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Optimized 25-hydroxyvitamin D analysis using liquid–liquid extraction with 2D separation with LC/MS/MS detection, provides superior precision compared to conventional assays

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Abstract

The analysis of 25-hydroxyvitamin D3 (25(OH)D3) and related metabolites represents a considerable challenge for both clinical and research laboratories worldwide. There is currently debate about the best methodology employed to assess vitamin D status and whether the 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3) should be separated and quantitated when measuring 25(OH)D3. Mass spectrometry techniques are generally regarded as the gold standard due to high specificity for vitamin D metabolites. However, many methods require high sample volumes for analysis. We have developed a new 2 dimensional (2D) ultra performance liquid chromatography (UPLC) separation coupled tandem mass spectrometry (MS/MS) detection to accurately quantitate 25(OH)D3, epi-25(OH)D3, and 25(OH)D2 in adults and children, requiring only 50 μL of human serum. The assay gives excellent separation of epi-25(OH)D3, and 25(OH)D2 from 25(OH)D3, has excellent precision with an intra-assay CV of 0.5 % at 74 nmol/L and can report down to 2 nmol/L for 25(OH)D3. Furthermore, the method shows excellent agreement with the vitamin D external quality assessment scheme (DEQAS) quality control program for vitamin D analysis. We present this approach as a candidate reference method for 25(OH)D3, epi-25(OH)D3, and 25(OH)D2 analysis in humans.

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References

  • Ding, S., Schoenmakers, I., Jones, K., Koulman, A., Prentice, A., & Volmer, D. (2010). Quantitative determination of vitamin D metabolites in plasma using UHPLC-MS/MS. Analytical and Bioanalytical Chemistry, 398, 779–789.

    Article  PubMed  CAS  Google Scholar 

  • El-Khoury, J. M., Reineks, E. Z., & Wang, S. (2011). Progress of liquid chromatography-mass spectrometry in measurement of vitamin D metabolites and analogues. Clinical Biochemistry, 44, 66–76.

    Article  PubMed  CAS  Google Scholar 

  • Farrell, C.-J. L., Martin, S., McWhinney, B., Straub, I., Williams, P., & Herrmann, M. (2012). State-of-the-art vitamin D assays: A comparison of automated immunoassays with liquid chromatography–tandem mass spectrometry methods. Clinical Chemistry, 58, 531–542.

    Article  PubMed  CAS  Google Scholar 

  • Grebe, S. K., & Singh, R. J. (2011). LC-MS/MS in the clinical laboratory—Where to from here? Clinical Biochemist Reviews, 32, 5–31.

    Google Scholar 

  • Herrmann, M., et al. (2010). A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D. Steroids, 75, 1106–1112.

    Article  PubMed  CAS  Google Scholar 

  • Hollis, B. W. (2008). Measuring 25-hydroxyvitamin D in a clinical environment: Challenges and needs. American Journal of Clinical Nutrition, 88, 507S–510S.

    PubMed  CAS  Google Scholar 

  • Kamao, M., et al. (2004). C-3 epimerization of vitamin D3 metabolites and further metabolism of C-3 epimers. Journal of Biological Chemistry, 279, 15897–15907.

    Article  PubMed  CAS  Google Scholar 

  • Kushnir, M. M., et al. (2010). Rapid analysis of 25-hydroxyvitamin D2 and D3 by liquid chromatography–tandem mass spectrometry and association of vitamin d and parathyroid hormone concentrations in healthy adults. American Journal of Clinical Pathology, 134, 148–156.

    Article  PubMed  CAS  Google Scholar 

  • Lensmeyer, G., Poquette, M., Wiebe, D., & Binkley, N. (2012). The C-3 epimer of 25-hydroxyvitamin D3 is present in adult serum. Journal of Clinical Endocrinology and Metabolism, 97, 163–168.

    Article  PubMed  CAS  Google Scholar 

  • Maunsell, Z., Wright, D. J., & Rainbow, S. J. (2005). Routine isotope-dilution liquid chromatography–tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3. Clinical Chemistry, 51, 1683–1690.

    Article  PubMed  CAS  Google Scholar 

  • Molnar, F., et al. (2011). 1 alpha,25(OH) 2–3-epi-vitamin D3, a natural physiological metabolite of vitamin D3: Its synthesis, biological activity and crystal structure with its receptor. PLoS One, 6, e18124.

    Article  PubMed  CAS  Google Scholar 

  • Moon, H.-W., et al. (2012). Comparison of four current 25-hydroxyvitamin D assays. Clinical Biochemistry, 45, 326–330.

    Article  PubMed  CAS  Google Scholar 

  • Plum, L. A., & DeLuca, H. F. (2010). Vitamin D, disease and therapeutic opportunities. Nat Rev Drug Discov, 9, 941–955.

