7)-BQB). BQB and d(7)-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 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 3.27 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. (C) 2011 Elsevier Inc. All rights reserved.
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