Metabolic profiling of major vitamin D metabolites using Diels-Alder derivatization and ultra-performance liquid chromatography-tandem mass spectrometry

Abstract

Biologically active forms of vitamin D are important analytical targets in both research and clinical practice. The current technology is such that each of the vitamin D metabolites is usually analyzed by individual assay. However, current LC-MS technologies allow the simultaneous metabolic profiling of entire biochemical pathways. The impediment to the metabolic profiling of vitamin D metabolites is the low level of 1alpha,25-dihydroxyvitamin D(3) in human serum (15-60 pg/mL). Here, we demonstrate that liquid-liquid or solid-phase extraction of vitamin D metabolites in combination with Diels-Alder derivatization with the commercially available reagent 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by ultra-performance liquid chromatography (UPLC)-electrospray/tandem mass spectrometry analysis provides rapid and simultaneous quantification of 1alpha,25-dihydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(2), 24R,25-dihydroxyvitamin D(3), 25-hydroxyvitamin D(3) and 25-hydroxyvitamin D(2) in 0.5 mL human serum at a lower limit of quantification of 25 pg/mL. Precision ranged from 1.6-4.8 % and 5-16 % for 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3), respectively, using solid-phase extraction.

Figures

Fig. 1

Metabolism of vitamin D. The metabolites measured in this study are highlighted in a darker font on the left. The two major forms of vitamin D and sites of hydroxylation are shown on the right

Fig. 2a–b

Improvement in the sensitivity of MS/MS analysis of 1α,25(OH)2D3 by derivatization with PTAD. Analysis of 1α,25(OH)2D3-PTAD, 1 ng injected on column (574.3>314.0 reaction trace), is shown in panel a, and that of native 1α,25(OH)2D3, 10 ng injected on column (399.4>135.2 reaction trace), is shown in panel b. Both chromatograms were scaled the same way, indicating a 100-fold increase in signal intensity for the derivatized 1α,25(OH)2D3. Standards were injected in 10 μL acetonitrile and separated using gradient elution on a 5-cm UPLC BEH C18 column

Fig. 3a–b

CID spectra of 1α,25(OH)2D3. Native 1α,25(OH)2D3 a; 1α,25(OH)2D3-PTAD b. Both product ion spectra were acquired for the dominant [M–18+H]+ ion. The general fragmentation reaction for derivatized vitamin D metabolites is shown. We observed a dominant fragment for PTAD derivatives at m/z 298 (m/z 314 for metabolites hydroxylated at position 1)

Fig. 4

Chromatographic separation of major vitamin D metabolites using the conditions described in the “Experimental” section

Fig. 5

Separation of 1α,25(OH)2D3 from coeluting interferences using a 10-cm UPLC BEH C18 column. The identity of the peak was supported by a standard addition experiment (see Table 5) and the use of the deuterated internal standard d6 1α,25(OH)2D3. Hexadeuterated surrogates of vitamin D metabolites were found to elute ~0.03 min earlier than their native analogs (see Fig. 4). Arrows indicate 1α,25(OH)2D3-PTAD. Detection was performed in MRM mode

Fig. 6

Selection of solvents for liquid–liquid extraction. Human serum was spiked with deuterated 1α,25(OH)2D3 and prepared as described in the “Experimental” section. Extraction was performed in two steps (st) with methyl tert-butyl ether (MTBE), dichloromethane (DCM) and ethyl acetate (EtAc). No second extraction was performed for DCM, which formed an emulsion that was difficult to separate. Data are normalized to MTBE recoveries in the first step. No d6 25(OH)D3 was available at the time of the experiment, and so native 25(OH)D3 was used to measure the relative recovery. Four samples were analyzed independently in each sample group (black bars, d6 1α,25(OH)2D3; gray bars, d6 25(OH)D3). Error bars represent standard deviations

Fig. 7a–c

Selection of solvents for sample loading, SPE wash, and elution. Supernatant from the protein precipitation was transferred into SPE cartridges (Oasis HLB) and diluted with given volume of water (W), 0.4 M K2HPO4 (K), or 0.4 M Na2HPO4 (Na). a SPE cartridges were washed with 50 % methanol and eluted with 1.5 mL ethyl acetate. b SPE cartridges loaded with diluted supernatant were washed with 2 mL 70% methanol, 2 mL 50% methanol, 2 mL 50% methanol and 2 mL hexane, 2 mL 30% methanol, and water and eluted with 1.5 mL ethyl acetate. c SPE cartridges were loaded with diluted supernatant, washed with 70% methanol and eluted from Oasis HLB cartridges with 3×1 mL of acetonitrile (MeCN), ethyl acetate (EtAc) or methanol (MeOH). Each 1 mL sample was spiked with calcipotriol to study the effect of matrix on derivatization efficiency. Four samples were analyzed independently in each sample group (black bars, d6 1α,25(OH)2D3; gray bars, d6 25(OH)D3; white bars, calcipotriol). All serum samples were spiked with deuterated surrogates prior to sample preparation. Error bars represent standard deviations

Fig. 8

Comparison of RIA (DiaSorin) and LC-MS data for 50 individual measurements of 25(OH)D in serum samples collected in the fall study. LC-MS data are plotted as the sum of the 25(OH)D2 and 25(OH)D3 concentrations

Fig. 9a–c

Metabolic profile of (a) 25(OH)D3, (b) 1α,25(OH)2D3 and (c) 24R,25(OH) 2D3 in HIV-positive (black bars; n=7) and -negative (gray bars; n=8) female subjects. Error bars represent standard deviations

Fig. 10a–c

Seasonal variations in (a) 25(OH)D3 [15.5 to 67.8 ng/mL; 8.0 to 50.7 ng/mL], (b) 1α,25(OH)2D3 [25 to 128 pg/mL; 25 to 108 pg/mL], and (c) 24R,25(OH) 2D3 [0.9 to 9.6 ng/mL; 0.3 to 4.3 ng/mL] in two different groups of healthy subjects studied in fall (black bars; n=17) and winter (gray bars; n=17). Values in the brackets shows the ranges of concentrations in fall and winter, respectively. Error bars represent standard deviations