Automated Radiosynthesis of [18F]Fluoromannitol for Clinical Research on a Commercially Available Trasis AllinOne Radiosynthesizer

Automated Radiosynthesis of [18F]Fluoromannitol for Clinical Research on a Commercially Available Trasis AllinOne Radiosynthesizer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Automated Radiosynthesis of [ 18 F]Fluoromannitol for Clinical Research on a Commercially Available Trasis AllinOne Radiosynthesizer Amy L. Vavere, Allison J. Clay, Arijit Ghosh, Joana Marie Almazan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7124066/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2025 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted 5 You are reading this latest preprint version Abstract Background : Infections pose a significant risk to immunocompromised individuals, and accurate, efficient diagnosis remain challenging. Current imaging methods like MRI and FDG PET lack pathogen specificity which complicate diagnosis and lead to overuse of antibiotics. Recent data shows that [ 18 F]fluoromannitol ([ 18 F]FMtl) is sensitive and specific to infection in vivo by exploiting the pathogen-specific mannitol transporter. This work aims to establish a reliable, automated method for producing [ 18 F]fluoromannitol to facilitate clinical research studies in human subjects. Results: This study optimized and automated the radiosynthesis of [¹⁸F]fluoromannitol ([¹⁸F]FMtl) on a Trasis AllinOne synthesizer. The 105-minute synthesis achieved an average yield of 11.0% (n=19) with >97% radiochemical purity, and the product remained stable for at least 8 hours. While yield was lower than the previously reported manual method, automation enabled reproducibility and sterility. Process improvements included optimizing evaporation steps and reaction temperature, which significantly increased fluorine incorporation and yield. The process was validated to meet USP regulatory requirements including full QC testing on three consecutive batches. Conclusions : An automated method for the radiochemical synthesis of [ 18 F]fluoromannitol was developed and optimized on a commercially available Trasis AllinOne radiosynthesizer. This method allows for the reliable production and global dissemination of [ 18 F]FMtl for use in clinical research trials. Radiosynthesis Automation Radiofluorination Fluoromannitol PET Infection Figures Figure 1 Figure 2 Figure 3 Background Infectious diseases have a major impact on the health and economies of countries worldwide, especially those with lower income. In the United States, the death rate from infection in cancer patients is nearly three times that of the general population (Zheng et al. 2021). In a study of pediatric cancer patients, infection accounted for half of treatment-related mortalities or death not due to progression of their disease (Loeffen et al. 2019). Diagnosis of infection relies on many variables, including clinical symptoms, invasive biopsies, laboratory tests, and pathology, all which can take several days. In the interim, patients are often given unnecessary cycles of antibiotics which the Center of Disease Control and Prevention (CDC) cites as the largest contributor of antibiotic resistance.The rising trend of antimicrobial resistance, compounded by a growing population of immuno-compromised individuals (HIV/AIDS, chemotherapy, organ transplant, diabetes), creates an enormous economic strain on the US healthcare system. Cost estimates range from $28-$45B annually, calling for an improvement in infectious disease management (Stone et al. 2009). Immunocompromised individuals (e.g. cancer treatment, HIV/AIDS, organ transplant, diabetes) are at increased susceptibility to opportunistic infectious diseases. In fact, infection is one of the most common complications of cancer and cancer treatment (Seo et al. 2020). In addition, these individuals often have atypical symptoms which not only cause issues with diagnosis, but may delay care (McGrath et al. 2020). In a decade-long study in the Netherlands encompassing almost 1800 pediatric cancer patients, infection was responsible for roughly 53% of treatment-related mortality across all diagnoses (hematological, solid tumor, and brain tumor) (Loeffen et al. 2019). A Danish study of 3255 patients over twenty years concluded that infection was the leading cause of treatment-related deaths at 37% (Sorenson et al. 2024). Infection can also cause extreme complications in patients with chronic conditions. Osteomyelitis is a significant complication for patients with sickle cell disease (Al Farii et al. 2020). These patients often also suffer from vaso-occlusive crises that can present in a similar fashion, and there is currently not a test or imaging method to clearly differentiate between the two (Berger et al. 2009; (Scruggs and Pateva et al. 2023). As a result, patients are often overprescribed antibiotics as a safeguard. Imaging can often be used to rule out other potential diagnoses but is not specific to identify infection. Magnetic Resonance Imaging (MRI) is often employed in the evaluation of musculoskeletal concerns, and has a 100% negative predictive value in excluding infection in these tissues (Weaver et al. 2022). However, differentiating infection from the host response is not possible due to many similar radiographic features (Salaffi et al. 2021). Positron emission tomography (PET) offers an imaging modality that follows function with radiolabeled molecules of interest to map processes within the body. The most common clinical PET tracer for imaging infectious diseases is [ 18 F]fluorodeoxyglucose (FDG), which allows interrogation of glucose metabolism over the entire body. FDG, while not specific for infection, has grown to be a valuable tool in the rapid detection of inflammation and infection, even gaining approval by CMS for reimbursement in these cases (Wahl et al. 2021). However, more specific tracers are essential, especially in confounding cases with differential diagnoses. In recent years, a significant amount of preclinical research targeting bacterial-specific metabolic pathways has proven worthwhile to increase specificity of PET tracers for infection (Calabria et al. 2024; (Kahts et al. 2024; (Kleynhans et al. 2023). As a testament to the outstanding need for an imaging agent capable of specifically diagnosing bacterial infection in vivo, many recently developed radiopharmaceuticals seek to exploit various bacteria-specific signatures such as sugar/sugar alcohol metabolism (Gowrishankar et al. 2017; (Kang et al. 2020; (Li et al. 2018; (Ning et al. 2014; (Takemiya et al. 2019; (Weinstein et al. 2014), folic acid biosynthesis (Mutch et al. 2018; (Zhang et al. 2018), D -amino acid metabolism (Neumann et al. 2017; (Parker et al. 2020), and labeled antibiotics (Gowrishankar et al. 2017; (Sellmeyer et al. 2017; (van Oosten et al. 2013; (Zhang et al. 2016) in both normal and infected cohorts (Britton et al. 2002; (Ordonez et al. 2020; (Sellmeyer et al. 2017; (Tucker et al. 2018). Despite these scientific advances, there is a persistent and dire need for imaging agents that meet the challenges of clinical infectious diseases practice. Recently, [ 18 F]fluoromannitol was shown to be highly specific for detection bacterial infection in vivo exploiting mannitol transport ( mtl A) not utilized by mammals. [ 18 F]FMtl successfully distinguished inflammation from bacterial infection caused by S. aureus , E. coli and A. baumannii in multiple models of infection. [ 18 F]FMtl also demonstrated the ability to monitor antibiotic efficacy of vancomycin treatment for S. aureus infections in vivo. Based on these promising results, [ 18 F]FMtl is being translated for clinical trials. As such, the goal of this work was to implement a routine, reproducible, and automated method for the production of [ 18 F]fluoromannitol for clinical research studies. We sought to achieve this by using a Trasis AllinOne radiosynthesizer to result in sufficient activity yields to support clinical trials and a production time of less than two hours using a single-use manifold. We present here an optimized and validated radiosynthesis method in accordance with the guidelines of USP , including quality control results from three consecutive successful syntheses as part of an investigational new drug (IND, #174021) application submission to the FDA. Methods General All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise noted. All aqueous solutions were prepared with ultrapure water (Milli-Q Integral Water Purification System, Millipore Corp.; 18.2 ΜΩ·cm resistivity) or sterile water for injection. The radiochemical precursor 4,6-O-Benzylidene-3-O-ethoxymethyl-2-O-trifluoromethanesulfonyl-1-O-methyl-β-D-glucopyranoside was obtained from ABX (Radeberg, Germany) and used as supplied. The [ 18 F]fluoromannitol ([ 18 F]FMtl) synthesis method described was developed and optimized using an automated Trasis AllinOne radiosynthesizer and three, 6-port research cassettes custom assembled in series with additional tubing, syringes, and vials (see Figure 1). The assembly shown was used for a single production. The final product line attached to the outlet of the manifold was cleaned and sterilized by rinsing with at least 10 mL sterile water, at least 10 mL sterile 70% ethanol, and then dried with nitrogen prior to each synthesis. All radioactive work was accomplished in a Comecer hot cell. The 18 F-labeled materials were measured for activity using a Capintec CRC-15PET dose calibrator. Analytical HPLC was performed on a 1200 Series Agilent LC system using diode array detection, evaporative light scattering detection, and a Bioscan Flow-Count radionuclide detector (Eckert & Ziegler - Bioscan). Thin layer chromatography was assessed for activity using a Bioscan/Eckert & Ziegler AR2000 counter. Analysis for volatile organic impurities by gas chromatography was carried out using an Agilent model 8890 GC system. Both the LC and GC equipment were controlled by Agilent’s OpenLab CDS ChemStation software. Radiosynthesis of [ 18 F]Fluoromannitol Briefly, [ 18 F]FMtl was produced by fluorination of a benzylidene and ethoxymethyl-protected precursor followed by deprotection to produce [ 18 ]fluoromannose. This was purified, reduced and the final [ 18 F]fluoromannitol was cartridge-purified following the work of Simpson et al. (Simpson et al. 2022) shown in Figure 2. Aqueous [ 18 F]fluoride, produced by the 18 O(p,n) 18 F nuclear reaction in an IBA Cyclone ® 18/9 cyclotron from 18 O-enriched water, was transferred from the target to a v-vial in a dose calibrator within the hot cell using argon flow (99.9999% high purity – Nexair). After measuring the activity value, the [ 18 F]fluoride solution was passed through a QMA cartridge (Myja Scientific) to trap the [ 18 F]fluoride and separate it from the [ 18 O]water. The [ 18 F]fluoride was then eluted from the QMA cartridge into the first reactor with a 1 mL solution of 9:1 acetonitrile-water containing 1 mg of potassium carbonate and 6 mg of Kryptofix (K222). The solvents were removed under nitrogen flow with reduced pressure and heating at 90°C for 4 minutes. A second azeotropic evaporation was performed by addition of 1 mL of acetonitrile followed by solvent removal under nitrogen flow with reduced pressure and heating at 90°C for 6 minutes. Anhydrous acetonitrile (1 mL) containing 3 ± 0.3 mg of 4,6-O-Benzylidene-3-O-ethoxymethyl-2-O-trifluoromethanesulfonyl-1-O-methyl-β-D-glucopyranoside was added to the reactor vessel containing the dried [ 18 F]KF. The vial was sealed and heated to 120°C for 20 min. After heating, the solvent was removed