A NEW METHOD FOR THE ANALYSIS OF VITAMIN D

描述（由申请人提供）：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在骨骼健康中的作用，越来越多的证据表明，它可能是慢性疾病的一个危险因素，从自身免疫性疾病和癌症到心血管疾病和II型糖尿病。正在进行的研究的一个并发症是缺乏可靠的测量技术，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。市售试剂盒检测提供25(OH)D的高通量分析，但不包括维生素D，且实验室间性能较差。此外，这些试剂盒无法准确地单独测量25(OH)D3和25(OH)D2，因为它们与分解代谢的24,25(OH)2D代谢物的交叉反应性相混淆。另一方面，HPLC与质谱联用（LC-MS）提供了更高的灵敏度和选择性，并最大限度地减少了复杂食品基质中常见的干扰。已有几种LC-MS/MS方法用于测定血浆中维生素D代谢物，其定量限（LOQ）范围为0.17 ~ 6.5 ng/mL。虽然有希望，但离子源中维生素D分子的过早断裂有助于这些方法的高可变性和高LOQ。为了克服这一问题，开发了一种基于Diels-Alder环加成的维生素D衍生化反应。利用PTAD衍生化和甲胺，对5种维生素D代谢物的loq达到了10 - 20pg /mL。虽然是一个显著的改进，但该方法没有达到免疫测定的灵敏度，也不允许在一次色谱运行中同时分析多个样品。解决这些问题的办法将在本提案中提出，即提高测定维生素D成分的选择性和灵敏度。这将通过一种新的液相色谱-质谱方法来实现，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，这种试剂既可以根据样品来源对成分进行不同的同位素编码，又可以大大提高它们的电离效率。综合起来，这将使多个样品能够在与免疫测定相当的水平上同时进行分析（1-5 pg/mL），同时仍然允许维生素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之间的分子区分。

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.