Development of a candidate reference measurement procedure for the determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry.

Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8392, USA.
Analytical Chemistry (Impact Factor: 5.83). 02/2010; 82(5):1942-8. DOI: 10.1021/ac9026862
Source: PubMed

ABSTRACT Vitamin D exists in two major forms, vitamin D(3) and vitamin D(2). Vitamin D helps the body absorb calcium and promote optimal bone health. Both forms of vitamin D are metabolized to 25-hydroxyvitamin D in the body, and the levels of 25-hydroxyvitamin D(3) [25(OH)D(3)] and 25-hydroxyvitamin D(2) [25(OH)D(2)] in serum are considered the best indicators of vitamin D status. A candidate reference measurement procedure for serum 25(OH)D(3) and 25(OH)D(2) has been developed and critically evaluated. The deuterated compounds 25(OH)D(3)-d(3) and 25(OH)D(2)-d(3) are used as internal standards for 25(OH)D(3) and 25(OH)D(2), respectively. The 25(OH)D(3) and 25(OH)D(2) and their respective labeled internal standards are simultaneously extracted from serum using liquid-liquid extraction prior to reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a cyano (CN) column for both 25(OH)D(3) and 25(OH)D(2). Atmospheric pressure chemical ionization (APCI) in the positive ion mode and multiple reaction monitoring (MRM) were used for LC-MS/MS. The accuracy of the method was evaluated by recovery studies of measuring 25-hydroxyvitamin D [25(OH)D] in spiked samples with known 25(OH)D levels. The recoveries of the added 25(OH)D(3) and 25(OH)D(2) ranged from 99.0% to 101.0%. The absolute recoveries with this method were 97% and 92% for 25(OH)D(3) and 25(OH)D(2), respectively. Excellent precision was obtained with between-set coefficients of variation (CVs) of 0.2-0.6% for 25(OH)D levels >1 ng/g and within 2% for the level of <1 ng/g. Chromatographic separation of 25(OH)D(3) and 25(OH)D(2) from their respective isomers 3-epi-25(OH)D(3) and 3-epi-25(OH)D(2) was achieved. The limit of detection at a signal-to-noise ratio of approximately 3 was 40 pg of 25(OH)D on column (or approximately 0.15 ng/g as expressed as a concentration). This candidate reference measurement procedure for serum 25(OH)D(3) and 25(OH)D(2) demonstrates good accuracy and precision and low susceptibility to interferences. It can be used to provide an accuracy base to which clinical methods for 25(OH)D(3) and 25(OH)D(2) can be compared and that will serve as a standard of higher order for measurement traceability.

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    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.
    Journal of Chromatography B 07/2014; 967:195–202. · 2.69 Impact Factor
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    ABSTRACT: Within a few years, 25-hydroxyvitamin D (25-OHD) 1 has emerged as a high volume test in many regions. Several analytical issues have been recognized regarding this analyte, in particular variable co-detection of metabolites (25-hydroxyvitamin D 2; 3-epi-25-hydroxyvitamin D; 24,25-dihydroxyvitamin D), variable release of the analyte from its protein bonds and matrix effects in automated ligand binding tests, contributing to an often unsatisfactory correlation of high-throughput assays with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Reference methods based on LC-MS/MS as well as reference materials have been introduced only very recently, achieving a truly significant improvement in the standardization of serum 25-OHD measurement. However, beyond these analytical issues in relation to 25-OHD, it should be scrutinized how biologically appropriate this inactive intermediate metabolite can actually describe vitamin D status as a surrogate marker -individually and at a population level. There is, for example, at present little knowledge regarding individual vitamin D requirement in relation to dietary calcium supply; the impact of genetic variation of vitamin D binding protein on serum concentrations; the impact of genetic variation in downstream metabolism and signaling of vitamin D on individual vitamin demand. Furthermore, there is no accepted approach to assess functional whole year vitamin D status, addressing the fundamental seasonal variation of endogenous generation of vitamin D in many regions. Consequently, additional functional markers should be considered when describing vitamin D status (parathyroid hormone, corrected serum calcium and phosphate, urinary calcium, well selected bone markers, etc.), with season-adapted sampling strategies. In conclusion, it should be recognized that there is substantial uncertainty in the currently used approach to characterize vitamin D status by singular measurement of 25-OHD using mainstream assays. It seems questionable to focus the worldwide debate on a widespread vitamin supplementation merely on cut-off results of this marker.
    LaboratoriumsMedizin 02/2014; 38(1):1-10. · 0.30 Impact Factor
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    ABSTRACT: The constantly increasing requests for the measurement of serum 25-hydroxyvitamin D over the last years has led reagent manufacturers to market different automated and semi-automated methods, that being unfortunately not fully harmonized, yield different results. Liquid chromatography coupled to tandem mass spectrometry has more recently been introduced. This approach allows the distinction between the two forms of 25-hydroxyvitamin D and to measure other metabolites. This approach also requires harmonization to curtail the differences between the different analytical methods. To meet this requirement, the American national institutes of health (NIH), the CDC (Center for disease control and prevention) in Atlanta, the NIST (National institute of standards and technology) and the vitamin D Reference laboratory of Ghent University have pooled their expertise to develop a standardization program. This article reviews the main elements and the difficulties of the automated and semi-automated methods for 25-hydroxyvitamin D, from sample preparation to the analytical phase, as well as those related to mass spectrometry. It also addresses the issues related to the clinical decision thresholds and the possibility of measurements in different biological liquids.
    Annales de biologie clinique 02/2015; 73(1):79-92. · 0.42 Impact Factor

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