Simultaneous determination of 24,25- and 25,26-dihydroxyvitamin D3 in serum samples with liquid-chromatography mass spectrometry - A useful tool for the assessment of vitamin D metabolism.
24,25-dihydroxyvitamin D; 25,26-dihydroxyvitamin D; LC-MS/MS; Vitamin D deficiency; Vitamin D metabolite ratio
Abstract :
[en] Vitamin D status is typically assessed by the measurement of 25-hydroxyvitamin D (25(OH)D). However, in selected patient groups the sole determination of 25(OH)D has been proven insufficient for this purpose. The simultaneous measurement of additional vitamin D metabolites may provide useful information for a better evaluation of the vitamin D status. Therefore, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 25(OH)D(3), 25(OH)D(2), 24,25(OH)(2)D(3) and additionally 25,26(OH)(2)D(3), which was identified with a synthesized pure substance. Pure and deuterated substances were used to prepare calibrators and internal standards for all target metabolites. Pre-analytical sample preparation comprised protein precipitation followed by liquid-liquid-extraction and derivatization with 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) using 50 µL sample volume. Samples were analyzed on an Agilent HPLC 1260 system equipped with a silica-based Kinetex® 5 µm F5 100 Å core-shell column (150 × 4.6 mm) coupled to a Sciex 4500 mass spectrometer. For all four metabolites, limit of detection (LoD) and limit of quantification (LoQ) ranged from 0.3 to 1.5 nmol/L and 1.0 to 3.1 nmol/L, respectively. Recovery varied between 76.1 % and 84.3 %. Intra- and inter-assay imprecision were <8.6 % and <11.5 %, respectively. The analysis of external and internal quality control samples showed good accuracy for 25(OH)D(3), 25(OH)D(2), 24(R),25(OH)(2)D(3) and 25,26(OH)(2)D(3). Method comparison studies with human samples that were also analyzed with two other LC-MS/MS methods showed close agreement. Finally, the present method has been shown capable of identifying patients with 24-hydroxylase deficiency, which proves its clinical utility.
Disciplines :
Laboratory medicine & medical technology
Author, co-author :
Zelzer, Sieglinde
Meinitzer, Andreas
Enko, Dietmar
Simstich, Sebastian
Le Goff, Caroline ; Université de Liège - ULiège > Département des sciences de la santé publique > Département des sciences de la santé publique
Cavalier, Etienne ; Université de Liège - ULiège > Département de pharmacie > Chimie médicale
Herrmann, Markus
Goessler, Walter
Language :
English
Title :
Simultaneous determination of 24,25- and 25,26-dihydroxyvitamin D3 in serum samples with liquid-chromatography mass spectrometry - A useful tool for the assessment of vitamin D metabolism.
Publication date :
November 2020
Journal title :
Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
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