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).

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.