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Endocrinology and Metabolism |
1 Department of Clinical Biochemistry, Northwick Park Hospital, North West London Hospitals NHS Trust, Harrow, United Kingdom.
aAddress correspondence to this author at: Department of Clinical Biochemistry, Northwick Park Hospital, North West London Hospitals NHS Trust, Watford Rd., Harrow HA1 3UL, United Kingdom. Fax 44-20-8869-2119; e-mail sandra.rainbow{at}nwlh.nhs.uk.
Background: Measurement of 25-hydroxyvitamin D2 and D3 (25-OH D2 and D3) is essential for investigating vitamin D deficiency. Competitive binding techniques are unable to distinguish between the 2 metabolites and suffer from interference from other hydroxy metabolites of vitamin D.
Methods: We used isotope-dilution liquid chromatography–tandem mass spectrometry (ID-LC-MS/MS) for routine determination of 25-OH D2 and D3 with a stable-isotope–labeled internal standard (IS). Serum samples (100 µL) were denatured with methanol–propanol containing IS, vortex-mixed, extracted into hexane, and dried under nitrogen. The reconstituted extract was chromatographed on a BDS C8 HPLC column, and the metabolites and IS were detected by electrospray ionization MS/MS in multiple-reaction monitoring mode.
Results: 25-OH D2 and D3 and the IS nearly coeluted, whereas 1
-hydroxyvitamin D3 was separated; total run time was 8 min. The interassay CVs for 25-OH D2 were 9.5% and 8.4% at 52 and 76 nmol/L, respectively, and for 25-OH D3 were 5.1% and 5.6% at 55 and 87 nmol/L, respectively. The detection limit of the present method was <4 nmol/L for both metabolites. Method comparison with a commercial RIA measuring total 25-hydroxyvitamin D showed good correlation: y = 0.97x – 2.7 nmol/L (r = 0.91). The analytical system can assay 100 samples in 12.5 h.
Conclusions: This simple robust interference-free LC-MS/MS assay is suitable for routine measurement of the 25-hydroxy metabolites of vitamins D2 and D3 in human serum. The assay has been in use for 9 months and has been used to assay more than 6000 routine samples.
The following articles in journals at HighWire Press have cited this article:
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  L. Rejnmark, A. Tietze, P. Vestergaard, L. Buhl, M. Lehbrink, L. Heickendorff, and L. Mosekilde Reduced Prediagnostic 25-Hydroxyvitamin D Levels in Women with Breast Cancer: A Nested Case-Control Study Cancer Epidemiol. Biomarkers Prev., October 1, 2009; 18(10): 2655 - 2660. [Abstract] [Full Text] [PDF]
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 L. Cleemann, B. E Hjerrild, A. L Lauridsen, L. Heickendorff, J. S Christiansen, L. Mosekilde, and C. H Gravholt Long-term hormone replacement therapy preserves bone mineral density in Turner syndrome Eur. J. Endocrinol., August 1, 2009; 161(2): 251 - 257. [Abstract] [Full Text] [PDF]
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 G. D. Carter 25-Hydroxyvitamin D Assays: The Quest For Accuracy Clin. Chem., July 1, 2009; 55(7): 1300 - 1302. [Full Text] [PDF]
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 S. Knox, J. Harris, L. Calton, and A M. Wallace A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry Ann Clin Biochem, May 1, 2009; 46(3): 226 - 230. [Abstract] [Full Text] [PDF]
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 C. Wejse, V. F. Gomes, P. Rabna, P. Gustafson, P. Aaby, I. M. Lisse, P. L. Andersen, H. Glerup, and M. Sodemann Vitamin D as Supplementary Treatment for Tuberculosis: A Double-blind, Randomized, Placebo-controlled Trial Am. J. Respir. Crit. Care Med., May 1, 2009; 179(9): 843 - 850. [Abstract] [Full Text] [PDF]
