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A simple HPLC—UV method for simultaneous determination of cysteine and cysteinylglycine in biological fluids

Głowacki R., Stachniuk J., Borowczyk K.

Acta Chromatographica

2016

Vol. 28, no. 3

333--346

A new and simple method based on high-performance liquid chromatography with ultraviolet detection (HPLC-UV) for the determination of cysteine (Cys) and cysteinylglycine (CysGly) in plasma and urine has been developed. The method involves reduction of disulfide bonds with tris(2-carboxyethyl)phosphine, derivatization of the analytes with 2-chloro-1-methylquinolinium tetrafluoroborate, and separation on Aeris PEPTIDE XB-C18 column (150 mm × 4.6 mm, 3.6 μm, Phenomenex) with UV detection at 355 nm. The calibration lines, obtained with human plasma and urine spiked with Cys- Gly and Cys, were linear in the range of 2.5–50 μmol L−1 and 20–300 μmol L−1, respectively. The intra- and inter-day precision values of the method, expressed as a relative standard deviation, were 0.25–11.1% and 0.71–12.3%, respectively. The analytical recovery varied from 89.7 to 112.3%. The LOQs for total Cys and CysGly were 1.5 pmol and 2.3 pmol in peak, respectively. The method was successfully applied to samples donated by apparently healthy individuals. Concentrations of Cys and CysGly in human plasma from 18 subjects varied from 141.6 to 217.8 μmol L−1 and from 21.1 to 50.9 μmol L−1, respectively. Their concentrations in urine samples (n = 14) ranged from 137.3 to 426.8 μmol L−1 and from 1.6 to 4.9 μmol L−1, respectively.

Analysis of major urinary aminothiols by high-performance liquid chromatography with ultraviolet detection

Kuśmierek K, Bald E.

Acta Chromatographica

2009

Vol. 21, no. 3

411--420

High-performance liquid chromatography has been used for measurement of the concentrations of total cysteine and cysteinylglycine in human urine. The method involved conversion of disulfides to their reduced counterparts by use of tris(2-carboxyethyl)phosphine hydrochloride, derivatization with 1-benzyl-2-chloropyridinium bromide, and ion-pairing reversed-phase high-performance liquid chromatographic separation with ultraviolet detection at 315 nm. The linearity of the method was validated in the ranges 50–300 and 5–50 µmol L -1 urine for cysteine and cysteinylglycine, respectively; regression coefficients were better than 0.999. The detection and quantitation limits were 0.2 and 0.5 µmol L -1, respectively, for both analytes. Intra-assay and inter-assay imprecision were below 6.0%, and accuracy was 98.95–100.80%. The method was applied to urine samples donated by apparently healthy volunteers.