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A candidate reference measurement procedure for quantifying serum concentrations of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 using isotope-dilution liquid chromatography-tandem mass spectrometry

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Abstract

The inaccuracy of routine serum 25-hydroxyvitamin D measurements hampers the interpretation of data in patient care and public health research. We developed and validated a candidate reference measurement procedure (RMP) for highly accurate quantitation of two clinically important 25-hydroxyvitamin D metabolites in serum, 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3]. The two compounds of interest together with spiked deuterium-labeled internal standards [d 3-25(OH)D2 and d 6-25(OH)D3] were extracted from serum via liquid-liquid extraction. The featured isotope-dilution LC-MS/MS method used reversed-phase chromatography and atmospheric pressure chemical ionization in positive ion mode. A pentafluorophenylpropyl-packed UHPLC column together with isocratic elution allowed for complete baseline resolution of 25(OH)D2 and 25(OH)D3 from their structural C-3 isomers within 12 min. We evaluated method trueness, precision, potential interferences, matrix effects, limits of quantitation, and measurement uncertainty. Calibration materials were, or were traceable to, NIST Standard Reference Materials 2972. Within-day and total imprecision (CV) averaged 1.9 and 2.0 % for 25(OH)D3, respectively, and 2.4 and 3.5 % for 25(OH)D2, respectively. Mean trueness was 100.3 % for 25(OH)D3 and 25(OH)D2. The limits of quantitation/limits of detection were 4.61/1.38 nmol/L for 25(OH)D3 and 1.46/0.13 nmol/L for 25(OH)D2. When we compared our RMP results to an established RMP using 40 serum samples, we found a nonsignificant mean bias of 0.2 % for total 25(OH)D. This candidate RMP for 25(OH)D metabolites meets predefined method performance specifications (≤5 % total CV and ≤1.7 % bias) and provides sufficient sample throughput to meet the needs of the Centers for Disease Control and Prevention Vitamin D Standardization Certification Program.

Bias assessment using NIST standard reference materials. Legend CDC mean mass fractions (ng/g) ± U 95 (6 replicates per mean). NIST-certified mass fractions (ng/g) ± U 95 from the Certificates of Analysis

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Acknowledgments

The authors thank Dr. Susan Tai from NIST for her guidance and advice during the method development. We would also like to thank Dr. Katleen Van Uytfanghe from the University of Ghent and Dr. Mary Bedner from NIST for their guidance on RMP measurement series acceptance criteria. In addition, we would like to thank all members of the hormone standardization research team at the CDC for providing logistic support.

Conflict of interest

No specific sources of financial support. The findings and conclusions in this report are those of the authors and do not necessarily represent the official views or positions of the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry.

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Authors and Affiliations

  1. Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Mail Stop F55, Atlanta, GA, 30341, USA

    Ekaterina M. Mineva, Rosemary L. Schleicher, Madhulika Chaudhary-Webb, Khin L. Maw, Julianne C. Botelho, Hubert W. Vesper & Christine M. Pfeiffer

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  1. Ekaterina M. Mineva
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  2. Rosemary L. Schleicher
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  3. Madhulika Chaudhary-Webb
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Correspondence to Rosemary L. Schleicher.

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Mineva, E.M., Schleicher, R.L., Chaudhary-Webb, M. et al. A candidate reference measurement procedure for quantifying serum concentrations of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 using isotope-dilution liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 407, 5615–5624 (2015). https://doi.org/10.1007/s00216-015-8733-z

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