Improved sensitivity of serum/plasma 1α,25-dihydroxyvitamin D quantification by DAPTAD derivatization
Improved sensitivity of serum/plasma 1α,25-dihydroxyvitamin D quantification by DAPTAD derivatization
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DOI：
10.1016/j.cca.2017.08.033
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发表时间：
2017-10-01
影响因子：
5
通讯作者：
Nomura, Fumio
中科院分区：
文献类型：
--
作者：
Ishige, Takayuki;Satoh, Mamoru;Nomura, Fumio
背景：虽然免疫测定法有一些局限性，如抗体的交叉反应性，但这种技术被广泛用于血清/血浆1,25(OH)(2)D的定量。需要一种精确的方法来测定1,25(OH)(2)D的状态。方法：采用LC-MS/MS设计血清/血浆1,25(OH)(2)D定量方法。采用免疫亲和萃取法（IE）和新开发的cookson型试剂4-（4′-二甲氨基苯基）-1,2,4-三唑啉3,5-二酮（DAPTAD）分别进行样品制备和衍生化。进行了分析和分析前验证。测定了232例健康日本人血清1,25(OH)(2)D-3的浓度。结果：1,25(OH)(2)D-3的组内和组间CVs分别为5.2%和7.0%。1,25(OH)(2)D-3的定量限为7.1 pg/ml。类风湿因子（RF）浓度低于517 IU/ml时不影响血清1,25(OH)(2)D分析。不同采血管、反复冻融循环、全血静置时间、血清保存时间均无显著差异。LC-MS/MS与放射免疫分析法（RIA）之间存在很强的相关性（r = 0.786），但RIA测定的血清1,25(OH)(2)D浓度比LC-MS/MS高2倍。LC-MS/MS检测血清1,25(OH)(2)D-3浓度为18.7 ~ 53.9 pg/ml。结论：采用IE和DAPTAD衍生化技术，建立了一种高灵敏度、高选择性的基于LC-MS/ ms的血清/血浆1,25(OH)(2)D定量方法。该方法将能够在临床环境中准确测定血清/血浆1,25(OH)(2)D浓度。
Background: Although immunoassays have several limitations such as the cross-reactivities of antibodies, such techniques are widely used for serum/plasma 1,25(OH)(2)D quantification. An accurate method is required for the determination of the 1,25(OH)(2)D status.Methods: We designed a serum/plasma 1,25(OH)(2)D quantification method using LC-MS/MS. Immunoaffinity extraction (IE) and the recently developed Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline3,5-dione (DAPTAD) were used for sample preparation and derivatization, respectively. Analytical and pre-analytical validations were performed. Serum 1,25(OH)(2)D-3 concentrations were determined in 232 healthy Japanese individuals.Results: The intra- and inter-assay CVs for 1,25(OH)(2)D-3 were 5.2% and 7.0%, respectively. The limit of quantification for 1,25(OH)(2)D-3 was 7.1 pg/ml. Rheumatoid factor (RF) at concentrations below 517 IU/ml did not affect serum 1,25(OH)(2)D analysis. No significant differences were observed for various blood collection tubes, repeated freeze thaw cycles, whole blood standing time, or serum storage time. A strong correlation between LC-MS/MS and radioimmunoassay (RIA) was observed (r = 0.786), but serum 1,25(OH)(2)D concentrations obtained from RIA were 2-fold higher than those obtained from LC-MS/MS. Serum 1,25(OH)(2)D-3 concentrations by LC-MS/MS were 18.7-53.9 pg/ml.Conclusion: A highly sensitive and selective LC-MS/MS-based serum/plasma 1,25(OH)(2)D quantification method was developed using IE and DAPTAD derivatization. This method will enable the accurate determination of serum/plasma 1,25(OH)(2)D concentrations in the clinical setting.