Automation of simplified two-step radiolabeling via ditosylate synthon for ¹⁸F-labeled radiotracers using AllinOne module

Abstract

Direct fluorination of a tosylate or mesylate precursor has been a wide-spread and reliable way for radiofluorination. This approach can be difficult to achieve when the precursor cannot be easily obtained or is chemically unstable. A possible alternative method is to radiolabel ethylene ditosylate or 1,3-propanediol di-ptosylate to form a radiofluorinated synthon. Here we present the automation of a simplified and reliable approach for the two-step fluorination using [¹⁸F]FP-TMP, an analog of antibacterial agent trimethoprim. We demonstrate the feasibility of purifying the fluorinated synthon via filtration, and one final HPLC purification on a commercially available Trasis AllinOne module. The overall reaction time for the two-step reaction is around 90 min andthe decay-corrected yield for more than fifty preparations of [¹⁸F]FP-TMP is 22 ± 5 % with high radiochemical purity (≥ 90 %) and specific activities (147 ± 107 GBq/μmol). All batches passed pre-established quality control specifications, demonstrating the utility of using this method in tracer syntheses that meet good manufacturing practice (GMP) requirement. This method can be adopted to the syntheses of other radiotracers, such as [¹⁸F]FE-TMP, (+)-[¹⁸F]F-PHNO and [¹⁸F]FFMZ.

1. Introduction

In the era of personalized precision medicine, positron emission tomography (PET) has emerged as the preferred molecular imaging procedure for assessing individual difference in drug target expression [1,2]. The recent advances in PET scanner design, such as the long axial field-of-view systems, and the FDA-approval of theranostic agents for treating cancer offers new opportunities for clinical research [3]. Improved methods for radiolabeling PET tracers that can be used in a clinical research setting has become increasingly important. To successfully supply a radiopharmaceutical for clinical PET studies, the syntheses needs to be reliable, reproducible and completed in reasonable synthesis time and with sufficient radiochemical yield [4]. Fully automated processes are necessary from many aspects, such as reducing personnel exposure, decreased variability, and increase regulatory compliance such as current Good Manufacturing Practice (cGMP) regulations.

The historic background and evolution of automated synthesis for radiopharmaceuticals and the overview of cassette-based modules are given in a number of excellent reviews [5-8], and a guidance on developing and usage of automated modules in EU has also been published [9]. Several recent publications focused on the automation of individual tracers further demonstrates the increased desire to produce radiotracers in a consistent and reliable manner. The recent advancement in using nucleophilic fluorination, followed by conversion on the sep-pak, to produce high molar activity [¹⁸F]F-DOPA has also been demonstrated by Neves and others using several different synthesis platforms [10-12]. Other radiotracers, such as [¹⁸F]FLUDA targeting A2A receptor and used to investigate neurological disorders, were also automated in the past two years [11,13-15]. A new approach reported the use of 3-D printing customized electronics, valves and other parts to assemble a synthesis module economically; the group also automated several common ¹¹C- and ¹⁸F-labeled tracers to demonstrate the versatility of this approach [16]. These examples further demonstrate the importance of automated radiosynthesis that satisfied the ever-increasing demands of multiple batches in a single day from high starting radioactivities.

Currently, there are a number of commercially-available automated synthesis modules on the market, including Synthera^® (IBA Radio Pharma solutions), FASTLab 2^™, TracerLab^™ FX2 N series (GE Healthcare), E-Z modules (Eckert & Ziegler modular lab), AllinOne (Trasis) and RNPlus series (Synthra). These automated modules are capable of carrying out 2–3 reaction steps, followed by either resin-based solid-phase extraction (SPE) and/or HPLC purification. Both cassette-based and non-cassette-based modules are widely used in the production of PET radiotracers. Typically, non-cassette-based modules have lower cost per synthesis and are versatile in the types of radiosynthesis they can accommodate. However, the cassette-based modules have several advantages, such as lower burden on chemical impurities and contamination from prior syntheses and more consistent and uniform inter-operator results. Furthermore, the use of a sterile cassette reduces the overall concern for product sterility and endotoxin content. Because of these advantages, we have focused on using a cassette-based module, the Trasis AllinOne, to investigate the radiolabeling yield and reliability of radiosynthesis using a two-step, two-vessel fluorinated-synthon. This procedure was built upon our previous effort in successfully automating the complex synthesis of [¹⁸F](2S,4R)4-Fluoroglutamine’ ([¹⁸F]F-Gln) on a Trasis module [17].

