High-performance liquid chromatographic method for measuring total plasma homocysteine levels

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7- fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.6562.3 and 8.962.4 mmol/ l, while post-methionine load gave tHcy levels of 19.965.5 and 26.865.5 mmol/ l, for women and men, respectively (n540).