2-氟-1-甲基吡啶鎓对甲苯磺酸盐：一种用于维生素 D 代谢物的新型 LC-MS/MS 衍生化试剂。,Journal of Lipid Research

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2-氟-1-甲基吡啶鎓对甲苯磺酸盐：一种用于维生素 D 代谢物的新型 LC-MS/MS 衍生化试剂。
Journal of Lipid Research ( IF 5.0 ) Pub Date : 2023-07-03 , DOI: 10.1016/j.jlr.2023.100409
Anastasia Alexandridou ₁ , Dietrich A Volmer ₁

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通过质谱法进行维生素 D 分析面临着一些分析挑战，包括维生素 D 化合物的低效电离、非特异性碎片模式、差向异构体、异构体和同量异位体的干扰，以及血液样本中某些代谢物的浓度水平非常低。衍生化已成为其中几个问题的解决方案。在本研究中，我们首次使用2-氟-1-甲基吡啶鎓对甲苯磺酸盐（FMP-TS）对维生素D ₃代谢物进行衍生化，目的是提高检测灵敏度并实现维生素D的全色谱分离异构体和差向异构体。UHPLC-MS/MS 用于测定人血清中五种主要维生素 D _{3代谢物。}与 Amplifex 和 4-苯基-1,2,4-三唑啉-3,5-二酮 (PTAD) 相比，FMP-TS 反应所需的时间更短（仅 15 分钟）。对方法的多个实验条件进行了优化和验证，保证了方法的准确度、精密度和可靠性。使用内部和市售的质量控制样品来确保 25-羟基维生素 D ₃结果的质量。该方法表现出非常好的线性度以及日内和日间准确度和精密度；决定系数(r ² )范围为0.9977-0.9992，相对回收率为95-111%，变异系数为0.9-11.3。稳定性测试表明，提取的衍生化血清样品在-20℃保存24小时后保持稳定。^此外，24,25(OH) ₂ D ₃和1,25(OH) ₂ D ₃ -FMP衍生物在1内保持稳定。 -80 ^℃保存一周。优化后的方法成功应用于健康个体样本中维生素D _{3 、25-羟基维生素D}₃和24,25-二羟基维生素D ₃两种差向异构体的定量测定。

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2-fluoro-1-methylpyridinium p-toluene sulfonate: a new LC-MS/MS derivatization reagent for vitamin D metabolites.

Vitamin D analysis by mass spectrometry faces several analytical challenges, including inefficient ionization of vitamin D compounds, non-specific fragmentation patterns, interferences from epimers, isomers and isobars, as well as very low concentration levels for some of the metabolites in blood samples. Derivatization has been a solution to several of these problems. In this study, we used 2-fluoro-1-methylpyridinium p-toluene sulfonate (FMP-TS) for the first time for derivatization of vitamin D₃ metabolites, with the aim of increasing detection sensitivity and allowing for full chromatographic separation of vitamin D isomers and epimers. UHPLC-MS/MS was used for the measurement of five major vitamin D₃ metabolites in human serum. Compared to Amplifex and 4-phenyl-1,2,4-triazolin-3,5-dion (PTAD), the FMP-TS reaction required less time to be performed (only 15 min). Multiple experimental conditions of the method were optimized and validated to ensure the accuracy, precision and reliability of the method. In-house and commercially available quality control samples were used to assure the quality of the results for 25-hydroxyvitamin D₃. The method showed very good linearity and intra- and inter-day accuracy and precision; coefficients of determination (r²) ranged between 0.9977-0.9992, relative recovery from 95-111% and coefficient of variation from 0.9-11.3. Stability tests showed that the extracted derivatized serum samples were stable for 24 h after storage at -20 ^oC. Additionally, 24,25(OH)₂D₃ and 1,25(OH)₂D₃-FMP derivatives were stable for 1 week when stored at -80 ^oC. The optimized method was successfully applied to human samples of healthy individuals for quantitative determination of vitamin D₃, the two epimers of 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃.

更新日期：2023-07-03

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