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October 2013, Volume 9, Issue 5, pp 1031–1040 | Cite as

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



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.


Vitamin D Mass spectrometry Epimer DEQAS Pediatric samples



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.



© Springer Science+Business Media New York 2013



Clarke, M.W., Tuckey, R.C., Gorman, S. et al. Metabolomics (2013) 9: 1031. https://doi.org/10.1007/s11306-013-0518-9