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A. M. Yates, A. Bowron, L. Calton, J. Heynes, H. Field, S. Rainbow, and B. Keevil Interlaboratory Variation in 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 Is Significantly Improved If Common Calibration Material Is Used Clin. Chem., December 1, 2008; 54(12): 2082 - 2084. [Full Text] [PDF]
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J. G. Lewis and P. A. Elder Serum 25-OH Vitamin D2 and D3 are Stable under Exaggerated Conditions Clin. Chem., November 1, 2008; 54(11): 1931 - 1932. [Full Text] [PDF]
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 B. W Hollis Measuring 25-hydroxyvitamin D in a clinical environment: challenges and needs Am. J. Clinical Nutrition, August 1, 2008; 88(2): 507S - 510S. [Abstract] [Full Text] [PDF]
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H. J. Roth, H. Schmidt-Gayk, H. Weber, and C. Niederau Accuracy and clinical implications of seven 25-hydroxyvitamin D methods compared with liquid chromatography-tandem mass spectrometry as a reference Ann Clin Biochem, March 1, 2008; 45(2): 153 - 159. [Abstract] [Full Text] [PDF]
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 E. Hypponen, S. Turner, P. Cumberland, C. Power, and I. Gibb Serum 25-Hydroxyvitamin D Measurement in a Large Population Survey with Statistical Harmonization of Assay Variation to an International Standard J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4615 - 4622. [Abstract] [Full Text] [PDF]
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C. Wejse, R. Olesen, P. Rabna, P. Kaestel, P. Gustafson, P. Aaby, P. L Andersen, H. Glerup, and M. Sodemann Serum 25-hydroxyvitamin D in a West African population of tuberculosis patients and unmatched healthy controls Am. J. Clinical Nutrition, November 1, 2007; 86(5): 1376 - 1383. [Abstract] [Full Text] [PDF]
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T. M. Annesley Methanol-Associated Matrix Effects in Electrospray Ionization Tandem Mass Spectrometry Clin. Chem., October 1, 2007; 53(10): 1827 - 1834. [Abstract] [Full Text] [PDF]
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A. R. Martineau, R. J. Wilkinson, K. A. Wilkinson, S. M. Newton, B. Kampmann, B. M. Hall, G. E. Packe, R. N. Davidson, S. M. Eldridge, Z. J. Maunsell, et al. A Single Dose of Vitamin D Enhances Immunity to Mycobacteria Am. J. Respir. Crit. Care Med., July 15, 2007; 176(2): 208 - 213. [Abstract] [Full Text] [PDF]
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J. A. Schmidt Measurement of 25-Hydroxyvitamin D Revisited Clin. Chem., December 1, 2006; 52(12): 2304 - 2305. [Full Text] [PDF]
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G. Lensmeyer, D. Wiebe, N. Binkley, and M. Drezner The authors of the article cited above respond: Clin. Chem., December 1, 2006; 52(12): 2305 - 2306. [Full Text] [PDF]
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J. Kuhn, C. Prante, S. Schon, C. Gotting, and K. Kleesiek Measurement of Fibrosis Marker Xylosyltransferase I Activity by HPLC Electrospray Ionization Tandem Mass Spectrometry Clin. Chem., December 1, 2006; 52(12): 2243 - 2249. [Abstract] [Full Text] [PDF]
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N. Binkley, M. K. Drezner, and B. W. Hollis Laboratory reporting of 25-hydroxyvitamin d results: potential for clinical misinterpretation. Clin. Chem., November 1, 2006; 52(11): 2124 - 2125. [Full Text] [PDF]
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G. L. Lensmeyer, D. A. Wiebe, N. Binkley, and M. K. Drezner HPLC Method for 25-Hydroxyvitamin D Measurement: Comparison with Contemporary Assays Clin. Chem., June 1, 2006; 52(6): 1120 - 1126. [Abstract] [Full Text] [PDF]
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