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 7.65+/-2.3 and 8.9+/-2.4 micromol/l, while post-methionine load gave tHcy levels of 19.9+/-5.5 and 26.8+/-5.5 micromol/l, for women and men, respectively (n=40).
Fermo
I
I
Laboratory of Chromatographic and Separative Techniques, IRCCS H San Raffaele, Milan, Italy. fermo.isabella@hsr.it
Arcelloni
C
C
Mazzola
G
G
D'Angelo
A
A
Paroni
R
R
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D008297:Male; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D006710:Homocysteine; C037802:7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate