Analytical and Bioanalytical Chemistry

, Volume 408, Issue 27, pp 7617–7627 | Cite as

Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC–MS/MS for clinical laboratory applications

  • Mamoru Satoh
  • Takayuki Ishige
  • Shoujiro Ogawa
  • Motoi Nishimura
  • Kazuyuki Matsushita
  • Tatsuya Higashi
  • Fumio NomuraEmail author
Research Paper
Part of the following topical collections:
  1. New Applications of Mass Spectrometry in Biomedicine


The quantification of serum 25-hydroxyvitamin D [25(OH)D] as an indicator of vitamin D status is currently primarily conducted by immunoassays, yet LC–MS/MS would allow more accurate determination. Furthermore, LC–MS/MS would allow simultaneous measurement of multiple analytes. The aim of this study was to develop and validate an LC–MS/MS method to simultaneously measure four vitamin D metabolites (25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3) in serum for clinical laboratory applications. Serum samples were first prepared in a 96-well supported liquid extraction plate and the eluate was derivatized using the Cookson-type reagent 4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), which rapidly and quantitatively reacts with the s-cis-diene structure of vitamin D metabolites. The derivatized samples were subjected to LC–MS/MS, ionized by electrospray ionization (positive-ion mode), and detected by selected reaction monitoring. The lower limits of quantification for 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 were 0.091, 0.020, 0.013, and 0.024 ng/mL, respectively. The accuracy values and the extraction recoveries for these four metabolites were satisfactory. Serum 25(OH)D levels determined by our LC–MS/MS were compared with those obtained by conventional radioimmunoassay (RIA) that cannot distinguish 25(OH)D3 and 25(OH)D2. The values obtained by the RIA method exhibited a mean bias of about 8.35 ng/mL, most likely as a result of cross reaction of the antibody with low-abundance metabolites, including 24,25(OH)2D3. Various preanalytical factors, such as long sample sitting prior to serum separation, repeated freeze–thaw cycles, and the presence of anticoagulants, had no significant effects on these determinations. This high-throughput LC–MS/MS simultaneous assay of the four vitamin D metabolites 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 required as little as 20 μL serum. This method will aid further understanding of low-abundance vitamin D metabolites, as well as the accurate determination of 25(OH)D3 and 25(OH)D2.


25(OH)D3 3-epi-25(OH)D3 24,25(OH)2D3 25(OH)D2 4-(4′-Dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) Clinical laboratory 



25-hydroxyvitamin D3


3-epi-25-hydroxyvitamin D3


(24R)-24,25-dihydroxyvitamin D3


25-hydroxyvitamin D2


coefficient of variation




electrospray ionization


internal standard


limit of detection


limit of quantification


lower limit of quantification


selected reaction monitoring



This research was partially supported by the Ministry of Education, Culture, Science, Sports and Science and Technology (MEXT) by a Grant-in-Aid for Scientific Research (C), 2015-2018, 15K08610 (M. Satoh) and a Grant-in-Aid for Research Activity Start-up, 15H06096 (T. Ishige).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

216_2016_9821_MOESM1_ESM.pdf (706 kb)
ESM 1 (PDF 705 kb)