The traditional way of two-step radiolabeling using ethylene ditosylate or 1,3-bis(tosyloxy)propane involved a semi-HPLC purification to obtain high purity fluorinated product for the second step [14,18]. This approach requires two HPLC purifications for the overall production, one for purification of synthon and the second one for the second step. The required special setup of the module and long production time made this approach not ideal for the radiotracer supply for clinical trials. In addition, this method is not favorable for automation as most of the commercially available modules cannot support two HPLC purifications. Recently, some advances have been reported to eliminate the use of second HPLC purification. The corresponding N-desmethyl precursor was successfully radiolabeled with [¹⁸F]fluoroethyl tosylate in the synthesis of [¹⁸F]FEDAC [19]. Khan [20] et al. also optimized the two-step synthesis of [¹⁸F]GE-179 via fluorination of ethylene ditosylate on a FastLab synthesis module. However, these approaches still required two HPLC purifications. Here, we have optimized this process to use only one HPLC purification step using [¹⁸F]FP-TMP as the model reaction. We show a new way of simplifying the radiolabeling via 1,3-bis(tosyloxy) propane, followed by alkylation on hydroxyl precursor and effectively skip the extra HPLC step by only working up the final product by HLPC.

Trimethoprim (TMP) is an antimicrobial that is used to treat and prevent bacterial infection. Both ¹¹C- and ¹⁸F-labeled TMP have been used for PET imaging of infection [21,22]. Using the affinity of ¹⁸F-labeled TMP to Escherichia coli protein, dihydrofolate reductase enzyme (eDHFR), this PET radiotracer has been expanded to other applications, such as the quantification of reporter gene expression and monitoring the trafficking of chimeric antigen receptor (CAR) T cells [23] [24]. Direct fluorinating of tosylate precursor of [¹⁸F]FP-TMP was shown to be problematic because of the multistep synthesis required for the corresponding precursor. Therefore, we focused our effort to develop a two-step reaction by first radiolabeling 1,3-bis(tosyloxy)propane, followed by alkylation of hydroxyl precursor. To reduce the overall radiolabeling time, we developed a simplified two-step, two-vessel, one-HPLC procedure that is compliant GMP standards.

2. Procedures

2.1. Materials

[¹⁸F]FP-TMP and [¹⁸F]FE-TMP [25] reference standards and their phenol precursor, 4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenol were made following previous procedures [26]. Cryptand, (+)-[¹⁸F]F-PHNO reference standard and its precursor, (+)-HNO hydrochloride, and [¹⁸F]FFMZ reference standard, fluoroethylflumazenil and its precursor flumazenil acid, were obtained from ABX (Radeberg, Germany).

1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD), 7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD) 1,8-Diazabicyclo[5.4.0]-7-undecene (DBU) were purchased from TCI America (Portland, USA). Both ditosylate precursors, 1,3-bis(tosyloxy)propane and 1,2-bis(tosyloxy) ethane, acetonitrile (MeCN, anhydrous for synthesis; HPLC grade), dimethylformamide (DMF, anhydrous), methanol (HPLC grade), ammonium formate (AMF, HPLC grade) were purchased from Sigma-Aldrich (St Louis, MO, USA). Water (HPLC grade) was purchased from Fisher Scientific (Pittsburg, PA, USA). Cartridges used for solid phase extractions, such as Sep-Pak Light Accell Plus QMA Carbonate Cartridges, Sep-Pak Dry Cartridges, Sep-Pak Light ^tC18 Cartridges, OASIS HLB Light Cartridges were purchased from Waters (Milford, MA, USA). USP grade sterile water for injection was purchased from B. Braun (Melsungen, Germany). USP grade sterile normal saline (0.9 % w/v) was purchased from Hospira Inc. (Lake Forest, IL, USA) and ethanol (200 proof, USP grade) was from Decon Labs (King of Prussia, PA, USA). O-18 water (>97 %) was purchased from Huayi Isotopes Co. (Changshu, China) or ABX (Radeberg, Germany). Low-protein binding 0.2 μm Millex^® FG sterile filters, and 0.45 μm nylon filters were obtained from Millipore (Bedford, USA). All other chemicals and solvents for radiosyntheses were obtained from Sigma-Aldrich or Fisher Scientific. These chemicals are at least HPLC grade and used without further purification. AllinOne synthesis module and all consumable parts were purchased from TRASIS (Ans, Belgium).