under reduced pressure with nitrogen flow at 120°C for 5 minutes. After the solvent was removed, 1 mL of 2.5 M hydrochloric acid was added to the reactor vial for deprotection, and the solution was heated to 150°C for 10 minutes to form the [ 18 F]fluoromannose intermediate. The reactor vial was removed from heating and cooled for 1 minute. Water was added to the reactor (2 mL), and the aqueous intermediate solution was passed through a cartridge purification system consisting of an Accell Plus CM plus short cartridge, followed by 3 g of AG ® 11 A8 resin [custom packed in a Phenomenex Strata C18-U (55 μm, 70 Å) cartridge], and a Sep-Pak Alumina N plus long cartridge all connected in series (previously conditioned with 20-30 mL of water per cartridge) and eluted to waste. An additional 6 mL of water was passed through the cartridge system and collected in a second reactor preloaded with 8 mg of sodium borohydride in 2 mL water. The reactor was sealed, and the aqueous solution was heated to 60°C for 30 minutes. The reactor vial was removed from the heating source and allowed to cool for 30 seconds. The aqueous solution was transferred from the second reactor vial through a cartridge purification system consisting of a Chromabond Set V (previously conditioned with 30 mL of water), followed by a SCX cartridge (previously conditioned with 10 mL of water), and QMA plus cartridge (previously conditioned with 3 mL 1M sodium bicarbonate and 5 mL of water) all connected in a series and, finally, through a sterilizing filter (0.22 μm) into the sterile final product vial assembly. Lastly, 2 mL of water was passed through the cartridge series and collected into the final product vial for a final volume of 10 mL. Quality Control Testing & Validation Several quality control tests were performed on the [ 18 F]FMtl product to assure the quality of the final dose in accordance with guidelines from USP Radiopharmaceuticals for Positron Emission Tomography-Compounding. The final product was confirmed to be clear and colorless with no visual evidence of cloudiness or particulate matter per USP Color and Achromicity. Due to the nature of this product and synthesis intermediates, a single analytical method cannot be used to quantify potential impurities, as some of the compounds interact with a C18 HPLC column, and some require a specialized ion exchange column specifically designed for sugars. For analysis with a standard C18 HPLC column, we used an Agilent Zorbax Eclipse XDB-C18 (4.6 x 150 mm) with an isocratic solvent system of 70% acetonitrile and 30% water at 1.0 mL/min. This allowed elution of precursor, partially deprotected precursor ((+)-(4,6-O-Benzylidene)methyl-α-D-glucopyranoside), benzaldehyde, and benzyl alcohol. For analysis of sugars and sugar-like impurities, we employed an evaporative light scattering detector (ELSD) to visualize compounds that do not contain chromophores for standard UV detection. We were able to get a reproducible signal for our standards using a HILIC ion exchange column (Shodex Asahipak NH2P-50 4E; 250 x 4.6 mm) in 85% acetonitrile/15% water solvent at a flow rate of 1 mL/min. The ELSD evaporator temperature was 90°C and the nebulizer temperature was 50°C with a gas flow rate of 1.20 SLM. To determine radiochemical identity, the HPLC ELSD peak of the fluoromannitol standard in the coinject solution was compared to the activity peak measured by the radioactive detector (see Supplementary Information for example). To determine radiochemical purity, the area of the radioactivity peak corresponding to [ 18 F]Fluoromannitol in the final product formulation represented not less than 90% of the total activity measured across the chromatogram for all other detected activity peaks. Three injections of a standard solution were performed on the same day, and the variation in area between injections was observed as verification of proper functioning of the HPLC instrument (system suitability). All standard areas had a relative standard deviation of ±10%. The ELSD spectrum of the final product formulation was used to estimate potential impurities. Since water has a spectrum on ELSD under these conditions, a water standard was injected. The sum of all ELSD peaks from the water standard were subtracted from the sum of all ELSD peaks in the final formulation. Any remaining area was compared to the average area of the three fluorodeoxymannose standards performed for system suitability to calculate an estimated mass of impurities. To quantify the volatile organic impurities/residual solvents in the final sample, a small aliquot of the [ 18 F]FMtl (0.5 µL) was analyzed by gas chromatography using a DB-WAX column (J & W 122-7032, 30 m x 250 µm x 0.25 µm). The oven temperature was set to 40°C for 1 minute, then ramped to 70°C at a rate of 20°C/min and held for 1 minute, then ramped to 80°C. The total run time was 3 minutes, and the front inlet heater was set to 250°C. The split ratio was 15:1, hydrogen flow was 40 mL/min, and the air flow was 400 mL/min. The GC peak retention times and areas were compared to standards of methanol (0.03%; RT ~ 1.68 min), ethanol (0.1%; RT ~ 1.88 min) and acetonitrile (0.03%; RT ~ 2.24 min). The amount of each component was calculated based on the ratio of peak areas for the samples vs. the standard. Residual Kryptofix (K222) was determined by color spot test on silica based on the method by Mock et al. (Mock et al. 1997). The FDA has proposed a maximum permissible level of 50 µg/mL of Kryptofix ® 222 in 2-[ 18 F]FDG, therefore we believe this maximum permissible level is appropriate for the [ 18 F]FMtl final product. A strip of plastic-backed silica TLC plate was prepared by soaking in an acidic iodoplatinate solution and dried. 2 µL each of final product, water, and 50 μg/mL K222 standard was spotted on the iodoplatinated test strip. Kryptofix ® 222 in the standard will stain the center of the spot a dark color with a blue ring while the water standard will remain light in the center. The product was compared to the standard to confirm that the color change is lighter than the standard limit of 50 μg/mL. In addition, the pH of the final formulation of [ 18 F]FMtl was tested by spotting on a narrow range pH indicator strip and comparing to the color range chart provided by the manufacturer. The final drug product was tested for the presence of bacterial endotoxins utilizing the Endosafe®-PTS™ unit (Charles River) to determine the endotoxin concentration in a sample. Sterility was tested using the direct inoculation method into trypticase soy broth and fluid thioglycolate media as recommended by the USP, and the samples were observed over 14 days. The radionuclidic identity of the final product was determined by observing the radioactive half-life (t ½ ) using a Capintec CRC-15PET dose calibrator with two measurements separated by an interval of 10 minutes. Radionuclidic purity was determined using a Canberra MCA System. Testing took place at least 8 days after production with an acquisition time of 12 hours, ensuring adequate spectral resolution for impurity identification. This approach follows standard industry practices, including USP Radioactivity, which requires sufficient decay time to detect potential long-lived impurities. The assessment was performed by the Cyclotron Facility at the Mallinckrodt Institute of Radiology, Washington University in St. Louis, using gamma spectrometry to compare the emission spectrum against known radionuclide decay schemes and calibration standards. Results Radiosynthesis of [ 18 F]Fluoromannitol The goal of this study was to optimize and automate the synthesis of [ 18 F]FMtl on a Trasis AllinOne radiosynthesizer, which, to our knowledge, has not been reported to-date. We based our synthesis script on the previously published method (Simpson et al. 2022 ) with some modifications and optimizations. Figure 3 shows a schematic representation of the steps in our radiosynthesis and purification of [ 18 F]fluoromannitol. The total synthesis time for [ 18 F]FMtl was approximately 105 minutes and produced product with an EOS average non-decay corrected activity yield of 11.0 ± 2.12% (n = 19). Quality Control Testing & Validation The synthesis routinely yielded product with > 97% radiochemical purity. Once all processes were optimized, three process validation syntheses were conducted to qualify the radiosynthesis for clinical research production and IND submission, and these results are shown in Table 1 . Table 1 Validation production results Test Specification Batch 1 Batch 2 Batch 3 Total activity @ EOS N/A 7733 MBq (209 mCi) 7696 MBq (208 mCi) 4399 MBq (118.9 mCi) Concentration @ EOS N/A 773 MBq/mL (20.9 mCi/mL) 769 MBq/mL (20.8 mCi/mL) 440 MBq/mL 11.9 mCi/mL Visual Appearance Clear, colorless, and free of particulates Pass Pass Pass pH 4.5–7.5 5.0 5.0 5.0 Radiochemical Identity & Purity ≥ 90% [ 18 F]FMtl RT within 15% of std. RCP: 98.23% RT Std: 13.515 min RT Rad: 13.389 min Difference: 0.93% RCP: 97.10% RT Std: 13.213 min RT Rad: 13.061 min Difference: 1.15% RCP: 100.0% RT Std: 13.567 min RT Rad: 13.420 min Difference: 1.08% Impurities (ELSD) ≤ 100 µg impurities /dose Impurities conc.: 23.71 µg/mL Max dose volume: 4.2 mL Impurities conc.: 21.07 µg/mL Max dose volume: 4.8 mL Impurities conc.: 20.96 µg/mL Max dose volume: 4.8 mL Radionuclidic Identity Observed t ½ is 105 to 115 minutes 111.66 min 111.61 min 108.7 min Volatile Organic Impurities Methanol ≤ 0.3% < 0.001% < 0.001% 0.004% Ethanol ≤ 0.5% 0.017% 0.019% 0.008% Acetonitrile ≤ 0.041% < 0.001% < 0.001% < 0.001% Bacterial Endotoxin (LAL) ≤ 175 EU/dose < 2.50 EU/mL < 2.50 EU/mL < 2.50 EU/mL Filter Integrity ≥ 50 psi breaking pressure 60.4 psi 60.0 psi 60.3 psi Sterility no growth observed over 14 days Pass Pass Pass Impurities (UV) ≤ 10 µg/mL < 0.01 µg/mL < 0.01 µg/mL 1.14 µg/mL Radionuclidic Purity ≥ 99.5% 18 F 100% 100% 100% In addition, the product was tested for stability over eight hours. Radiochemical and chemical purity analysis showed no discernable change over that time with all values remaining stable, as well as meeting all QC acceptance criteria at the conclusion of this time. Discussion We based our synthesis protocol on previously published methods (Simpson et al. 2022 ) with some modifications (see Table 2 .) to support automation. Final yield of the optimized synthesis of 11.0% is lower than reported manual synthesis yield of 23%, however the current reported method has the benefit of being fully automated and utilizing disposable, sterile cassettes. Table 2 Optimizations made to synthesis in comparison to previous work Synthesis Step Trasis AiO – Current Work Manual Method (Simpson et al. 2022 ) Additional azeotropic distillation 1–1 mL MeCN 2–1 mL MeCN Acid deprotection 150°C 140°C Intermediate transfer - 2 mL added to HCl to transfer to cartridge purification - No 0.45 µm filter - HCl solution passed directly through cartridge purification - 0.45 filter post purification Reduction - 8 mg NaBH 4 - No stirring - 5.5 mg NaBH 4 - Stirring Final purification Chromabond Set V cartridge + SCX cartridge + QMA cartridge Chromabond Set V cartridge During our optimization testing, we assessed whether the two additional azeotropic evaporations performed in the manual synthesis would result in higher initial fluorination on this system. In results not shown, we achieved a 25% increase in fluorine incorporation by adding a second azeotropic evaporation, but that benefit was lost with a third azeotropic evaporation. As a result, we added one additional evaporation. Preliminary optimization to reaction temperatures showed a doubling of overall activity