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Determination of 25-Hydroxyvitamin D in Human Plasma Using High-Performance Liquid Chromatography−Tandem Mass Spectrometry

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Department of Hygienic Sciences, Kobe Pharmaceutical University, 4-19-1, Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
Cite this: Anal. Chem. 2005, 77, 9, 3001–3007
Publication Date (Web):April 2, 2005
https://doi.org/10.1021/ac048249c
Copyright © 2005 American Chemical Society

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    Abstract

    We report here the development of a precise and sensitive method to determine 25-hydroxyvitamin D (25-OH-D2/ -D3) in human plasma using high-perfomance liquid chromatography−tandem mass−mass spectrometry with atomospheric pressure chemical ionization (LC-APCI-MS/MS). The method involves the use of deuterated 25-OH-D3 as an internal standard compound for 25-OH-D2/-D3, which was synthesized in our laboratory, and the selection of a precursor and product ion with a MS/MS multiple reaction monitoring method. The average intraassay and interassay variation values (relative standard deviation) were 5.7 and 2.5%, respectively, for 25-OH-D3 and 4.5 and 5.1%, respectively, for 25-OH-D2. The average spiked recoveries from authentic compounds added to normal human plasma samples for 25-OH-D3 and 25-OH-D2 were 103.8 and 98.8%, respectively. Mean plasma concentrations of 25-OH-D3 and 25-OH-D2 in healthy subjects were 20.5 and 0.4 ng/mL, respectively. We conclude that this novel LC-APCI-MS/MS method would be useful for the evaluation of the vitamin D status in postmenopausal women and elderly subjects and provide useful information in the diagnosis of vitamin D insufficiency/deficiency, as well as for the treatment and prevention of osteoporosis with vitamin D.

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     To whom correspondence should be addressed. E-mail:  t-okano@ kobepharma-u.ac.jp.

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    Calibration curves for 25-OH-D3, 25-OH-D2, and 24,25(OH)2D3. This material is available free of charge via the Internet at http://pubs.acs.org.

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