A NEW METHOD FOR THE ANALYSIS OF VITAMIN D

维生素 D 分析的新方法

• 批准号: 8122498
• 负责人: Fred E Regnier
• 金额: $ 9.95万
• 依托单位: NOVILYTIC, LLC
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8122498
• 关键词: Acetonitriles; Address; Alder plant; Antibodies; Autoimmune Diseases; Biological Assay; Blood; Cardiovascular Diseases; Cholecalciferol; Chronic Disease; Code; Complex; Complication; Coupled; Detection; Discrimination; Disease; Family; Flame Ionization; Food; Goals; Health; High Pressure Liquid Chromatography; Hormones; Institutes; Laboratories; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methods; Methylamines; Molecular; Non-Insulin-Dependent Diabetes Mellitus; Performance; Phase; Physiological; Plasma; Proteins; Reaction; Reagent; Relative (related person); Reporting; Reproducibility; Research; Risk Factors; Role; Running; Sampling; Scheme; Secosteroids; Serum; Solutions; Source; Specificity; Technology; Time; Tissue Sample; Tissues; Variant; Vitamin D; Vitamin D2; Vitamins; Work; base; bone health; cross reactivity; cycloaddition; diene; high throughput analysis; ion source; ionization; liquid chromatography mass spectrometry; methylamine; pi bond; premature; programs; reversed phase chromatography; scale up

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.

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 在骨骼健康方面的既定功能之外，越来越多的证据表明，它可能是多种慢性疾病的危险因素，包括自身免疫性疾病和癌症、心血管疾病和 II 型糖尿病。正在进行的研究面临的一个难题是缺乏具有足够选择性和灵敏度的可靠测量技术来确定组织、血液和食物中维生素 D 及其代谢物的生理水平。市售试剂盒检测可提供 25(OH)D 的高通量分析，但不能分析维生素 D，且实验室间性能较差。此外，这些试剂盒无法准确、单独地测量 25(OH)D3 和 25(OH)D2，因为它们与分解代谢 24,25(OH)2D 代谢物存在交叉反应。另一方面，HPLC 与质谱 (LC-MS) 结合可提高灵敏度和选择性，并最大限度地减少复杂食品基质中常见的干扰。现已开发出多种用于测量血浆中维生素 D 代谢物的 LC-MS/MS 方法，其定量限 (LOQ) 范围为 0.17 至 6.5 ng/mL。虽然前景广阔，但离子源中维生素 D 分子的过早碎裂导致这些方法具有更高的可变性和高 LOQ。为了克服这个问题，开发了一种基于 Diels-Alder 环加成的特定维生素 D 衍生反应。使用 PTAD 衍生化和甲胺，五种维生素 D 代谢物的 LOQ 达到 10 - 20 pg/mL。尽管有显着的改进，但该方法并未达到免疫测定的灵敏度，并且不允许在单次色谱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方法是提高维生素 D 成分测定的选择性和灵敏度。这将通过一种新的液相色谱-质谱法来实现，其中维生素 D 的 9,10- 类固醇成分用一种新试剂衍生化，该试剂既可以根据样品来源对成分进行同位素差异编码，又可以大大提高其电离效率。总而言之，这将能够以与免疫测定 (1-5 pg/mL) 相当的水平同时分析多个样品，同时仍允许对维生素 D2、维生素 D3、25(OH)D2、25(OH)D3 进行分子区分、1,25(OH)2D2、1,25(OH)2D3、24,25(OH)2D2、24,25(OH)2D3。 公共健康相关性：正在开发一种用于分析维生素 D 及其不同形式的新定量策略和试剂，这将有助于更广泛地了解这种激素样维生素特有的各种生物调节功能。