yield by increasing the deprotection temperature from 140°C to 150°C (4.7 to 9.6%, n = 3–6). The reactor seal was compromised by raising the temperature to 160°C. Addition of a dose calibrator to measure exact F-18 starting activity was essential to allow an accurate determination of activity yield, which confirmed that we were previously underestimating. On a couple of occasions, we observed unexplainable, low trapping of F-18 on the QMA cartridge at the initial transfer to the radiosynthesizer resulting in a lower yield, however, final product concentration still allowed for patient dose preparation. We opted not to include molar activity determination as previous work shows very low mass of fluoromannitol. In those studies, the measured mass concentration of fluoromannitol in the final product was 7.31 ± 0.25 µg/mL (n = 14). (Simpson et al. 2022 ) This meets the FDA microdose definition and is suitable for clinical research studies. D -mannitol is non-toxic to humans and the agent is FDA-approved for intravenous administration of concentrations as high as 25 g/100 mL of water, more than 34,000 times the mass reported in the final formulation of the radiopharmaceutical. It should be noted that radiochemical purity and identity as well as chemical purity quality control testing of the final formulation of [ 18 F]fluoromannitol are complicated by the fact that mannitol and fluoromannitol do not absorb UV light and are not retained on typical reversed-phase HPLC columns. Unfortunately, mannitol and fluoromannitol elute with the solvent front on these columns and therefore no separation of the product from [ 18 F]F - is possible to determine radiochemical purity. While other detector options are available, such as refractive index and evaporative light scattering detectors, they are not common in radiopharmaceutical labs. In addition, they often suffer from baseline drift, noisy background, and sensitivity to mobile phases making reproducibility challenging. In the context of quality control for release of PET tracers, these options offered many concerns. Initially, we considered a method whereby we confirmed identity via TLC analysis by comparing the R f of our drug product (radioactivity detection) to a standard using TLC of copper ion-impregnated silica that allows retention of the fluoromannitol. Visualization of the standard was possible by treating the plate with a common stain (basic potassium permanganate) and heating to develop. For determination of radiochemical purity, we used the more sensitive method of HPLC using an ion chromatography column for sugars and sugar alcohols and used the primary radioactivity peak of the now confirmed fluoromannitol (by TLC). However, quantification of impurities was hindered by our original methods. We ultimately opted to introduce an evaporative light scattering detector (ELSD). During initial analyses, we did observe a significant unknown impurity. To remedy this, we added an additional SCX Sep-Pak to the final purification that successfully eliminated the impurity, but this lowered the pH below acceptable limits. The final QMA cartridge (pretreated with sodium bicarbonate) was added to raise the final product pH to levels required for injection. While analysis by reversed phase chromatography with UV detection is not useful for determining the identity or purity of the final product, we thought it essential to assess for any impurities that could be visualized in this manner and not by the HILIC HPLC with ELSD. There are several purification cartridges in this synthesis, so we did not expect to observe impurities, and this was confirmed through several (n = 25) analyses. In all cases, virtually no evidence of impurity peaks was observed in the final product by elution on standard C18 column and reversed-phase system (see Supplementary Information). With confidence from these tests, we elected not include this analysis in routine quality control testing, however, will perform annually and did include in our validation syntheses. As outlined in FDA’s guidance on solvents in pharmaceutical products, acetonitrile and methanol are Class 2 solvents and are limited to 0.041% for acetonitrile and 0.3% for methanol; acetonitrile is used in this synthesis and methanol is a possible side product. Ethanol is a class 3 solvent and is limited to 0.5%. Ethanol is used to sterilize the radiosynthesis system and is a possible side product. Organic solvents are used by commercial vendors in the preparation of purification substrates and cartridges used in this synthesis; while they remove > 99% of residual solvents, we observed ethanol, methanol, acetone and isopropanol in our product during synthesis optimization. Although in very small quantities, they were confirmed to be due specifically to residual solvents that eluted from these cartridges with the product. This was confirmed by GC analysis of water passed through each cartridge prior to preparation for synthesis compared to solvent standards. Our results showed < 0.01% ethanol and isopropanol on the Alumina N cartridge, < 0.01% methanol and isopropanol on the 11 A8 + C18-U cartridge, and no quantifiable solvent amounts from the Accell CM. The Chromabond cartridge contained trace amounts of acetone, < 0.01% ethanol, and 0.5–1% methanol in our analysis. Additional volume of water rinses in preparation of the purification cartridges eliminated virtually all of the solvents. Conclusions In summary, an automated method for the radiochemical synthesis of [ 18 F]fluoromannitol was developed and optimized on a commercially available Trasis AllinOne radiosynthesizer. This method allows for the reliable production of [ 18 F]FMtl passing USP quality control testing for use in clinical research trials in less than two hours. Abbreviations CMS Centers for Medicare & Medicaid Services ELSD Evaporative light scattering detector EOS End of synthesis FDA Food and Drug Administration FDG Fluorodeoxyglucose FDS Fluorodeoxysorbitol FPV Final product vial FMTL Fluoromannitol H 2 O Water HCl Hydrochloric acid HILIC Hydrophilic interaction liquid chromatography HPLC High performance liquid chromatography IND Investigational New Drug K222 Kryptofix® 2.2.2 K 2 CO 3 Potassium carbonate KF Potassium fluoride LAL Limulus amebocyte lysate M1PDH Mannitol-1-phosphate dehydrogenase MeCN Acetonitrile MRI Magnetic resonance imaging NaBH 4 Sodium borohydride PABA para -aminobenzoic acid PET Positron emission tomography RT Retention time TLC Thin layer chromatography USP United States Pharmacopeia UV Ultra-violet Declarations Supplementary Information The online version contains supplementary material available at: Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no conflict of interest with regard to this study. Funding This study was funded by ALSAC-St. Jude Children’s Research Hospital, NIH/NIBIB R01AI177976-01, and NIH/NIAID R01AI192221-01. Authors’ Contributions ALV, AJC, AG, JMA, MJB, SS, and KDN designed the studies. AJC, AG, JMA, MJB, and SS carried out the experiments. ALV prepared the manuscript. All authors read, edited and approved the final manuscript. Acknowledgements The authors would like to thank the staff of the Molecular Imaging Core @ St. Jude Children’s Research Hospital. Data Availability The data used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Al Farii H, Zhou S, Albers A. Management of Osteomyelitis in Sickle Cell Disease: Review Article. Journal of the American Academy of Orthopaedic Surgeons Global Research Review. 2020;4:e20.00002. Berger E, Saunders N, Wang L. Sickle Cell Disease in Children: Differentiating Osteomyelitis from Vaso-occulisive Crisis. JAMA Pediatrics. 2009;163:251-5. Britton K, Wareham D, Das S, Solanki K, Amaral H, Bhatnagar A, et al. Imaging bacterial infection with 99m Tc-ciprofloxacin (Infecton). Journal of Clinical Pathology. 2002;55:817-23. Calabria F, Gaudagnino G, Cimini A, Leporace M. PET/CT Imaging of Infectious Diseases: Overview of Novel Radiopharmaceuticals. Diagnostics. 2024;14:1-20. Gowrishankar G, Hardy J, Wardak M, Namavari M, Reeves RE, Neofytou E, et al. Specific Imaging of Bacterial Infection Using 6″- 18 F-Fluoromaltotriose: A Second-Generation PET Tracer Targeting the Maltodextrin Transporter in Bacteria. Journal of Nuclear Medicine. 2017;58:1679-84. Kahts M, Summers B, Gutta A, Pilloy W, Ebenhan T. Recently developed radiopharmaceuticals for bacterial infection imaging. EJNMMI Radiopharmacy and Chemistry. 2024;9:1-38. Kang S-R, Jo EJ, Nguyen VH, Zhang Y, Yoon HS, Pyo A, et al. Imaging of tumor colonization by Escherichia coli using 18 F-FDS PET. Theranostics. 2020;10:4958-66. Kleynhans J, Machaba Sathekge M, Ebenhan T. Preclinical Research Highlighting Contemporary Targeting Mechanisms of Radiolabelled Compounds for PET Based Infection Imaging. Seminars in Nuclear Medicine. 2023;53:630-43. Li J, Zheng H, Fodah R, Warawa JW, Ng CK. Validation of 2- 18 F-Fluorodeoxysorbitol as a Potential Radiopharmaceutical for Imaging Bacterial Infection in the Lung. Journal of Nuclear Medicine. 2018;59:134-9. Loeffen EAH, Knops RRG, Boerhof J, Feijen EAML, Merks JHM, Reedijk AMJ, et al. Treatment-related mortality in children with cancer: Prevalence and risk factors. European Journal of Cancer. 2019;121:113-22. McGrath B, Broadhurst M, Roman C. Infectious disease considerations in immunocompromised patients. Journal of the American Academy of Physician Assistants. 2020;33:16-25. Mock BH, Winkle W, Vavrek MT. A color spot test for the detection of Kryptofix 2.2.2 in [ 18 F]FDG preparations. Nuclear Medicine and Biology. 1997;24:193-5. Mutch CA, Ordonez AA, Qin H, Parker M, Bambarger LE, Villanueva-Meyer JE, et al. [ 11 C]Para-Aminobenzoic Acid: A Positron Emission Tomography Tracer Targeting Bacteria-Specific Metabolism. ACS Infectious Diseases. 2018;4:1067-72. Neumann KD, Villanueva-Meyer JE, Mutch CA, Flavell RR, Blecha JE, Kwak T, et al. Imaging Active Infection in vivo Using D-Amino Acid Derived PET Radiotracers. Scientific Reports. 2017;7:7903. Ning X, Seo W, Lee S, Takemiya K, Rafi M, Feng X, et al. PET imaging of bacterial infections with fluorine-18-labeled maltohexaose. Angewandte Chemie International Edition. 2014;53:14096-101. Ordonez AA, Wang H, Magombedze G, Ruiz-Bedoya CA, Srivastava S, Chen A, et al. Dynamic imaging in patients with tuberculosis reveals heterogeneous drug exposures in pulmonary lesions. Nature Medicine. 2020;26:529-34. Parker MF, Luu JM, Schulte B, Huynh TL, Stewart MN, Sriram R, et al. Sensing Living Bacteria in Vivo Using D ‑Alanine-Derived 11 C Radiotracers. 2020;6:155-65. Salaffi F, Ceccarelli L, Carottie M, Di Carlo M, Polonara G, Facchini G, et al. Differentiation between infectious spondylodiscitis versus inflammatory or degenerative spinal changes: How can magnetic resonance imaging help the clinician? La Radiologia Medica. 2021;126:843-59. Scruggs M, Pateva I. Multifocal osteomyelitis in a child with sickle cell disease and review of the literature regarding best diagnostic approach. Clinical Case Reports. 2023;11:e7288. Sellmeyer MA, Lee I, Hou C, Weng C-C, Li S, Lieberman BP, et al. Bacterial infection imaging with [ 18 F]fluoropropyl-trimethoprim. Proceedingd of the National Academy of Sciences of the United States of America. 2017;114:8372-7. Seo SK, Liu C, Dadwal SS. Infectious Disease Complications in Patients with Cancer. Critical Care Clinics. 2020;37:69-84. Simpson SR, Kesterson AE, Wilde JH, Qureshi Z, Kundu BK, Simons MP, et al. Imaging Diverse Pathogenic Bacteria in vivo with [18F]fluoromannitol Positron Emission Tomography. The Journal of Nuclear Medicine. 