Development of a Candidate Reference Measurement Procedure for the Determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in Human Serum Using Isotope-Dilution Liquid Chromatography/Tandem Mass Spectrometry

Published

February 5, 2010

Author(s)

Susan S. Tai, Mary Bedner, Karen W. Phinney

Abstract

Vitamin D exists in two major forms, vitamin D3 and vitamin D2. Vitamin D helps the body absorb calcium and promote optimal growth. Both forms of vitamin D are metabolized to 25-hydroxyvitamin D in the body, and the levels of 25-hydroxyvitamin D3 (25-(OH)D3) and 25-hydroxyvitamin D2 (25-(OH)D2) in serum are the best indicators of vitamin D status. A Candidate reference measurement procedure for serum 25(OH)D3 and 25(OH)D2 has been developed and critically evaluated. Deuterated labeled compounds of 25(OH)D3-d3 and 25(OH)D2-d3 are used as internal standards for 25(OH)D3 and 25(OH) D2, respectively. The 25(OH)D3 and 25(OH)D2 along with their internal standards are simultaneously extracted from serum using liquid-liquid extraction prior to reversed-phase LC/MS/MS. Chromatographic separation was performed using a CN column for both 25(OH)D3 and 25(OH)D2. Atmosphere pressure chemical ionization (APCI) in the positive ion mode and multiple rection monitoring (MRM) were used for LC/MS/MS. The accuracy of the method was evaluated by recovery studies of measuring 25-hydroxyvitamin D (25(OH)D) in spiked samples with known 25(OH)D levels. The recoveries of the added 25(OH)D3 and 25(OH)D2 ranged from 99.0% to 101.0%. The absolute recoveries with this method were 97% and 92% for 25(OH)D3 and 25(OH)D2, respectively. Excellent precision was obtained with between-set CVs of 0.2% - 0.6% for 25(OH)D levels > 1 ng/g and within 2% for the level of < 1 ng/g. LC separation of 25(OH)D3 and 25(OH)D2 with their respective isomers of 3-epi-25(OH)D3 and 3-epi-25(OH)D2 was achieved. The limit of detection at a signal-to-noise ration of ~ 3 was 40 pg of 25(OH)D. This reference measurement procedure for serum 25(OH)D3 and 25(OH)D2, which demonstrates good accuracy and precision and low susceptibility to interferences, can be used to provide an accuracy base to which the methods for 25(OH)D3 and 25(OH)D2 used in the clinical laboratories can be compared.

Citation

Analytical Chemistry

Volume

82

Pub Type

Journals

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Keywords

25-hydroxyvitamin D3, 25(OH)D3, 25-hydroxyvitamin D2, 25(OH)D2, Reference measurement procedure, Atmospheric pressure chemical ionization, Isotope dilution, Liquid chromatography/tandem mass spectrometry, LC-MS/MS, Liquid-liquid extraction.

Citation

Tai, S. , Bedner, M. and Phinney, K. (2010), Development of a Candidate Reference Measurement Procedure for the Determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in Human Serum Using Isotope-Dilution Liquid Chromatography/Tandem Mass Spectrometry, Analytical Chemistry, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=903827 (Accessed April 29, 2024)