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Troubleshooting for F-18 Fluorocholine Synthesis: An Institutional Experience
Address for correspondence: Dr. Rakhee Vatsa, Department of Nuclear Medicine and PET, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012, India. E-mail: rakheevatsa@gmail.com
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This article was originally published by Medknow Publications & Media Pvt Ltd and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Choline is a natural substrate for phospholipid synthesis. F-18 labeled fluorocholine nowadays is routinely used for imaging brain tumors, parathyroid adenoma, and prostate cancer. It is synthesized through nucleophilic substitution reaction using dibromomethane and N, N-dimethylaminoethanol as primary and secondary precursors, respectively. However, sometimes, failures are encountered in F-18 fluorocholine production. Few problems and troubleshooting during synthesis are discussed here.
Keywords
F-18 fluorocholine
failure
synthesis
troubleshooting
Introduction
Choline is a naturally occurring water-soluble vitamin which comes under the class of quaternary ammonium salts.[12] It is used for synthesis of phospholipids in cells. Enhanced cell procreation and increased cell membrane metabolism result in increased choline uptake in tumor cells. C-11 and F-18 labeled choline have been explored for imaging prostate, brain and lung tumors.[34] In the present study, F-18 was produced through 18O(p, n) 18F reaction in an on-site medical cyclotron (PETtrace 4 GE Healthcare). F-18 florocholine (F-18 FCH) was synthesized by nucleophilic substitution reaction as proposed by DeGrado et al.,[5] using cassette-based automated synthesis module Neptis virtual synthesizer®, ORA [Figure 1]. Dibromomethane was used as a primary precursor and N, N-dimethylaminoethanol (DMAE) in dimethyl sulfoxide, preloaded on C-18 cartridge was used as a secondary precursor. Postsynthesis purification was accomplished using ready to use cartridges (silica and CM), based on solid-phase extraction chromatography. The final product (F-18 FCH) was eluted in 0.9% NaCl. Although the synthesis is automated, there are still possibilities of mechanical/manual errors leading to production failure. The difficulties faced during F-18 FCH synthesis are herein reported case wise.
Case Reports
Case 1: Reduced F-18 florocholine – Vacuum leak
The usual yield of F-18 FCH was 10%–15%. During one of the syntheses, the obtained yield was less than usual yield (2%). All the reagents released during the synthesis were in order. During weekly self-tests, the vacuum leak was detected as shown in Figure 2. The leak was traced on the waste bottle connection and was repaired.
Case 2: No F-18 florocholine production – Cartridge malfunction
During another F-18 FCH production, no product was obtained after completion of synthesis. F-18 was observed to be normally trapped and released from QMA on real-time synthesis graph and was delivered efficaciously into the reaction vial. While comparing the postsynthesis reports with the previous successful synthesis reports, it was noticed that there was no flow between 27 and 42 min on flow curve as shown in Figure 3. On the following day, radioactivity was also detected in the silica cartridge (top). Further, when checked with N2 gas, one of the silica cartridges (top) was found to be a defective causing hindrance in the purification of floralbromomethane and subsequently in further reaction with the secondary precursor. The cartridge slot was later on replaced by providers for upcoming synthesis.
Case 3: No production of F-18 florocholine – Reagent malfunction
In another case of failure, error 299 was spotted at 45 min during synthesis. The flow curve showed normal flow during 27–42 min. However, no volume of F-18 FCH was obtained at the end of the synthesis. On observing the cassette on following day, the right syringe in the third manifold of cassette was found to be deformed as shown in Figure 4. The cartridges were checked with N2 gas flow, before the synthesis. However, postsynthesis CM cartridge was found to be blocked. Rest all other cartridges were working well. It was inferred as there may be problem in reaction with DMAE that led the formation of some intermediates that hindered the flow from CM cartridge during the synthesis.
Case 4: No production – Over tightened check valves connecting cartridge
In another case of failure, no volume of F-18 FCH was observed postsynthesis in the mother vial. All presynthesis tests were passed successfully. All the cartridges were in order as tested on N2 gas flow test before synthesis. On observing the cassette next day postsynthesis, leakage was detected in the line connecting C-18 cartridge as shown in Figure 5. Later on, it was noticed that the check valves connecting C-18 cartridge were over tightened restricting the free flow of reaction intermediates and leading to failure of synthesis.
Another production failure was noticed due to overtightened valves connecting C-18 cartridge. The flow meter was destroyed due to backflow of reaction mixture in the nitrogen lines as faulty readings were observed on the flow curve as shown in Figure 6. The flow meter was later on replaced with a new one.
Troubleshooting for F-18 fluorocholine synthesis
Gas flow check
Nitrogen
As nitrogen is the carrier gas for the synthesis, it should be very pure (99.9995 ppm or UHP 5.5). Before synthesis nitrogen flow should be checked by opening high flow (HF) valve in the maintenance mode, because a reduced pressure of nitrogen gas may lead to the leakage or poor output of the product. It is wise to have an external calibrated flow meter to check the nitrogen flow in case of production failure and also during planned maintenance. Change the flow meter of the synthesis module if the flow is less than required or if the analog display of the flow meter is not working properly.