2022;64:809-15. Sorenson MCL, Andersen MM, Rostgaard K, Schmiegelow K, Mikkelsen TS, Wehner PS, et al. Treatment-related mortality among children with cancer in Denmark during 2001-2021. Acta Oncologica. 2024;63:294-302. Stone P. Economic burden of healthcare-associated infections: an American perspective. Expert Review of Pharmaeconomics & Outcomes Research. 2009;9:417-22. Takemiya K, Ning X, Seo W, Wang X, Mohammad R, Joseph G, et al. Novel PET and near infrared imaging probes for the specific detection of bacterial infections associated with cardiac devices. JACC Cardiovasc Imaging. 2019;12:875-86. Tucker EW, Guglieri-Lopez B, Ordonez AA, Ritchie B, Klunk MH, Sharma R, et al. Noninvasive 11 C-rifampin positron emission tomography reveals drug biodistribution in tuberculous meningitis. Science Translational Medicine. 2018;10:470. van Oosten M, Schaefer T, Gazendam JA, Ohlsen K, Tsompanidou E, de Goffau MC, et al. Real-time in vivo imaging of invasive- and biomaterial-associated bacterial infections using fluorescently labelled vancomycin. Nature Communications. 2013;4:2584. Wahl RL, Disizian V, Pelstro CJ. At Last, 18 F-FDG for Inflammation and Infection! The Journal of Nuclear Medicine. 2021;62:1048-9. Weaver JS, Omar IM, Mar WA, Klauser AS, Winegar BA, Mlady GW, et al. Magnetic resonanace imaging of musculoskeletal infections. Polish Journal of Radiology. 2022;87:e141-e62. Weinstein EA, Ordonez AA, DeMarco VP, Murawski AM, Pokkali S, MacDonald EM, et al. Imaging Enterobacteriaceae infection in vivo with 18F-fluorodeoxysorbitol positron emission tomography. Science Translational Medicine. 2014;6:259ra146. Zhang XM, Zhang HH, McLeroth P, Berkowitz RD, Mont MA, Stabin MG, et al. [ 124 I]FIAU: Human dosimetry and infection imaging in patients with suspected prosthetic joint infection. Nuclear Medicine and Biology. 2016;43:273-9. Zhang Z, Ordonez AA, Wang H, Li Y, Gogarty KR, Weinstein EA, et al. Positron Emission Tomography Imaging with 2-[ 18 F]F-p-Aminobenzoic Acid Detects Staphylococcus aureus Infections and Monitors Drug Response. ACS Infectious Diseases. 2018;4:1635-44. Zheng Y, Chen Y, Yu K, Yang Y, Wang X, Yang X, et al. Fatal Infections Among Cancer Patients: A Population-Based Study in the United States. Infectious Diseases and Therapy. 2021;10:871-95. Cite Share Download PDF Status: Published Journal Publication published 28 Oct, 2025 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted Editorial decision: Major revision 08 Aug, 2025 Reviewers agreed at journal 28 Jul, 2025 Reviewers invited by journal 28 Jul, 2025 Editor assigned by journal 21 Jul, 2025 First submitted to journal 18 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7124066","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":491900461,"identity":"6b5a2c7d-7159-417c-8894-560b029e1c67","order_by":0,"name":"Amy L. Vavere","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBACNiBm5gEzmQ9//mNzuM6+gfn4hx94tPAjtLClSSTwHE42ADIke/BokWyAa+ExYwBqSdwAZEjjc5jBtTMGjDNqDsuZt/d8I1LL7RyglmOHjWXOnN38AaRlPwOP+WfCWtjSEmdI5G6QgNpiflgGjxZ7oBaGD//S6mfIv3kA02KWzEPAFoaPbTYJEhI8DBIJPRAtxvi1pBUcSOyzMZzBk2YmkXAjLdmAmUAgG9xO3vgg4ZuEvAT74ccfEgps6uzbm49J4ItKBgYOgwOoAsx4lYMA+wOCSkbBKBgFo2CEAwBkw1KBnQMK4gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0008-2011-8790","institution":"St Jude Children's Research Hospital","correspondingAuthor":true,"prefix":"","firstName":"Amy","middleName":"L.","lastName":"Vavere","suffix":""},{"id":491900462,"identity":"0cfce83f-43bb-4658-acd4-d49f73627f97","order_by":1,"name":"Allison J. Clay","email":"","orcid":"","institution":"St Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Allison","middleName":"J.","lastName":"Clay","suffix":""},{"id":491900463,"identity":"cda1042d-9f6d-4132-bd6d-bf3fdd6dce3e","order_by":2,"name":"Arijit Ghosh","email":"","orcid":"","institution":"St Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Arijit","middleName":"","lastName":"Ghosh","suffix":""},{"id":491900464,"identity":"003f98c9-4e0b-42c9-b272-91f6ac1a6c9f","order_by":3,"name":"Joana Marie Almazan","email":"","orcid":"","institution":"St Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Joana","middleName":"Marie","lastName":"Almazan","suffix":""},{"id":491900465,"identity":"c8efc4ad-4e64-41c7-85a4-d2f78ade6a34","order_by":4,"name":"Melissa J. Brown","email":"","orcid":"","institution":"St Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Melissa","middleName":"J.","lastName":"Brown","suffix":""},{"id":491900466,"identity":"a7588f78-fe95-4a34-a59e-d8521caaced2","order_by":5,"name":"Spenser Simpson","email":"","orcid":"","institution":"St Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Spenser","middleName":"","lastName":"Simpson","suffix":""},{"id":491900467,"identity":"9780d2f7-3bc9-4238-a13e-6bfbe79a5491","order_by":6,"name":"Kiel D. Neumann","email":"","orcid":"","institution":"St Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kiel","middleName":"D.","lastName":"Neumann","suffix":""}],"badges":[],"createdAt":"2025-07-14 19:36:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7124066/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7124066/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41181-025-00388-x","type":"published","date":"2025-10-28T15:58:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88093476,"identity":"1ee104ba-1b50-4c0f-b8b3-ee0a9a6ad21a","added_by":"auto","created_at":"2025-08-01 10:39:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":507940,"visible":true,"origin":"","legend":"\u003cp\u003eCustom manifold layout for Trasis AllinOne synthesis of [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7124066/v1/5cfec77b738db0f3615b003c.png"},{"id":88093477,"identity":"79b60780-bb0c-4ad1-920f-9e84174525fd","added_by":"auto","created_at":"2025-08-01 10:39:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98517,"visible":true,"origin":"","legend":"\u003cp\u003eRadiosynthesis scheme of [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7124066/v1/7cacacbbb8e29870c036d289.png"},{"id":88094342,"identity":"0708cfe2-1951-43e5-90d5-0f1b8ad12b2c","added_by":"auto","created_at":"2025-08-01 10:48:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":809232,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the steps in the preparation of [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7124066/v1/711573ae0a512e9cc75d7eee.png"},{"id":95040684,"identity":"dbe5e7f5-c347-481a-9ce2-d5d256cd2409","added_by":"auto","created_at":"2025-11-03 16:10:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2602727,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7124066/v1/159fae6d-eac0-4483-8d3d-bd3b32e5c26d.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAutomated Radiosynthesis of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]Fluoromannitol for Clinical Research on a Commercially Available Trasis AllinOne Radiosynthesizer\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eInfectious diseases have a major impact on the health and economies of countries worldwide, especially those with lower income. In the United States, the death rate from infection in cancer patients is nearly three times that of the general population\u0026nbsp;(Zheng et al. 2021). In a study of pediatric cancer patients, infection accounted for half of treatment-related mortalities or death not due to progression of their disease\u0026nbsp;(Loeffen et al. 2019). Diagnosis of infection relies on many variables, including clinical symptoms, invasive biopsies, laboratory tests, and pathology, all which can take several days. In the interim, patients are often given unnecessary cycles of antibiotics which the Center of Disease Control and Prevention (CDC) cites as the largest contributor of antibiotic resistance.The rising trend of antimicrobial resistance, compounded by a growing population of immuno-compromised individuals (HIV/AIDS, chemotherapy, organ transplant, diabetes), creates an enormous economic strain on the US healthcare system. Cost estimates range from $28-$45B annually, calling for an improvement in infectious disease management\u0026nbsp;(Stone et al. 2009).\u003c/p\u003e\n\u003cp\u003eImmunocompromised individuals (e.g. cancer treatment, HIV/AIDS, organ transplant, diabetes) are at \u0026nbsp; increased susceptibility to opportunistic infectious diseases. In fact, infection is one of the most common complications of cancer and cancer treatment\u0026nbsp;(Seo et al. 2020). In addition, these individuals often have atypical symptoms which not only cause issues with diagnosis, but may delay care\u0026nbsp;(McGrath et al. 2020). In a decade-long study in the Netherlands encompassing almost 1800 pediatric cancer patients, infection was responsible for roughly 53% of treatment-related mortality across all diagnoses (hematological, solid tumor, and brain tumor)\u0026nbsp;(Loeffen et al. 2019). A Danish study of 3255 patients over twenty years concluded that infection was the leading cause of treatment-related deaths at 37%\u0026nbsp;(Sorenson et al. 2024).\u003c/p\u003e\n\u003cp\u003eInfection can also cause extreme complications in patients with chronic conditions. Osteomyelitis is a significant complication for patients with sickle cell disease\u0026nbsp;(Al Farii et al. 2020). These patients often also suffer from vaso-occlusive crises that can present in a similar fashion, and there is currently not a test or imaging method to clearly differentiate between the two\u0026nbsp;(Berger et al. 2009; (Scruggs and Pateva et al. 2023). As a result, patients are often overprescribed antibiotics as a safeguard. Imaging can often be used to rule out other potential diagnoses but is not specific to identify infection.\u003c/p\u003e\n\u003cp\u003eMagnetic Resonance Imaging (MRI) is often employed in the evaluation of musculoskeletal concerns, and has a 100% negative predictive value in excluding infection in these tissues\u0026nbsp;(Weaver et al. 2022). However, differentiating infection from the host response is not possible due to many similar radiographic features\u0026nbsp;(Salaffi et al. 2021). Positron emission tomography (PET) offers an imaging modality that follows function with radiolabeled molecules of interest to map processes within the body. The most common clinical PET tracer for imaging infectious diseases is [\u003csup\u003e18\u003c/sup\u003eF]fluorodeoxyglucose (FDG), which allows interrogation of glucose metabolism over the entire body. FDG, while not specific for infection, has grown to be a valuable tool in the rapid detection of inflammation and infection, even gaining approval by CMS for reimbursement in these cases\u0026nbsp;(Wahl et al. 2021). However, more specific tracers are essential, especially in confounding cases with differential diagnoses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn recent years, a significant amount of preclinical research targeting bacterial-specific metabolic pathways has proven worthwhile to increase specificity of PET tracers for infection\u0026nbsp;(Calabria et al. 2024; (Kahts et al. 2024; (Kleynhans et al. 2023). As a testament to the outstanding need for an imaging agent capable of specifically diagnosing bacterial infection in vivo, many recently developed radiopharmaceuticals seek to exploit various bacteria-specific signatures such as sugar/sugar alcohol metabolism\u0026nbsp;(Gowrishankar et al. 2017; (Kang et al. 2020; (Li et al. 2018; (Ning et al. 2014; (Takemiya et al. 2019; (Weinstein et al. 2014), folic acid biosynthesis\u0026nbsp;(Mutch et al. 2018; (Zhang et al. 2018), \u003cem\u003eD\u003c/em\u003e-amino acid metabolism\u0026nbsp;(Neumann et al. 2017; (Parker et al. 2020), and labeled antibiotics\u0026nbsp;(Gowrishankar et al. 2017; (Sellmeyer et al. 2017; (van Oosten et al. 2013; (Zhang et al. 2016) in both normal and infected cohorts\u0026nbsp;(Britton et al. 2002; (Ordonez et al. 2020; (Sellmeyer et al. 2017; (Tucker et al. 2018). Despite these scientific advances, there is a persistent and dire need for imaging agents that meet the challenges of clinical infectious diseases practice.