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

维生素D由一组副类固醇化合物组成，其中维生素D2和D3是最重要的生物活性变体。维生素D在骨骼健康中起着至关重要的作用，但也与许多其他疾病有关，如癌症和慢性肝病。为了确定人体内维生素D的状态，有几种可用于测量最重要的维生素D标志物25-羟基维生素D的自动化临床检测方法。不幸的是，这些检测方法往往缺乏特异性和准确性。因此，LC-MS/MS技术是当今的首选，因为它们能够区分维生素D变体，提高了灵敏度和选择性。然而，开发维生素D的LC-MS/MS检测方法需要大量的专业知识来克服各种固有的限制和潜在的干扰。这一建议解决了其中的一些局限性，并建议改进测量维生素D代谢物的技术。该方法包括两个技术进步领域；(1)提高灵敏度和选择性的化学标记技术；(2)结合差分离子迁移率(DMS)/质谱法分离等压/异构体物质，从而减少潜在的系统误差。尽管这不是该项目的主要目的，但我们希望对明确定义的患者样本的维生素D代谢分布的测量将为我们提供代谢物指纹与疾病表型相关的初步证据。第一个主题解决了维生素D分析的重要局限性；那就是检测灵敏度有限和定量问题，目前大多数分析都局限于主要的维生素D代谢物。这就排除了使用低丰度代谢物更精细的诊断应用。我们打算开发一种差分定量技术，使用新的同位素编码标签用于LC-MS/MS，能够对维生素D代谢物水平的纵向上下调节进行比较测量，例如在补充研究过程中或用于比较疾病阶段。我们还建议使用一种包含镧系螯合部分的金属编码标签，使用ICP-MS分析，它将允许在比目前可能的低得多的浓度水平下进行分析。第二个主题解决了LC-MS/MS对干扰内源性成分的有限选择性。在这个提议中，我们建议在质谱之前使用DMS气相分离步骤。DMS通常可以分离等压线和同分异构体，因为差分迁移率在很大程度上与质量电荷比无关，而是取决于构象、电荷分布等。在此，我们建议在维生素D分析过程中使用DMS-MS去除等压物质和消除共洗脱干扰，以降低检测限并避免系统误差。我们还想研究DMS-MS是否适合分离C-3外显子，以区分这些物种的贡献。

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.