Accessible Requires Authentication Published by De Gruyter September 19, 2018

Comparison of serum 25-hydroxyvitamin D levels between radioimmunoassay and liquid chromatography-tandem mass spectrometry in infants and postpartum women

Kaori Hara ORCID logo, Kazushige Ikeda, Yuhei Koyama, Yasuhiro Wada and Tomonobu Hasegawa



Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold standard for the measurement of serum 25-hydroxyvitamin D (25(OH)D) levels instead of the conventional method, radioimmunoassay (RIA). However, there was no study that compared RIA and LC-MS/MS for measuring serum 25(OH)D levels in infants and their mothers. The aim of this study was to assess the agreement of RIA and LC-MS/MS for measuring the serum levels in infants and postpartum women.


This study enrolled 70 preterm infants, 113 term infants (134 samples), and 120 postpartum women. Serum concentration of 25(OH)D was measured by RIA and LC-MS/MS. We evaluated the correlation between RIA and LC-MS/MS. Also, we evaluated the bias between RIA and LC-MS/MS using Bland-Altman analysis.


Sixty percent of preterm infants had serum 25(OH)D levels below the lower limit of quantification (LOQ) (4 ng/mL) and 90% of them were classified as vitamin D deficient. The serum 25(OH)D levels measured by RIA were significantly correlated with those measured by LC-MS/MS in all groups. According to the Bland-Altman plot, the serum 25(OH)D levels of infants measured by RIA had constant positive bias (mean±standard deviation [SD] [95% confidence interval, CI], preterm: +4.8± 2.4 ng/mL [4.2–5.4], term: +5.8±4.0 [5.1–6.5]) and proportional bias (preterm: r=0.44, p<0.01, term: r=0.50, p<0.01) compared with LC-MS/MS. The serum 25(OH)D levels of postpartum women measured by RIA had constant positive bias compared with LC-MS/MS, but no proportional bias was found.


RIA demonstrated falsely high 25(OH)D levels when used for infants and postpartum women.

Corresponding author: Kaori Hara, MD, Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan, Phone: +81-3-3353-1211, Fax: +81-3-5379-1978

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None to declared.

  3. Employment of readership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: None declared.


1. Feldman D. Vitamin D, 3rd ed. London: Academic Press, 2011:5–6. Search in Google Scholar

2. Munns CF, Shaw N, Kiely M, Specker BL, Thacher TD, et al. Global consensus recommendations on prevention and management of nutritional rickets. J Clin Endocrinol Metab 2016;101:394–415. Search in Google Scholar

3. Siafarikas A, Deishi A, Jahreis G, Pieplow A, Vogel H, et al. Cross-sectional analysis of universal vitamin D supplementation in former East Germany during the first year of life. J Pediatr Endocrinol Metab 2017;30:395–404. Search in Google Scholar

4. Anguana SK, Angurana RS, Mahajan G, Kumar N, Mahajan V. Prevalence of vitamin D deficiency in apparently healthy children in north India. J Pediatr Endocrinol Metab 2014;27:1151–6. Search in Google Scholar

5. 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 epimer and isobars. Nutrition J 2011;10:46. Search in Google Scholar

6. Hollis BW. The determination of circulating 25-hydroxyvitamin D: no easy task. J Clin Endocr Metab 2004;89:3149–51. Search in Google Scholar

7. Schottker B, Jansen EH, Haug U, Schomburg L, Kohrle J, et al. Standardization of misleading immunoassay based 25-hydroxyvitamin D levels with liquid chromatography tandem-mass spectrometry in a large cohort study. PLoS One 2012;7:e48774. Search in Google Scholar

8. van den Ouweland JM, Beijers AM, van Daal H, Elisen MG, Steen G, et al. Evaluation of 3-epi-25-hydroxyvitamin D3 cross-reactivity in the Roche Elecsys vitamin D total protein binding assay. Clin Chem Lab Med 2014;52:373–80. Search in Google Scholar

9. 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;34:2–23. Search in Google Scholar

10. Farrell CL, Martin S, McWhinney B, Straub I, Williams P, et al. State-of-the art vitamin D assays: a comparison of automated immunoassays with liquid chromatography-tandem mass spectrometry methods. Clin Chem 2012;58:531–42. Search in Google Scholar

