Search tips
Search criteria 


Logo of clinbiorevLink to Publisher's site
Clin Biochem Rev. 2005 February; 26(1): 33–36.
PMCID: PMC1240027

Improving the Measurement of 25-hydroxyvitamin D


25-hydroxyvitamin D (25OHD) determination is of diagnostic importance for the investigation of vitamin D deficiency and much more rarely, intoxication. Despite the name, vitamin D is a pre-hormone, being endogenously synthesised provided there is adequate sunlight. Its biological function, exerted through the active form 1,25 dihydroxyvitamin D3 (1,25(OH)2D) is to maintain calcium and phosphate levels in the blood.1 In addition, it has important roles in immune regulation.2

Demand for 25OHD and 1,25(OH)2D assays has increased substantially worldwide since the introduction of commercial kit assays, but performance is relatively poor with only just over half of laboratories achieving acceptable performance.3 A recently emerging problem is that some immunoassays underestimate 25-hydroxyvitamin D2 metabolites due to differences in affinity between the antibodies or D-binding proteins employed.4

It is likely that achievement of reliable, clinically appropriate vitamin D results will require a combination of careful use of commercial systems by skilled operators, validated reference ranges, good quality control schemes and the availability of reference methods of analysis.

Vitamin D Metabolism

Vitamin D3 (cholecalciferol) is formed in the skin by photolysis of 7-dehydrocholesterol by ultraviolet radiation from sunlight. Cholecalciferol is transported in the circulation bound to vitamin D binding protein and is hydroxylated in the liver to 25OHD. A further hydroxylation reaction occurs in the kidney to form the active hormone 1,25(OH)2D. This reaction is tightly regulated by induction of the CYP27B1 enzyme which is stimulated by PTH and inhibited by hyperphosphataemia and 1,25(OH)2D.5

Ergocalciferol (vitamin D2) is produced commercially by ultraviolet irradiation of a provitamin D sterol (ergosterol) that occurs in plants.6 It differs from vitamin D3 in having an additional methyl group at C24 and a double bond at C22-23. It undergoes the same hydroxylation reactions as cholecalciferol to form 25OHD2 and 1,25(OH)2D2. Ergocalciferol is the only prescription pharmaceutical available in Australia7 and the only high dose preparation available in the USA8 so D2 metabolites can represent a significant fraction of the circulating hormone in patients treated with these preparations (see later).


Notwithstanding the International Union of Pure and Applied Chemistry (IUPAC) recommendation9 for the use of the trivial names calcidiol (25-hydroxyvitamin D, 25OHD) and calcitriol (1,25-dihydroxyvitamin D, 1,25(OH)2D), these two terms have not come into widespread use. As a consequence the abbreviations 25OHD and 1,25(OH)2D are used in this review.

Vitamin D Status

Although cholecalciferol and 1,25(OH)2D can be measured in the circulation, the best estimates of vitamin D status are provided by measurement of 25OHD 10,11 This is due to its long serum half-life (approximately 3 weeks) and because the 25-hydroxylation step is unregulated, thus reflecting substrate availability. A number of commercial kit assays are available for the clinical laboratory and will be discussed further.

In contrast, cholecalciferol has a short half-life (approximately 24 h) so that serum levels depend on recent sunlight exposure and vitamin D ingestion. The assay is difficult due to the lipophilic nature of the molecule and no commercial versions are available.12

Since production of 1,25(OH)2D is tightly regulated and serum half life is 4–6 h, circulating levels provide limited information about nutritional vitamin D status. Although commercial radioimmunoassays and ELISAs are now available, measurement of 1,25(OH)2D is principally of interest only in renal disease and remains the preserve of specialist laboratories.

Measurement of 25OHD

Approximately 85% of 25OHD is bound to D binding protein (DBP), 15% to albumin, and 0.03% free.13 Thus chromatographic separation requires an extraction step to release 25OHD from its binding proteins and this can be subject to variable co-precipitation.14 On the other hand, non-extracted immunoassay methods may be susceptible to matrix effects, particularly due to the lipophilic nature of 25OHD.15 Taken together with the low (nanomolar) levels of vitamin D metabolites in serum, these factors have made the routine measurement of 25OHD an analytical challenge. Clinical assessment of vitamin D status in patients receiving ergocalciferol requires measurement that is equally reactive to both the 25OHD3 and the 25OHD2 metabolites. It has recently become clear that not all of the currently available commercial immunoassays will detect 25OHD2 with adequate sensitivity.15 Although D2 is used in the USA and Australia, D3 is the form given in Europe and so these problems may be confined to Australia and the USA. However, these analytical issues may be exacerbated by the less efficient conversion of D2 to 25OHD2 compared to the D3 forms.16 Furthermore, the half life of 25OHD2 is shorter and clinical potency is less than one third.8 Together, this means that the clinical picture in an ergocalciferol-treated patient may be highly uncertain.

