二羟基化维生素D3的色谱分离及人血清中1α，25(OH)2D3的准确定量

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis Pub Date : 2025-08-18 DOI:10.1016/j.jpba.2025.117125

Koji Takahashi , Masaki Takiwaki , Rino Tsutsumi , Ryota Sakamoto , Kazuo Nagasawa , Yutaka Kuroda , Seketsu Fukuzawa

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引用次数: 0

摘要

二羟化维生素D （VD）最具生物活性的形式是1α，25-二羟维生素D3 (1α,25(OH)2D3)，它与各种生理过程和疾病发展有关。因此，准确定量1α，25(OH)2D3对于临床诊断、vd相关疾病管理和感染控制至关重要。然而，生物样品中的1α，25(OH)2D3的定量具有挑战性，因为其浓度低且受到其他二羟基化VD代谢物的干扰。在这项研究中，我们基于液相色谱-串联质谱（LC-MS /MS），结合蛋白质沉淀、固相萃取（SPE）和14-（4-二甲氨基苯基）-9-苯基-9,10-二氢-9,10-[1,2]吡唑蒽-13,15-二酮（DAP-PA）衍生化VD代谢物，建立了一种新的1α，25(OH)2D3定量分析方法。在衍生化过程中，以1:1的比例得到1α，25(OH)2D3的6个 R和6S异构体，并使用五氟苯-十八烷基硅基混合柱进行定量分离和检测。该方法使1α，25(OH)2D3与其他二羟基化vd3完全分离，包括1β，25-二羟维生素D3 (1β,25(OH)2D3), 3-epi-1α，25-二羟维生素D3 (3-epi-1α,25(OH)2D3), 2α，25-二羟维生素D3 (2α,25(OH)2D3), 2β，25-二羟维生素D3 (2β,25(OH)2D3), 4α，25-二羟维生素D3 (4α,25(OH)2D3), 4β，25-二羟维生素D3 （4β,25(OH)2D3）和4β，25-二羟维生素D3 （4β,25(OH)2D3）。为了证明该优化方法的适用性，我们对人血清进行了分析，并将结果与广泛使用的基于免疫亲和提取的LC-MS /MS法对1α，25(OH)2D3进行了比较，结果显示定量值相等。优化后的LC-MS /MS方法运行时间为7.7 min，定量下限为2.5 pg/mL，样品血清体积为100 µL。该方法灵敏度高，精度高，具有高通量分析能力，为准确定量1α，25(OH)2D3提供了一种可靠的方法，同时有效地分离干扰二羟基化VD3异构体。

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Chromatographic separation of dihydroxylated vitamin D3 and accurate quantification of 1α,25(OH)2D3 in human serum

The most bioactive form of dihydroxylated vitamin D (VD) is 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃), which is implicated in various physiological processes and disease development. Therefore, accurate quantification of 1α,25(OH)₂D₃ is essential for clinical diagnosis, VD-related disease management, and infection control. However, 1α,25(OH)₂D₃ quantification in biological samples is challenging because of its low concentration and interference from other dihydroxylated VD metabolites. In this study, we developed a novel assay for 1α,25(OH)₂D₃ quantification, based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) in combination with protein precipitation, solid-phase extraction (SPE), and derivatization of VD metabolites with 14-(4-dimethylaminophenyl)-9-phenyl-9,10-dihydro-9,10-[1,2]epitriazoloanthracene-13,15-dione (DAP-PA). In the derivatization process, the 6 R and 6S isomers of 1α,25(OH)₂D₃ obtained in a 1:1 ratio were quantitatively separated and detected using a pentafluorophenyl-octadecylsilyl mix mode column. This approach enabled the complete separation of 1α,25(OH)₂D₃ from other dihydroxylated VD₃s, including 1β,25-dihydroxyvitamin D₃ (1β,25(OH)₂D₃), 3-epi-1α,25-dihydroxyvitamin D₃ (3-epi-1α,25(OH)₂D₃), 2α,25-dihydroxyvitamin D₃ (2α,25(OH)₂D₃), 2β,25-dihydroxyvitamin D₃ (2β,25(OH)₂D₃), 4α,25-dihydroxyvitamin D₃ (4α,25(OH)₂D₃), and 4β,25-dihydroxyvitamin D₃ (4β,25(OH)₂D₃). To demonstrate the applicability of this optimized method, we analyzed human serum and compared the results with a widely used immunoaffinity extraction-based LC–MS/MS assay for 1α,25(OH)₂D₃, showing equivalent quantitative values. The optimized LC–MS/MS method achieved a run time of 7.7 min, with a lower limit of quantification of 2.5 pg/mL using a sample serum volume of 100 µL. This method is highly sensitive, precise, and capable of high-throughput analysis, providing a robust approach for accurate quantification of 1α,25(OH)₂D₃ while effectively separating interfering dihydroxylated VD₃ isomers.

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来源期刊

Journal of pharmaceutical and biomedical analysis 医学-分析化学

CiteScore

6.70

自引率

5.90%

发文量

588

审稿时长

37 days

期刊介绍： This journal is an international medium directed towards the needs of academic, clinical, government and industrial analysis by publishing original research reports and critical reviews on pharmaceutical and biomedical analysis. It covers the interdisciplinary aspects of analysis in the pharmaceutical, biomedical and clinical sciences, including developments in analytical methodology, instrumentation, computation and interpretation. Submissions on novel applications focusing on drug purity and stability studies, pharmacokinetics, therapeutic monitoring, metabolic profiling; drug-related aspects of analytical biochemistry and forensic toxicology; quality assurance in the pharmaceutical industry are also welcome. Studies from areas of well established and poorly selective methods, such as UV-VIS spectrophotometry (including derivative and multi-wavelength measurements), basic electroanalytical (potentiometric, polarographic and voltammetric) methods, fluorimetry, flow-injection analysis, etc. are accepted for publication in exceptional cases only, if a unique and substantial advantage over presently known systems is demonstrated. The same applies to the assay of simple drug formulations by any kind of methods and the determination of drugs in biological samples based merely on spiked samples. Drug purity/stability studies should contain information on the structure elucidation of the impurities/degradants.