Determination of 25-hydroxyvitamin D3 in human serum by fluorescence labelling and high-performance liquid chromatography.
A method is described for the determination of 25-hydroxyvitamin D3 in human blood serum. The problems of sensitivity and selectivity encountered with previous techniques were avoided by the formation of a highly fluorescent Diels-Alder adduct following solid-phase extraction of the vitamin. After excess of reagent had been eliminated, quantification was achieved by high-performance liquid chromatography. The recovery of the vitamin from serum was 76.4 +/- 1.76%. The precision of the method was determined, and the relative standard deviations were 8.38% at a concentration of 47.0 x 10(-9) mol dm-3, 6.74% at a concentration of 99.8 x 10(-9) mol dm-3 and 3.79% at a concentration of 146.8 x 10(-9) mol dm-3. The detection limit for the adduct was 2.93 x 10(-14) mol injected, for a signal-to-noise ratio of 3:1, and serum concentrations of 0.25 x 10(-9) mol dm-3 could easily be quantified. No interference from endogenous or exogenous substances was observed.
Jordan
P H
PH
Department of Chemistry, University of Exeter, UK.
Read
G
G
Hargreaves
T
T
D002112:Calcifediol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents; D013050:Spectrometry, Fluorescence / Q000379:methods
D007202:Indicators and Reagents; D002112:Calcifediol