Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC–MS/MS methods not separating both metabolites as determined by an LC–MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum

doi:10.1016/j.jchromb.2014.07.021

Journal of Chromatography B

Volume 967, 15 September 2014, Pages 195-202

https://doi.org/10.1016/j.jchromb.2014.07.021 Get rights and content

Highlights

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We report an LC–MS/MS method for measurement of (3-epi-)25(OH)D₃, and 25(OH)D₂.
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Separate calibrators and SIL-IS for each compound ensure accurate measurement.
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Structurally-related 3-epi-25(OH)D₃ and 25(OH)D₃ differ in ionisation efficiency.
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This leads to overestimation of 25(OH)D₃ concentration in C₁₈-based LC–MS/MS methods.

Abstract

Background

An LC–MS/MS method was developed for simultaneous quantification of 25-hydroxyvitamin D₃ (25(OH)D₃), 3-epi-25(OH)D₃, and 25(OH)D₂ in human serum. Methods: Sample preparation consisted of protein precipitation followed by off-line SPE. Calibration curves for each vitamin D metabolite were constructed in phosphate-buffered saline with 60 g/L albumin including its corresponding stable isotope labelled (SIL) internal standard. A pentafluorophenyl (PFP) analytical column was used to resolve 25(OH)D₃ from 25(OH)D₂ and 3-epi-25(OH)D₃, followed by SRM registration using positive ESI-MS/MS. Accuracy was assessed from measurement of samples with NIST reference method procedure (RMP) assigned values. The PFP LC–MS/MS method was compared to an in-house C₁₈ column LC–MS/MS method, not resolving 25(OH)D₃ from 3-epi-25(OH)D₃, using adult and newborn samples. Results: Intra-assay and inter-assay coefficients of variation were less than 4% and 7.5%, respectively for all three vitamin D metabolites; lower limits of quantification were 1, 1 and 2 nmol/L and linearity of methods were 1–500, 1–200 and 2–500 nmol/L for 25(OH)D₃, 3-epi-25(OH)D₃ and 25(OH)D₂, respectively. The PFP LC–MS/MS method showed minimal bias to the NIST RMP. Method comparison revealed that in the C₁₈ LC–MS/MS method, the 3-epi-25(OH)D₃ concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional 30–40% higher ionisation efficiency of 3-epi-25(OH)D₃ when compared to 25(OH)D₃. Conclusion: This accurate LC–MS/MS method allows the simultaneous measurement of 25(OH)D₃, 3-epi-25(OH)D₃, and 25(OH)D₂ in human serum. Due to increased ionisation efficiency, the contribution of the 3-epi-25(OH)D₃ metabolite to the total 25(OH)D₃ concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D₃ from 25(OH)D₃ and may compromise its use in infant samples known to have significant amounts of 3-epi-25(OH)D₃.

Introduction

At present, over 10% of clinical laboratories use liquid chromatography–tandem mass spectrometry (LC–MS/MS) for routine analysis of 25-hydroxyvitamin D (25(OH)D) in human serum [1]. One of the potentially interfering vitamin D metabolites is 3-epi-25(OH)D₃ (or C₃-epimer), a stereoisomer that differs from 25(OH)D₃ only at the C₃ position where the hydroxyl group is in the alpha orientation rather than in the beta orientation. Since mass and fragmentation patterns of the C₃-epimer equal those for 25(OH)D₃ its presence results in overestimation of 25(OH)D₃ when both metabolites are not chromatographically resolved. Most of the laboratories reporting LC–MS/MS results for 25(OH)D use a method not capable of resolving 3-epi-25(OH)D₃ from 25(OH)D₃, and as a result overestimate the true 25(OH)D₃ concentration [2], [3]. The 3-epi-25(OH)D₃ metabolite represents on average 4% of total 25(OH)D with incidental higher values up to 22% in adult samples [4], [5]. In the first 3 months of age, 3-epi-25(OH)D₃ concentrations are considerably higher representing up to 60% of total 25(OH)D [6], [7], [8]. The biological importance of 3-epi-25(OH)D₃ remains to be clarified, making the importance of its detection in clinical samples a topic for debate [4]. Quantification of both compounds might be of added value, in particular when measuring infant samples. In addition, an LC–MS/MS method for accurate, quantitative measurement of 3-epi-25(OH)D₃ can be useful for evaluating the degree of cross-reactivity to 3-epi-25(OH)D₃ in immunoassays for 25(OH)D analysis [9], [10].

