Radio Ion Chromatography

Positron emission tomography (PET) is one of the most powerful non-invasive diagnostic tools for tracing organ functioning. Quality control of the short-livedradiopharmaceuticals is challenging, not least because of the tough time limits, the radiation issue and the near nanomole radiotracer quantities.

This article presents a likewise rugged and versatile multichannel radio IC system that controls the production of the radionuclide [¹⁸F]fluoride (precursor) and the two radiotracers synthesized from it, [¹⁸F]fluorodeoxyglucose and [¹⁸F]fluorocholine, in accordance with pharmacopoeial regulations.

Principles of Positron Emission Tomography (PET)

Radiopharmaceuticals are radioactive substances used in nuclear medicine to diagnose, treat or prevent disease. They contain a radioactive isotope, a so-calledradionuclide, attached to a biologically inert or active molecule.

Radionuclides are unstable isotopes that have an excess of either neutrons or protons and, therefore, radioactively decay, resulting in the emission of gamma rays orsubatomic particles. In proton-rich nuclides, a proton changes to a neutron, whereby a positron (antiparticle of the electron or an electron with a positive charge, also calledβ⁺ particle) is emitted together with a neutrino (υ) according to

proton (p) → neutron (n) + positron ( β⁺ ) + neutrino (υ).

While travelling in the surrounding media, the released positron loses its kinetic energy and then combines with an electron. The encounter annihilates both positronand electron and results in two photons (gamma rays) each with an energy of 0.511 MeV that are emitted in opposite directions (Figure 1).

m_e⁺ + m_e^– → 2γ

Sophisticated scanners (detector) can detect such pairs of photons by coincidence detection. From the data collected, threedimensional images of tissue structuresare then calculated. The most commonly used short-lived, cyclotron-produced radionuclides in radiopharmacy are ¹¹C, ¹³N,⁵O and ¹⁸F. Their respective half-lives are 20.38, 9.96, 2.03 and 109.7 min.

Radiopharmaceuticals

To administer the radionuclide to a living human or animal, it is either incorporated in a biologically inert molecule (e.g., the blood flow tracers[¹⁵ O]water or [¹⁵ O]butanol) or in a biologically active molecule that is absorbed by the organ of interest.

Figure 1: Principle of positron emission tomography.

After the radiopharmaceutical is concentrated in the tissue of interest, the patient is placed in the PET scanner. By tracking the photons, computers withsophisticated software generate threedimensional images of the source of the photons. This allows the study of physiological, biochemical and pharmacological functions at amolecular level. Illnesses such as cancer, cardiovascular disease and even neurological disorders can be detected long before symptoms appear.

Production of PET Radiopharmaceuticals

Radionuclides used in PET experiments such as ¹¹C, ¹³N, ¹⁵ O and¹⁸ F are artificially produced in a cyclotron, where a beam of accelerated charged particles irradiates a prepared target. Subsequently, the resultingradionuclides are isolated and synthetically incorporated into the radiotracer. Since the fluorine atom is similar in size to the hydrogen atom, it acts as a pseudohydrogen andis therefore ideally suited for replacing hydrogen atoms in organic molecules. The positron emitter ¹⁸F is thus one of the most important imagingradionuclides in diagnostic nuclear medicine. It is produced by proton bombardment of an ¹⁸O-enriched water target. In an¹⁸O(p, n)¹⁸F reaction, highly accelerated protons (p) react with the ¹⁸O atomic nucleus to emita neutron (n) and ¹⁸F, which immediately decays by positron emission with a half-life of 109.7 min. The product of the ¹⁸Fdecay is the stable isotope ¹⁸O. After isolation of ¹⁸F from the target water, the radionuclide is incorporated into thechemical compound required. To this end, the preparation of reactive fluorine radionuclides in organic solvents is an important prerequisite for the synthesis of aliphaticcarbon-fluorine bonds.