Reference Methods

Definitive methods employ GC coupled with mass spectrometry detection.17,18 Recently a candidate reference method using LC-tandem mass spectrometry was published.14 Whilst these reference methods are suitable for validating the recovery and accuracy of routine methods, their complexity and derivatisation requirements mitigate against regular use.


A large number of HPLC methods for vitamin D determination have been published. Chromatographic separation is suitable as a reference method, since it is capable of resolving D2 and D3 forms as well as the 25OH, 1,25(OH)2 and 24,25(OH)2 metabolites. Early assays utilised normal phase separation19,20 and these have been followed by reverse phase separation.21 Many adaptations and variations have been published (reviewed15,22). The most widely adopted methods use liquid-liquid or liquid-solid pre-sample cleanup with UV detection after column separation. A C18 reverse phase column with isocratic or gradient elution using acetonitrile/water is now the standard procedure and diode array detection following hexane extraction gives results of sufficient clinical sensitivity.23 Thus determination of 25OHD by HPLC with UV detection can be considered the gold standard method.15 However, it is accepted that these methods are unsuitable for routine, clinical laboratory use.


The earliest immunoassay method used DBP and 3H-25OHD tracer.24 An RIA using antibody that is cospecific to both 25OHD2 and 25OHD3 was developed by Hollis25 and was improved with the use of 125I-labelled tracer.26 More recently, chemiluminescent assays have utilised both DBP27 and antibody-based binding.28 A range of immunoassays is now commercially available (in Australia, the DiaSorin, IDS, and Nichols Institute assays comprise 90% of the market29).

Diasorin RIA

The Hollis assay25,26 was released commercially with FDA approval and has become widely used. Acetonitrile extraction is followed by competitive radioimmunoassay using 125I-labelled 25OHD and antibody to 25OHD. A second antibody is used as precipitating agent. Although Hollis found that the primary antibody recognises 25OHD2 and 25OHD3 equally,4 other authors have published data indicating that the assay under-recognises 25OHD2.30

IDS Gamma-B

The sample obtained after acetonitrile extraction is incubated with antibody to 25OHD in competition with 125I-labelled 25OHD. A second antibody coupled to cellulose is used for separation of bound radioactivity. The manufacturers state that there is 75% cross reactivity of the antibody with 25OHD2 compared to 100% for the 25OHD3 form.31 Interestingly, data from the DEQAS indicate that the EIA version of this IDS assay performed better than the RIA, despite using the same antibody.32

Nichols Advantage

This assay first separates 25OHD from DBP using a denaturing agent. Competition is then established in the same sample well between the sample 25OHD and 25OHD on magnetic particles for human DBP. Separation utilises the magnetic particles and detection is by chemiluminescence using acridinium-ester.27

Despite the Nichols Advantage manufacturers earlier claims for 100% reactivity with 25OHD2, it has become apparent that the assay is unable to measure samples containing substantial amounts of 25OHD2 reliably.15 Recently, data from the UK quality assurance program has shown that the assay displays a positive bias (~31%) with 25OHD3 and a negative one with 25OHD2.32 This under-recovery of 25OHD2 in some patient samples is now acknowledged in the information supplied with the kit.

Diasorin Liaison

This chemiluminescent assay has recently become available. Serum is incubated with antivitamin-D coated microparticles and isoluminol derivative-conjugated 25OHD before measurement of the chemiluminescent signal.28 The antibody is said to be same as that used in the DiaSorin RIA33 and the results correlate well with the radioimmunoassay.34 However, this assay was found to recognise 25OHD2 more than 25OHD3,35 an anomaly that remains to be explained.

Comparability of Assays

Standardization of results between methods and laboratories remains a significant problem.30,36,37 This may be due to variability with temperature of antigen-antibody or protein-binding protein interactions as well as to differences in recognition of the 25OHD2 and 25OHD3 forms. Furthermore, the Diasorin RIA showed good correlation with HPLC in experienced hands but compared poorly in a laboratory that had not validated the method,37 emphasising the need for good quality assurance. The Nichols Advantage showed poor comparison with HPLC and overestimated basal 25OHD levels whilst underestimating exogenous 25OHD3. Recently this kit underwent a re-standardisation exercise due to the results having drifted downwards in the Australian QAP. This resulted in values increasing by 17% in an unselected patient population (Martin P, 2004 personal communication).

Aspects of Quality Control

An international quality assurance scheme (DEQAS, UK) has been in operation since 1989.3 At present there are 16 Australian laboratories participating (DEQAS, personal communication). Additionally in Australia, the RCPA-AACB QAP has 36 laboratories participating in the Vitamin D Endocrine program.29 Results from the DEQAS scheme have highlighted the 25OHD2 detection problem32 as well as confirming that only half the participants are achieving acceptable performance (80% of results within 30% of All Laboratories Trimmed Mean).


