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Vitamin D compounds are secosteroids, which are best known for their role in bone health. More recent studies have shown that vitamin D metabolites and catabolites such as dihydroxylated species (e.g., 1,25- and 24,25-dihydroxyvitamin D) play key roles in the pathologies of various diseases. Identification of these isomers by mass spectrometry is challenging and currently relies on liquid chromatography, as the isomers exhibit virtually identical product ion spectra under collision induced dissociation conditions. Here, we developed a simple MALDI-CID method that utilizes ion activation of reactive analyte/matrix adducts to distinguish isomeric dihydroxyvitamin D species, without the need for chromatography separation or chemical derivatization techniques. Specifically, reactive 1,5-diaminonaphthalene adducts of dihydroxyvitamin D compounds formed during MADI were activated and specific cleavages in the secosteroid's backbone structure were achieved that produced isomer-diagnostic fragment ions. [ABSTRACT FROM AUTHOR]

Vitamin D measurements in biological fluids by mass spectrometry are challenging at very low concentration levels. As a result, chemical derivatization is often employed to enhance the ionization properties of low abundant vitamin D compounds. Cookson-type reagents such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or similar derivatives work well but require careful, water-free experimental conditions, as traces of water inactivate the reagent and inhibit or stop the derivatization reactions, thus making quantitative measurements in aqueous samples impossible. We describe a novel electrospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for determining 25-hydroxyvitamin D (25(OH)D) directly in aqueous cellular systems using a new derivatization reagent, the ionic liquid 12-(maleimidyl)dodecyl-tri- n-butylphosphonium bromide (MDBP). The proof-of-concept for the MDBP assay was demonstrated by measuring the levels of 25(OH)D in four different human cell types, namely T cells, helper T cells, B cells, and macrophages. In addition to the ability to determine the levels of 25(OH)D directly in aqueous samples, the cellular integrity was maintained in our application. We show the time-dependent uptake of 25(OH)D into the investigated cells to demonstrate the applicability of the new label. Furthermore, the MDBP derivatization technique may be equally useful in imaging mass spectrometry, where it could be used for response enhancements of spatially localized vitamin D metabolites on wet tissue surfaces, without destroying the integrity of the tissue surface. [ABSTRACT FROM AUTHOR]

While the biological function of the 3α epimer of 25-hydroxyvitamin D 3 (25(OH)D 3 ) remains unknown, its presence needs to be accurately captured and separated from the main 3β epimer, to avoid positive bias in vitamin D status analyses. Several recent LC-MS/MS assays for 25(OH)D 3 successfully separate the 3α and 3β epimers by chromatography. Unfortunately, none of the existing LC-MS/MS assays, which utilize dried blood spots (DBS) as sampling/storage vessels, is able to quantify the individual epimers. DBS are often used for analysis of infant blood, however, and these samples are particularly likely to contain significant levels of interfering 3α epimer. Furthermore, proper calibration of DBS samples is much more difficult to achieve than for liquid serum or plasma samples. We addressed this important issue by creating an artificial vitamin D -free whole blood for calibration and then quantified 3α- and 3β-25(OH)D 3 levels from DBS. After chemical derivatization, the vitamin D epimers were separated on a PFP column and concentrations determined by electrospray ionization LC-MS/MS on a triple quadrupole mass spectrometer. Calibration with artificial whole blood showed improved precision over standard addition (7.6 versus 31.5% RSD for 3β-25(OH)D 3 ). The limits of quantification for 3β-25(OH)D 3 and for 3α-25(OH)D 3 were 1.0 and 0.1 ng/mL, respectively. Excellent intra/interday precisions between 2.1 and 2.2% CV (intra) and 4.4–5.3% CV (inter) were established for 3β-25(OH)D 3 and 3α-25(OH)D 3 . For 3β-25(OH)D 3 , only small concentration-independent bias and deviation of <3.3 ng/mL were seen between serum LC-MS/MS and DBS-LC-MS/MS measurements; analyses of 3α-25(OH)D 3 showed deviations of <0.8 ng/mL in all experiments. [ABSTRACT FROM AUTHOR]