Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 877, Issue 28, Pages 3400-3404Publisher
ELSEVIER
DOI: 10.1016/j.jchromb.2009.06.012
Keywords
Cysteine; Cysteinylglycine; Glutathione; Homocysteine; HPLC; On-line sample preparation; Chemical derivatization
Funding
- University of Lodz
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A fully automated HPLC method for the simultaneous determination of total thiols in plasma samples has been developed. The method involves reductive conversion of disulfides to their reduced counterparts with the use of tris(2-carboxyethyl)phosphine. After reduction the newly formed sulfhydryl groups are reacted with 2-chloro-1-methylquinolinium tetrafluoroborate to form 2-S-quinolinium derivatives followed by deproteinization by dialysis. The reaction products are separated by reversed-phase HPLC, detected and quantified by UV absorbance detection at 355 nm. The recommended HPLC procedure enables measurement of four main plasma aminothiols cysteine, cysteinylglycine,glutathione, and homocysteine with low imprecision (mean relative standard deviations within calibration range, 3.47%, 5.34%, 4.25% and 3.26%, respectively) and good sensitivity. Accuracy, expressed as the mean measured amount as percentage of added amount, was within 97.5-103.0%, 98.3-102.5%, 96.3-99.5% and 97.1-99.1%, respectively. The lower limit of quantification for all thiols was 0.5 mu M. The whole unattended instrument acquisition time amounts 13 min. (C) 2009 Elsevier B.V. All rights reserved
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