Serum is the preferred specimen although plasma (EDTA and Li-heparin) samples are satisfactory.34 Vitamin D analytes have been shown to be stable for up to 2 weeks at 30°C3 and to be unaffected by up to 4 freeze-thaw cycles.38 Storage of frozen serum samples at −20°C for up to one year has also been reported to cause no loss in vitamin D metabolites.12


Measurement of 25OHD by immunoassay will remain the method of choice for reasons of convenience, speed, turnaround and cost. Although the current generation of commercial assays have shortcomings in defining the vitamin D status of patients being treated with ergocalciferol, now that this problem has been recognised, performance will undoubtedly improve with the development of better assays. Given the problems caused by ergocalciferol outlined here, it may also be reasonable to recommend a change to using D3 as the pharmaceutical of choice.

Continuing efforts to improve laboratory performance and vigilance with quality assurance programs are required. Laboratories offering vitamin D assays are urged to participate in the local and international programs.

The establishment and maintenance of reference assays for 25OHD are required and have been supported by the AACB. An HPLC assay is being established at RMIT to provide estimation of both 25OHD2 and 25OHD3 metabolites in plasma samples so that the performance of routine immunoassays on quality assurance samples containing mixtures of these metabolites can be monitored.


The contents of articles or advertisements in The Clinical Biochemist – Reviews are not to be construed as official statements, evaluations or endorsements by the AACB, its official bodies or its agents. Statements of opinion in AACB publications are those of the contributors. Print Post Approved - PP255003/01665.

No literary matter in The Clinical Biochemist – Reviews is to be reproduced, stored in a retrieval system or transmitted in any form by electronic or mechanical means, photocopying or recording, without permission. Requests to do so should be addressed to the Editor. ISSN 0159 – 8090.


