BIOLOGICAL APPLICATIONS OF THERMOSPRAY LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY

热喷雾液相色谱/质谱的生物学应用

• 批准号: 3857161
• 负责人: A L YERGEY
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3857161
• 关键词: 25 hydroxycholecalciferol; biomedical equipment development; calcium metabolism; child (0-11); cholecalciferol; dietary calcium; dietary fiber; high performance liquid chromatography; human subject; laboratory rat; liquid chromatography; mass spectrometry; nutrition related tag; stable isotope; vitamin D; vitamin metabolism

Tracer Studies. Measurement of the endogenous production rate of 25-OH- vitamin D3 (25OHD) has been accomplished using 6,19,19-trideutero-25-OH- vitamin D3 as a tracer. The 25OHD measurements were done in conjunction with a study investigating the effects of dietary fiber on calcium absorption and 25OHD production rates. We have determined that half like of 25OHD to be about 9.5 days in 5 healthy young men. In contrast to earlier studies using radiolabelled tracers, the present results show: a) half life that is reduced by about 50% from earlier results (mean 20 days); b) no perturbation of the half life by high levels of dietary fiber, and c) reduction of the fractional absorption of dietary calcium by the high fiber diet. Vitamin D metabolites. We have demonstrated the HPLC separation and thermospray LC/MS analysis of the two major metabolic end products of 24,25-dihydroxy vitamin D, which is the principal unregulated pathway used for the disposition of 25OHD. The two water soluble oxidations products, 23-carboxy-24,25,27-tetranor vitamin D (C23 acid) and 24-carboxy-25,26,27- trinor vitamin D (C24 acid), are separated with a typical reverse phase column using 70% methanol solvent at pH 2.5 from acetic acid. We have observed a 4:1 ratio of C23 acid to C24 acid, produced by a perfused rat kidney model. This is the inverse of what had been observed previously by GC/MS. Instrumentation Development. An electrospray mass spectrometer interface for an existing quadrupole mass filter is being developed. The electrospray instrument will permit characterization of molecules in the 20,000 to 50,000 dalton range of molecular weights. Results to date include demonstrating that the vacuum system can accommodate flows of a 1:1 water methanol mixture flowing at 0.002ml/min through a 3 kV potential between a syringe pump needle and the sampling capillary orifice. This yields an ion beam of 60 picoamps at the center of the quadrupole entrance lens, 30 cm from the capillary entrance. Such high currents suggest that fmole sensitivity for certain large molecules may be anticipated.

示踪剂研究。 测量25-OH-的内源性生产率 维生素D3（25OHD）已使用6,19,19-Trideutero-25-OH-完成 维生素D3作为示踪剂。 25OHD测量是通过结合完成的 通过研究饮食纤维对钙的影响 吸收和25OHD的生产率。 我们已经确定一半喜欢 在5名健康的年轻男性中，有25天约9.5天。 与 使用放射性标记的示踪剂的早期研究，目前的结果表明：a） 与早期结果相比（平均20天）降低了约50％的半衰期； b）高水平的饮食纤维和c）没有扰动半衰期 降低高纤维饮食钙的分数吸收 饮食。 维生素D代谢物。 我们已经证明了HPLC分离和 热涂LC/MS分析的两个主要代谢终端产物 24,25-二羟基维生素D，这是所使用的主要不管制途径 对于25ohd的处置。 两种水溶性氧化产物， 23-羧基-24,25,27-四烷维生素D（C23酸）和24-羧基-25,26,27- Trinor维生素D（C24酸），用典型的反相分离 从乙酸pH 2.5时使用70％甲醇溶剂的列。 我们有 观察到C23酸与C24酸的比率为4：1，由灌注大鼠产生 肾脏模型。这是先前观察到的倒数 GC/MS。 仪器开发。 电喷雾质谱仪界面 对于现有的四极杆质量过滤器，正在开发。 这 电喷雾仪器将允许表征分子 20,000至50,000道尔顿分子量。 结果迄今为止 包括证明真空系统可以容纳1：1的流量 水甲醇混合物以0.002ml/min的流动通过3 kV电势 在注射器泵针和采样毛细管之间。这 在四极入口的中心产生一个60 picoamps的离子束 镜头，距毛细管入口30厘米。 如此高的电流表明 可以预期对某些大分子的Fmole敏感性。