\u003c/p\u003e\n\u003cp\u003eRecently, [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol was shown to be highly specific for detection bacterial infection in vivo exploiting mannitol transport (\u003cem\u003emtl\u003c/em\u003eA) not utilized by mammals. [\u003csup\u003e18\u003c/sup\u003eF]FMtl successfully distinguished inflammation from bacterial infection caused by \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e in multiple models of infection. [\u003csup\u003e18\u003c/sup\u003eF]FMtl also demonstrated the ability to monitor antibiotic efficacy of vancomycin treatment for \u003cem\u003eS. aureus\u003c/em\u003e infections in vivo.\u003c/p\u003e\n\u003cp\u003eBased on these promising results, \u0026nbsp;[\u003csup\u003e18\u003c/sup\u003eF]FMtl is being translated for clinical trials. As such, the goal of this work was to implement a routine, reproducible, and automated method for the production of [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol for clinical research studies. We sought to achieve this by using a Trasis AllinOne radiosynthesizer to result in sufficient activity yields to support clinical trials and a production time of less than two hours using a single-use manifold. We present here an optimized and validated radiosynthesis method in accordance with the guidelines of USP \u0026lt;823\u0026gt;, including quality control results from three consecutive successful syntheses as part of an investigational new drug (IND, #174021) application submission to the FDA.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eGeneral\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise noted. All aqueous solutions were prepared with ultrapure water (Milli-Q Integral Water Purification System, Millipore Corp.; 18.2 \u0026Mu;Ω\u0026middot;cm resistivity) or sterile water for injection. The radiochemical precursor 4,6-O-Benzylidene-3-O-ethoxymethyl-2-O-trifluoromethanesulfonyl-1-O-methyl-\u0026beta;-D-glucopyranoside was obtained from ABX (Radeberg, Germany) and used as supplied.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol ([\u003csup\u003e18\u003c/sup\u003eF]FMtl) synthesis method described was developed and optimized using an automated Trasis AllinOne radiosynthesizer and three, 6-port research cassettes custom assembled in series with additional tubing, syringes, and vials (see Figure 1). The assembly shown was used for a single production.\u003c/p\u003e\n\u003cp\u003eThe final product line attached to the outlet of the manifold was cleaned and sterilized by rinsing with at least 10 mL sterile water, at least 10 mL sterile 70% ethanol, and then dried with nitrogen prior to each synthesis. All radioactive work was accomplished in a Comecer hot cell. The \u003csup\u003e18\u003c/sup\u003eF-labeled materials were measured for activity using a Capintec CRC-15PET dose calibrator. Analytical HPLC was performed on a 1200 Series Agilent LC system using diode array detection, evaporative light scattering detection, and a Bioscan Flow-Count radionuclide detector (Eckert \u0026amp; Ziegler - Bioscan). Thin layer chromatography was assessed for activity using a Bioscan/Eckert \u0026amp; Ziegler AR2000 counter. Analysis for volatile organic impurities by gas chromatography was carried out using an Agilent model 8890 GC system. Both the LC and GC equipment were controlled by Agilent\u0026rsquo;s OpenLab CDS ChemStation software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRadiosynthesis of [\u003csup\u003e18\u003c/sup\u003eF]Fluoromannitol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBriefly, [\u003csup\u003e18\u003c/sup\u003eF]FMtl was produced by fluorination of a benzylidene and ethoxymethyl-protected precursor followed by deprotection to produce [\u003csup\u003e18\u003c/sup\u003e]fluoromannose. This was purified, reduced and the final [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol was cartridge-purified following the work of Simpson et al. (Simpson et al. 2022) shown in Figure 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAqueous [\u003csup\u003e18\u003c/sup\u003eF]fluoride, produced by the \u003csup\u003e18\u003c/sup\u003eO(p,n)\u003csup\u003e18\u003c/sup\u003eF nuclear reaction in an IBA Cyclone\u003csup\u003e\u0026reg;\u003c/sup\u003e 18/9 cyclotron from \u003csup\u003e18\u003c/sup\u003eO-enriched water, was transferred from the target to a v-vial in a dose calibrator within the hot cell using argon flow (99.9999% high purity \u0026ndash; Nexair). After measuring the activity value, the [\u003csup\u003e18\u003c/sup\u003eF]fluoride solution was passed through a QMA cartridge (Myja Scientific) to trap the [\u003csup\u003e18\u003c/sup\u003eF]fluoride and separate it from the [\u003csup\u003e18\u003c/sup\u003eO]water.\u0026nbsp;The [\u003csup\u003e18\u003c/sup\u003eF]fluoride was then eluted from the QMA cartridge into the first reactor with a 1 mL solution of 9:1 acetonitrile-water containing 1 mg of potassium carbonate and 6 mg of Kryptofix (K222). The solvents were removed under nitrogen flow with reduced pressure and heating at 90\u0026deg;C for 4 minutes. A second azeotropic evaporation was performed by addition of 1 mL of acetonitrile followed by solvent removal under nitrogen flow with reduced pressure and heating at 90\u0026deg;C for 6 minutes.\u003c/p\u003e\n\u003cp\u003eAnhydrous acetonitrile (1 mL) containing 3 \u0026plusmn; 0.3 mg of 4,6-O-Benzylidene-3-O-ethoxymethyl-2-O-trifluoromethanesulfonyl-1-O-methyl-\u0026beta;-D-glucopyranoside was added to the reactor vessel containing the dried [\u003csup\u003e18\u003c/sup\u003eF]KF. The vial was sealed and heated to 120\u0026deg;C for 20 min. After heating, the solvent was removed under reduced pressure with nitrogen flow at 120\u0026deg;C for 5 minutes. After the solvent was removed, 1 mL of 2.5 M hydrochloric acid was added to the reactor vial for deprotection, and the solution was heated to 150\u0026deg;C for 10 minutes to form the [\u003csup\u003e18\u003c/sup\u003eF]fluoromannose intermediate. The reactor vial was removed from heating and cooled for 1 minute.\u003c/p\u003e\n\u003cp\u003eWater was added to the reactor (2 mL), and the aqueous intermediate solution was passed through a cartridge purification system consisting of an Accell Plus CM plus short cartridge, followed by 3 g of AG\u003csup\u003e\u0026reg;\u003c/sup\u003e 11 A8 resin [custom packed in a Phenomenex Strata C18-U (55 \u0026mu;m, 70 \u0026Aring;) cartridge], and a Sep-Pak Alumina N plus long cartridge all connected in series (previously conditioned with 20-30 mL of water per cartridge) and eluted to waste. An additional 6 mL of water was passed through the cartridge system and collected in a second reactor preloaded with 8 mg of sodium borohydride in 2 mL water. The reactor was sealed, and the aqueous solution was heated to 60\u0026deg;C for 30 minutes. The reactor vial was removed from the heating source and allowed to cool for 30 seconds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe aqueous solution was transferred from the second reactor vial through a cartridge purification system consisting of a Chromabond Set V (previously conditioned with 30 mL of water), followed by a SCX cartridge (previously conditioned with 10 mL of water), and QMA plus cartridge (previously conditioned with 3 mL 1M sodium bicarbonate and 5 mL of water) all connected in a series and, finally, through a sterilizing filter (0.22 \u0026mu;m) into the sterile final product vial assembly. Lastly, 2 mL of water was passed through the cartridge series and collected into the final product vial for a final volume of 10 mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuality Control Testing \u0026amp; Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral quality control tests were performed on the [\u003csup\u003e18\u003c/sup\u003eF]FMtl product to assure the quality of the final dose in accordance with guidelines from USP \u0026lt;823\u0026gt; Radiopharmaceuticals for Positron Emission Tomography-Compounding. The final product was confirmed to be clear and colorless with no visual evidence of cloudiness or particulate matter per USP \u0026lt;631\u0026gt; Color and Achromicity.\u003c/p\u003e\n\u003cp\u003eDue to the nature of this product and synthesis intermediates, a single analytical method cannot be used to quantify potential impurities, as some of the compounds interact with a C18 HPLC column, and some require a specialized ion exchange column specifically designed for sugars. For analysis with a standard C18 HPLC column, we used an Agilent Zorbax Eclipse XDB-C18 \u0026nbsp;(4.6 x 150 mm) with an isocratic solvent system of 70% acetonitrile and 30% water at 1.0 mL/min. This allowed elution of precursor, partially deprotected precursor ((+)-(4,6-O-Benzylidene)methyl-\u0026alpha;-D-glucopyranoside), benzaldehyde, and benzyl alcohol. For analysis of sugars and sugar-like impurities, we employed an evaporative light scattering detector (ELSD) to visualize compounds that do not contain chromophores for standard UV detection. We were able to get a reproducible signal for our standards using a HILIC ion exchange column (Shodex Asahipak NH2P-50 4E; 250 x 4.6 mm) in 85% acetonitrile/15% water solvent at a flow rate of 1 mL/min. The ELSD evaporator temperature was 90\u0026deg;C and the nebulizer temperature was 50\u0026deg;C with a gas flow rate of 1.20 SLM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine radiochemical identity, the HPLC ELSD peak of the fluoromannitol standard in the coinject solution was compared to the activity peak measured by the radioactive detector (see Supplementary Information for example). To determine radiochemical purity, the area of the radioactivity peak corresponding to [\u003csup\u003e18\u003c/sup\u003eF]Fluoromannitol in the final product formulation represented not less than 90% of the total activity measured across the chromatogram for all other detected activity peaks. Three injections of a standard solution were performed on the same day, and the variation in area between injections was observed as verification of proper functioning of the HPLC instrument (system suitability). All standard areas had a relative standard deviation of \u0026plusmn;10%. The ELSD spectrum of the final product formulation was used to estimate potential impurities. Since water has a spectrum on ELSD under these conditions, a water standard was injected. The sum of all ELSD peaks from the water standard were subtracted from the sum of all ELSD peaks in the final formulation. Any remaining area was \u0026nbsp;compared to the average area of the three fluorodeoxymannose standards performed for system suitability to calculate an estimated mass of impurities.\u003c/p\u003e\n\u003cp\u003eTo quantify the volatile organic impurities/residual solvents in the final sample, a small aliquot of the [\u003csup\u003e18\u003c/sup\u003eF]FMtl (0.5 \u0026micro;L) was analyzed by gas chromatography using a DB-WAX column (J \u0026amp; W 122-7032, 30 m x 250 \u0026micro;m x 0.25 \u0026micro;m). The oven temperature was set to 40\u0026deg;C for 1 minute, then ramped to 70\u0026deg;C at a rate of 20\u0026deg;C/min and held for 1 minute, then ramped to 80\u0026deg;C. The total run time was 3 minutes, and the front inlet heater was set to 250\u0026deg;C. The split ratio was 15:1, hydrogen flow was 40 mL/min, and the air flow was 400 mL/min. The GC peak retention times and areas were compared to standards of methanol (0.03%; RT ~ 1.68 min), ethanol (0.1%; RT ~ 1.88 min) and acetonitrile (0.03%; RT ~ 2.24 min).