2.2. General information

In a typical procedure, [¹⁸F]F⁻ was produced by direct proton bombardment of a target containing H₂ [18]O water, through the radiochemical reaction ¹⁸O(p,n)¹⁸F, using an IBA Cyclone 18/9 cyclotron. The radioactivity was transferred and trapped onto an ion exchange cartridge on the synthesis module. After eluting the ¹⁸F from the cartridge, the first step of the two-step reaction was the radiolabeling of ditosylate precursor with [¹⁸F]F⁻.

The AllinOne synthesis module used in the radiolabeling procedure is a 30-valve configuration with two reaction vessels. The synthesis cassette consists of five of 6-valve manifolds, 2 reactors, and various tubing, cartridges and syringes. Purification of all fluorine-18 PET tracers included semi-preparative HPLC purification and SPE purification and concentration. HPLC purification was performed with the built-in HPLC system equipped with a radioactivity detector, Smartline UV detector 200 (Knauer, Berlin, Germany) and a pump P4.1.5 (Knauer, Berlin, Germany). Columns used for the purification were Phenomenex Luna^® C18 column, 5 μm, 100 Å, 250 × 10 mm, (Torrance, CA, USA); Agilent Zorbax StableBond SB-C18 column, 5 μm 80 Å, 100 × 9.4 mm (Santa Clara, CA, USA); or Waters SunFire C18 semi-preparative column, 5 μm 250 × 10 mm (Milford, MA, USA). SPE purification was carried out with Waters OASIS Light HLB cartridges (Milford, MA, USA). Waters Sep-Pak Dry cartridges were used to further remove water.

Analytical HPLC analysis was performed using Waters Alliance e2695 HPLC systems equipped with a 2489 UV/VIS detector (Milford, MA, USA) and a photomultiplier scintillation radio-detector (Eckert&Ziegler, Berlin Germany). An Agilent Zorbax XDB-C18, 5 μm 80 Å 150 × 4.6 mm, column (Santa Clara, CA, USA) was used for chemical and radiochemical purity testing for [¹⁸F]FP-TMP and [¹⁸F]FE-TMP. Waters Acquity UPLC^® H Class with a TUV or PDA UV detector and a photomultiplier scintillation radio-detector and a Waters column, BEH C18 1.7 μm 2.1 × 50 mm, was used for chemical purity and radiochemical purity testing of (+)-[¹⁸F]F-PHNO and [¹⁸F]FFMZ.

Residual solvent analysis was performed with Agilent gas chromatography (GC) system, 6890 or 7890 series. All GC and HPLC were controlled with Waters Empower software.

2.2.1. Radiolabeling of [¹⁸F]FP-TMP

After trapping [¹⁸F]F⁻ from an Accell^® Light QMA Carbonate cartridge, the radioactivity was eluted with 1 mL eluent containing 0.85 mL MeCN, 0.15 mL H₂O, 7 mg K222 and 1 mg K₂CO₃. The radioactivity was initially dried under vacuum and heat (100 °C) for 2 min, followed by addition of 1 mL MeCN to further drying the activity for additional 5 min. After the drying was completed, an aliquot of 5 mg ditosylate precursor, 1,3-bis(tosyloxy)propane, in 1 mL acetonitrile was added into the first reaction vessel. The reaction was carried out at 110 °C for 10 min and then cooled to 60 °C. After the completing of fluorination, 20 mL water was introduced into the first reaction vessel and the reaction mixture was then passed through a 33 mm 0.45 μm nylon filter to remove excess ditosylate precursor. The fluorinated synthon, 3-(fluoro-¹⁸F)propyl 4-emthylbenzenesulfonate, [¹⁸F]FPOTs, was concentrated on an OASIS Light HLB cartridge. The cartridge was washed with additional 10 mL of water. The [¹⁸F]FPOTs was eluted with 2 mL acetonitrile through a Sep-Pak Dry cartridge into the second reaction vessel. The second step was carried out by adding 0.8 mL DMF containing 4 mg TMP-OH and 10 mg TBD into the second reaction vessel. The mixture was heated at 100 °C for 9 min and cooled down to 40 °C. The reaction was quenched with mobile phase before being loaded to HPLC loop for purification. A semi-preparative HPLC with an Agilent Zorbax SB-C18 column (5 μm, 100 × 9.4 mm) was used. The mobile phase contained 33 % methanol in 0.1 M AMF solution and flow rate was 5 mL/min. The desired product was eluted around 15 min and the fraction was collected in a 20 mL syringe and then diluted with 15 mL water. The product was concentrated on a tC18 Light cartridge. The final product was eluted with 1 mL ethanol and passed through a 0.2 μm sterile Millex^® FG filter into final production vial. Afterward, 10 mL saline was also added.

