For short-lived radioisotopes, where the half life is less than a few years, decay measurements are efficient methods of isotope quantification if performed for an appreciable fraction of the mean life or if the sample is large enough to provide a statistically significant number of decays. For isotopes with long mean lives however, these conditions are seldom met; less than 0.012% of a radiocarbon sample with a mean life of 5740 years decays in one year.
Isotope quantification for stable or long-lived isotopes can be achieved using mass spectrometry (MS) which is used to determine isotope ratios (the concentration of rare isotope to the concentration of total element) as low as 10−9 in microgram to nanogram samples. Natural abundances of isotopes with mean lives from tens to millions of years are 10−9 to 10−9 times elemental abundances and cannot be detected efficiently by ‘conventional’ isotope ratio MS (IRMS). Accelerator mass spectrometry (AMS) which is used for the efficient detection of long-lived isotopes at part-per-quadrillion sensitivities must be employed.
AMS uses the high energy (from 1-30 million volts) of a tandem Van de Graaff electrostatic accelerator to produce positive ions of elements such as carbon, calcium, iodine, chlorine, tritium and aluminium. The system operates initially on negative ions produced from ionisation of a small button of solid sample in a caesium sputter ion source. The tandem accelerator has a collision cell at elevated positive electrical potential in which the accelerated negative ions lose one or more electrons through a collision process and are converted to positive ions. For measurement of the three isotopes of carbon (14C, 13C and 12C), AMS separates these by virtue of their different masses, charge and energy, providing data on the isotope ratios to parts per quadrillion sensitivity i.e., attomole to zeptomole levels with high precision.
The use of AMS in radiocarbon dating is well known The technique also has proven applications in oceanography, the food and agrochemical industries and other diverse research areas. The most important application of AMS to the pharmaceutical or biotechnology industry stems from the requirement of using radio-labelled drugs in various aspects of drug research and development, especially in clinical trials of potential new medicines.
Present use of radiolabelled drugs for research in human subjects owing to the radiation exposure is strictly regulated, restricting, wasteful on preliminary safety experiments in animals, time-consuming and costly. Use of radiolabelled drugs in some cases, such as dermal, buccal, vaginal, anal, subcutaneous or nasal application or administration by inhalation, is difficult and sometimes impossible. AMS can substitute high dose (microCuries) for low dose (nanoCuries) radioactive human studies and allows for the acquisition of similar data. The overwhelming advantage of AMS is that it can quantify, with relatively short analytical times, levels of radioactivity that are so low that the dose needed to be administered to a human subject fills below the stipulated levels of radioactivity which require regulatory review.
The ability to obtain human data much earlier in the drug development process is important. Approximately one third of development drugs have to be dropped because of some metabolism problem. Early discovery of these problems will save development time and costs. AMS also offers the advantage that it is possible to obtain measurements in very small samples ranging from micrograms or less of tissue or cells to a few microlitres of blood. The latter has important ethical implications in paediatric studies since only minimal amounts of blood need be drawn. In the case of laboratory rodents this means that instead of using several animals per group, a single animal can be used at each dose level and sequential blood samples removed for analysis.
The problem with any analysis which involves the use of AMS is the bottleneck at the sample preparation end prior to the analysis. In order to analyse elemental isotopes by AMS, the material to be tested must be converted to a form that can yield negative ions within the instrument's ion source. Carbon-containing materials are converted to graphite, halides to silver salts, aluminium to the oxide and calcium to a dihalide or dihydride. Preparation of the samples for use in AMS represents a tedious process which typically requires two days labour.
Current AMS ion sources require samples that are thermally and electrically conductive solids. This material is further constrained by effective chemical isolation of the sample from its matrix. The isolation process must be nonfractionating, efficient and protected from contamination by isobars or unexpected concentrations of the rare isotope in or on laboratory equipment. Uniformity and comparability between samples and standards are ensured by reducing all samples to a homogenous state from which the final target material is prepared. Carbon samples (containing 14C) are oxidised to C2 before reduction to graphite, commonly produced by the reduction of the C2 by hydrogen or zinc over an iron or cobalt catalyst or binder (see Vogel J S (1992) Rapid production of graphite without contamination for biomedical AMS. Radiocarbon, 34, 344-350).
Oxidation of carbon samples occurs in a sealed tube which is heated in a furnace at temperatures of up to 900° C. with an oxidant such as copper oxide. This process which can lead to sample losses through explosion of glass tubes at this temperature, together with a cooling period, lasts approximately eight hours. The resulting CO2 is reduced to graphite in a second step, after cryogenic transfer which can also result in losses, using a reducing agent such as zinc and titanium hydride and cobalt as a catalyst at temperatures of up to about 500° C. This step is particularly time consuming and lasts approximately eighteen hours with cooling. The prepared cobalt/graphite sample is then compressed into tablet form in a cylindrical aluminium cathode before elemental isotopic ratio analysis in the accelerator mass spectrometer.
U.S. Pat. Nos. 5,209,919 and 5,366,721 disclose conventional methods of sample preparation for AMS analysis. U.S. Pat. No. 5,209,919 combines the reducing abilities of hydrogen and zinc to achieve a more rapid and complete reaction at a lower temperature which produces filamentous graphite with low isotopic fractionation and high ion current output. Although this method is completed in 5 hours instead of the 12-26 hours required when zinc is used alone, this is still a lengthy process when compared with a typical six minute sample analysis time upon the AMS machine.
AMS is an enabling technology with huge potential for speeding up drug development. There remains however a real need for a method of sample preparation which will alleviate the bottleneck in AMS analysis at the sample preparation end.
USPTO Backgrounds
USPTO Backgrounds