Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 911; pp. 15 - 20
• Main Authors: Ferin, Rita, Pavão, Maria Leonor, Baptista, José
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 12.12.2012
• Subjects: acetonitrile; Adult; biomarkers; blood plasma; Chromatography, High Pressure Liquid - methods; Chromatography, Reverse-Phase; Cysteine; Cysteine - blood; Cysteinylglycine; Dipeptides - blood; Diseases; Female; fluorescence; Glutathione; Glutathione - blood; Homocysteine; Homocysteine - blood; HPLC; Humans; Linear Models; Linearity; Male; Methodology; Middle Aged; Phosphates; Plasma aminothiols; Reproducibility of Results; reversed-phase high performance liquid chromatography; Run time (computers); screening; Sensitivity and Specificity; Thiols; Cysteine; Cysteinylglycine; Homocysteine; Plasma aminothiols; HPLC; Glutathione
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2012.10.022

► Rapid and simultaneous determination of four total aminothiols levels in plasma. ► RP-HPLC isocratic elution within 6min of all analytes, including the IS. ► Validated method exhibits an excellent precision and recovery for all aminothiols. ► Method appropriated for high-throughput routine clinical analysis. Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53mm×7mm I.D., 3μm) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r2) ≥0.99. The LOD for the four plasma thiols was lower than 0.10μmol/L, while LOQ varied from 0.5 to 15μmol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6min. By reducing the total run time, a larger number of analysis can be performed daily.