Analytical Methods

The specific detection of radioactive isotopes is straightforward, with liquid scintillation counting (LSC) being the main method by which long-lived radiolabels are quantitatively detected. LSC uses a photomultiplier tube to detect light emissions from the fluor; a fluor is a fluorescent molecule that undergoes excitation by the absorption of radiation and releases light when it relaxes to the ground state. The amount of light emitted by a specified amount of radioactive material can be directly correlated to the amount of radioactivity present. Accelerator mass spectrometry has also been used to detect low levels of radioactivity in biological samples but is infrequently used due to the expense of the equipment and the difficulty in sample preparation.³⁾ Scintillation-based methods have typically been preferred for the detection of radiolabels both due to the sensitivity, and also due to the difficulties in handling contamination from sample spillage inside an instrument such as an NMR spectrometer.

In radiation-based imaging techniques, the radiolabeled compound is administered to the patient and emissions are recorded on film or digitally using a counter array.

The single-photon emission computed tomography (SPECT) and positron emission tomography (PET) medical imaging techniques employ substrates labeled with radioactive isotopes that have relatively short half-lives to visualize biochemical processes in animals and humans. SPECT imaging involves the use of a gamma scintillation camera where multiple images are taken, typically encompassing 180° or 360°. PET imaging is based on positron-electron annihilations that yield pairs of 511 kEV photons, which are minimally attenuated or scattered while passing through soft tissues due to their high energy. The PET imaging system captures and registers photons arising from annihilation precisely at the same time, thereby providing exceptional sensitivity. By contrast, SPECT isotopes emit photons in the lower range of 60-400 kEV, which are more likely to be scattered or absorbed, and thus image quality is worse the further the target is from the detector. However, SPECT has the advantage that since the various tracers emit photons with a range of energy peaks, it is possible to image more than one target in the same session by configuring the instrument to separate signals of different energies. PET isotopes all produce photons of the same energy, making it necessary to perform separate imaging sessions if two different probes are used to study the same subject. Both SPECT and PET systems use computer algorithms to reconstruct multiple tomograms in coronal, sagittal, and transverse projections.

The selective concentration of such a tracer at a site of interest is based either on high-affinity binding to a specific target, such as a hormone receptor, or on a specific chemical modification that traps it within a cell, such as phosphorylation. When new applications are developed, the feasibility and behavior of the tracer is often first checked in an in vitro model using analogous labeled compounds that contain the longer-lived ³H or ¹⁴C before moving to the diagnostic scenario.⁴⁾ The organic isotopes used in PET are: ¹¹C, ¹⁵O, ¹³N, ¹⁸F, ⁷⁶Br, and ¹²⁴I; while the one used in SPECT is ¹²³I.

A variety of physical techniques can be used in the detection of stable isotopes, such as high resolution mass spectroscopy, Raman spectroscopy had been more challenging until the development of nuclear magnetic resonance, especially the far more sensitive instruments developed in the past several decades. Chromatographic methods per se cannot distinguish isotopically labeled compounds from the parent compounds, but once the compounds, fragments or reaction products have been separated they can be detected by scintillation, mass spectroscopy, NMR, and other techniques.

All three hydrogen isotopes (¹H, ²H, and ³H) are magnetically active, but in practical terms only proton and deuteron NMR are used because of the risk of contamination with the radioactive triteron. The other organic isotope used very frequently for NMR is ¹³C, and to a lesser extent the ¹⁵N, ¹⁷O, and ³³S are used.