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

Shujing Ding; Schoenmakers, Inez; Jones, Kerry; Koulman, Albert; Prentice, Ann; Volmer, Dietrich A.
September 2010
Analytical & Bioanalytical Chemistry;Sep2010, Vol. 398 Issue 2, p779
Academic Journal
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, 25-OH D, 24,25-(OH) D, 1,25-(OH) D, and 1,25-(OH) D, 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 = 0.9738) for DEQAS samples, demonstrating the high degree of comparability of the new UHPLC-MS/MS technique to existing methods.


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