\u0026nbsp;The amount of each component was calculated based on the ratio of peak areas for the samples vs. the standard.\u003c/p\u003e\n\u003cp\u003eResidual Kryptofix (K222) was determined by color spot test on silica based on the method by Mock et al.\u0026nbsp;(Mock et al. 1997). The FDA has proposed a maximum permissible level of 50 \u0026micro;g/mL of Kryptofix\u003csup\u003e\u0026reg;\u003c/sup\u003e 222 in 2-[\u003csup\u003e18\u003c/sup\u003eF]FDG, therefore we believe this maximum permissible level is appropriate for the [\u003csup\u003e18\u003c/sup\u003eF]FMtl final product. A strip of plastic-backed silica TLC plate was prepared by soaking in an acidic iodoplatinate solution and dried. 2 \u0026micro;L each of final product, water, and 50 \u0026mu;g/mL K222 standard was spotted on the iodoplatinated test strip. Kryptofix\u003csup\u003e\u0026reg;\u003c/sup\u003e 222 in the standard will stain the center of the spot a dark color with a blue ring while the water standard will remain light in the center. The product was compared to the standard to confirm that the color change is lighter than the standard limit of 50 \u0026mu;g/mL. In addition, the pH of the final formulation of [\u003csup\u003e18\u003c/sup\u003eF]FMtl was tested by spotting on a narrow range pH indicator strip and comparing to the color range chart provided by the manufacturer. The final drug product was tested for the presence of bacterial endotoxins utilizing the Endosafe\u0026reg;-PTS\u0026trade; unit (Charles River) to determine the endotoxin concentration in a sample. Sterility was tested using the direct inoculation method into trypticase soy broth and fluid thioglycolate media as recommended by the USP, and the samples were observed over 14 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe radionuclidic identity of the final product was determined by observing the radioactive half-life (t\u003csub\u003e\u0026frac12;\u003c/sub\u003e) using a Capintec CRC-15PET dose calibrator with two measurements separated by an interval of 10 minutes. Radionuclidic purity was determined using a Canberra MCA System. Testing took place at least 8 days after production with an acquisition time of 12 hours, ensuring adequate spectral resolution for impurity identification. This approach follows standard industry practices, including USP \u0026lt;1821\u0026gt; Radioactivity, which requires sufficient decay time to detect potential long-lived impurities. The assessment was performed by the Cyclotron Facility at the Mallinckrodt Institute of Radiology, Washington University in St. Louis, using gamma spectrometry to compare the emission spectrum against known radionuclide decay schemes and calibration standards.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eRadiosynthesis of [\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF]Fluoromannitol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe goal of this study was to optimize and automate the synthesis of [\u003csup\u003e18\u003c/sup\u003eF]FMtl on a Trasis AllinOne radiosynthesizer, which, to our knowledge, has not been reported to-date. We based our synthesis script on the previously published method (Simpson et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e) with some modifications and optimizations. Figure\u0026nbsp;3 shows a schematic representation of the steps in our radiosynthesis and purification of [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol.\u003c/p\u003e\n\u003cp\u003eThe total synthesis time for [\u003csup\u003e18\u003c/sup\u003eF]FMtl was approximately 105 minutes and produced product with an EOS average non-decay corrected activity yield of 11.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12% (n\u0026thinsp;=\u0026thinsp;19).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuality Control Testing \u0026amp; Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesis routinely yielded product with \u0026gt;\u0026thinsp;97% radiochemical purity. Once all processes were optimized, three process validation syntheses were conducted to qualify the radiosynthesis for clinical research production and IND submission, and these results are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eValidation production results\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTest\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecification\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBatch 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBatch 2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBatch 3\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal activity @ EOS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e7733 MBq\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(209 mCi)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e7696 MBq\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(208 mCi)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4399 MBq\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(118.9 mCi)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration @ EOS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e773 MBq/mL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(20.9 mCi/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e769 MBq/mL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(20.8 mCi/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e440 MBq/mL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e11.9 mCi/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eVisual Appearance\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClear, colorless, and free of particulates\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePass\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePass\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePass\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.5\u0026ndash;7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRadiochemical Identity \u0026amp; Purity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;\u0026thinsp;90% [\u003csup\u003e18\u003c/sup\u003eF]FMtl\u003c/p\u003e\n \u003cp\u003eRT within 15% of std.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRCP: \u003cstrong\u003e98.23%\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eRT Std: \u003cstrong\u003e13.515 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eRT Rad: \u003cstrong\u003e13.389 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eDifference: \u003cstrong\u003e0.93%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRCP: \u003cstrong\u003e97.10%\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eRT Std: \u003cstrong\u003e13.213 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eRT Rad: \u003cstrong\u003e13.061 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eDifference: \u003cstrong\u003e1.15%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRCP: \u003cstrong\u003e100.0%\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eRT Std: \u003cstrong\u003e13.567 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eRT Rad: \u003cstrong\u003e13.420 min\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eDifference: \u003cstrong\u003e1.08%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eImpurities (ELSD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;100 \u0026micro;g impurities /dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eImpurities conc.: \u003cstrong\u003e23.71 \u0026micro;g/mL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMax dose volume:\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4.2 mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eImpurities conc.: \u003cstrong\u003e21.07 \u0026micro;g/mL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMax dose volume:\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4.8 mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eImpurities conc.: \u003cstrong\u003e20.96 \u0026micro;g/mL\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMax dose volume:\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4.8 mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRadionuclidic Identity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eObserved t\u003csub\u003e\u0026frac12;\u003c/sub\u003e is 105 to 115 minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e111.66 min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e111.61 min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e108.7 min\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eVolatile Organic Impurities\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethanol\u0026thinsp;\u0026le;\u0026thinsp;0.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.004%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthanol\u0026thinsp;\u0026le;\u0026thinsp;0.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.017%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.019%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.008%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetonitrile\u0026thinsp;\u0026le;\u0026thinsp;0.041%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial Endotoxin (LAL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;175 EU/dose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;2.50 EU/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;2.50 EU/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;2.50 EU/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFilter Integrity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;\u0026thinsp;50 psi breaking pressure\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e60.4 psi\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e60.0 psi\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e60.3 psi\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSterility\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eno growth observed over 14 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePass\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePass\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePass\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eImpurities (UV)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026le;\u0026thinsp;10 \u0026micro;g/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.01 \u0026micro;g/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.01 \u0026micro;g/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.14 \u0026micro;g/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRadionuclidic Purity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ge;\u0026thinsp;99.5% \u003csup\u003e18\u003c/sup\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e100%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn addition, the product was tested for stability over eight hours. Radiochemical and chemical purity analysis showed no discernable change over that time with all values remaining stable, as well as meeting all QC acceptance criteria at the conclusion of this time.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe based our synthesis protocol on previously published methods (Simpson et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) with some modifications (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.) to support automation. Final yield of the optimized synthesis of 11.0% is lower than reported manual synthesis yield of 23%, however the current reported method has the benefit of being fully automated and utilizing disposable, sterile cassettes.