1. Morris HA. Vitamin D: a hormone for all seasons- how much is enough? Clin Biochem Rev. 2005;26:21–33. [PMC free article] [PubMed]
2. Deluca HF, Cantorna MT. Vitamin D: its role and uses in immunology. Faseb J. 2001;15:2579–85. [PubMed]
3. Carter GD, Carter CR, Gunter E, et al. Measurement ofVitaminDmetabolites:aninternationalperspective on methodology and clinical interpretation. J Steroid Biochem Mol Biol 2004;89–90:467–71. [PubMed]
4. Hollis BW. Comparison of commercially available (125)I-based RIA methods for the determination of circulating 25-hydroxyvitamin D. Clin Chem. 2000;46:1657–61. [PubMed]
5. OmdahlJL, MorrisHA, MayBK Hydroxylaseenzymes of the vitamin D pathway: expression, function, and regulation. Annu Rev Nutr. 2002;22:139–66. [PubMed]
6. Horst RL, Reinhardt TA, Russell JR, Napoli JL. The isolation and identification of vitamin D2 and vitamin D3 from Medicago sativa (alfalfa plant) Arch Biochem Biophys. 1984;231:67–71. [PubMed]
7. Glendenning P. Vitamin D deficiency and multicultural Australia. Med J Aust 2002;176:242–3; author reply 243. [PubMed]
8. Armas LA, Hollis BW, Heaney RP. Vitamin D2 is much less effective than vitamin D3 in humans. J Clin Endocrinol Metab. 2004;89:5387–91. [PubMed]
9. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN): Nomenclature of vitamin D. Recommendations 1981. Eur J Biochem. 1982;124:223–7. [PubMed]
10. Iqbal SJ. Vitamin D metabolism and the clinical aspects of measuring metabolites. Ann Clin Biochem. 1994;31(( Pt 2)):109–24. [PubMed]
11. Holick MF. The use and interpretation of assays for vitamin D and its metabolites. J Nutr. 1990;120(Suppl 11):1464–9. [PubMed]
12. Zerwekh JE. The measurement of vitamin D: analytical aspects. Ann Clin Biochem. 2004;41:272–81. [PubMed]
13. Bikle DD, Gee E, Halloran B, Kowalski MA, Ryzen E, Haddad JG. Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein. J Clin Endocrinol Metab. 1986;63:954–9. [PubMed]
14. Vogeser M, Kyriatsoulis A, Huber E, Kobold U. Candidate reference method for the quantification of circulating 25-hydroxyvitamin D3 by liquid chromatography-tandem mass spectrometry. Clin Chem. 2004;50:1415–7. [PubMed]
15. Hollis BW. Editorial: The determination of circulating 25-hydroxyvitamin D: no easy task. J Clin Endocrinol Metab. 2004;89:3149–51. [PubMed]
16. Trang HM, Cole DE, Rubin LA, Pierratos A, Siu S, Vieth R. Evidence that vitamin D3 increases serum 25-hydroxyvitamin D more efficiently than does vitamin D2. Am J Clin Nutr. 1998;68:854–8. [PubMed]
17. Schmidt-Gayk H, Bouillon R, Roth HJ, Seamark DA, Trafford DJ, Makin HL. Measurement of vitamin D and its metabolites (calcidiol and calcitriol) and their clinical significance. Scand J Clin Lab Invest Suppl. 1997;227:35–45. [PubMed]
18. Coldwell RD, Porteous CE, Trafford DJ, Makin HL. Gas chromatography-mass spectrometry and the measurement of vitamin D metabolites in human serum or plasma. Steroids. 1987;49:155–96. [PubMed]
19. Eisman JA, Shepard RM, DeLuca HF. Determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma using high-pressure liquid chromatography. Anal Biochem. 1977;80:298–305. [PubMed]
20. Gilbertson TJ, Stryd RP. High-performance liquid chromatographic assay for 25-hydroxyvitamin D3 in serum. Clin Chem. 1977;23:1700–4. [PubMed]
21. Aksnes L. A simplified high-performance liquid chromatographic method for determination of vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum. Scand J Clin Lab Invest. 1992;52:177–82. [PubMed]
22. Luque de Castro MD, Fernandez-Romero JM, Ortiz-Boyer F, Quesada JM. Determination of vitamin D3 metabolites: state-of-the-art and trends. J Pharm Biomed Anal. 1999;20:1–17. [PubMed]
23. Turpeinen U, Hohenthal U, Stenman UH. Determination of 25-hydroxyvitamin D in serum by HPLC and immunoassay. Clin Chem. 2003;49:1521–4. [PubMed]
24. Haddad JG, Chyu KJ. Competitive protein-binding radioassay for 25-hydroxycholecalciferol. J Clin Endocrinol Metab. 1971;33:992–5. [PubMed]
25. Hollis BW, Napoli JL. Improved radioimmunoassay for vitamin D and its use in assessing vitamin D status. Clin Chem. 1985;31:1815–9. [PubMed]
26. Hollis BW, Kamerud JQ, Selvaag SR, Lorenz JD, Napoli JL. Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer. Clin Chem. 1993;39:529–33. [PubMed]
27. Nichols Institute Diagnostics. Nichols Advantage 25hydroxyvitamin D Directional Insert, 2004.
28. Sackrison JL, Ersfield DL, Miller AB, Olson GT, MacFarlane GD. Development of a sensitive automatednon-extracteddirectLiaisonimmunoassay for 25 OH Vitamin D. Clin Chem. 2002;48:A122.
29. RCPA-AACB QAP. Endocrine Program, End of Cycle Report, Cycle 22. 2004.
30. Glendenning P, Noble JM, Taranto M, et al. Issues of methodology, standardization and metabolite recognition for 25-hydroxyvitamin D when comparing the DiaSorin radioimmunoassay and the Nichols Advantage automated chemiluminescence protein-binding assay in hip fracture cases. Ann Clin Biochem. 2003;40:546–51. [PubMed]
31. IDS. Product Insert Gamma-B 25-Hydroxy Vitamin D RIA, 2005.
32. Carter GD, Carter R, Jones J, Berry J. How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme. Clin Chem. 2004;50:2195–7. [PubMed]
33. Souberbielle JC, Fayol V, Sault C, Lawson-Body E, KahanA, Cormier C.Assay-Specific Decision Limits for Two New Automated Parathyroid Hormone and 25-Hydroxyvitamin D Assays. Clin Chem (in press doi:10.1373/clinchem.2004.037606 2005.) [PubMed]
34. Ersfeld DL, Rao DS, Body JJ, et al. Analytical and clinical validation of the 25 OH vitamin D assay for the LIAISON automated analyzer. Clin Biochem. 2004;37:867–74. [PubMed]
35. Barnes SC, Berry JL, Davies M, Wheeler MJ. Vitamin D: HPLC, manual RIA or automated chemiluminescent immunoassay? Proc ACB National Meeting 2004:125.
36. Lips P, Chapuy MC, Dawson-Hughes B, Pols HA, Holick MF. An international comparison of serum 25-hydroxyvitamin D measurements. Osteoporos Int. 1999;9:394–7. [PubMed]
37. Binkley N, Krueger D, Cowgill CS, et al. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. J Clin Endocrinol Metab. 2004;89:3152–7. [PubMed]
38. Antoniucci DM, Black DM, Sellmeyer DE. Serum 25-hydroxyvitamin D is unaffected by multiple freeze-thaw cycles. Clin Chem. 2005;51:258–61. [PubMed]

Articles from The Clinical Biochemist Reviews are provided here courtesy of The Australian Association of Clinical Biochemists