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Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC–MS/MS for clinical laboratory applications

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Abstract

The quantification of serum 25-hydroxyvitamin D [25(OH)D] as an indicator of vitamin D status is currently primarily conducted by immunoassays, yet LC–MS/MS would allow more accurate determination. Furthermore, LC–MS/MS would allow simultaneous measurement of multiple analytes. The aim of this study was to develop and validate an LC–MS/MS method to simultaneously measure four vitamin D metabolites (25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3) in serum for clinical laboratory applications. Serum samples were first prepared in a 96-well supported liquid extraction plate and the eluate was derivatized using the Cookson-type reagent 4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), which rapidly and quantitatively reacts with the s-cis-diene structure of vitamin D metabolites. The derivatized samples were subjected to LC–MS/MS, ionized by electrospray ionization (positive-ion mode), and detected by selected reaction monitoring. The lower limits of quantification for 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 were 0.091, 0.020, 0.013, and 0.024 ng/mL, respectively. The accuracy values and the extraction recoveries for these four metabolites were satisfactory. Serum 25(OH)D levels determined by our LC–MS/MS were compared with those obtained by conventional radioimmunoassay (RIA) that cannot distinguish 25(OH)D3 and 25(OH)D2. The values obtained by the RIA method exhibited a mean bias of about 8.35 ng/mL, most likely as a result of cross reaction of the antibody with low-abundance metabolites, including 24,25(OH)2D3. Various preanalytical factors, such as long sample sitting prior to serum separation, repeated freeze–thaw cycles, and the presence of anticoagulants, had no significant effects on these determinations. This high-throughput LC–MS/MS simultaneous assay of the four vitamin D metabolites 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 required as little as 20 μL serum. This method will aid further understanding of low-abundance vitamin D metabolites, as well as the accurate determination of 25(OH)D3 and 25(OH)D2.

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Abbreviations

25(OH)D3 :

25-hydroxyvitamin D3

3-epi-25(OH)D3 :

3-epi-25-hydroxyvitamin D3

24,25(OH)2D3 :

(24R)-24,25-dihydroxyvitamin D3

25(OH)D2 :

25-hydroxyvitamin D2

CV:

coefficient of variation

DAPTAD:

4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione

ESI:

electrospray ionization

IS:

internal standard

LOD:

limit of detection

LOQ:

limit of quantification

LLOQ:

lower limit of quantification

SRM:

selected reaction monitoring

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Acknowledgments

This research was partially supported by the Ministry of Education, Culture, Science, Sports and Science and Technology (MEXT) by a Grant-in-Aid for Scientific Research (C), 2015-2018, 15K08610 (M. Satoh) and a Grant-in-Aid for Research Activity Start-up, 15H06096 (T. Ishige).

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    Authors and Affiliations

    1. Division of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan

      Mamoru Satoh & Fumio Nomura

    2. Division of Laboratory Medicine, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan

      Takayuki Ishige, Motoi Nishimura & Kazuyuki Matsushita

    3. Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba, 278-8510, Japan

      Shoujiro Ogawa & Tatsuya Higashi

    4. Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan

      Motoi Nishimura & Kazuyuki Matsushita

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    1. Mamoru Satoh
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    Published in the topical collection New Applications of Mass Spectrometry in Biomedicine with guest editors Fumio Nomura Mitsutoshi Setou, and Toshimitsu Niwa.

    Mamoru Satoh and Takayuki Ishige contributed equally to this work.

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    Satoh, M., Ishige, T., Ogawa, S. et al. Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC–MS/MS for clinical laboratory applications. Anal Bioanal Chem 408, 7617–7627 (2016). https://doi.org/10.1007/s00216-016-9821-4

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    • DOI: https://doi.org/10.1007/s00216-016-9821-4

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