    Article  PubMed  CAS  Google Scholar 

  • Saenger, A. K., Laha, T. J., Bremner, D. E., & Sadrzadeh, S. M. H. (2006). Quantification of serum 25-hydroxyvitamin D2 and D3 using HPLC–tandem mass spectrometry and examination of reference intervals for diagnosis of vitamin D deficiency. American Journal of Clinical Pathology, 125, 914–920.

    Article  PubMed  CAS  Google Scholar 

  • Schleicher, R. L., Encisco, S. E., Chaudhary-Webb, M., Paliakov, E., McCoy, L. F., & Pfeiffer, C. M. (2011). Isotope dilution ultra performance liquid chromatography-tandem mass spectrometry method for simultaneous measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in human serum. Clinica Chimica Acta, 412, 1594–1599.

    Article  CAS  Google Scholar 

  • Shah, I., James, R., Barker, J., Petroczi, A., & Naughton, D. P. (2011). Misleading measures in vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars. Nutrition Journal, 10, 46.

    Article  PubMed  CAS  Google Scholar 

  • Singh, R. J., Taylor, R. L., Reddy, G. S., & Grebe, S. K. G. (2006). C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. Journal of Clinical Endocrinology and Metabolism, 91, 3055–3061.

    Article  PubMed  CAS  Google Scholar 

  • Stepman, H. C. M., Vanderroost, A., Van Uytfanghe, K., & Thienpont, L. M. (2011). Candidate reference measurement procedures for serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 by using isotope-dilution liquid chromatography–tandem mass spectrometry. Clinical Chemistry, 57, 441–448.

    Article  PubMed  CAS  Google Scholar 

  • Tai, S. S. C., Bedner, M., & Phinney, K. W. (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, 82, 1942–1948.

    Article  PubMed  CAS  Google Scholar 

  • Thibeault, D., Caron, N., Djiana, R., Kremer, R., & Blank, D. (2012). Development and optimization of simplified LC–MS/MS quantification of 25-hydroxyvitamin D using protein precipitation combined with on-line solid phase extraction (SPE). Journal of Chromatography B, 883–884, 120–127.

    Article  Google Scholar 

  • van den Ouweland, J. M. W., Beijers, A. M., Demacker, P. N. M., & van Daal, H. (2010). Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay. Journal of Chromatography B, 878, 1163–1168.

    Article  Google Scholar 

  • van den Ouweland, J. M. W., Beijers, A. M., & van Daal, H. (2011). Fast separation of 25-hydroxyvitamin D3 from 3-epi-25-hydroxyvitamin D3 in human serum by liquid chromatography–tandem mass spectrometry: Variable prevalence of 3-epi-25-hydroxyvitamin D3 in infants, children, and adults. Clinical Chemistry, 57, 1618–1619.

    Article  PubMed  Google Scholar 

  • Vogeser, M., Kyriatsoulis, A., Huber, E., & Kobold, U. (2004). Candidate Reference Method for the Quantification of Circulating 25-Hydroxyvitamin D3 by Liquid Chromatography–Tandem Mass Spectrometry. Clinical Chemistry, 50, 1415–1417.

    Article  PubMed  CAS  Google Scholar 

  • Wagner, D., et al. (2011). The ratio of serum 24,25-dihydroxyvitamin D3 to 25-hydroxyvitamin D3 is predictive of 25-hydroxyvitamin D3 response to vitamin D3 supplementation. The Journal of Steroid Biochemistry and Molecular Biology, 126, 72–77.

    Article  PubMed  CAS  Google Scholar 

  • Wallace, A. M., Gibson, S., de la Hunty, A., Lamberg-Allardt, C., & Ashwell, M. (2010). Measurement of 25-hydroxyvitamin D in the clinical laboratory: Current procedures, performance characteristics and limitations. Steroids, 75, 477–488.

    Article  PubMed  CAS  Google Scholar 

  • Yetley, E. A., et al. (2010). NHANES monitoring of serum 25-hydroxyvitamin D: A roundtable summary. The Journal of Nutrition, 140, 2030S–2045S.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors wish to thank Bill McConnell and Sydney Sacks from Clinipath Laboratories, Perth, Western Australia for providing adult serum samples used in this study. We also wish to thank Annabel Mitchell and Chris Fouracre from Agilent Technologies for their input into the method development.

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Correspondence to Michael W. Clarke.

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Clarke, M.W., Tuckey, R.C., Gorman, S. et al. Optimized 25-hydroxyvitamin D analysis using liquid–liquid extraction with 2D separation with LC/MS/MS detection, provides superior precision compared to conventional assays. Metabolomics 9, 1031–1040 (2013). https://doi.org/10.1007/s11306-013-0518-9

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  • DOI: https://doi.org/10.1007/s11306-013-0518-9

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