Compressed air
Compressed air is used to open the pneumatic valves of the cassette. Always assure that there is required air pressure for the synthesis module. Check all the necessary pneumatic 3-way actuators (valves) on cassette before using the synthesis module for the hot run. Furthermore, ensure the supply of moisture-free filtered air with a flow rate set as per the machine requirement.
Air filter
Notably assure while placing the purification filter for the nitrogen flow for its expiry and nature (hydrophilic/hydrophobic). This is an important component not only for supplying the sterile nitrogen but also to avoid any backflow of liquid into the flow meter from the faulty distillation accidents.
Size of waste bottle
Always verify that appropriate volume waste bottle is used for collection of waste. A smaller bottle can lead to extra pressure and accidental loosening during the synthesis and vice versa for the larger size bottle (lesser/reduced vacuum throughout the synthesis).
Leak in connector of the waste bottle
Minor leak in the waste bottle may lead to decreased production yield. Always reconfirm that the connectors are well tighten and “O-ring” should always be present inside the lid of the bottle. Leak on the waste bottle can also be present at the back of synthesis module where tubings are connected to the module. To locate the leak, enter a set point of −0.8 bar. Once reached, remove the set point. Pinch the upper waste tubing (which goes to the waste bottle) and check if the vacuum is proper or if the leak is still present. Pinch the lower waste tubing and check if the vacuum is maintained or if the leak is still present.
Silica cartridges block
Due to blockage in silica cartridge, the intermediate product will get struck on the first silica cartridge and it will not be available for further reaction with DMAE on C-18 cartridge leading to production failure. To locate the silica cartridge blockage, check the flow of individual cartridge before synthesis on HF valve. The normal flow rate values are shown in Figure 7. If one of the cartridges has a flow value too low (10% less than the mentioned values), it should be replaced with a new cartridge for smooth run. Similar test should also be performed before using CM and C-18 cartridges to assure they are also working properly.
Blockage of check valves connecting C-18/CM cartridges
Over tightened check valves (connected on the C18/CM cartridges) into the cartridges may obstruct the flow of gases and the liquid through them. This may lead to faulty distillation or no distillation at all. Hence, check valves should always be tightened carefully.
Mechanical glitches
Physically inspect all the tubing and connections for any blockage. Make sure the cassette syringes are properly clamped. Because this may disable the rinsing of the syringes and there will be no volume in the final product vial at all. While loading the chemicals also cross verify their position in the synthesis module.
Other common possible causes of failure
No or partial transfer of F-18 to module can lead to either synthesis failure or less yield. Transfer lines may get clogged after using for long time or leak may also be present in the lines. Before F-18 production, it is always recommended to fill the target with H216O followed by drying of transfer line with helium. The collected volume must be measured to rule out any possibility of obstruction/leakage. This will also help in checking the delivery path to correct hot cell.
No trapping of F-18 in the quaternary methyl ammonium (QMA) anion exchange column is one of the root causes for failed synthesis. Always ensure that the QMA is properly conditioned and dried before installation in the cassette.
No release of F-18 from QMA is another source for the failure of synthesis. As eluent position is slightly out of the way, each time confirm that the eluent is placed at respective position and is pricked properly in the eluent needle. The eluent line should also be pinched during synthesis. Before each run line must be washed with water and dried to rule out any obstruction.
Always observed for the color change of the QMA column resin and recovered O-18 water in the recovery bottle next day of synthesis. There will be a color change from pale yellow to brown if the irradiated O-18 water has added impurities of the target body. This will lead to poor retention/release of the F-18 ions by the QMA column.
Software problem
Sometimes, it is witnessed that even after passing all the tests, the synthesis is failed. Later on, it is observed that some valves did not open during the hot run due to which the reagents are not released at required steps. This may occur due to the problem in software. It is always recommended to perform the self-test for the machine at least once in a month to reassure that all the valves, syringe actuators, vacuum pump, and reactor heater are working well.
In addition to these, always ensure the expiry and storage condition requirement of the chemicals and precursor. Keep a track on the graph values for the pressure, temperature, and radioactivity. This will aid in the easy identification of the possible cause in case of failure/reduced yield.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
The author gratefully acknowledges the support given by Guillaume Villeret for solving the synthesis issues as and when needed.
References
- Choline. 2016. Human Metabolome Database. Edmonton, Canada: The Metabolomics Innovation Centre, University of Alberta; Available from: http://www.hmdb.ca/metabolites/HMDB0000097
- [Google Scholar]
- Institute of Medicine: Food and Nutrition Board. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin. In: Vitamin B6, Folate, Vitamin B12, Pantotheni Acid, Biotin and Choline. Washington, DC: National Academy Press; 1998.
- [Google Scholar]
- Fully automated [18F]fluorocholine synthesis in the tracerLab MX FDG coincidence synthesizer. Nucl Med Biol. 2008;35:255-60.
- [Google Scholar]
- Imaging of prostate cancer with PET/CT using (18)F-fluorocholine. Am J Nucl Med Mol Imaging. 2015;5:96-108.
- [Google Scholar]
- Synthesis and evaluation of (18)F-labeled choline analogs as oncologic PET tracers. J Nucl Med. 2001;42:1805-14.
- [Google Scholar]