The Joint Committee for Traceability in Laboratory Medicine (JCTLM) (http://www.bipm.org/en/committees/jc/jctlm/) has listed two isotope dilution (ID) MS reference methods for measurement of 25(OH)D₃ and 25(OH)D₂, but not for measurement of their respective C₃-epimers in human serum: one is an LC–MS/MS method developed by the National Institute of Standards and Technology (NIST) (http://www.nist.gov/index.html) [11] and the other being the University of Ghent reference measurement procedure (RMP) [12]. Both reference methods do separate 25(OH)D₃ and 3-epi-25(OH)D₃ chromatographically. Of the NIST reference material available, NIST972(a) level 4 is the only sample with an assigned 3-epi-25(OH)D₃ level. Unfortunately, no details are available on how the 3-epi-25(OH)D₃ metabolite is quantified, e.g. which stable isotopically labelled internal standard (SIL-IS) (deuterated 3-epi-25(OH)D₃ or 25(OH)D₃) has been used. Also, it should be kept in mind that the 3-epi-25(OH)D₃ content in SRM972(a) level 4 originates from fortification and may show different behaviour when compared to patient material carrying endogenous 3-epi-25(OH)D₃.

Of all published LC–MS/MS methods for simultaneous measurement of 25(OH)D₃, 25(OH)D₂ and their respective C₃-epimers [6], [7], [8], [13], [14], [15], [16], [17], [18], [19], [20] it has not always been clear how the C₃-epimer concentration is calculated. Some reported relative percentages ([C₃-epi peak area/Σ(C₃-epi + 25(OH)D) peak areas] × 100%) as 3-epi-25(OH)D₃ calibration curves were lacking [7], [19], others reported absolute concentrations 3-epi-25(OH)D₃, however, without giving details on whether separate 3-epi-25(OH)D₃ calibrators were used [6], [8], [13], [14], [15], [16], [17], [18], [20]. Furthermore, as a SIL-IS for 3-epi-25(OH)D₃ was not commercially available until recently, d₆-25(OH)D₃ [6], [7], [8], [13], [14], [15], [16], [19] and in minor cases d₃-25(OH)D₂ [20] or d₃-stanozol [17] have been used as surrogate SIL-IS for 3-epi-25(OH)D₃ quantification. When using a SIL-IS that is non-identical to the analyte, one should be aware of the fact that ionisation efficiency may differentially affect the analyte and its SIL-IS, particularly in case of (slight) differences in retention time between both components.

We here describe a fast and accurate LC–MS/MS method for simultaneous measurement of 25(OH)D₃, 3-epi-25(OH)D₃, and 25(OH)D₂. Accurate measurement is achieved by the use of separate calibration curves and corresponding SIL-IS for each vitamin D metabolite. Using this method we could show that due to the increased ionisation efficiency of 3-epi-25(OH)D₃, the contribution of this metabolite to the total 25(OH)D₃ concentration is overestimated by approximately 30–40% in ESI-LC-MS/MS methods that do not resolve 3-epi-25(OH)D₃ from 25(OH)D₃.

Section snippets

Materials and chemicals

25(OH)D₃, 25(OH)D₂ and 3-epi-25(OH)D₃ (≥98% purity) and bovine serum albumin (BSA) were purchased from Sigma–Aldrich (Zwijndrecht, The Netherlands). SIL-IS 26,26,26,27,27,27-hexadeuterium labelled 25(OH)D₃ and 6,19,19-trideuterium labelled 25(OH)D₂ were from Medical Isotopes (Pelham, NH, USA), 6,19,19-trideuterium labelled 3-epi-25(OH)D₃ was from Isosciences (King of Prussia, PA, USA). Water was prepared by USF-ELGA Rossmark (Ede, The Netherlands) and LC/MS grade acetonitrile (ACN), methanol

Method development

Recent concern regarding the importance of removing 3-epi-25(OH)D₃ interference from 25(OH)D₃ measurement prompted us to replace our routine C₁₈-RP based LC–MS/MS method [21] with a method using PFP as stationary phase. The PFP column enables baseline separation of 25(OH)D₃ from 3-epi-25(OH)D₃ and 25(OH)D₂ as is shown in Fig. 1, with 25(OH)D₃(-d₆) eluting at about 2.83 min, and 25(OH)D₂(-d₃) and 3-epi-25(OH)D₃(-d₃) co-eluting at 2.99 min. Assay calibration was achieved by means of five-point

Conclusion

We have developed a fast and reliable LC–MS/MS method for simultaneous measurement of 3-epi-25(OH)D₃, 25(OH)D₃ and 25(OH)D₂ in human serum. Accurate measurement is achieved by use of separate calibration curves and SIL-IS for each compound, effectively eliminating relative matrix effect liability. In LC–MS/MS methods, not separating 3-epi-25(OH)D₃ from 25(OH)D₃, the 3-epi-25(OH)D₃ concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional

Acknowledgements

We thank the DEQAS organization for providing us with additional samples having values assigned by the NIST RMP. The VitDQAP organizers are acknowledged for sharing 3-epi-25(OH)D₃ results with us prior to publication.

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Highlights

Abstract

Background

Introduction

Section snippets

Materials and chemicals

Method development

Conclusion

Acknowledgements

Clin. Chim. Acta

Clin. Biochem.

Clin. Nutr.

Clin. Chim. Acta

Clin. Biochem.

Clin. Chim. Acta

Clin. Biochem.

J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.

Steroids

J. Clin. Endocrinol. Metab.