Liquid chromatographic determination of total homocysteine in blood plasma with photometric detection

A rapid and sensitive method for quantification of homocysteine total forms and glutathione levels in blood plasma via HPLC was developed. Dithiotreitol as a water soluble agent has been used as a reductant for both protein and nonprotein disulphides. Dithiotreitol reacts with the mixed disulphides under 60 °C treatment within 10 min. Reduced aminothiols and homocystein were easily derivated with 5,5′-dithiobis-(2-nitrobenzoic acid) and the resultant ultraviolet absorbance within 330 nm was detected by the HPLC method. The concentration of total plasma homocysteine was significantly higher in groups of patients: with the end stage of renal disease: 45.5±40.9 μmol/l (n=79), with cerebral vascular disorders 12.3±7.0 μmol/l (n=65), and with coronary atherosclerosis 15.4±10.9 μmol/l (n=15) than that in healthy subjects (6.2±1.74 μmol/l, n=20). Some major advantages of the method include: simultaneous measurement of both total homocysteine and total glutathione, no loss of oxidized form during processing of blood plasma for aminothiols measurement, use of protein-bound aminothiols solution as a calibrator.