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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• 作者：Mamoru Satoh ; Takayuki Ishige ; Shoujiro Ogawa…
• 关键词：25(OH)D3 ; 3 ; epi ; 25(OH)D3 ; 24 ; 25(OH)2D3 ; 25(OH)D2 ; 4 ; (4′-Dimethylaminophenyl) ; 1 ; 2 ; 4 ; triazoline ; 3 ; 5 ; dione (DAPTAD) ; Clinical laboratory
• 刊名：Analytical and Bioanalytical Chemistry
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：408
• 期：27
• 页码：7617-7627
• 全文大小：902 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Analytical Chemistry
  Food Science
  Inorganic Chemistry
  Physical Chemistry
  Monitoring, Environmental Analysis and Environmental Ecotoxicology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1618-2650
• 卷排序：408

文摘

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.