11. van den Ouweland JM, Beijers AM, Demacker P, van Daal H. Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay. J Choromatogr B 2010;878:1163–8. Search in Google Scholar

12. Yun C, Chen J, Yang C, Li Y, Piao J, et al. Comparison of two 25-hydroxyvitamin D immunoasssays to liquid chromatography-tandem mass spectrometry in assessing samples from the Chinese population. Clin Chim Acta 2015;448:22–6. Search in Google Scholar

13. 25-Hydroxyvitamin D 125I RIA kit instruction manual. Diasorin, REF 68100E. Search in Google Scholar

14. Molloy B, Calton L, Cooper D. An offline automated solid-phase extraction method for measurement of serum 25-hydroxyvitamin D for clinical research. 2014. Waters, Application Note 72003139EN. Search in Google Scholar

15. Holick MF. Vitamin D deficiency. New Engl J Med 2007;357:266–81. Search in Google Scholar

16. Aghajafari F, Field CJ, Rabi D, Kaplan BJ, Maggiore JA, et al. Plasma 3-epi-25-hydroxycholecalciferol can alter the assessment of vitamin D status using the current reference ranges for pregnant women and their newborns. J Nutr 2016;146:70–5. Search in Google Scholar

17. Bailey D, Perumal N, Yazdanpanah M, Al Mahmud A, Baqui AH, et al. Maternal-fetal-infant dynamics of the C3-epimer of 25-hydroxyvitamin D. Clin Biochem 2014;47:816–22. Search in Google Scholar

18. Hara K, Ikeda L, Koyama Y, Wada Y, Hasegawa T. Serum 25-hydroxyvitamin D3 levels of one-month old term infants in Tokyo using liquid chromatography tandem mass spectrometry. Acta Paediatr 2018;107:532–3. Search in Google Scholar

19. Strathmann FG, Sadilkova K, Laha TJ, LeSourd SE, Bornhorst JB, et al. 3-epi-25 hydroxyvitamin D concentrations are not correlated with age in a cohort of infants and adults. Clin Chim Acta 2012;413:203–6. Search in Google Scholar

20. Granado-Lorencio F, Garcia-Heras LM, Blanco-Navarro I, Perez-Sacristan B. Assessment of 3-epi-25-OH-D3 in preterm and full term infant samples and its relationship to demographic, anthropometric and biochemical determinants. Clin Biochem 2014;47:853–6. Search in Google Scholar

21. Bartoszewicz Z, Kondracka A, Jazwiec R, Popow M, Dadlez M, et al. Can we accurately measure the concentration of clinically relevant vitamin D metabolites in the circulation? The problems and their consequences. Endokrynol Pol 2013;64:238–45. Search in Google Scholar

22. Higashi T, Goto A, Murohashi M, Ogawa A, Komatsu K, et al. Development and validation of a method for determination of plasma 25-hydroxyvitamin D3 3-sulfate using liquid chromatograph/tandem mass spectrometry. J Chromatgr B Analyt Technol Biomed Life Sci 2014;969:230–4. Search in Google Scholar

23. Higashi T, Yokota M, Goto A, Komatsu K, Sugiura T, et al. A method for simultaneous determination of 25-hydroxyvitamin D3 and its 3-sulfate in newborn plasma by LC/ESI-MS/MS after derivatization with a proton-affinitive cookson-type reagent. Mass Spectrom 2016;5:S0051. Search in Google Scholar

24. Nakamura K, Nashimoto M, Yamamoto M. Summer/winter differences in the serum 25-hydroxyvitamin D3 and parathyroid hormone levels of Japanese women. Int J Biometeorol 2000;44:186–9. Search in Google Scholar

25. Ono Y, Suzuki A, Kotake M, Zhang X, Nishiwaki-Yasuda K, et al. Seasonal changes of serum 25-hydroxyvitamin D and intact parathyroid hormone levels in a normal Japanese population. J Bone Miner Metab 2005;23:147–51. Search in Google Scholar

Received: 2018-06-23
Accepted: 2018-08-27
Published Online: 2018-09-19
Published in Print: 2018-10-25

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