A NEW METHOD FOR THE ANALYSIS OF VITAMIN D

描述(申请人提供)：经过近一个世纪的研究，关于维生素D在健康和疾病中的作用仍然存在关键问题。除了维生素D在骨骼健康中公认的功能外，越来越多的证据表明，它可能是从自身免疫性疾病到癌症、心血管疾病和II型糖尿病等慢性疾病的风险因素。正在进行的研究中的一个复杂问题是缺乏可靠的测量技术，该技术具有足够的选择性和灵敏度来确定组织、血液和食物中维生素D及其代谢物的生理水平。市面上可买到的试剂盒检测提供了25(OH)D的高通量分析，但不能提供维生素D的分析，而且实验室间的性能很差。此外，这些试剂盒无法准确和单独地测量25(OH)D3和25(OH)D2，因为它们与分解代谢的24，25(OH)2D代谢物存在交叉反应。另一方面，高效液相色谱质谱联用(LC-MS)提供了更高的灵敏度和选择性，并最大限度地减少了常见的复杂食品基质的干扰。已建立了几种LC-MS/MS测定血浆中维生素D代谢物的方法，其定量限(LOQ)在0.17~6.5 ng/mL之间。虽然前景看好，但离子源中维生素D分子的过早碎裂导致了这些方法的更高可变性和高LOQ。为了克服这一问题，开发了一种基于Diels-Alder环加成反应的特异性维生素D衍生化反应。利用PTAD衍生化和甲胺，5种维生素D代谢物的检出限可达到10-20pg/mL。虽然这是一个显著的改进，但该方法不能达到免疫分析的灵敏度，并且不允许在一次层析运行中同时分析多个样品。本提案中提出的解决这些问题的办法是提高测定维生素D组分的选择性和灵敏度。这将通过一种新的液-质联用方法实现，在这种方法中，维生素D的9，10-秒类固醇成分用一种新的试剂衍生化，这种新试剂根据样品来源对成分进行不同的等位编码，并极大地提高它们的电离效率。综上所述，这将使多个样品能够在与免疫分析相当的水平上同时进行分析(1-5pg/mL)，同时仍然允许对维生素D2、维生素D3、25(OH)D2、25(OH)D3、1，25(OH)2D2、1，25(OH)2D3、24，25(OH)2D2、24，25(OH)2D3进行分子区分。 与公共卫生相关：正在开发一种新的量化战略和试剂，用于分析维生素D及其不同形式，这将有助于更广泛地了解这种荷尔蒙类维生素特有的各种生物调节功能。

DESCRIPTION (provided by applicant): After almost a century of research there are still critical questions regarding the role of vitamin D in health and disease. Beyond the well established function of vitamin D in bone health, evidence is emerging that it may be a risk factor in chronic diseases ranging from autoimmune diseases and cancer to cardiovascular disease and type II diabetes. One complication in ongoing research is the lack of reliable measurement technology with sufficient selectivity and sensitivity to determine physiological levels of vitamin D and its metabolites in tissues, blood, and food. Commercially available kit assays provide high throughput analysis of 25(OH)D, but not of vitamin D, and inter-laboratory performance is poor. Additionally, these kits are unable to accurately and separately measure 25(OH)D3 and 25(OH)D2, as they are confounded by cross-reactivity with catabolic 24,25(OH)2D metabolites. On the other hand, HPLC coupled with mass spectrometry (LC-MS) offers both increased sensitivity and selectivity and minimizes interferences commonly seen from complex food matrices. Several LC-MS/MS methods have been developed for measuring vitamin D metabolites in plasma that reported Limits of Quantification (LOQ) ranging from 0.17 to 6.5 ng/mL. While promising, the premature fragmentation of vitamin D molecules in ion sources contributes to the higher variability of these methods and high LOQ. To overcome this problem, a specific vitamin D derivatization reaction based on Diels-Alder cycloaddition was developed. Using PTAD derivatization and methylamine, LOQs of 10 - 20 pg/mL have been accomplished for five vitamin D metabolites. Although a significant improvement, this method does not achieve the sensitivity of immuno- assays and does not allow for simultaneous analysis of multiple samples in a single chromatographic run. A solution to these problems that will be developed in this proposal is to increase both the selectivity and sensitivity with which the components of vitamin D are determined. This will be achieved with a new liquid chromatography-mass spectrometry approach in which the 9,10- secosteroid components of vitamin D are derivatized with a new reagent that both differentially codes components isotopically according to sample origin and greatly increases their ionization efficiency. Taken together this will enable multiple samples to be analyzed simultaneously at the level comparable to immuno-assays (1-5 pg/mL) while still allowing molecular discrimination between vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3. PUBLIC HEALTH RELEVANCE: A new quantification strategy and reagents for the analysis of vitamin D and its different forms is being developed that will facilitate broader understanding of the various bio- regulatory functions unique to this hormone-like vitamin.