  1. 1.
    DeLuca HF. The vitamin D story: a collaborative effort of basic science and clinical medicine. FASEB J. 1988;2(3):224–36.Google Scholar
  2. 2.
    Seo E-G, Norman AW. Three-fold induction of renal 25-hydroxyvitamin D3-24-hydroxylase activity and increased serum 24,25-dihydroxyvitamin D3 levels are correlated with the healing process after chick tibial fracture. J Bone Miner Res. 1997;12(4):598–606.CrossRefGoogle Scholar
  3. 3.
    Wagner D, Hanwell HE, Schnabl K, Yazdanpanah M, Kimball S, Fu L, et al. The ratio of serum 24,25-dihydroxyvitamin D(3) to 25-hydroxyvitamin D(3) is predictive of 25-hydroxyvitamin D(3) response to vitamin D(3) supplementation. J Steroid Biochem Mol Biol. 2011;126(3-5):72–7.CrossRefGoogle Scholar
  4. 4.
    Higashi T, Ogasawara A, Shimada K. Investigation of C-3 epimerization mechanism of 24,25-dihydroxyvitamin D3 in rat using liquid chromatography/mass spectrometry. Anal Sci. 2000;16:477–82.CrossRefGoogle Scholar
  5. 5.
    Kamao M, Tatematsu S, Hatakeyama S, Sakaki T, Sawada N, Inouye K, et al. C-3 epimerization of vitamin D3 metabolites and further metabolism of C-3 epimers: 25-hydroxyvitamin D3 is metabolized to 3-epi-25-hydroxyvitamin D3 and subsequently metabolized through C-1alpha or C-24 hydroxylation. J Biol Chem. 2004;279(16):15897–907.CrossRefGoogle Scholar
  6. 6.
    Higashi T, Suzuki M, Hanai J, Inagaki S, Min JZ, Shimada K, et al. A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3. J Sep Sci. 2011;34(7):725–32.CrossRefGoogle Scholar
  7. 7.
    Strathmann FG, Sadilkova K, Laha TJ, LeSourd SE, Bornhorst JA, Hoofnagle AN, et al. 3-epi-25 Hydroxyvitamin D concentrations are not correlated with age in a cohort of infants and adults. Clin Chim Acta. 2012;413(1–2):203–6.CrossRefGoogle Scholar
  8. 8.
    Mochizuki A, Kodera Y, Saito T, Satoh M, Sogawa K, Nishimura M, et al. Preanalytical evaluation of serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 measurements using LC-MS/MS. Clin Chim Acta. 2013;420:114–20.CrossRefGoogle Scholar
  9. 9.
    Ogawa S, Ooki S, Morohashi M, Yamagata K, Higashi T. A novel Cookson-type reagent for enhancing sensitivity and specificity in assessment of infant vitamin D status using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2013;27(21):2453–60.CrossRefGoogle Scholar
  10. 10.
    Shimizu M, Yamada S. New fluorescence-labeling reagent targeting conjugated dienes: application to the fluorometric analysis of vitamin D and A metabolites. Vitamins (Japan). 1994;68:15–30.Google Scholar
  11. 11.
    van den Ouweland JM, Vogeser M, Bacher S. Vitamin D and metabolites measurement by tandem mass spectrometry. Rev Endocr Metab Disord. 2013;14(2):159–84.CrossRefGoogle Scholar
  12. 12.
    Meunier C, Blondelle D, Faure P, Baguet JP, Le Goff C, Chabre O, et al. Development and validation of a method using supported liquid extraction for aldosterone determination in human plasma by LC-MS/MS. Clin Chim Acta. 2015;447:8–15.CrossRefGoogle Scholar
  13. 13.
    Rositano J, Harpas P, Kostakis C, Scott T. Supported liquid extraction (SLE) for the analysis of methylamphetamine, methylenedioxymethylamphetamine and delta-9-tetrahydrocannabinol in oral fluid and blood of drivers. Forensic Sci Int. 2016;265:125–30.CrossRefGoogle Scholar
  14. 14.
    Geib T, Meier F, Schorr P, Lammert F, Stokes CS. Volmer DA A simple micro-extraction plate assay for automated LC-MS/MS analysis of human serum 25-hydroxyvitamin D levels. J Mass Spectrom. 2015;50(1):275–9.CrossRefGoogle Scholar
  15. 15.