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOptimizations made to synthesis in comparison to previous work\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSynthesis Step\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTrasis AiO \u0026ndash; Current Work\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eManual Method (Simpson et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdditional azeotropic distillation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u0026ndash;1 mL MeCN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u0026ndash;1 mL MeCN\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcid deprotection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e150\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e140\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIntermediate transfer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e- 2 mL added to HCl to transfer to cartridge purification\u003c/p\u003e\u003cp\u003e- No 0.45 \u0026micro;m filter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e- HCl solution passed directly through cartridge purification\u003c/p\u003e\u003cp\u003e- 0.45 filter post purification\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReduction\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e- 8 mg NaBH\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e- No stirring\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e- 5.5 mg NaBH\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e- Stirring\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFinal purification\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eChromabond Set V cartridge +\u003c/p\u003e\u003cp\u003eSCX cartridge +\u003c/p\u003e\u003cp\u003eQMA cartridge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChromabond Set V cartridge\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDuring our optimization testing, we assessed whether the two additional azeotropic evaporations performed in the manual synthesis would result in higher initial fluorination on this system. In results not shown, we achieved a 25% increase in fluorine incorporation by adding a second azeotropic evaporation, but that benefit was lost with a third azeotropic evaporation. As a result, we added one additional evaporation. Preliminary optimization to reaction temperatures showed a doubling of overall activity yield by increasing the deprotection temperature from 140\u0026deg;C to 150\u0026deg;C (4.7 to 9.6%, n\u0026thinsp;=\u0026thinsp;3\u0026ndash;6). The reactor seal was compromised by raising the temperature to 160\u0026deg;C. Addition of a dose calibrator to measure exact F-18 starting activity was essential to allow an accurate determination of activity yield, which confirmed that we were previously underestimating. On a couple of occasions, we observed unexplainable, low trapping of F-18 on the QMA cartridge at the initial transfer to the radiosynthesizer resulting in a lower yield, however, final product concentration still allowed for patient dose preparation.\u003c/p\u003e\u003cp\u003eWe opted not to include molar activity determination as previous work shows very low mass of fluoromannitol. In those studies, the measured mass concentration of fluoromannitol in the final product was 7.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 \u0026micro;g/mL (n\u0026thinsp;=\u0026thinsp;14). (Simpson et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) This meets the FDA microdose definition and is suitable for clinical research studies. \u003cem\u003eD\u003c/em\u003e-mannitol is non-toxic to humans and the agent is FDA-approved for intravenous administration of concentrations as high as 25 g/100 mL of water, more than 34,000 times the mass reported in the final formulation of the radiopharmaceutical.\u003c/p\u003e\u003cp\u003eIt should be noted that radiochemical purity and identity as well as chemical purity quality control testing of the final formulation of [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol are complicated by the fact that mannitol and fluoromannitol do not absorb UV light and are not retained on typical reversed-phase HPLC columns. Unfortunately, mannitol and fluoromannitol elute with the solvent front on these columns and therefore no separation of the product from [\u003csup\u003e18\u003c/sup\u003eF]F\u003csup\u003e-\u003c/sup\u003e is possible to determine radiochemical purity. While other detector options are available, such as refractive index and evaporative light scattering detectors, they are not common in radiopharmaceutical labs. In addition, they often suffer from baseline drift, noisy background, and sensitivity to mobile phases making reproducibility challenging. In the context of quality control for release of PET tracers, these options offered many concerns. Initially, we considered a method whereby we confirmed identity via TLC analysis by comparing the R\u003csub\u003ef\u003c/sub\u003e of our drug product (radioactivity detection) to a standard using TLC of copper ion-impregnated silica that allows retention of the fluoromannitol. Visualization of the standard was possible by treating the plate with a common stain (basic potassium permanganate) and heating to develop. For determination of radiochemical purity, we used the more sensitive method of HPLC using an ion chromatography column for sugars and sugar alcohols and used the primary radioactivity peak of the now confirmed fluoromannitol (by TLC). However, quantification of impurities was hindered by our original methods. We ultimately opted to introduce an evaporative light scattering detector (ELSD). During initial analyses, we did observe a significant unknown impurity. To remedy this, we added an additional SCX Sep-Pak to the final purification that successfully eliminated the impurity, but this lowered the pH below acceptable limits. The final QMA cartridge (pretreated with sodium bicarbonate) was added to raise the final product pH to levels required for injection.\u003c/p\u003e\u003cp\u003eWhile analysis by reversed phase chromatography with UV detection is not useful for determining the identity or purity of the final product, we thought it essential to assess for any impurities that could be visualized in this manner and not by the HILIC HPLC with ELSD. There are several purification cartridges in this synthesis, so we did not expect to observe impurities, and this was confirmed through several (n\u0026thinsp;=\u0026thinsp;25) analyses. In all cases, virtually no evidence of impurity peaks was observed in the final product by elution on standard C18 column and reversed-phase system (see Supplementary Information). With confidence from these tests, we elected not include this analysis in routine quality control testing, however, will perform annually and did include in our validation syntheses.\u003c/p\u003e\u003cp\u003eAs outlined in FDA\u0026rsquo;s guidance on solvents in pharmaceutical products, acetonitrile and methanol are Class 2 solvents and are limited to 0.041% for acetonitrile and 0.3% for methanol; acetonitrile is used in this synthesis and methanol is a possible side product. Ethanol is a class 3 solvent and is limited to 0.5%. Ethanol is used to sterilize the radiosynthesis system and is a possible side product. Organic solvents are used by commercial vendors in the preparation of purification substrates and cartridges used in this synthesis; while they remove\u0026thinsp;\u0026gt;\u0026thinsp;99% of residual solvents, we observed ethanol, methanol, acetone and isopropanol in our product during synthesis optimization. Although in very small quantities, they were confirmed to be due specifically to residual solvents that eluted from these cartridges with the product. This was confirmed by GC analysis of water passed through each cartridge prior to preparation for synthesis compared to solvent standards. Our results showed\u0026thinsp;\u0026lt;\u0026thinsp;0.01% ethanol and isopropanol on the Alumina N cartridge, \u0026lt; 0.01% methanol and isopropanol on the 11 A8\u0026thinsp;+\u0026thinsp;C18-U cartridge, and no quantifiable solvent amounts from the Accell CM. The Chromabond cartridge contained trace amounts of acetone, \u0026lt; 0.01% ethanol, and 0.5\u0026ndash;1% methanol in our analysis. Additional volume of water rinses in preparation of the purification cartridges eliminated virtually all of the solvents.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, an automated method for the radiochemical synthesis of [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol was developed and optimized on a commercially available Trasis AllinOne radiosynthesizer. This method allows for the reliable production of [\u003csup\u003e18\u003c/sup\u003eF]FMtl passing USP\u0026thinsp;\u0026lt;\u0026thinsp;823\u0026thinsp;\u0026gt;\u0026thinsp;quality control testing for use in clinical research trials in less than two hours.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCMS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Centers for Medicare \u0026amp; Medicaid Services\u003c/p\u003e\n\u003cp\u003eELSD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Evaporative light scattering detector\u003c/p\u003e\n\u003cp\u003eEOS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;End of synthesis\u003c/p\u003e\n\u003cp\u003eFDA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Food and Drug Administration\u003c/p\u003e\n\u003cp\u003eFDG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fluorodeoxyglucose\u003c/p\u003e\n\u003cp\u003eFDS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fluorodeoxysorbitol\u003c/p\u003e\n\u003cp\u003eFPV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Final product vial\u003c/p\u003e\n\u003cp\u003eFMTL\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Fluoromannitol\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Water\u003c/p\u003e\n\u003cp\u003eHCl\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hydrochloric acid\u003c/p\u003e\n\u003cp\u003eHILIC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hydrophilic interaction liquid chromatography\u003c/p\u003e\n\u003cp\u003eHPLC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;High performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eIND\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Investigational New Drug\u003c/p\u003e\n\u003cp\u003eK222\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Kryptofix\u0026reg; 2.2.2\u003c/p\u003e\n\u003cp\u003eK\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Potassium carbonate\u003c/p\u003e\n\u003cp\u003eKF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Potassium fluoride\u003c/p\u003e\n\u003cp\u003eLAL\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Limulus amebocyte lysate\u003c/p\u003e\n\u003cp\u003eM1PDH\u0026nbsp; \u0026nbsp;\u0026nbsp;Mannitol-1-phosphate dehydrogenase\u003c/p\u003e\n\u003cp\u003eMeCN\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Acetonitrile\u003c/p\u003e\n\u003cp\u003eMRI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Magnetic resonance imaging\u003c/p\u003e\n\u003cp\u003eNaBH\u003csub\u003e4\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sodium borohydride\u003c/p\u003e\n\u003cp\u003ePABA \u003cem\u003epara\u003c/em\u003e-aminobenzoic acid\u003c/p\u003e\n\u003cp\u003ePET\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Positron emission tomography\u003c/p\u003e\n\u003cp\u003eRT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Retention time\u003c/p\u003e\n\u003cp\u003eTLC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Thin layer chromatography\u003c/p\u003e\n\u003cp\u003eUSP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; United States Pharmacopeia\u003c/p\u003e\n\u003cp\u003eUV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Ultra-violet\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest with regard to this study.