2.2.2. Radiolabeling of [¹⁸F]FE-TMP

[¹⁸F]FE-TMP was synthesized on AllinOne using a similar procedure as described for [¹⁸F]FP-TMP. The precursor, 1,2-bis(tosyloxy)ethane (5 mg in MeCN), was added to the dried [¹⁸F]F⁻/K222 complex in the reaction vessel and the solution was heated at 110 °C for 10 min to obtain 2-fluoro-¹⁸F)ethyl 4-methylbenzenesulfonate, [¹⁸F]FEOTs. Afterward, [¹⁸F]FEOTs was concentrated on the sep-pak and eluted to the second reaction vial before adding 4 mg TMP-OH and 10 mg TBD in DMF. the reaction was carried out at 100 °C for 9 min before the mixture was quenched and purified by a semi-preparative HPLC using an Agilent SB-C18 column (5 μm, 100 × 9.4 mm) and the mobile phase of 28 % MeOH in 100 mM ammonium formate solution at 5 mL/min flow rate. The product was eluted at around 10 min.

2.2.3. Radiolabeling of (+)-[¹⁸F]F-PHNO

(+)-[¹⁸F]F-PHNO was synthesized on AllinOne using similar procedures as described of [¹⁸F]FP-TMP. After eluting and transferring [¹⁸F]FPOTs into the second reaction vessel, the eluent was allowed to react with 4 mg precursor (+)HNO with 10 mg of TBD (base). The coupling was carried out at 100 °C for 15 min. The semi-preparative HPLC purification used a Phenomenex Luna^® C18, 5 μm 250 × 10 mm column and mobile phase of 25 % MeCN in 100 mM ammonium bicarbonate with flow rate of 7 mL/min. The collected product, eluted at 18 min, was enriched on a ^tC₁₈ Sep-Pak cartridge and the final product was formulated with ≤10 % ethanol in saline.

2.2.4. Radiolabeling of [¹⁸F]FFMZ

The synthesis of [¹⁸F]FFMZ on AllinOne was similar to the procedures of [¹⁸F]FE-TMP. Dried [¹⁸F]FEOTs was transferred into the second reaction vessel and reacted with 4 mg precursor flumazenil acid at the presence of 2 mg base TBD. The esterification was carried out at 100 °C for 9 min. After completing of the reaction, the reaction vessel was cooled, and the reaction mixture was quenched with mobile phase before injected onto a semi-preparative HPLC for purification. An Agilent Zorbax SB-C18 column (5 μm, 100 × 9.4 mm) was used and the mobile phase was composed of 45 % MeOH and 55 % 100 mM ammonium formate solution. When the system was run at 5 mL/min and the retention time of the product was at 7 min. The collected product was enriched with a ^tC18 Sep-Pak cartridge before eluted with ethanol.

3. Results and discussion

We tested the two-step radiolabeling method by first fluorinating the ditosylate to form synthons, [¹⁸F]FEOTs or [¹⁸F]FPOTs, followed by alkylation to form the final product. Instead of using HPLC to purify the synthon, we took advantage of the poor water-soluble property of 1,3-bis(tosyloxy)propane and added water into the first reaction vessel at the end of fluorination to precipitate excess unreacted ditosylate. The mixture was first filtered with a 33 mm, 0.45 μm nylon filter and then enriched onto a Waters Oasis HLB Light cartridge. The radiolabeled synthon was then eluted with acetonitrile and passed through a Sep-Pak Dry cartridge to remove excess of water before transferred into the second reaction vessel for the second-step synthesis. The reaction conditions of all four compounds are listed in Fig. 1. The general AllinOne software interface for the set-up of these two-step reactions is shown in Fig. 2a. The actual module set up is shown in Fig. 2b.

Fig. 1.

Synthesis scheme of four radiotracers employing two-step reaction.

Fig. 2.

The module configuration for two-step two-vessel reactions. a. AllinOne user software interface (left); b. Set up with cassettes and reagents (right).

