Improved quantitative LC-MS/MS analysis of vitamin D metabolites in serum after one-pot double derivatization

In this study, we report a one-pot double derivatization scheme, which used acetylation after a Diels-Alder reaction using 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to improve separation efficiency and provide baseline separations of the five vitamin D metabolites 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 24,25-dihydroxyvitamin D3 (24,25(OH)₂D₃), 3β-25-hydroxyvitamin D₃ (3β-25(OH)D₃), 3α-25-hydroxyvitamin D₃ (3α-25(OH)D₃) and vitamin D₃ on a C-18 stationary phase. Vitamin D metabolites are often very challenging to measure quantitatively using mass spectrometry, due to their low serum concentration levels and low ionization efficiencies. Moreover, some of these species are isomers with virtually identical mass spectral dissociation behavior. To overcome the low ionization efficiency and unspecific fragmentation behavior, derivatization using Diels-Alder reactions with Cookson-type reagents such as PTAD are common. These derivatization reactions generally result in more complicated liquid chromatography separations, because both 6R- and 6S-isomers are formed during Diels-Alder reactions. It has been shown that separations have been particularly challenging for the 3α-25(OH)D₃ and 3β-25(OH)D₃ epimers. Here, we optimized the PTAD derivatization and the esterification using acetic anhydride. By utilizing the esterification catalyst 4-dimethylaminopyridine, we avoided quenching and evaporation between the two derivatization steps, but were also able to perform the esterification at room temperature without heating. The optimized one-pot double derivatization LC-MS/MS assay was validated with respect to inter/intra-day precision, accuracy, recovery and linear dynamic range and applied to metabolic fingerprinting of vitamin D₃ metabolites in serum samples. The metabolites 3α-25(OH)D₃, 3β-25(OH)D₃ and 24,25(OH)₂D₃, were readily quantified in all investigated samples. The method was, in principle, also fit for purpose for quantification of the native vitamin D₃ species; the relatively high blank concentration of the commercial vitamin D-depleted serum used for calibration, however, limited the limits of quantification for this metabolite. The method provided insufficient limits of quantification for serum levels of 1,25(OH)₂D₃.