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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.

Abstract

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)D3, 25(OH)D2, 24,25(OH)2D3 and additionally 25,26(OH)2D3, 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)D3, 25(OH)D2, 24(R),25(OH)2D3 and 25,26(OH)2D3. 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.

Authors+Show Affiliations

Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria.Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria.Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria; Institute of Clinical Chemistry and Laboratory Medicine, General Hospital Hochsteiermark, Leoben, Austria.Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria.Department of Clinical Chemistry, University of Liege, Liege, Belgium.Department of Clinical Chemistry, University of Liege, Liege, Belgium.Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria. Electronic address: markus.herrmann@medunigraz.at.Institute of Chemistry, University of Graz, Graz, Austria.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

33091679

Citation

Zelzer, Sieglinde, et al. "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." Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, vol. 1158, 2020, p. 122394.
Zelzer S, Meinitzer A, Enko D, et al. 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. J Chromatogr B Analyt Technol Biomed Life Sci. 2020;1158:122394.
Zelzer, S., Meinitzer, A., Enko, D., Simstich, S., Le Goff, C., Cavalier, E., Herrmann, M., & Goessler, W. (2020). 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. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 1158, 122394. https://doi.org/10.1016/j.jchromb.2020.122394
Zelzer S, et al. 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. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Nov 20;1158:122394. PubMed PMID: 33091679.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Zelzer,Sieglinde, AU - Meinitzer,Andreas, AU - Enko,Dietmar, AU - Simstich,Sebastian, AU - Le Goff,Caroline, AU - Cavalier,Etienne, AU - Herrmann,Markus, AU - Goessler,Walter, Y1 - 2020/10/06/ PY - 2020/06/09/received PY - 2020/09/06/revised PY - 2020/09/23/accepted PY - 2020/10/23/pubmed PY - 2021/4/27/medline PY - 2020/10/22/entrez KW - 24,25-dihydroxyvitamin D KW - 25,26-dihydroxyvitamin D KW - LC-MS/MS KW - Vitamin D deficiency KW - Vitamin D metabolite ratio SP - 122394 EP - 122394 JF - Journal of chromatography. B, Analytical technologies in the biomedical and life sciences JO - J Chromatogr B Analyt Technol Biomed Life Sci VL - 1158 N2 - 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)D3, 25(OH)D2, 24,25(OH)2D3 and additionally 25,26(OH)2D3, 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)D3, 25(OH)D2, 24(R),25(OH)2D3 and 25,26(OH)2D3. 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. SN - 1873-376X UR - https://www.unboundmedicine.com/medline/citation/33091679/Simultaneous_determination_of_2425__and_2526_dihydroxyvitamin_D3_in_serum_samples_with_liquid_chromatography_mass_spectrometry___A_useful_tool_for_the_assessment_of_vitamin_D_metabolism_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1570-0232(20)31270-8 DB - PRIME DP - Unbound Medicine ER -