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.
Highlights
Mass spectrometric quantification of small molecules in routine laboratory testing utilizes liquid chromatography coupled to low mass resolution triple-quadrupole mass spectrometers (QQQs)
We used a hybrid instrument quadrupole-Orbitrap that offers high mass resolution with up to 140,000 at m/z 200. Based on their high specificity, these instruments have been applied in full scan or targeted selected ion monitoring (SIM) [15]
We decided to used MS/highresolution mass spectrometry (HR-MS) with a mass resolution setting of 35,000 that permits separation of quasi-isobaric product ions at m/z 383 generated from 25(OH)D3 and D6-25(OH)D2
Summary
Mass spectrometric quantification of small molecules in routine laboratory testing utilizes liquid chromatography coupled to low mass resolution triple-quadrupole mass spectrometers (QQQs). We introduce highresolution 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. High mass resolution was advantageously applied to separate a quasi-isobaric interference of the internal standard D625(OH)D2 with 3-epi-25(OH)D3. Quantification of 25(OH)D by LCMS/HR-MS is applicable for routine testing and holds promise for highly specific quantification of other small molecules.—Liebisch, G., and S. Accurate and reliable quantification of 25-hydroxy-vitamin D species by liquid chromatography high-resolution tandem mass spectrometry.
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