Rapid measurement of total plasma homocysteine by HPLC.
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 micro mol/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.
Frick
Barbara
B
Institute for Medical Chemistry and Biochemistry, University of Innsbruck, A-6020, Innsbruck, Austria.
Schröcksnadel
Katharina
K
Neurauter
Gabriele
G
Wirleitner
Barbara
B
Artner-Dworzak
Erika
E
Fuchs
Dietmar
D
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents; D010084:Oxidation-Reduction; D010720:Phosphines; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000097:blood
D007202:Indicators and Reagents; D010720:Phosphines; D013438:Sulfhydryl Compounds; D006710:Homocysteine