Poster Topical Area: Vitamins and Minerals

Location: Hall D

Poster Board Number: 504

P26-043 - 24R,25(OH)2D3 as potential interferent in measurement of 25-hydroxyvitamin D

Sunday, Jun 10
8:00 AM – 6:00 PM


  1. Objectives: Serum total 25(OH)D is a functional biomarker of vitamin D status. Tandem liquid chromatography-mass spectrometry is the preferred technique for quantitative analysis because of sensitivity and specificity; however, immunoassays are widely used in the clinic because of their high-throughput. Antibodies commonly used in clinical 25(OH)D assays are reported to cross-react with 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]. Serum concentrations of 24,25(OH)2D range from 2 to 20% of the total 25(OH)D, thus the interference can be substantial. Limited data showed in six widely used immunoassays a positive 25(OH)D bias ranging from 2 to 23% likely due to 24R,25(OH)2D3. We developed a new LC-MS/MS method for quantitation of 24R,25(OH)2D3, which will  provide data to two major vitamin D accuracy-based programs (CDC Vitamin D Standardization-Certification Program and the UK Vitamin D External Quality Assessment Scheme) to help identify the 24R,25(OH)2D3 cross-reactivity in immunoassay 25(OH)D measurements. The data will also inform immunoassay users about assay specificity.


  2. Methods:
    We developed an isotope-dilution LC-MS/MS method quantitation from serum using liquid-liquid extraction and positive APCI ionization.


  3. Results:
    The method features highly accurate measurements; the mean bias from standard reference materials was 0.2%, and the mean recovery from spiking of three different levels of 24R,25(OH)2D3 (5-15 nmol/L) into two different serum materials was 98 ±5%. Inter-assay imprecision in three QC materials with endogenous concentrations of 2.9, 5.9 and 9.7 nmol/L was 3.4, 2.9 and 3.4% CV, respectively. 24R,25(OH)2D3 was stable in two serum materials 1) at room temperature for 24 h, 2) for 3 freeze/thaw cycles, and 3) for long-term storage at -70 oC. Individual serum donors were screened (n=60), and 24R,25(OH)2D3 was detected in 80% of the samples with concentrations ranging from LOD (0.9 nmol/L) to 12.2 nmol/L.


  4. Conclusions:
    We have developed a specific and sensitive LC-MS/MS method, which provides accurate measurements of 24R,25(OH)2D3, without complex sample preparation and derivatization. It will provide assay manufacturers with data about this common cross-reacting metabolite.



CoAuthors: Gwendolyn Gabey – CDC Foundation; Rosemary Scheicher, PhD – NCEH./CDC

Ekaterina Mineva

Senior Research Scientist
CDC
Atlanta, Georgia