On-line post-column Diels-Alder derivatization for the determination of vitamin D3 and its metabolites by liquid chromatography/thermospray mass spectrometry.

Liquid chromatography/thermospray mass spectrometry (LC/TSP MS) has been used for the determination of vitamin D3 and some of its metabolites, i.e. 1 alpha(OH) vitamin D3, 25(OH) vitamin D3, 1 alpha,25(OH)2 vitamin D3 and 24,25(OH)2 vitamin D3, using positive and negative ion detection. Using these two modes positional isomers can be identified. Detection in the negative ion mode was preferred because of the slightly higher sensitivity. The limits of detection, using multiple ion detection, are 50-100 nM (6-12 pmol injected). On-line post-column derivatization based on [4 + 2] cyclo-addition (Diels-Alder reaction) proceeds within 1 min at room temperature. If this step is included in LC/TSP MS, the detection limits of the analytes can be improved 7-70-fold depending on the analyte tested. The best results (detection limits down to 1 nM, i.e. 0.12 pmol injected) are obtained with discharge ionization in the negative ion mode.