A validated HPLC-fluorescence method with a semi-micro column for routine determination of homocysteine, cysteine and cysteamine, and the relation between the thiol derivatives in normal human plasma (original) (raw)
Related papers
Journal of Chromatography A, 2002
In recent papers, we presented a new analytical method for thiol quantification in serum. This method was developed with capillary electrophoresis (CE) and laser-induced fluorescence (LIF) to analyze thiol-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results for homocysteine, glutathione, cysteinylglycine, and cysteine were presented (Causse E., et al., Clin. Chem. 45 (1999) 412). An exhaustive comparison of the quantitation of homocysteine in plasma, using high-performance liquid chromatography with either conventional fluorescence detection or fluorescence polarizatioń immunoassay was also reported (Causse E., et al., Electrophoresis 21 . Sample preparation prior to derivatization with IAF had never been investigated. Recently we studied protein precipitation in serum with different organic agents (Causse E., et al., J. Chromatogr. A 895 (2000) 173). In this work, we evaluated the conditions of protein precipitation in function of the amounts of acetonitrile and their influence on quantitation and quality of the electropherograms. Then, we looked at the variation of thiol concentrations in the haemolysis states and studied the thiol stability of blood samples cooled on ice.
Homocysteine and other thiols in plasma and urine: automated determination and sample stability
Clinical Chemistry, 1993
We have developed a modified version of our fully automated column-switching HPLC method for determining total plasma homocysteine based on single-column (reversed-phase) separation. Homocysteine, cysteine, and cysteinylglycine in plasma (total concentrations), acid-precipitated plasma (non-protein-bound concentrations), and urine can be determined. The derivatization and chromatography were performed automatically by a sample processor. The successful separation of all thiol species (within 15 min) was accomplished by accurate adjustment of the pH of the mobile phase to 3.65 (plasma) or 3.50 (acid-precipitated plasma, urine). Maximal fluorescence yield of cysteine, cysteinylglycine, and, to a lesser degree, homocysteine was dependent on optimal concentrations of EDTA and dithioerythritol during reduction (with NaBH4) and derivatization (with monobromobimane). The method is sensitive (detection limit approximately 0.05 pmol) and has a high degree of precision (CV < 5%). The sampl...
Clinical chemistry, 1998
We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine--cysteine, homocysteine, cysteinylglycine, and glutathione--as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97-107%, with little difference between plasma and urine samples. The detection limit was approximately 50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (a...
Rapid measurement of total plasma homocysteine by HPLC
Clinica Chimica Acta, 2003
Background: Determination of plasma homocysteine has gained increasing interest during the past few years. Several HPLC methods for determination of homocysteine are available. Based on these methods, we developed a new HPLC assay for rapid and sensitive measurement of total plasma homocysteine. Methods: As a reducing reagent tris-(2-carboxylethyl)-phosphine is used, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate serves as the derivatization agent. Separation is performed by reversed-phase HPLC using a precolumn and a 55-mm RP 18 cartridge; mobile phase: 0.1 mol/l KH 2 PO 4 with 5% methanol, adjusted to pH 2.7 with ortho-phosphoric acid, flow-rate 1.1 ml/min. Results: Homocysteine is clearly separated from other thiols, the retention time being 2.2 min, total analysis time is 6 min. Tests for linearity, recovery and precision are satisfactory, as well as the comparison with a commercial available assay method. Detection limit of the method is 0.5 Amol/l, it could be further enhanced for measurements of even lower homocysteine concentrations in, e.g., cell culture supernatants. Conclusions: The described method is well suited for analysis of thiols in blood specimens. It is more convenient and more rapid than methods described earlier. D
Journal of Chromatography B, 2014
Homocysteine thiolactone (Hcy-thiolactone), an intramolecular thioester, easily acylates free-amino groups in proteins, which impairs or alters the protein's biological function. Here, we describe new capillary electrophoresis assay for the determination of Hcy-thiolactone in human urine based on a field amplified sample injection and sweeping MEKC with UV detection. The two steps procedure relies on sample liquid-liquid extraction followed by CE separation and UV detection at 240 nm. The Hcy-thiolactone standard added to the urine before the extraction step shows that the response of the detector is linear within the range studied, from 0.1 to 1 mol L-1 urine. The intra-and interday precision and recovery were 3.2-14.4 % (average 5.1 % and 9.3 %) and 92.5-112.6 % (average 99.8 % and 99.1 %), respectively. The lower limit of quantification was 0.09 nmol Hcy-thiolactone in 1 mL of urine. The proposed method was applied for the analysis of 15 urine samples donated by apparently healthy volunteers. The average concentration of the analyte was 0.170±0.029 mol L-1 .
Visual Detection of Cysteine and Homocysteine
Journal of The American Chemical Society, 2004
Homocysteine is a risk factor for disorders including cardiovascular 1 and Alzheimer's disease. 2 Cysteine deficiency is involved in slowed growth, hair depigmentation, edema, lethargy, liver damage, muscle and fat loss, skin lesions, and weakness. 3 The detection of important biological thiols including cysteine and homocysteine has been the focus of numerous research efforts. The majority of the reported methods are based on redox chemistry or derivatization with chromophores or fluorophores. The determination of specific thiols is often carried out in conjunction with HPLC or capillary electrophoresis separations or via immunoassays. 4 Recent reports describe a need for much simpler methods that employ stable, nontoxic reagents 5a,b which are less sensitive to pH 5c and afford the requisite sensitivity 5d,6a as well as high selectivity. 6b
Electrophoresis, 2017
A rapid and selective method has been developed for highly sensitive determination of total cysteine and homocysteine levels in human blood plasma and urine by capillary electrophoresis (CE) coupled with liquid-liquid extraction. Analytes were first derivatized with 1,1'-thiocarbonyldiimidazole and then samples were purified by chloroform-ACN extraction. Electrophoretic separation was performed using 0.1 M phosphate with 30 mM triethanolamine, pH 2, containing 25 μM CTAB, 2.5 μM SDS, and 2.5% polyethylene glycol 600. Samples were injected into the capillary (with total length 32 cm and 50 μm i.d.) at 2250 mbar*s and subsequent injection was performed for 30 s with 0.5 M KОН. The total analysis time was less than 9 minutes, accuracy was 98%, and precision was <2.6%. The LOD was 0.2 μM for homocysteine and 0.5 μM for cysteine. The use of liquid-liquid extraction allowed the precision and sensitivity of the CE method to be significantly increased. The validated method was applie...