Assay of plasma homocysteine: light sensitivity of the fluorescent 7-benzo-2-oxa-1, 3-diazole-4-sulfonic acid derivative, and use of appropriate calibrators

Abstract

The recognition of homocysteine as a vascular risk factor has led to increased clinical interest in assaying plasma homocysteine concentrations. Our aim was to improve the reliability of a widely used assay based on HPLC of the fluorescent 7-benzo-2-oxa-1, 3-diazole-4-sulfonic acid (SBD) derivative. We found that SBD derivatives of homocysteine, cysteine, and N-acetylhomocysteine were highly unstable in light but essentially stable in the dark for several hours at either 0 degree C or 25 degrees C. As our primary calibrator, we chose homocystine added to human serum for more consistent results than homocysteine or homocystine in an aqueous buffer. N-acetylcysteine was effective as an internal recovery standard. We observed a previously unreported peak with a prolonged elution time in some plasma samples from subjects who had ingested methionine. Our findings suggest improvements in this and other assay procedures for plasma homocysteine.