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Rapid measurement of total plasma homocysteine by HPLC
Barbara Frick1, Katharina Schröcksnadel, Gabriele Neurauter
1Institute for Medical Chemistry and Biochemistry, University of Innsbruck, A-6020, Innsbruck, Austria.
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.