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.
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.
Dudman
N P
NP
Center for Thrombosis and Vascular Research, University of New South Wales, Prince Henry Hospital, Little Bay, NSW, Australia. nickdud@ozemail.com.au
Guo
X W
XW
Crooks
R
R
Xie
L
L
Silberberg
J S
JS
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D003545:Cysteine; D004355:Drug Stability; D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D006710:Homocysteine / Q000031:analogs & derivatives / Q000097:blood*; D006711:Homocystine; D006801:Humans; D008027:Light; D011786:Quality Control; D015203:Reproducibility of Results
D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D006710:Homocysteine; D006711:Homocystine; C025584:N-acetylhomocysteine; C037802:7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; D003545:Cysteine