Several factors were important for the success of these automation efforts to synthesize radiotracers with high reliability and reproducibility. The first factor is the selection of a filter after the fluorination step. Filters made of different material, such as polytetrafluoroethylene (PTFE), cellulose and nylon were tested. Based on the need and capacity of the filter, the 33 mm 0.45 μm configuration was selected. Nylon filters were selected based on their low retention of radioactivity and sufficient flow rates in the filtration step without pressure building on the filter. The final amount of ditosylate used for the first step was set at 5 mg based on the filter capacity and amount of fluorinated synthon obtained.

The other consideration in this optimization effort was the selection of general base used in the alkylation step. For O-alkylation or N-alkylation, triethylamine or diisopropylethylamine are typically used as the organic bases of choice whereas cesium carbonate is the inorganic base of choice. However, low radiolabeling yield or no product was found when we tested the radiolabeling of [¹⁸F]FP-TMP using the above bases. Other inorganic bases, such as potassium carbonate, sodium hydride, and sodium methoxide, also gave similar unsatisfactory results. We subsequently found that the use of an organic base, such as 1,8-diazabicyclo[5,4,0]unden-7-ene, triazabicyclodecene (TBD) or 7-methyl-1,5,7-triazabicyclo[4,4,0]dec-5-ene, gave higher overall reaction yields, around 20 % (see Table 1). TBD, a base in solid form, was our final choice for [¹⁸F]FP-TMP because of the reliability and reproducibility of the reactions.

Table 1.

Base selection for O-alkylation of [¹⁸F]FP-TMP.

Base (4.5 eq)	pKa (ACN)	Yield	n
Potassium carbonate	na	0	1
Cesium carbonate	na	0	1
Sodium Hydride	na	0	1
Triethylamine	18.82	0	1
Sodium Methoxide	na	8–10 %	2
1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU)	24.34	18–22 %	2
7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD)	25.49	20–21 %	2
1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD)	26.03	22 ± 5 %	35

We also investigated the HPLC purification method. Compared with HPLC purification for removing un-reacted ditosylate, using filtration results in only a partial removal of ditosylate, which can also react in the second step. This by-product can co-elute with the radiolabeled compound from semi-prep purification and results in an excessive amount of mass impurities in the final product. Therefore, we spent additional effort in optimizing the semi-prep purification conditions. For example, using 35 % methanol in water as mobile phase for the purification of [¹⁸F]FP-TMP gave the product with few mass impurities if the fluorinated synthon was purified by HPLC. However, mass impurities were found if the fluorinated synthon was purified via filtration. After additional effort, we found that using 100 mM ammonium formate solution versus water as the mobile phase, the mass impurity was well-separated and was eliminated from final product. See Fig. 3 for an example of the semi-prep purification chromatogram.

Fig. 3.

Chromatography of Semi-Prep HPLC for [¹⁸F]FP-TMP (Top: Radioactivity; Bottom: UV).

After the optimization efforts were concluded, relevant regulatory documents were generated. Three consecutive validation runs were performed to demonstrate the reliability and reproducibility of the process. A first-in-human imaging trial using [¹⁸F]FP-TMP for infection imaging is currently ongoing. The decay-corrected yield for more than fifty preparations is 22 ± 5 % with high radiochemical purity (≥ 90 %) and specific activities (147 ± 107 GBq/μmol) at the end of synthesis (EOS).

The radiolabeling product passed all QC specifications prior to be released for clinical research studies. The QC tests included filter integrity test via bubble test, appearance via visual test, pH test via pH paper, radionuclide identity via half-life measurement, radiochemical purity and identity test via analytical HPLC, chemical purity and impurity via HPLC, residue solvent analysis via GC, K222 spot test, pyrogenicity, and sterility. An example of QC specifications is listed in Table 2.

Table 2.

[¹⁸F]FP-TMP QC release specification.

