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 2PO 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 μmol/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.