[Determination of plasma homocysteine by high performance liquid chromatography with fluorescence detection]

This article report a highly sensitive method specific for the determination of homocysteine in plasma by high performance liquid chromatography with fluorescence detection. Half mL plasma with 100 microL 0.11 mol/L sodium borohydride in 50 mmol/L Tris-HCl (pH 9.0) was kept at 30 degrees C for 30 min, and then 0.5 mL 0.5 mol/L perchloric acid was added. After the mixture was kept at room temperature for 10 min and centrifuged at 15,000 r/min for 10 min, 0.5 mL aliquots of the supernatant solution was pipetted into another vial containing 0.1 mL 3 mmol/L Bromobimane in 1.0 mol/L Na2EDTA (pH 7.0) and 0.7 mL of 90 mmol/L ammonium bicarbonate buffer containing 1.43 mol/L Na2EDTA, pH 8.0. The content was mixed, kept at 37 degrees C for 30 min and centrifuged at 15,000 r/min for 10 min. Then 10 microL aliquots of the supernatant solution was injected into a high performance liquid chromatograph with fluorescence detector. The chromatographic conditions were as follows: an ODS column (4.6 mm i.d. x 150 mm, 5 microns), was eluted with a flow rate of 1.0 mL/min. The fluorescence detector was operated at lambda ex 365 nm and lambda em 475 nm. Mobile phase frompump A was 3% methanol containing 0.25% acetic acid. In gradient elution program the methanol from pump B was as follows: 0-8 min, 5%; 8-15 min, 5%-12%; 15-20 min, 12%; 20-30 min, 12%-20%; 30-32 min, 20%; 32-35 min, 20%-100%; 35-40 min, 100%; 40-43 min, 100%-5%; 43-45 min, 5%. The method proved to be linear in the range of 2.5-80.0 mumol/L with a regression coefficient of 0.9988. The minimum detection limit was 0.5 mumol/L, the recoveries were 94.0%-112.0%, and the RSD values were less than 5.6%.