MSACL 2019 EU Abstract

Establishment of a Liquid-Chromatography Tandem Mass-Spectrometry Method for Vitamin D Metabolites to Detect 24-Hydroxylase Deficiency

Sieglinde Zelzer (1), Andreas Meinitzer (1), Dietmar Enko (2), Sebastian Simstich (1), Walter Goessler (3), Markus Herrmann (1)
(1) Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria (2) Institute of Clinical Chemistry and Laboratory Medicine, General Hospital Hochsteiermark, Austria (3) Institute of Chemistry, University of Graz, 8010 Graz, Austria

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Sieglinde Zelzer (Presenter) Medical University Graz >> POSTER (PDF)

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION: Most clinical laboratories measure total 25-hydroxy vitamin D by immunoassays with variable analytical performance. Other relevant metabolites, such as 25-hydroxy vitamin D2 (25(OH)D2), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 25,26-dihydroxyvitamin D3 (25,26(OH)2D3) may provide additional information beyond the simple measurement of 25(OH)D. Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) allows the simultaneous measurement of multiple vitamin D metabolites with high sensitivity and specificity.
OBJECTIVES: Hence, we established an in-house LC-MS/MS method, for the determination of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3 and 25,26(OH)2D3 in order to detect individuals with 24-hydroxylase deficiency.
METHODS: Our method is based on the derivatization with 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) after protein precipitation and liquid-liquid-extraction. Chromatographic separation was performed on an Agilent HPLC 1260 system using a Zorbax C18Eclipse column and a gradient method with 2 mobile phases. The eluate was introduced into a Sciex 4500 MS/MS instrument for the detection of all four metabolites with a run time of 18 min.
This method was assessed by linearity, limit of detection (LOD), limit of quantification (LOQ), imprecision and recovery. Accuracy was analyzed by method comparison and the reliability of this method was evaluated in four patients, which were referred to our laboratory.
RESULTS: The analytical performance of our new method showed the following results: the within- and between-run precisions ranged between 1.8 and 10.4% and were within the acceptance criteria of <15 %. The LOD was 1.5 nmol/L for 25(OH)D3, and 0.3 nmol/L for 25(OH)2, 24,25(OH)2D3 and 25,26(OH)2D3. The LOQ was 3.1 nmol/L for 25(OH)D3, and 1.0 nmol/L for 25(OH)2, 24,25(OH)2D3 and 25,26(OH)2D3, respectively. In all four subjects a total 25(OH)D (=25(OH)D3 + 25(OH)D2) serum concentration of >100 nmol/L was measured. One subject had distinct lower 24,25(OH)2D3 and 25,26(OH)D3 concentrations of 0.16 nmol/L and 2.35 nmol/L, respectively. Subsequent sequencing of the 24-hydroxylase gene (CYP 24A1) confirmed an inactivating mutation.
CONCLUSION: Our in-house method showed a good and reliable analytical performance for all four tested vitamin D metabolites. Additionally, the establishment of this vitamin D assay enabled to detect a defect of the CYP24A1 gene coding for 24-hydroxylase, which was confirmed with CYP24A1 sequencing.