    Trufelli H, Palma P, Famiglini G, Cappiello A. An overview of matrix effects in liquid chromatography–mass spectrometry. Mass Spectrom Rev. 2011;30(3):491–509.CrossRefGoogle Scholar
  16. 16.
    Ismaiel OA, Halquist MS, Elmamly MY, Shalaby A, Karnes HT. Monitoring phospholipids for assessment of matrix effects in a liquid chromatography-tandem mass spectrometry method for hydrocodone and pseudoephedrine in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;859(1):84–93.CrossRefGoogle Scholar
  17. 17.
    Bylda C, Thiele R, Kobold U, Volmer DA. Recent advances in sample preparation techniques to overcome difficulties encountered during quantitative analysis of small molecules from biofluids using LC-MS/MS. Analyst. 2014;139(10):2265–76.CrossRefGoogle Scholar
  18. 18.
    Singh RJ, Taylor RL, Reddy GS, Grebe SK. 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. J Clin Endocrinol Metab. 2006;91(8):3055–61.CrossRefGoogle Scholar
  19. 19.
    Shah I, James R, Barker J, Petroczi A, Naughton DP. Misleading measures in Vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars. Nutr J. 2011;10(1):1–9.CrossRefGoogle Scholar
  20. 20.
    Qi Y, Geib T, Schorr P, Meier F, Volmer DA. On the isobaric space of 25-hydroxyvitamin D in human serum: potential for interferences in liquid chromatography/tandem mass spectrometry, systematic errors and accuracy issues. Rapid Commun Mass Spectrom. 2015;29(1):1–9.CrossRefGoogle Scholar
  21. 21.
    Ogawa S, Ooki S, Shinoda K, Higashi T. Analysis of urinary vitamin D(3) metabolites by liquid chromatography/tandem mass spectrometry with ESI-enhancing and stable isotope-coded derivatization. Anal Bioanal Chem. 2014;406(26):6647–54.CrossRefGoogle Scholar
  22. 22.
    Müller MJ, Volmer DA. Mass spectrometric profiling of vitamin D metabolites beyond 25-hydroxyvitamin D. Clin Chem. 2015;61(8):1033–48.CrossRefGoogle Scholar
  23. 23.
    Volmer DA, Mendes LR, Stokes CS. Analysis of vitamin D metabolic markers by mass spectrometry: current techniques, limitations of the "gold standard" method, and anticipated future directions. Mass Spectrom Rev. 2015;34(1):2–23.CrossRefGoogle Scholar
  24. 24.
    Baecher S, Leinenbach A, Wright JA, Pongratz S, Kobold U, Thiele R. Simultaneous quantification of four vitamin D metabolites in human serum using high performance liquid chromatography tandem mass spectrometry for vitamin D profiling. Clin Biochem. 2012;45(16-17):1491–6.CrossRefGoogle Scholar
  25. 25.
    French D. Development and validation of a serum total testosterone liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay calibrated to NIST SRM 971. Clin Chim Acta. 2013;415:109–17.CrossRefGoogle Scholar
  26. 26.
    Pauwels S, Antonio L, Jans I, Lintermans A, Neven P, Claessens F, et al. Sensitive routine liquid chromatography-tandem mass spectrometry method for serum estradiol and estrone without derivatization. Anal Bioanal Chem. 2013;405(26):8569–77.CrossRefGoogle Scholar
  27. 27.
    Singh RJ. Are clinical laboratories prepared for accurate testing of 25-hydroxy vitamin D? Clin Chem. 2008;54(1):221–3.CrossRefGoogle Scholar
  28. 28.
    Cashman KD, Hayes A, Galvin K, Merkel J, Jones G, Kaufmann M, et al. Significance of serum 24,25-dihydroxyvitamin D in the assessment of vitamin D status: a double-edged sword? Clin Chem. 2015;61(4):636–45.CrossRefGoogle Scholar
  29. 29.
    Bailey D, Veljkovic K, Yazdanpanah M, Adeli K. Analytical measurement and clinical relevance of vitamin D(3) C3-epimer. Clin Biochem. 2013;46(3):190–6.CrossRefGoogle Scholar
  30. 30.
