Novel analytical approaches to quantify low-abundant dynamic metabolites in the vitamin D metabolic cascade

量化维生素 D 代谢级联中低丰度动态代谢物的新颖分析方法

• 批准号: 242744369
• 负责人: Professor Dr. Dietrich Volmer
• 金额: --
• 依托单位: Arbeitsgruppe Bioanalytische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/242744369?language=en
• 关键词: Novel; analytical; approaches; quantify; low

Vitamin D comprises a group of secosteroid compounds, of which vitamin D2 and D3 are the most important bioactive variants. Vitamin D plays a crucial role in bone health but has also been linked to many other diseases such as cancer and chronic liver disease. To determine vitamin D status in humans, several automated clinical assays are available, which measure the most important vitamin D marker, 25-hydroxyvitamin D. Unfortunately, these assays often lack specificity and accuracy. As a result, LC-MS/MS techniques are preferred today because of their ability to distinguish between vitamin D variants, their improved sensitivity and selectivity. Development of LC-MS/MS assays for vitamin D requires significant expertise to overcome the various inherent limitations, however, and the potential for interferences. This proposal addresses some of these limitations and suggests improved techniques for measurement of vitamin D metabolites. The approach comprises two areas of technical advancements; (1) chemical labeling techniques for increased sensitivity and selectivity, and (2) combined differential ion mobility (DMS)/mass spectrometry for separating isobaric/isomeric species, thus reducing potential systematic errors. Even though it is not the primary aim of this project, we are hoping that measurement of vitamin D metabolic distributions of well-defined patient samples will give us preliminary evidence for correlations of metabolite fingerprints with disease phenotypes. The first theme addresses important limitations of vitamin D analysis; that is limited detection sensitivity and issues of quantification, which currently restrict analysis to the main vitamin D metabolite in most assays. This precludes more elaborate diagnostic applications using low abundant metabolites. We intend to develop a differential quantification technique using new isotope-coded tags for LC-MS/MS, enabling comparative measurement of longitudinal up- and down-regulation of vitamin D metabolite levels, e.g. over the course of a supplementation study or for comparing stages of disease. We are also suggesting a metal-coded tag incorporating a lanthanide-chelating moiety, which will permit analysis at much lower concentration levels than currently possible, using analysis by ICP-MS.The second theme addresses the limited selectivity of LC-MS/MS from interfering endogenous components. In this proposal, we suggest a gas-phase separation step using DMS prior to mass spectrometry. DMS can often separate isobars and isomers because differential mobilities are largely independent of mass-to-charge-ratios but rather depend on conformation, charge distribution etc. Here we propose the use of DMS-MS for removing isobaric species and elimination of co-eluting interferences during vitamin D analysis, to lower detection limits and avoid systematic errors. We also want to examine whether DMS-MS is suited for separation of the C-3 epimers, to distinguish contributions of these species.

维生素 D 包含一组类固醇化合物，其中维生素 D2 和 D3 是最重要的生物活性变体。维生素 D 在骨骼健康中发挥着至关重要的作用，但也与癌症和慢性肝病等许多其他疾病有关。为了确定人类维生素 D 的状态，可以使用几种自动化临床检测方法来测量最重要的维生素 D 标记物 25-羟基维生素 D。不幸的是，这些检测方法通常缺乏特异性和准确性。因此，LC-MS/MS 技术因其区分维生素 D 变体的能力、更高的灵敏度和选择性而成为当今的首选。然而，维生素 D 的 LC-MS/MS 检测方法的开发需要大量的专业知识来克服各种固有的限制以及潜在的干扰。该提案解决了其中一些局限性，并提出了改进维生素 D 代谢物测量技术的建议。该方法包括两个技术进步领域； (1) 化学标记技术可提高灵敏度和选择性，(2) 组合微分离子淌度 (DMS)/质谱法可分离同量异位/同分异构体，从而减少潜在的系统误差。尽管这不是该项目的主要目标，但我们希望对明确患者样本的维生素 D 代谢分布进行测量将为我们提供代谢物指纹与疾病表型相关性的初步证据。第一个主题解决了维生素 D 分析的重要局限性；这是有限的检测灵敏度和定量问题，目前在大多数测定中限制了对主要维生素 D 代谢物的分析。这妨碍了使用低丰度代谢物进行更复杂的诊断应用。我们打算开发一种使用新的 LC-MS/MS 同位素编码标签的差异定量技术，从而能够比较测量维生素 D 代谢物水平的纵向上调和下调，例如维生素 D 代谢物水平的纵向上调和下调。在补充研究过程中或用于比较疾病阶段。我们还建议使用包含镧系元素螯合部分的金属编码标签，这将允许使用 ICP-MS 分析以比目前低得多的浓度水平进行分析。第二个主题解决了 LC-MS/MS 对干扰内源成分的有限选择性。在此提案中，我们建议在质谱分析之前使用 DMS 进行气相分离步骤。 DMS 通常可以分离同量异位素和异构体，因为不同的迁移率在很大程度上与质荷比无关，而是取决于构象、电荷分布等。在这里，我们建议使用 DMS-MS 去除同量异位物质并消除维生素 D 分析过程中的共洗脱干扰，以降低检测限并避免系统误差。我们还想检查 DMS-MS 是否适合分离 C-3 差向异构体，以区分这些物种的贡献。