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Rapid Quantitation of 25-Hydroxyvitamin D2 and D3 in Human Serum Using Liquid Chromatography/drift Tube Ion Mobility-Mass Spectrometry

submitted on 10.06.2019, 01:57 and posted on 11.06.2019, 17:05 by Nicholas R. Oranzi, Jiajun Lei, Robin H. J. Kemperman, Christopher D. Chouinard, Brett Holmquist, Timothy J. Garrett, Richard Yost

Ion mobility was integrated with liquid chromatography/high resolution mass spectrometry (LC/IM-HRMS) to quantify 25-hydroxyvitamin D (25OHD) in human serum. It has previously been shown that 25OHD adopts two gas-phase conformations which are resolved using ion mobility; in contrast, the inactive epimer, 3-epi-25-hydroxyvitamin D (epi25OHD) only adopts one. Interference from epi25OHD was eliminated by filtering the chromatogram to retain the drift time that corresponds to the unique gas-phase conformation of 25OHD. Although ion mobility separates the epimers, some chromatography is required to separate compounds which interfere with ionization or fall at the same nominal m/z. Standards were prepared in 4% albumin solutions and compared against commercial serum quality controls. Standards and quality controls were analyzed and validated using a two-minute LC/IM-MS method. 25-hydroxyvitamin D3 and D2 were quantified over the range between 2 and 500 ng/mL with bias and precision within 15%. When epi25OHD was spiked into quality control samples, no significant bias was introduced, and analysis of 30 patient samples shows good agreement between this LC/IM-MS and traditional LC/MS/MS methods. This work shows that ion mobility can be incorporated with liquid chromatography and mass spectrometry for rapid quantitation of 25OHD in human serum.


National Institute of Health #U24DK097209

Wellspring Clinical Lab



Email Address of Submitting Author


University of Florida


United States

ORCID For Submitting Author


Declaration of Conflict of Interest