Quantitative determination of vitamin D metabolites in plasma using UHPLC-MS/MS.
Summary of "Quantitative determination of vitamin D metabolites in plasma using UHPLC-MS/MS."
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.
Affiliation
Medical Research Council-Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge, CB1 9NL, UK.
Journal Details
This article was published in the following journal.
Name: Analytical and bioanalytical chemistry
ISSN: 1618-2650
Pages:
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/20628873
- DOI: http://dx.doi.org/10.1007/s00216-010-3993-0
Medical and Biotech [MESH] Definitions
Vitamin D Deficiency
A nutritional condition produced by a deficiency of VITAMIN D in the diet, insufficient production of vitamin D in the skin, inadequate absorption of vitamin D from the diet, or abnormal conversion of vitamin D to its bioactive metabolites. It is manifested clinically as RICKETS in children and OSTEOMALACIA in adults. (From Cecil Textbook of Medicine, 19th ed, p1406)
Vitamin D-binding Protein
An alpha-globulin found in the plasma of man and other vertebrates. It is apparently synthesized in the liver and carries vitamin D and its metabolites through the circulation and mediates the response of tissue. It is also known as group-specific component (Gc). Gc subtypes are used to determine specific phenotypes and gene frequencies. These data are employed in the classification of population groups, paternity investigations, and in forensic medicine.
Radioligand Assay
Quantitative determination of receptor (binding) proteins in body fluids or tissue using radioactively labeled binding reagents (e.g., antibodies, intracellular receptors, plasma binders).
Vitamin K
A lipid cofactor that is required for normal blood clotting. Several forms of vitamin K have been identified: VITAMIN K 1 (phytomenadione) derived from plants, VITAMIN K 2 (menaquinone) from bacteria, and synthetic naphthoquinone provitamins, VITAMIN K 3 (menadione). Vitamin K 3 provitamins, after being alkylated in vivo, exhibit the antifibrinolytic activity of vitamin K. Green leafy vegetables, liver, cheese, butter, and egg yolk are good sources of vitamin K.
Quantitative Trait Loci
Locations, on the GENOME, of GENES or other genetic elements that encode or control the expression of a quantitative trait (QUANTITATIVE TRAIT, HERITABLE).
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