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was funded by ALSAC-St. Jude Children\u0026rsquo;s Research Hospital, NIH/NIBIB R01AI177976-01, and NIH/NIAID R01AI192221-01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eALV, AJC, AG, JMA, MJB, SS, and KDN designed the studies. AJC, AG, JMA, MJB, and SS carried out the experiments. ALV prepared the manuscript. All authors read, edited and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank the staff of the Molecular Imaging Core @ St. Jude Children\u0026rsquo;s Research Hospital.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAl Farii H, Zhou S, Albers A. Management of Osteomyelitis in Sickle Cell Disease: Review Article. Journal of the American Academy of Orthopaedic Surgeons Global Research Review. 2020;4:e20.00002.\u003c/li\u003e\n\u003cli\u003eBerger E, Saunders N, Wang L. Sickle Cell Disease in Children: Differentiating Osteomyelitis from Vaso-occulisive Crisis. JAMA Pediatrics. 2009;163:251-5.\u003c/li\u003e\n\u003cli\u003eBritton K, Wareham D, Das S, Solanki K, Amaral H, Bhatnagar A, et al. Imaging bacterial infection with \u003csup\u003e99m\u003c/sup\u003eTc-ciprofloxacin (Infecton). Journal of Clinical Pathology. 2002;55:817-23.\u003c/li\u003e\n\u003cli\u003eCalabria F, Gaudagnino G, Cimini A, Leporace M. PET/CT Imaging of Infectious Diseases: Overview of Novel Radiopharmaceuticals. Diagnostics. 2024;14:1-20.\u003c/li\u003e\n\u003cli\u003eGowrishankar G, Hardy J, Wardak M, Namavari M, Reeves RE, Neofytou E, et al. Specific Imaging of Bacterial Infection Using 6\u0026Prime;-\u003csup\u003e18\u003c/sup\u003eF-Fluoromaltotriose: A Second-Generation PET Tracer Targeting the Maltodextrin Transporter in Bacteria. Journal of Nuclear Medicine. 2017;58:1679-84.\u003c/li\u003e\n\u003cli\u003eKahts M, Summers B, Gutta A, Pilloy W, Ebenhan T. Recently developed radiopharmaceuticals for bacterial infection imaging. 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Treatment-related mortality in children with cancer: Prevalence and risk factors. European Journal of Cancer. 2019;121:113-22.\u003c/li\u003e\n\u003cli\u003eMcGrath B, Broadhurst M, Roman C. Infectious disease considerations in immunocompromised patients. Journal of the American Academy of Physician Assistants. 2020;33:16-25.\u003c/li\u003e\n\u003cli\u003eMock BH, Winkle W, Vavrek MT. A color spot test for the detection of Kryptofix 2.2.2 in [\u003csup\u003e18\u003c/sup\u003eF]FDG preparations. Nuclear Medicine and Biology. 1997;24:193-5.\u003c/li\u003e\n\u003cli\u003eMutch CA, Ordonez AA, Qin H, Parker M, Bambarger LE, Villanueva-Meyer JE, et al. [\u003csup\u003e11\u003c/sup\u003eC]Para-Aminobenzoic Acid: A Positron Emission Tomography Tracer Targeting Bacteria-Specific Metabolism. ACS Infectious Diseases. 2018;4:1067-72.\u003c/li\u003e\n\u003cli\u003eNeumann KD, Villanueva-Meyer JE, Mutch CA, Flavell RR, Blecha JE, Kwak T, et al. Imaging Active Infection in vivo Using D-Amino Acid Derived PET Radiotracers. Scientific Reports. 2017;7:7903.\u003c/li\u003e\n\u003cli\u003eNing X, Seo W, Lee S, Takemiya K, Rafi M, Feng X, et al. PET imaging of bacterial infections with fluorine-18-labeled maltohexaose. Angewandte Chemie International Edition. 2014;53:14096-101.\u003c/li\u003e\n\u003cli\u003eOrdonez AA, Wang H, Magombedze G, Ruiz-Bedoya CA, Srivastava S, Chen A, et al. Dynamic imaging in patients with tuberculosis reveals heterogeneous drug exposures in pulmonary lesions. Nature Medicine. 2020;26:529-34.\u003c/li\u003e\n\u003cli\u003eParker MF, Luu JM, Schulte B, Huynh TL, Stewart MN, Sriram R, et al. Sensing Living Bacteria \u003cem\u003ein Vivo\u003c/em\u003e Using \u003cem\u003eD\u003c/em\u003e‑Alanine-Derived \u003csup\u003e11\u003c/sup\u003eC Radiotracers. 2020;6:155-65.\u003c/li\u003e\n\u003cli\u003eSalaffi F, Ceccarelli L, Carottie M, Di Carlo M, Polonara G, Facchini G, et al. Differentiation between infectious spondylodiscitis versus inflammatory or degenerative spinal changes: How can magnetic resonance imaging help the clinician? La Radiologia Medica. 2021;126:843-59.\u003c/li\u003e\n\u003cli\u003eScruggs M, Pateva I. Multifocal osteomyelitis in a child with sickle cell disease and review of the literature regarding best diagnostic approach. Clinical Case Reports. 2023;11:e7288.\u003c/li\u003e\n\u003cli\u003eSellmeyer MA, Lee I, Hou C, Weng C-C, Li S, Lieberman BP, et al. Bacterial infection imaging with [\u003csup\u003e18\u003c/sup\u003eF]fluoropropyl-trimethoprim. Proceedingd of the National Academy of Sciences of the United States of America. 2017;114:8372-7.\u003c/li\u003e\n\u003cli\u003eSeo SK, Liu C, Dadwal SS. Infectious Disease Complications in Patients with Cancer. Critical Care Clinics. 2020;37:69-84.\u003c/li\u003e\n\u003cli\u003eSimpson SR, Kesterson AE, Wilde JH, Qureshi Z, Kundu BK, Simons MP, et al. Imaging Diverse Pathogenic Bacteria in vivo with [18F]fluoromannitol Positron Emission Tomography. The Journal of Nuclear Medicine. 2022;64:809-15.\u003c/li\u003e\n\u003cli\u003eSorenson MCL, Andersen MM, Rostgaard K, Schmiegelow K, Mikkelsen TS, Wehner PS, et al. Treatment-related mortality among children with cancer in Denmark during 2001-2021. Acta Oncologica. 2024;63:294-302.\u003c/li\u003e\n\u003cli\u003eStone P. Economic burden of healthcare-associated infections: an American perspective. Expert Review of Pharmaeconomics \u0026amp; Outcomes Research. 2009;9:417-22.\u003c/li\u003e\n\u003cli\u003eTakemiya K, Ning X, Seo W, Wang X, Mohammad R, Joseph G, et al. Novel PET and near infrared imaging probes for the specific detection of bacterial infections associated with cardiac devices. JACC Cardiovasc Imaging. 2019;12:875-86.\u003c/li\u003e\n\u003cli\u003eTucker EW, Guglieri-Lopez B, Ordonez AA, Ritchie B, Klunk MH, Sharma R, et al. Noninvasive \u003csup\u003e11\u003c/sup\u003eC-rifampin positron emission tomography reveals drug biodistribution in tuberculous meningitis. Science Translational Medicine. 2018;10:470.\u003c/li\u003e\n\u003cli\u003evan Oosten M, Schaefer T, Gazendam JA, Ohlsen K, Tsompanidou E, de Goffau MC, et al. Real-time\u003cem\u003e in vivo\u003c/em\u003e imaging of invasive- and biomaterial-associated bacterial infections using fluorescently labelled vancomycin. Nature Communications. 2013;4:2584.\u003c/li\u003e\n\u003cli\u003eWahl RL, Disizian V, Pelstro CJ. At Last, \u003csup\u003e18\u003c/sup\u003eF-FDG for Inflammation and Infection! The Journal of Nuclear Medicine. 2021;62:1048-9.\u003c/li\u003e\n\u003cli\u003eWeaver JS, Omar IM, Mar WA, Klauser AS, Winegar BA, Mlady GW, et al. Magnetic resonanace imaging of musculoskeletal infections. Polish Journal of Radiology. 2022;87:e141-e62.\u003c/li\u003e\n\u003cli\u003eWeinstein EA, Ordonez AA, DeMarco VP, Murawski AM, Pokkali S, MacDonald EM, et al. Imaging Enterobacteriaceae infection in vivo with 18F-fluorodeoxysorbitol positron emission tomography. Science Translational Medicine. 2014;6:259ra146.\u003c/li\u003e\n\u003cli\u003eZhang XM, Zhang HH, McLeroth P, Berkowitz RD, Mont MA, Stabin MG, et al. [\u003csup\u003e124\u003c/sup\u003eI]FIAU: Human dosimetry and infection imaging in patients with suspected prosthetic joint infection. Nuclear Medicine and Biology. 2016;43:273-9.\u003c/li\u003e\n\u003cli\u003eZhang Z, Ordonez AA, Wang H, Li Y, Gogarty KR, Weinstein EA, et al. Positron Emission Tomography Imaging with 2-[\u003csup\u003e18\u003c/sup\u003eF]F-p-Aminobenzoic Acid Detects \u003cem\u003eStaphylococcus aureus\u003c/em\u003e Infections and Monitors Drug Response. ACS Infectious Diseases. 2018;4:1635-44.\u003c/li\u003e\n\u003cli\u003eZheng Y, Chen Y, Yu K, Yang Y, Wang X, Yang X, et al. Fatal Infections Among Cancer Patients: A Population-Based Study in the United States. Infectious Diseases and Therapy. 2021;10:871-95.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Radiosynthesis, Automation, Radiofluorination, Fluoromannitol, PET, Infection","lastPublishedDoi":"10.21203/rs.3.rs-7124066/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7124066/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Infections pose a significant risk to immunocompromised individuals, and accurate, efficient diagnosis remain challenging. Current imaging methods like MRI and FDG PET lack pathogen specificity which complicate diagnosis and lead to overuse of antibiotics. Recent data shows that [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol ([\u003csup\u003e18\u003c/sup\u003eF]FMtl) is sensitive and specific to infection in vivo by exploiting the pathogen-specific mannitol transporter. This work aims to establish a reliable, automated method for producing [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol to facilitate clinical research studies in human subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e This study optimized and automated the radiosynthesis of [¹⁸F]fluoromannitol ([¹⁸F]FMtl) on a Trasis AllinOne synthesizer. The 105-minute synthesis achieved an average yield of 11.0% (n=19) with \u0026gt;97% radiochemical purity, and the product remained stable for at least 8 hours. While yield was lower than the previously reported manual method, automation enabled reproducibility and sterility. Process improvements included optimizing evaporation steps and reaction temperature, which significantly increased fluorine incorporation and yield. The process was validated to meet USP \u0026lt;823\u0026gt; regulatory requirements including full QC testing on three consecutive batches.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: An automated method for the radiochemical synthesis of [\u003csup\u003e18\u003c/sup\u003eF]fluoromannitol was developed and optimized on a commercially available Trasis AllinOne radiosynthesizer. This method allows for the reliable production and global dissemination of [\u003csup\u003e18\u003c/sup\u003eF]FMtl for use in clinical research trials.\u003c/p\u003e","manuscriptTitle":"Automated Radiosynthesis of [18F]Fluoromannitol for Clinical Research on a Commercially Available Trasis AllinOne Radiosynthesizer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-01 10:39:55","doi":"10.21203/rs.3.rs-7124066/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2025-08-08T09:16:12+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-28T10:40:48+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-28T10:36:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-21T05:42:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Radiopharmacy and Chemistry","date":"2025-07-18T15:47:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b01a99f0-5240-4cdf-a34b-3c8a8fb0e436","owner":[],"postedDate":"August 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:07:41+00:00","versionOfRecord":{"articleIdentity":"rs-7124066","link":"https://doi.org/10.1186/s41181-025-00388-x","journal":{"identity":"ejnmmi-radiopharmacy-and-chemistry","isVorOnly":false,"title":"EJNMMI Radiopharmacy and Chemistry"},"publishedOn":"2025-10-28 15:58:27","publishedOnDateReadable":"October 28th, 2025"},"versionCreatedAt":"2025-08-01 10:39:55","video":"","vorDoi":"10.1186/s41181-025-00388-x","vorDoiUrl":"https://doi.org/10.1186/s41181-025-00388-x","workflowStages":[]},"version":"v1","identity":"rs-7124066","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7124066","identity":"rs-7124066","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}