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Fast separation of 25-hydroxyvitamin [D.sub.3] from 3-Epi-25-hydroxyvitamin [D.sub.3] in human serum by liquid chromatography-tandem mass spectrometry: variable prevalence of 3-epi-25-hydroxyvitamin [D.sub.3] in infants, children, and adults.

To the Editor:

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is becoming increasingly popular for the measurement of 25-hydroxyvitamin D [25(OH)D] in human serum. A limitation of most LC-MS/MS methods is the potential interference from coeluting isomeric compounds having identical elemental composition but different structure, leading to overestimation of true 25(OH)D concentrations. Of particular interest is the C3 epimer of 25(OH)D, 3-epi-25(OH)D, which can be present at relatively high concentrations in sera from infants, but can also be found in sera from adults, albeit at lower concentrations (1,2). To separate 3-epi-25(OH)D from 25(OH)D, current procedures require lengthy chromatographic run times varying from 12 to 40 min (3), which makes these methods unsuitable for clinical laboratories that must deal with increasing numbers of vitamin D requests.

We describe a modification of an established LC-MS/MS method for measurement of 25(OH)[D.sub.3] and 25(OH)[D.sub.2] (4) that allows fast separation of 25(OH)[D.sub.3] from 3epi-25(OH)[D.sub.3] in human serum. Sample preparation, assay calibration, and instrument operation were carried out as described (4), with minor modifications. 25(OH)[D.sub.3,] 25(OH)[D.sub.2], and 3-epi-25(OH)[D.sub.3] were from Sigma Aldrich, and the internal standard (IS) 26,27-hexadeuterium-labeled 25(OH)[D.sub.3] was from Synthetica AS.

Calibrators, controls, and patient sera were treated with sodium hydroxide to release vitamin D metabolites from the binding protein before protein precipitation. Subsequent off-line solid-phase extraction was followed by chromatographic separation performed by use of a pentafluorophenyl-propyl (PFP) column (Acquity UPLC CSH[TM] fluoro-phenyl 1.7 [micro]m, 2.1 X 100 mm; Waters). The relative rigidity of the fluorinated bonded phase provides enhanced shape selectivity (5). Mobile phases A and B consisted of 1 mL/L formic acid in ammonium acetate (2 mmol/L), and 3 mL/L formic acid in methanol, respectively. A flow rate of 0.35 mL/min was used, with reduction to 0.30 mL/min in the final step, by using a gradient to 85% B (0-5 min), 85% B rinse (5.0-5.3 min), and reversion to 50% B (5.3-5.4 min), followed by 50% B (5.4-6.5 min). Detection was by registration m/z transitions 401.5[right arrow]159.2 for 25(OH)[D.sub.3] and 3-epi-25(OH)[D.sub.3], 413.4 [right arrow] 159.2 for 25(OH)[D.sub.2], and 407.5 [right arrow] 159.2 for the IS. The percentage of 3-epi25(OH)[D.sub.3] was calculated relative to the total 25(OH)[D.sub.3] content. In terassay CVs for 3 concentrations of 25(OH)[D.sub.3] control sera (39, 92, and 127 nmol/L; Chromsystems) (n = 4) were 4.2%, 3.5%, and 2.8%, respectively. 25(OH)[D.sub.3] and IS eluted at about 4.32 min, with 3-epi-25(OH)[D.sub.3] eluting at 4.42 min, near base-line separation from 25(OH)[D.sub.3] (Fig. 1). 25(OH)[D.sub.2] eluted at 4.42 min (result not shown).

