Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC-MS/MS for clinical laboratory applications
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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DOI：
10.1007/s00216-016-9821-4
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发表时间：
2016-11-01
影响因子：
4.3
通讯作者：
Nomura, Fumio
中科院分区：
文献类型：
--
作者：
Satoh, Mamoru;Ishige, Takayuki;Nomura, Fumio
血清25-羟基维生素D [25（OH）D]作为维生素D状态的指标的定量目前主要通过免疫测定法进行，但LC-MS/MS将允许更准确的测定。此外，LC-MS/MS将允许同时测量多种分析物。本研究的目的是开发和验证LC-MS/MS方法，以同时测量血清中的四种维生素D代谢物（25（OH）D-3、3-epi-25（OH）D-3、25（OH）D-2和24，25（OH）（2）D-3），用于临床实验室应用。首先在96孔支撑的液体萃取板中制备血清样品，并使用Cookson型试剂4-（4'-二甲基氨基苯基）-1，2，4-三唑啉-3，5-二酮（DAPPl）衍生化DAPPl，其与维生素D代谢物的s-顺式-二烯结构快速定量反应。对衍生化样品进行LC-MS/MS，通过电喷雾电离（正离子模式）电离，并通过选择反应监测进行检测。25（OH）D-3、3-epi-25（OH）D-3、25（OH）D-2和24，25（OH）（2）D-3的定量下限分别为0.091、0.020、0.013和0.024 ng/mL。这四种代谢物的准确度值和提取回收率均符合要求。将我们的LC-MS/MS测定的血清25（OH）D水平与无法区分25（OH）D-3和25（OH）D-2的常规放射免疫测定法（RIA）测定的血清25（OH）D水平进行比较。通过RIA方法获得的值显示平均偏倚约为8.35 ng/mL，最可能是抗体与低丰度代谢物（包括24，25（OH）（2）D-3）交叉反应的结果。各种预分析因素，如血清分离前长时间放置样品、重复冻融循环和抗凝剂的存在，对这些测定结果无显著影响。四种维生素D代谢物25（OH）D-3、3-epi-25（OH）D-3、25（OH）D-2和24，25（OH）（2）D-3的这种高通量LC-MS/MS同时测定仅需要20 μ L血清。该方法将有助于进一步了解低丰度维生素D代谢物，以及准确测定25（OH）D-3和25（OH）D-2。
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)D-3, 3-epi-25(OH)D-3, 25(OH)D-2, and 24,25(OH)(2)D-3) 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)D-3, 3-epi-25(OH)D-3, 25(OH)D-2, and 24,25(OH)(2)D-3 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)D-3 and 25(OH)D-2. 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)(2)D-3. 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)D-3, 3-epi-25(OH)D-3, 25(OH)D-2, and 24,25(OH)(2)D-3 required as little as 20 mu L serum. This method will aid further understanding of low-abundance vitamin D metabolites, as well as the accurate determination of 25(OH)D-3 and 25(OH)D-2.