Fully automated method for simultaneous determination of total cysteine, cysteinylglycine, glutathione and homocysteine in plasma by HPLC with UV absorbance detection

Głowacki, Rafał; Bald, Edward

doi:10.1016/j.jchromb.2009.06.012

Cited by 95 publications

(55 citation statements)

References 33 publications

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“…Blood was collected by venipuncture from apparently healthy subjects in a fasting state to the K3 EDTA Vacutainer tubes, cooled on ice and centrifuged at 800×g for 15 min at room temperature within 30 min after collection. The plasma supernatant was stored at −80 °C or analyzed within 2 h. Both plasma and urine samples were prepared according to modified procedure published previously [19]. A 24 μL of appropriate biological fluid was diluted with 24 μL of 0.2 mol L −1 pH 7.8 phosphate buffer and treated with 2 μL of 0.25 mol L −1 TCEP solution for 15 min in order to reduce disulfide bonds.…”

Section: Biological Samples Collection and Pretreatmentmentioning

confidence: 99%

“…In the present study, for simultaneous determination of total Cys and CysGly, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), which reacts with all disulfides at room temperature, was used. Our earlier study has shown that TCEP reduces all dimer and mixed disulfides, efficiently making them accessible to derivatization reagent [9,17,19]. In the present methodology, for signal enhancement and labile sulfhydryl group protection, we exploited CMQT, a highly reactive and thiol specific UV derivatization reagent [18].…”

Section: Sample Preparationmentioning

confidence: 99%

“…Among a variety of assays designed to determination of thiols in biological fluids, most of them depend on derivatization followed by chromatographic separation and ultraviolet [7][8][9] or fluorescence detection [10][11][12][13][14][15]. Most of chromatographic procedures dedicated to hydrophilic thiols measurements exploit reversed phase (RP)-HPLC and buffered eluents containing ion-pairing reagents, such as alkyl sulphonates or trichloroacetic acid [7][8][9][10][11][12][13][14][15][16][17][18][19][20]. Unfortunately, such approach requires a long equilibration time of a chromatographic column and significantly increases the total costs of analysis.…”

Section: Introductionmentioning

confidence: 99%

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

Głowacki¹,

Stachniuk²,

Borowczyk³

2016

Acta Chromatographica

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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.

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Section: Biological Samples Collection and Pretreatmentmentioning

confidence: 99%

Section: Sample Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Głowacki¹,

Stachniuk²,

Borowczyk³

2016

Acta Chromatographica

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“…As well, mass spectrometry detection needs special instrumentation with high cost. Therefore, GSH measurements have always proven to be difficult because of the interference of the coexisting compounds, difficulties in sample preparation, the necessity of derivatization, and lack of sufficient sensitivity to the traditional methods, such as enzymatic or HPLCcolorimetric methods [21][22][23][24][25]. Electrochemical methods, however, present the advantages of simplicity, high sensitivity, low expense, and ease of miniaturization.…”

Section: Introductionmentioning

confidence: 99%

Novel sensor based on carbon paste/Nafion® modified with gold nanoparticles for the determination of glutathione

2012

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Several problems for the direct electrochemical oxidation of reduced glutathione (GSH) challenge the usage of electroanalytical techniques for its determination. In this work, the electrochemical oxidation of GSH catalyzed by gold nanoparticles electrodeposited on Nafion modified carbon paste electrode in 0.04 mol L(-1) universal buffer solution (pH 7.4) is proved successful. The effect of various experimental parameters including pH, scan rate and stability on the voltammetric response of GSH was investigated. At the optimum conditions, the concentration of GSH was determined using differential pulse voltammetry (DPV) in two concentration ranges: 0.1 × 10(-7) to 1.6 × 10(-5) mol L(-1) and 2.0 × 10(-5) to 2.0 × 10(-4) mol L(-1) with correlation coefficients 0.9988, 0.9949 and the limit of detections (LOD) are 3.9 × 10(-9) mol L(-1) and 8.2 × 10(-8) mol L(-1), respectively, which confirmed the sensitivity of the electrode. The high sensitivity, wide linear range, good stability and reproducibility, and the minimal surface fouling make this modified electrode useful for the determination of spiked GSH in urine samples and in tablet with excellent recovery results obtained.

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“…To accomplish this, it is necessary to both quench any artifactual oxidation of GSH to GSSH by direct in situ derivatization as illustrated above, and to exhaustively reduce GSSG. For the latter, commonly used reducing agents include tris(2-carboxyethyl)phosphine hydrochloride (TCEP•HCl) [156,157], dithiothreitol [158],trialkylphosphines [159], NaBH 4 [160], and occasionally sulfites, cyanides or some enzymes. We selected the TCEP•HCl reagent (Scheme 2.4.)…”

Section: Quantification Of Cellular Glutathione (Aim E)mentioning

confidence: 99%

Chemoselective reactions and their applications in metabolite isolation and analysis.

Gori¹

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Fully automated method for simultaneous determination of total cysteine, cysteinylglycine, glutathione and homocysteine in plasma by HPLC with UV absorbance detection

Cited by 95 publications

References 33 publications

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

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

Novel sensor based on carbon paste/Nafion® modified with gold nanoparticles for the determination of glutathione

Chemoselective reactions and their applications in metabolite isolation and analysis.

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