title: Accurate and reliable quantification of 25-hydroxy-vitamin D species by liquid chromatography high-resolution tandem mass spectrometry.
abstract: In general, mass spectrometric quantification of small molecules in routine laboratory testing utilizes liquid chromatography coupled to low mass resolution triple-quadrupole mass spectrometers (QQQs). Here we introduce high-resolution tandem mass spectrometry (quadrupole-Orbitrap) for the quantification of 25-hydroxy-vitamin D [25(OH)D], a marker of the vitamin D status, because the specificity of 25(OH)D immunoassays is still questionable and mass spectrometric quantification is becoming increasingly important. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/HR-MS) was used to quantify 25-hydroxy-cholecalciferol [25(OH)D3], 25-hydroxy-ergocalciferol [25(OH)D2], and their C3-epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2. The method has a run time of 5 min and was validated according to the US Food and Drug Administration and the European Medicines Agency guidelines. High mass resolution was advantageously applied to separate a quasi-isobaric interference of the internal standard D6-25(OH)D2 with 3-epi-25(OH)D3. All analytes showed an imprecision of below 10% coefficient of variation (CV), trueness between 90% and 110%, and limits of quantification below 10 nM. Concentrations measured by LC-MS/HR-MS are in good agreement with those of the National Institute of Standards and Technology reference methods using LC-MS/MS (QQQ). In conclusion, quantification of 25(OH)D by LC-MS/HR-MS is applicable for routine testing and also holds promise for highly specific quantification of other small molecules.
authors.affiliation: Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Liebisch
authors.affiliation: Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Matysik
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014481:United States; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D014807:Vitamin D; C104450:25-hydroxyvitamin D
keywords: Orbitrap; cholecalciferol; clinical chemistry; epimers; ergocalciferol; laboratory medicine; routine testing
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