Simultaneous measurement of plasma vitamin D(3) metabolites, including 4β,25-dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry

Abstract

Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that permits the quantification of major circulating vitamin D(3) metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid-liquid extraction, and Diels-Alder derivatization procedure prior to LC-MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D(3) peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] concentrations. This interfering metabolite has been identified as 4β,25-dihydroxyvitamin D(3) [4β,25(OH)(2)D(3)] and was found at concentrations comparable to 1α,25(OH)(2)D(3). Quantification of 1α,25(OH)(2)D(3) in plasma required complete chromatographic separation of 1α,25(OH)(2)D(3) from 4β,25(OH)(2)D(3). An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD(3), 24R,25(OH)(2)D(3), 1α,25(OH)(2)D(3), and 4β,25(OH)(2)D(3) in healthy individuals. The LC-MS/MS method developed and described here could result in considerable improvement in quantifying 1α,25(OH)(2)D(3) as well as monitoring the newly identified circulating metabolite, 4β,25(OH)(2)D(3).

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Figure 1

Structures of 1α,25(OH)2D3, 24R,25(OH)2D3, 25OHD3 and 4β,25(OH)2D3.

Figure 2

Product ion mass spectra of the derivatized vitamin D3 metabolites. A) 1α,25(OH)2D3; B) 24R,25(OH)2D3; and C) 25OHD3. The product ion spectra were acquired from the dominant [M-H2O+H]+ precursor ion. Proposed fragmentation reaction is shown in figure D. Multiple reaction monitoring (MRM) channel m/z 574 → 314 indicates two hydroxyl groups on the A-ring.

Figure 3

Representative ion chromatograms of the derivatized vitamin D3 metabolites by LC-MS/MS. The MRM chromatograms were obtained from a standard solution containing d6-1α,25(OH)2D3 (1 ng/mL), 1α,25(OH)2D3 (0.4 ng/mL), 24R,25(OH)2D3 (8 ng/mL), d6-25OHD3 (1 ng/mL) and 25OHD3 (20 ng/mL). The deuterated internal standards of vitamin D3 metabolites were found to elute ~0.1 min earlier than their natural analogs.

Figure 4

Chromatographic separation of 1α,25(OH)2D3 from an unknown dihydroxyvitamin D3 metabolite in human plasma. Varying amounts of 1α,25(OH)2D3 were separately spiked into plasma for validation. An arrow indicates the interfering peak with two hydroxyl groups on the A-ring, which could co-elute with 1α,25(OH)2D3 under certain conditions, e.g., 70% acetonitrile.

Figure 5

The MRM (m/z 547 → 314) chromatograms of 1α,25(OH)2-3-epi-D3 and periodate cleavage of the unknown dihydroxyvitamin D3 in human plasma extracts. A) Chromatograms of 1α,25(OH)2D3 and 1α,25(OH)2-3-epi-D3 ; B) Chromatogram of plasma extracts after periodate cleavage. The disappearance of the unknown metabolite peak indicated vicinal hydroxyl groups on the A-ring, subsequently identified as 4β,25(OH)2D3 [41]. As expected, no significant changes was observed with the peak for 1α,25(OH)2D3.

Figure 6

Plasma concentrations of vitamin D3 metabolites in healthy volunteers (n = 25). Four vitamin D3 metabolites [1α,25(OH)2D3, 4β,25(OH)2D3, 24R,25(OH)2D3, and 25OHD3] were measured by this newly developed LC-MS/MS method. Each data point represents the mean of two determinations.