MSACL 2018 US Abstract

Topic: Endocrinology

LC-MS/MS Method for Measurement of Free 25-Hydroxy Vitamin D

Mark Kushnir (Presenter)
ARUP Institute for Clinical and Experimental Patho

Bio: Mark Kushnir, Ph.D. Senior Scientist ARUP Institute for Clinical and Experimental Pathology Education: PhD in Analytical Chemistry from Uppsala University (Sweden). Affiliations: • Adjunct Assistant Professor, Department of Pathology, University of Utah Publications and presentations: • Author/coauthor of over 100 publications in scientific Journals • Over 130 scientific presentations at scientific conferences • 8 patents • Author/coauthor of 8 book chapters • Citation index 29 Reviewer for Journals: Clinical Chemistry, Clinical Biochemistry, Clin Chim Acta, Rapid Communications in Mass Spectrometry, Journal of American Society of Mass Spectrometry, Journal of Chromatography, Bioanalysis.

Authorship: Mark M. Kushnir (1), Alan L. Rockwood (2), Joely A. Straseski (3).
(1) ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, USA; (2) Rockwood Scientific Consulting, Salt Lake City, USA; (3) Department of Pathology, University of Utah, Salt Lake City, USA.

Short Abstract

25-hydroxy vitamin D (25OHD) is widely used as biochemical marker for assessment of status of vitamin D. It was demonstrated that the majority of cells in the human body respond to free rather than total 25OHD, therefore measurement of free 25OHD should be more relevant than total 25OHD to assess the physiological actions of vitamin D. We developed an LC-MS/MS method that allows accurate measurement of free 25OHD in biological samples and evaluated its performance. Sensitivity and specificity of the method are sufficient to quantify free 25OHD in samples of healthy and pathologic individuals.

Long Abstract


Serum 25-hydroxy vitamin D (25OHD) is widely used as biochemical marker to assess vitamin D storage. 25OHD is highly lipophilic and is tightly bound to vitamin D binding protein (VDBP); a smaller fraction of 25OHD is weakly bound to albumin. Similar to the mechanism of action of several other hormones, it has been demonstrated that the majority of cells in the human body respond to free rather than total 25OHD. Therefore measurement of free 25OHD is likely more relevant than total 25OHD for assessment of the local physiological action of vitamin D. Two methods for determining free 25OHD concentrations are currently available: an ELISA (Future Diagnostics, The Netherlands) and a calculation-based method utilizing measurement of serum concentrations of total 25OHD, VDBP and albumin, and the corresponding binding constants.


We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the measurement of free 25OHD. Sample preparation was performed as follows: free 25OHD2 and 25OHD3 were separated from the protein bound fraction using size exclusion based separation, stable isotope labeled internal standard was added to the samples; 25OHD was derivatized and analyzed by LC-MS/MS. Two mass transitions were monitored for each analyte and the internal standard; the ratio of the primary and the secondary mass transitions for the analytes and the internal standards was used to assess specificity of the analysis.


The lower limit of quantification and upper limit of linearity of the assay for free 25OHD2 and 25OHD3 were 0.005 ng/mL and 3 ng/mL, respectively. Total imprecision of triplicate measurements in serum samples over five days was < 18%. Reasonably good correlation (r2=0.787, n=62) was observed with a commercial ELISA (Future Diagnostics), while concentrations measured by ELISA were on average 6.2 times lower than by the LC-MS/MS method. One likely explanation for the lower measured concentrations is irreversible binding of free 25OHD to the labware used in the ELISA. Using this method we established reference intervals for free 25OHD3 in serum samples of self-reported healthy adults (n=120; age 20-63). A nonparametric reference interval for free 25OHD3 was 0.024-0.073 ng/mL and for percent free 25OHD3 was 0.080-0.18%. In the reference interval study samples, we observed better association of PTH with the measured concentrations of free 25OHD (p=0.0024) than with total 25OHD (p=0.082).

Conclusions & Discussion

Our data suggest that the presented method is specific to measurement of free 25OHD2 and 25OHD3, and its performance characteristics are acceptable for use in clinical diagnostic applications. Sensitivity of the method is sufficient for quantitative measurements of free 25OHD in serum and plasma samples at concentrations expected in both, health and pathology.

References & Acknowledgements:

Financial Disclosure

Board MemberyesCLSI committee on proposed document C64 (measurement of proteins by LCMS)

IP Royalty: no

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