a)[¹⁸ F]Fluorodeoxyglucose

[¹⁸F]Fluorodeoxyglucose, commonly abbreviated [¹⁸F]FDG, or simply FDG, is a glucose analogue in which thehydroxyl group

at the 2' position of the glucose molecule is substituted by [¹⁸ F]fluorine (Figure 2a). It throws light on the use and metabolism ofglucose in heart, lungs and brain. Additionally, it is used in oncology to determine abnormal glucose metabolism to characterize different tumour types. After administering[¹⁸F]FDG to the patient, it is incorporated into the cells by the same transport mechanism as the normal glucose, but unlike this, once inside thecell, it is not metabolized and thus remains in the cell allowing PET tomographic imaging. Not least because of its many diagnostic uses, the high number of existing labellingprocedures and its advantageous halflife of approximately two hours, which allows the transport to sites that have no cyclotron, [¹⁸F]FDG is actuallythe most frequently used organic PET radiopharmaceutical.

b) [¹⁸ F]Fluorocholine

In cells, choline is used as a precursor for the biosynthesis of phospholipids. As the latter are essential cell membrane components and because tumours revealincreased metabolismof cell membrane constituents and increased choline uptake, radiolabelled choline tracers are invaluable diagnostic tools for cancer detection.[¹⁸F]Fluorocholine (Figure 2b) is a recently developed PET radiotracer that allows choline metabolism to be imaged in vivo. It isbased on the tumour-detecting radiotracer [¹¹C]choline. The driving force of the production of the ¹⁸F-labelled derivativewas the substantially longer half-life, which allows the distribution of this tracer to PET institutions without on-site cyclotron.

Figure 2: Chemical structures of two PET radiopharmaceuticals. (a) In [18F]FDG, the hydroxyl group at the 2’ position of normalglucose is substituted by 18F. (b) In [18F]fluorocholine, a [18F]fluoroalkyl group is attachedto the nitrogen atom of N,N-dimethylaminoethanol (DMæ).

Radio Ion Chromatography (IC)

Radio IC is a powerful, fast and sensitive tool for the quality control of a wide range of PET pharmaceuticals. It aims at determining the radiochemical purity ofradiopharmaceuticals after radiosynthesis with cyclotron-produced nuclides. To this end, Radio IC uses a flowthrough mass and radioactivity detector.

Besides the accuracy and reproducibility of the analytical results, high throughput is a must. One and the same multichannel radio IC takes over the quality controlof three production lines. The analytical unit provides the following benefits:

a) Flexibility of the System

The ion chromatography system installed at the ITP (Instituto Tecnológico PET) in Spain combines three quality control systems for PET pharmaceuticals in one.From the very same injection system, the flow can be automatically directed to the three channels. By selecting between an array of different columns, mobile phases anddetectors, [¹⁸F]fluoride, [¹⁸F]FDG and [¹⁸F]fluorocholine can be separately determined (Table1). All aspects of system operation and data acquisition are controlled by MagIC Net™ software.

Figure 3: (a) Conductivity and (b) radioactivity chromatogram of cyclotron-produced [18F]fluoride. In the subsequent radiosynthesis(nucleophilic fluorination), tracer (i.e., very low) quantities of [18F]fluoride ions are used to form carbon-fluorine bonds. The IC software convertsthe radiation units, counts per second (cps), to mV. Chromatographic conditions are shown in Table 1.

b) Safety

The automated sample injection with Metrohm's patented Dosino technology allows the aspiration of very low sample volumes with accuracy and precision. By usingthe MagIC Net™

are completely automated without any carryover. The system's modular design supports the installation of the required lead shielding and thus guarantees usersafety. The injection valve is placed inside a 5 cm thick tailor-made lead housing, while radiation from the radiotracers in the separation columns is attenuated to a safe levelby a sufficiently thick lead shielding. In addition, a lead sample holder avoids user exposure to gamma radiation.

c) Comprehensive Quality Control Using Multichannel Radio IC

Radiopharmaceuticals have unique characteristics and require special tests described in numerous pharmacopoeias. The quality control includes testing for bothchemical and radiochemical purity before the radiotracer is administered to the patient. The radiochemical purity of a radiotracer – the ratio of the radionuclide in boundform (e.g., [¹⁸ F]FDG) to the radionuclide in unbound form (e.g., [¹⁸ F]fluoride) – guarantees the image quality ofthe PET scan and protects the patient from unnecessary radiation.