    Wientroub S, Price PA, Reddi AH. The dichotomy in the effects of 1,25 dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 on bone γ-carboxyglutamic acid-containing protein in serum and bone in vitamin D-deficient rats. Calcif Tissue Int. 1987;40(3):166–72.CrossRefGoogle Scholar
  31. 31.
    van Driel M, Koedam M, Buurman CJ, Roelse M, Weyts F, Chiba H, et al. Evidence that both 1alpha,25-dihydroxyvitamin D3 and 24-hydroxylated D3 enhance human osteoblast differentiation and mineralization. J Cell Biochem. 2006;99(3):922–35.CrossRefGoogle Scholar
  32. 32.
    Wehmeier KR, Alamir A-R, Sultan S, Haas MJ, Wong NCW, Mooradian AD. 24, 25-Dihydroxycholecalciferol but not 25-hydroxycholecalciferol suppresses apolipoprotein A-I gene expression. Life Sci. 2011;88(1–2):110–6.CrossRefGoogle Scholar
  33. 33.
    Wehmeier K, Onstead-Haas LM, Wong NCW, Mooradian AD, Haas MJ. Pro-inflammatory signaling by 24,25-dihydroxyvitamin D3 in HepG2 cells. J Mol Endocrinol. 2016;57(2):87–96.CrossRefGoogle Scholar
  34. 34.
    Schlingmann KP, Kaufmann M, Weber S, Irwin A, Goos C, John U, et al. Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia. N Engl J Med. 2011;365(5):410–21.CrossRefGoogle Scholar
  35. 35.
    Dauber A, Nguyen TT, Sochett E, Cole DE, Horst R, Abrams SA, et al. Genetic defect in CYP24A1, the vitamin D 24-hydroxylase gene, in a patient with severe infantile hypercalcemia. J Clin Endocrinol Metab. 2012;97(2):E268–74.CrossRefGoogle Scholar
  36. 36.
    Ketha H, Kumar R, Singh RJ. LC-MS/MS for identifying patients with CYP24A1 mutations. Clin Chem. 2016;62(1):236–42.CrossRefGoogle Scholar
  37. 37.
    Wiebe D, Binkley N. Case report: three patients with substantial serum levels of 3-epi-25(OH)D including one with 3-epi-25(OH)D2 while on high-dose ergocalciferol. J Clin Endocrinol Metab. 2014;99(4):1117–21.CrossRefGoogle Scholar
  38. 38.
    Reddy GS, Rao DS, Siu-Caldera M-L, Astecker N, Weiskopf A, Vouros P, et al. 1α,25-Dihydroxy-16-ene-23-yne-vitamin D3 and 1α,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3: analogs of 1α,25-dihydroxyvitamin D3 that resist metabolism through the C-24 oxidation pathway are metabolized through the C-3 epimerization pathway. Arch Biochem Biophys. 2000;383(2):197–205.CrossRefGoogle Scholar
  39. 39.
    Molnár F, Sigüeiro R, Sato Y, Araujo C, Schuster I, Antony P, et al. 1α,25(OH)(2)-3-epi-Vitamin D(3), a natural physiological metabolite of vitamin D(3): its synthesis, biological activity and crystal structure with its receptor. PLoS One. 2011;6(3):e18124.CrossRefGoogle Scholar
  40. 40.
    Wang C, Shiraishi S, Leung A, Baravarian S, Hull L, Goh V, et al. Validation of a testosterone and dihydrotestosterone liquid chromatography tandem mass spectrometry assay: Interference and comparison with established methods. Steroids. 2008;73(13):1345–52.CrossRefGoogle Scholar
  41. 41.
    Garg U, Munar A, Frazee C, Scott D. A simple, rapid atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry method for the determination of 25-hydroxyvitamin D2 and D3. J Clin Lab Anal. 2012;26(5):349–57.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Mamoru Satoh
    • 1
  • Takayuki Ishige
    • 2
  • Shoujiro Ogawa
    • 3
  • Motoi Nishimura
    • 2
    • 4
  • Kazuyuki Matsushita
    • 2
    • 4
  • Tatsuya Higashi
    • 3
  • Fumio Nomura
    • 1
    Email author
  1. 1.Division of Clinical Mass SpectrometryChiba University HospitalChiba-shiJapan
  2. 2.Division of Laboratory MedicineChiba University HospitalChiba-shiJapan
  3. 3.Faculty of Pharmaceutical SciencesTokyo University of ScienceNoda-shiJapan
  4. 4.Department of Molecular Diagnosis, Graduate School of MedicineChiba UniversityChiba-shiJapan

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