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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.
Anal Bioanal Chem. 2016 Nov; 408(27):7617-7627.AB

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.

Authors+Show Affiliations

Division of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan.Division of Laboratory Medicine, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan.Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba, 278-8510, Japan.Division of Laboratory Medicine, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan. Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan.Division of Laboratory Medicine, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan. Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan.Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba, 278-8510, Japan.Division of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260-8670, Japan. fnomura@faculty.chiba-u.jp.

Pub Type(s)

Journal Article
Validation Study

Language

eng

PubMed ID

27526091

Citation

Satoh, Mamoru, et al. "Development and Validation of the Simultaneous Measurement of Four Vitamin D Metabolites in Serum By LC-MS/MS for Clinical Laboratory Applications." Analytical and Bioanalytical Chemistry, vol. 408, no. 27, 2016, pp. 7617-7627.
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. 2016;408(27):7617-7627.
Satoh, M., Ishige, T., Ogawa, S., Nishimura, M., Matsushita, K., Higashi, T., & Nomura, F. (2016). Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC-MS/MS for clinical laboratory applications. Analytical and Bioanalytical Chemistry, 408(27), 7617-7627.
Satoh M, 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. 2016;408(27):7617-7627. PubMed PMID: 27526091.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC-MS/MS for clinical laboratory applications. AU - Satoh,Mamoru, AU - Ishige,Takayuki, AU - Ogawa,Shoujiro, AU - Nishimura,Motoi, AU - Matsushita,Kazuyuki, AU - Higashi,Tatsuya, AU - Nomura,Fumio, Y1 - 2016/08/15/ PY - 2016/04/13/received PY - 2016/07/21/accepted PY - 2016/07/06/revised PY - 2016/8/16/pubmed PY - 2018/1/20/medline PY - 2016/8/16/entrez KW - 24,25(OH)2D3 KW - 25(OH)D2 KW - 25(OH)D3 KW - 3-epi-25(OH)D3 KW - 4-(4′-Dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) KW - Clinical laboratory SP - 7617 EP - 7627 JF - Analytical and bioanalytical chemistry JO - Anal Bioanal Chem VL - 408 IS - 27 N2 - 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. SN - 1618-2650 UR - https://www.unboundmedicine.com/medline/citation/27526091/Development_and_validation_of_the_simultaneous_measurement_of_four_vitamin_D_metabolites_in_serum_by_LC_MS/MS_for_clinical_laboratory_applications_ DB - PRIME DP - Unbound Medicine ER -