Quantitative Measurement of Isotope Ratios by TOF-SIMS MS

描述（由申请人提供）：本项目旨在开发和演示一种新型多级飞行时间质谱仪的性能，该质谱仪可在极低水平下精确测量同位素的相对丰度。该仪器最终将提供至少等同于采用加速器质谱仪的既定方法所取得的性能，而成本仅为加速器质谱仪的一小部分。所提出的台式仪器体积小，自动化程度高，适合医院或医学研究实验室中相对未受过培训的操作人员使用。这项工作的主要重点是那些需要在十亿分之一以下的水平上测量特定同位素的应用。这些应用通常涉及半衰期很长的放射性同位素（100 - 1000年）。具体的例子包括用于放射性碳测年和生物示踪研究的14C，以及用于监测人类患者骨长期代谢状态的41Ca示踪剂。这些应用通常需要精确测定10-10以下和10-15以下水平的同位素相对丰度。目前，这些测量需要使用一个非常大而昂贵的“加速器质谱仪”（AMS），通常位于中心设施中。加速器质谱法已经证明了长寿命放射性同位素作为生物示踪剂的效用，但由于中央设施内的复杂仪器成本相对较高且难以获得，其应用受到限制。然而，在美国国立卫生研究院、制药行业和几家基于AMS的企业的支持下，2006年FDA发布了一份指导声明，其中包括基于AMS的14C微剂量（即药物药代动力学和药效动力学的首次人体“0期”研究），以及用于癌症诊断的41Ca的AMS研究，这些都得到了美国国立卫生研究院的早期支持。该技术仅限于固体样品沉积在合适的目标上，并且似乎为AMS开发的样品制备程序可以在建议的仪器中进行很少或不进行修改。除了开发一种经济的替代AMS用于测量ppt水平的放射性同位素外，该项目还将提供一种非常强大的方法来精确测量更丰富的同位素，如2H， 13C, 15N, 17O, 18O， 33S和34S，以及各种各样的金属，即使存在于非常复杂的基质中。SIMS已经被证明应用于生物组织、微生物和其他复杂的有机和无机样品，使用传统的SIMS仪器。新型多级TOF分析仪具有高质量分辨率、高速度和高灵敏度，可为这些应用提供卓越的结果，并允许同时精确测量亚皮摩尔水平的稳定同位素水平，用于LC分离的组分。提出了一种用串联TOF质谱法同时测定各组分同位素比值的方法。飞行时间质谱法的两项重大创新使该项目成为可能。一个是多级TOF质谱仪的发展，与早期的仪器相比，它极大地提高了丰度灵敏度。另一个是发明和实施了一种新的飞行时间质谱法原理，该原理可以通过脉冲或连续电离源同时提供空间和速度聚焦。后者能够使用Cs+ SIMS离子源，该离子源提供了几个数量级的电离速率，这可能与早期工作中使用的脉冲激光器相同。

DESCRIPTION (provided by applicant): This project is aimed at developing and demonstrating the performance of a new multi-stage time-of-flight mass spectrometer that provides accurate measurement of the relative abundance of isotopes at very low levels. This instrument will ultimately provide performance at least equal to that achieved by established methods employing accelerator mass spectrometers at a very small fraction of the cost. The proposed bench top instrument is small, highly automated and suitable for use by relatively untrained operators in a hospital or medical research laboratory. The major focus in this work is on those applications that require measurements of specific isotopes at levels below the part-per-billion level. These applications generally involve radioactive isotopes with very long half-lives (>1000 years). Specific examples include 14C for radiocarbon dating and biological tracer studies, and 41Ca used as a tracer for monitoring bone long term metabolic status in human patients. These applications often require precise determination of the relative abundance of isotopes at levels below 10-10 and extending down to 10-15. At present these measurements require use of a very large and expensive "accelerator mass spectrometer" (AMS) generally located in a central facility. Accelerator mass spectrometry has demonstrated the utility of long-lived radioisotopes as biological tracers, but applications have been limited by the relatively hig cost and inaccessibility of complex instruments housed in central facilities. Nevertheless, support from the NIH, the pharmaceutical industry, and several AMS-based businesses led to a 2006 FDA guidance statement including AMS-based 14C micro dosing (i.e., first-in-human "Phase 0" studies) of drug pharmacokinetics and pharmacodynamics, and AMS studies of 41Ca for cancer diagnostics have also seen early support from the NIH. The technique is limited to solid samples deposited on a suitable target, and it appears that the sample preparation procedures that have been developed for AMS can be employed with little or no modification in the proposed instrument. In addition to developing an economical alternative to AMS for measuring radioactive isotopes at ppt levels, this project will provide a very powerful method for accurate measurement of more abundant isotopes such as 2H, 13C, 15N, 17O, 18O, 33S, and 34S, as well as a wide variety of metals even when present in very complex matrices. SIMS has already been demonstrated for applications to biological tissues, microorganisms, and other complex organic and inorganic samples using conventional SIMS instrumentation. The high mass resolving power, high speed, and high sensitivity available with the new multi-stage TOF analyzer should provide superior results for these applications, and will allow simultaneous accurate measurements of stable isotope levels at sub-picomole levels for components separated by LC. An approach to identification of components in LC fractions by tandem TOF MS in parallel with simultaneous measurements of isotope ratios is presented. Two major innovations in time of flight mass spectrometry make this project feasible. One is the development of a multi-stage TOF mass spectrometer that dramatically improves the abundance sensitivity compared to earlier instruments. The other is invention and implementation of a new principle in time of flight mass spectrometry that provides simultaneous space and velocity focusing with either pulsed or continuous sources of ionization. The latter enables the use of a Cs+ SIMS ion source that provides several orders of magnitude greater ionization rates that are possible with the pulsed lasers used in earlier work. PUBLIC HEALTH RELEVANCE: The introduction of biomedical applications of AMS in the early 1990's brought the possibility of significant changes in strategies employed for drug development. The ability of AMS to distinguish 14C-labeled compounds from their unlabeled counterparts and to quantify exceedingly small amounts of these compounds in any biological matrix enabled "micro dosing" studies to obtain detailed information on metabolic pathways long before safety and efficacy trials in humans. Measurement of 41Ca/Ca in urine and serum enables early detection and improved clinical management of osteoporosis, multiple myeloma, and cancer metastatic to the bone. This project will enable broader application of these state-of-the-art research and diagnostic methods by providing inexpensive instruments with competitive performance that are suitable for routine use in clinical and medical research laboratories.