Quantitative determination of vitamin D metabolites in plasma using UHPLC-MS/MS

Abstract

Vitamin D is an important determinant of bone health at all ages. The plasma concentrations of 25-hydroxy vitamin D (25-OH D) and other metabolites are used as biomarkers for vitamin sufficiency and function. To allow for the simultaneous determination of five vitamin D metabolites, 25-OH D(3), 25-OH D(2), 24,25-(OH)(2) D(3), 1,25-(OH)(2) D(3), and 1,25-(OH)(2) D(2), in low volumes of human plasma, an assay using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established. Plasma samples were spiked with isotope-labeled internal standards and pretreated using protein precipitation, solid-phase extraction (SPE) and a Diels-Alder derivatization step with 4-phenyl-1,2,4-triazoline-3,5-dione. The SPE recovery rates ranged from 55% to 85%, depending on the vitamin D metabolite; the total sample run time was <5 min. Mass spectrometry was conducted using positive ion electrospray ionization in the multiple reaction monitoring mode on a quadrupole-quadrupole-linear ion trap instrument after pre-column addition of methylamine to increase the ionization efficiency. The intra- and inter-day relative standard deviations were 1.6-4.1% and 3.7-6.8%, respectively. The limit of quantitation for these compounds was determined to be between 10 and 20 pg/mL. The 25-OH D results were compared with values obtained for reference materials (DEQAS). In addition, plasma samples were analyzed with two additional Diasorin antibody assays. All comparisons with conventional methods showed excellent correlations (r(2) = 0.9738) for DEQAS samples, demonstrating the high degree of comparability of the new UHPLC-MS/MS technique to existing methods.

Figures

Fig. 1

Recovery of spiked components from plasma suing different sample pretreatment methods (sequence: protein precipitation → extraction → derivatization). LLE, 200 μL of plasma, dried and extracted with 200 μL ethyl acetate and 100 μLH2O after protein precipitation. SPE H2O, after protein precipitation, acetonitrile was evaporated and water was added. SPE NaOH, SPE with 20 μL 1 M NaOH added before protein precipitation. SPE NaOH HCl, SPE with 20 μL 1 M NaOH added before protein precipitation followed by addition of HCl to pH 5 and SPE. Derivatization before SPE, plasma sample were dried and derivatized with 50 μL of PTAD, addition of 800 μL of H2O before SPE

Fig. 2

Collision-induced dissociation spectra of 1,25-(OH)2 D3 ((M + H-H2O)+ at m/z 399, top spectrum) and PTAD-derivatized 1,25-(OH)2 D3 (M + CH3NH3+ at m/z 623, bottom spectrum). The transition m/z 623 → 314 was utilized for MRM

Fig. 3

MRM signal intensities obtained for different mobile phase compositions: solvent 1 and 3, ion currents for transition derivatized precursor ion/fragment ion; solvent 2 and 4, ion currents for transition methylamine adduct of derivatized precursor ion/fragment ion. (Solvent 1: A, 0.1% formic acid in H2O and B, 0.1% formic acid in methanol. Solvent 2: A, 0.1% formic acid, 5 mM methylamine in H2O, and B, 0.1% formic acid in methanol. Solvent 3: A, 10 mM ammonium formate in H2O and B, 0.1% formic acid in methanol. Solvent 4: A, 10 mM ammonium formate, 5 mM methylamine in H2O, and B, 0.1% formic acid in methanol)

Fig. 4

UHPLC-MRM chromatograms for 5 vitamin D metabolites

Fig. 5

Cross-calibration diagrams for 25-OH D: a in-house UHPLC-MS/MS versus DEQAS LC-MS and DEQAS ALTM values for 12 DEQAS samples; b Bland–Altman plots (bottom)

Fig. 6

Regression analysis of 25-OH D plasma concentrations in 42 subjects measured by in-house UHPLC-MS/MS and Diasorin Liaison and RIA methods (left). Bland–Altman plot (right)

Fig. 7

MRM chromatograms of vitamin D metabolites in plasma after SPE and PTAD derivatization using sequential MRM time windows (the concentration levels are given in parentheses). a Vitamin D metabolites 24,25-(OH)2 D3, 25-OH D3 and 25-OH D2; b low abundant vitamin D metabolite 1,25-(OH)2 D3