Determination of 25-hydroxyvitamin D3 in human serum by fluorescence labelling and high-performance liquid chromatography

Abstract

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 × 10^–9 mol dm^–3, 6.74% at a concentration of 99.8 × 10^–9 mol dm^–3 and 3.79% at a concentration of 146.8 × 10^–9 mol dm^–3. The detection limit for the adduct was 2.93 × 10^–14 mol injected, for a signal-to-noise ratio of 3 : 1, and serum concentrations of 0.25 × 10^–9 mol dm^–3 could easily be quantified. No interference from endogenous or exogenous substances was observed.