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

Abstract

Summary.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 CysGly 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.

Simultaneous Determination of Human Serum Albumin and Low-Molecular-Weight Thiols after Derivatization with Monobromobimane

“…Detailed analytical data for total Cys, Hcy, GSH, Cys-Gly, and HSA are shown in Table 3 . These results are mostly in agreement with those previously reported [ 5 , 11 , 30 , 39 , 56 , 57 ]. The elaborated method can be successfully applied in large populations to monitor changes in thiol concentration in different physiological conditions.…”

“…In biological systems, thiols occur mainly in oxidized or protein-bound forms, and therefore the reduction of disulfide bonds to thiol groups must be performed prior to the derivatization reaction. NaBH 4 [ 5 , 41 , 54 ], dithiothreitol (DTT) [ 42 , 43 , 52 ], tris(2-carboxyethyl)phosphine (TCEP) [ 4 , 30 , 39 , 57 ], or 2-mercaptoethanol (2-ME) [ 6 , 52 ] are the most commonly used substances to reduce disulfide bonds. The choice of an appropriate reductant depends on the analytical procedure to be followed, especially on the type of the derivatizing reagent used and the detection method.…”

Fluorescent cysteine probe based on a signal amplification unit, a catalyzed hairpin assembly reaction and Förster resonance energy transfer

“…Conventional techniques for cysteine detection include high-performance liquid chromatography (HPLC) [5] and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [6]. Despite being highly sensitive and selective, they are expensive, complicated, and involve multiple steps of sample preparation.…”