We further investigated the prevalence of the 3-epi-25(OH)[D.sub.3] in leftover serum samples from infants (<1 year of age, n = 51), children (1-10 years of age, n = 74), and adults (>18 years of age, n = 104). The samples were treated in agreement with local ethics guidelines. 25(OH)[D.sub.3] concentrations ranged from 4.3 to 300 nmol/L. No relevant concentrations of 25(OH)[D.sub.2] were measured in these samples. We could detect the presence of 3-epi25(OH)[D.sub.3] in all sera from infants and children and in 75% of sera from adults. The mean (median; range) percentages were 11.1% (9.3%; 2.3%-49.2%) in infants, 6.2% (5.7%; 2.5%-20.0%) in children, and 3.5% (3.1%; <2%-10.6%) in adults. No correlation was found between the relative content of 3-epi25(OH)[D.sub.3] and the absolute amount of 25(OH)[D.sub.3]. Percentages of 3-epi25(OH)[D.sub.3] exceeding 10% were mainly found in 18 (39%) of 46 infants < 3 months of age, consistent with previous findings (1), although higher percentages (10%-20%) were found in 4 (5.4%) of 74 children and 1 (1.0%) of 104 adults as well, confirming recent findings of considerable amounts of 3-epi 25(OH)[D.sub.3] in adults (2). In adult sera, the new LC-MS/MS method gives a mean 4% lower concentration for 25(OH)[D.sub.3] compared with our previous method, for which we used C18 as stationary phase (4) [Passing and Bablock regression: PFP LC-MS/MS = 0.94 (95% CI: 0.92-0.97) X C18 LC-MS/MS + 0.31 (95% CI: -0.42 to 0.96); r = 0.995; n = 104] due to exclusion of 3-epi-25(OH)[D.sub.3]. The comeasurement of 3-epi-25(OH)D is likely to contribute to the positive bias of many current LC-MS/MS assays compared to the NIST candidate reference measurement procedure (3). Evidently, further investigations are needed to elucidate the biological significance of the 3-epi-25(OH)D metabolites, the conditions that favor C3 epimerization of 25(OH)D, and to what extent separate reporting of 3-epi-25(OH)[D.sub.3] might be of clinical relevance.

In conclusion, the presence of 3-epi-25(OH)[D.sub.3] in nearly all human sera necessitates the use of an LC-MS/MS method that separates 3-epi-25(OH)[D.sub.3] from 25(OH)[D.sub.3] for accurate detection of 25(OH)[D.sub.3]. By using a PFP column, 25(OH)[D.sub.3] and the 3-epi25(OH)[D.sub.3] can be separated within a total run time of6.5 min, making this method fast and attractive for routine measurement of25(OH)D in clinical laboratories.


Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

DOI: 10.1373/clinchem.2011.170282


(1.) 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;91:3055-61.

(2.) Stepman HCM, Vanderroost A, Stockl D, Thienpont LM. Full-scan mass spectral evidence for 3-epi-25-hydroxyvitamin [D.sub.3] in serum of infants and adults. Clin Chem Lab Med 2011;49:253-6.

(3.) Tai SS, Bedner M, Phinney KW. Development of a candidate reference measurement procedure for the determination of 25-hydroxyvitamin [D.sub.3] and 25-hydroxyvitamin [D.sub.2] in human serum using isotope-dilution liquid chromatographytandem mass spectrometry. Anal Chem 2010;82: 1942-8.

(4.) Van den Ouweland JM, Beijers AM, Demacker PN, 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 Chromatogr B Analyt Technol Biomed Life Sci 2010;878:1163-8.

(5.) Marchand DH, Croes K, Dolan JW, Snyder LR, Henry RA, Kallury KM, et al. Column selectivity in reversed-phase liquid chromatography. VIII. Phenylalkyl and fluoro-substituted columns. J Chromatogr A 2005;1062:65-78.

Johannes M.W. van den Ouweland * Antonius M. Beijers Henny van Daal

Department of Clinical Chemistry Canisius-Wilhelmina Hospital Nijmegen, The Netherlands

* Address correspondence to this author at: Department of Clinical Chemistry Canisius Wilhelmina Hospital Weg door Jonkerbos 100 6532 SZ Nijmegen, the Netherlands Fax +31-24-3658671 E-mail
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Title Annotation:Letters to the Editor
Author:van den Ouweland, Johannes M.W.; Beijers, Antonius M.; van Daal, Henny
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Nov 1, 2011
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