In the first instance, the concentration of the cyclotron-produced radionuclide, the ¹⁸ F, which is used as a precursor in the subsequentradiosynthesis, has to be determined (Figure 3).

Figure 4: (a) IC-PAD chromatogram with the glucose precursor, the carrier-free [18F]FDG and the impurity chlorodeoxyglucose. (b)Radioactivity chromatogram of the [18F]FDG. The IC software converts the radiation units, counts per second (cps), to mV. Chromatographic conditionsare shown in Table 1

Subsequently, the appropriate chemical purity – as for any other pharmaceutical preparation – and the concentration of the synthesizedradiopharmaceutical, the ¹⁸ F, which mostly is in the nanomolar range, have to be determined. In doing so, excessive precursors andradiolabelling-derived impurities have to be quantified also. Figure 4 shows the chromatogram with the glucose precursor, the carrier-free [¹⁸ F]FDGand the chlorodeoxyglucose impurity. The analysis is completed in less than 10 min.

Figure 5 shows the chromatogram of the reaction mixture of [¹⁸ F]fluorocholine synthesis. Besides nanomole quantities of the[¹⁸ F]fluorocholine radiotracer, considerable amounts of the reactant N,N-dimethylaminoethanol and trace-levels of calcium impurities were detected inthe reaction mixture.

Figure 5: (a) Conductivity and (b) radioactivity chromatogram of the [18F]fluorocholine reaction mixture.[18F]Fluorocholine is synthesized by 18F-fluoroalkylation of N,N-dimethylaminoethanol (DMæ) using gaseous18F-fluorobromomethane. This labelling reaction results in high levels of residual DMæ. Other potential byproducts such as bromocholine (notdetected) can additionally be determined. The IC software converts the radiation units, counts per second (cps), to mV. Chromatographic conditions are shown in Table1.

d) Analysis Time

As most positron-emitting radiopharmaceuticals are characterized by short half-lives, there is a strong drive to reduce the time spent on quality control. Fast andprecise analyses are guaranteed by optimally harmonized and computer-controlled determination and rinsing sequences for detector pathways and the sample injection circuit.

Table 1: Chromatographic conditions for the quality control of [18F]fluoride, [18F]FDG and [18F]fluorocholine.

Conclusion

Metrohm's highly customizable chromatography system copes with the tough requirements of the radiopharmaceutical industry and pharmacopoeial regulations. Onesingle multichannel radio IC meets the quality control requirements of various production lines. Besides the high quality, the Metrohm IC presented ensures user safety, lowmaintenance costs and outstanding ruggedness.

Acknowledgements

The authors thank the entire group of the Ion Chromatography Department of Gomensoro for their outstanding support and fruitful discussions during this project.

References

(1) D. Slaets, S. De Bruyne, C. Dumolyn, L. Moerman, K. Mertens and F. De Vos, Reduced dimethylaminoethanol in [¹⁸ F]fluoromethylcholine:an important step towards enhanced tumour visualization, Europ. J. Nucl. Med. Mol. Imaging 37(11), 2136–2145 (2010).

(2) D. Kryza, V. Tadino, M. Azuzurra Filannino, G. Villeret and L. Lemoucheux, Fully automated [¹⁸ F]fluorocholine synthesis in theTracerLab MXFDG Coincidence synthesizer, Nucl. Med. Biol. 35, 255–260 (2008).

(3) J. Passchier, Fast high-performance liquid chromatography in PET quality control and metabolite analysis, Q. J. Nucl. Med. Mol. Imaging 53, 411–416 (2009).

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