Test	Acceptance criteria
Filter membrane integrity test	Following manufacturer's specification
pH	4.5–7.5
Appearance	Clear, colorless, and particle-free
Strength (@EOS)	4–25 mCi/mL
Radionuclide identity (half -life)	105.0 min to 115.0 min
Radiochemical purity	≥ 90 %
Radiochemical identity	RT difference of radioactivity and reference peaks ≤10 %
Drug mass	≤ 10 μg/ injection dose
Chemical impurity	≤ 10 μg/ injection dose
Kryptofix	≤ 50 μg/mL
Bacterial Endotoxin	≤ 175 EU/ total dose
Residue solvent: MeCN	≤ 0.41 mg/mL (4.1 mg/day max)
Residue solvent: MeOH	≤ 3 mg/mL (30 mg/day max)
Residue solvent: EtOH	≤ 100 mg/mL
Residue solvent: DMF	≤ 0.88 mg/mL (8.8 mg/day max)
Sterility (results after 14 days)	Sterile (no visible growth)
Radionuclidic purity (multi-channel analyzer, annual test)	≥ 99.5 %

After the successful optimization of this two-step two-vessel one-HPLC radiolabeling on Trasis AllinOne module, we wanted to investigate the versatility of this method for other similar two-step reactions. The first reaction was to test whether ditosylate ethane can be fluorinated using the same procedure. In addition, we tested this method on structurally different compounds. Three additional F-18 labeled radiotracers were produced with full automation on the Trasis AllinOne module without any hardware modification. We found that the fluorination condition used for radiolabeling of [¹⁸F]FPOTs can also be used for [¹⁸F]FEOTs without additional modification. The radiolabeling results of [¹⁸F]FE-TMP were comparable to [¹⁸F]FP-TMP, with decay-corrected yield of 23 ± 5 % and molar activity of 182 ± 60 GBq/mol at EOS. (+)-[¹⁸F]F-PHNO was chosen as the model reaction to test whether this method can be used for N-alkylation. The method gave a lower yield, between 11 ± 1 % but good molar activity (148 ± 6.4 GBq/mol at EOS) and radiochemical purity (> 90 %). The radiolabeling of [¹⁸F]FFMZ was used to test the formation of an ester using this method. The method gave a lower yield, between 10 ± 1 % but good molar activity (150 ± 20.5 GBq/mol at EOS) and radiochemical purity (> 90 %). All the processes gave good yields, product purities and specific activities. The summary of labeling method, decay-corrected radiochemical yield, and specific activity at EOS and run time of all four compounds is listed in Table 3.

Table 3.

Two-step fluorination methods and radiolabeling yields.

Radiotracer	No. of Runs	Yield (d.c. %)	Specific Activity GBq/mol at EOS	Run Time* (min)	Reaction
[18F]FP-TMP	50	22 ± 5	147 ± 107	70	O-alkylation
[18F]FE-TMP	5	23 ± 5	182 ± 60	65	O-alkylation
(+)-[18F]F-PHNO	2	11 ± 1	148 ± 6.4	75	N-alkylation
[18F]FFMZ	2	10 ± 1	150 ± 20.5	63	Esterification

• Starting activity ranged from 30 mCi to 800 mCi.

^*Run time was defined as the time lapse between the start of synthesis (radioactivity measured on QMA after radioactivity delivery) to end of synthesis (product formulated in final product vial), including the drying [¹⁸F]F⁻, two-step reactions, purification and formulation.

Additional notes to the above test reactions is that additional optimization will be needed for the radiolabeling of (+)-[¹⁸F]F-PHNO and [¹⁸F]FFMZ. We noticed that when TBD was used for carboxylate alkylation, the amount of base amount should be decreased to 1 eq. Other organic bases may also serve better for these reactions.

4. Conclusion

The two-step, two-vessel radiolabeling of [¹⁸F]FP-TMP has been successfully developed with the combination of filtration and a one semi-prep HPLC purification on the Trasis AllinOne. Over 50 runs of successful GMP syntheses of [¹⁸F]FP-TMP synthesis demonstrated the reliability and reproducibility of this approach.

We also have demonstrated that this method can also be used for preparation of [¹⁸F]FEOTs and its use in subsequent reactions. The versatility of the method was shown in the successful labeling of N-alkylation and O-alkylation reactions. All tracers were produced with good radiochemical yield with simplified setup. This is a proven method and can be used for supplying radiotracers used in clinical studies.

Abbreviations

PET

Position Emission Tomography

HPLC

High Pressure Liquid Chromatography

GC

Gas Chromatography

QC

Quality control

UV

Ultraviolet

SPE

Solid Phase Extraction

EOS

End of Synthesis

USP

United States Pharmacopeia

cGMP

current Good Manufacturing Practice

DMF

Dimethylformamide

MeCN

acetonitrile

RV

Reaction vessel