State of the art procedures towards reactive [18F]fluoride in PET tracer synthesis

Background Positron emission tomography (PET) is a powerful, non-invasive preclinical and clinical nuclear imaging technique used in disease diagnosis and therapy assessment. Fluorine-18 is the predominant radionuclide used for PET tracer synthesis. An impressive variety of new ‘late-stage’ radiolabeling methodologies for the preparation of 18F-labeled tracers has appeared in order to improve the efficiency of the labeling reaction. Main body Despite these developments, one outstanding challenge into the early key steps of the process remains: the preparation of reactive [18F]fluoride from oxygen-18 enriched water ([18O]H2O). In the last decade, significant changes into the trapping, elution and drying stages have been introduced. This review provides an overview of the strategies and recent developments in the production of reactive [18F]fluoride and its use for radiolabeling. Conclusion Improved, modified or even completely new fluorine-18 work-up procedures have been developed in the last decade with widespread use in base-sensitive nucleophilic 18F-fluorination reactions. The many promising developments may lead to a few standardized drying methodologies for the routine production of a broad scale of PET tracers.


Background
Molecular imaging is the in vivo visualization of biological processes at the molecular level via the interaction of a probe with its target (Gu et al. 2011).Within the field of molecular imaging, positron emission tomography (PET) is a powerful, non-invasive preclinical and clinical nuclear imaging modality.The use of PET requires administration of a tracer radiolabeled with usually short-lived positron-emitting nuclides such as 15 O, 13 N, 11 C, 18 F, 64 Cu, 68 Ga or 89 Zr.Although more positron emitting radionuclides find their way into PET applications, fluorine-18 remains the most frequently used radionuclide due to its favorable chemical and physical properties.A convenient half-life of 109.7 min, a clean decay profile (97% positron emission) and a low positron energy (maximum 0.635 MeV) resulting in high-resolution PET images (Van Der Born et al. 2017).
Several radiosynthetic methods are currently available for incorporation of fluorine-18 into biologically active compounds.In recent years, the unique and distinctive radiochemistry has been excellently reviewed (Van Der Born et al. 2017;Deng et al. 2019;Preshlock et al. 2016b).Although many different techniques have been implemented to improve the efficiency of the labeling reaction, only a few have dealt with the early key steps of the process, which consist of the removal of [ 18 F]fluoride from its oxygen-18 enriched water ([ 18 O]H 2 O) matrix and the generation of its reactive form.The landmark paper by Hamacher et al., describing a high yielding synthesis of 2-[ 18 F]fluoro-2-deoxy-D-glucose ([ 18 F]FDG), more or less defined the conventional route of producing reactive [ 18 F]fluoride (Hamacher et al. 1986).This method starts with the production of fluorine-18 by proton irradiation of [ 18 O]H 2 O in a cyclotron via the 18 O(p,n) 18 F nuclear reaction.The radionuclide is obtained as strongly hydrated [ 18 F]fluoride ions typically ranging from 50 to 200 GBq and with high molar activity (up to 1 TBq/µmol) (Aerts et al. 2010;Brichard and Aigbirhio 2014;Pees et al. 2018;Wessmann et al. 2012).The [ 18 F]fluoride is trapped on an anion-exchange resin, typically a Waters Sep-Pak QMA, allowing the removal and recovery of expensive target [ 18 O]H 2 O.The radioactivity is then eluted with a ~ 10% aqueous acetonitrile (MeCN) solution containing an inorganic base and cryptand, typically K 2 CO 3 /K 222 (20 : 40 µmol).Subsequent drying of the eluate by repeated azeotropic distillation with MeCN provides the [ 18 F]fluoride starting material.Finally, the residual anhydrous [ 18 F]KF/ K 222 complex is taken up in a polar aprotic solvent suitable for the subsequent labeling reaction.
Although optimized, the overall procedure is time-consuming in the context of 18 F-radiochemistry (around 10-15 min) and can lead to losses of significant amounts of radioactivity (up to 30%) due to unspecific adsorption on the reactor surface during azeotropic drying (Brichard and Aigbirhio 2014;Pees et al. 2018;Wessmann et al. 2012).This procedure is also complex to automate and miniaturize, e.g. for the use in microfluidic devices, and it is hard to control the precise hydration state of fluoride leading to irreproducible results.Most importantly, substantial amounts of base are needed to isolate [ 18 F]fluoride from the cartridge thereby limiting its synthetic utility in the subsequent radiofluorination.The principles of the conventional work-up still remain in modern radiofluorination methods, but significant changes into especially the elution step, but also the trapping and drying stages have been introduced in the last decade (Fig. 1).In this review, we provide an overview of the strategies and recent developments in the production of reactive [ 18 F]fluoride and their applications in PET tracer synthesis in the last decade.To demonstrate that these isolation and activation protocols are indeed valuable assets, the technical ease, efficiency (radiochemical yield (RCY), activity yield (AY), radiochemical conversion (RCC), molar activity (A m ) and time will be discussed in each case (Coenen et al. 2017;Herth et al. 2021).Where applicable, the proof-of-concept application and routine production of representative Fig. 1 Overview of the conventional route for the purification and drying of aqueous [ 18 F]fluoride with an anion-exchange cartridge and clinically relevant 18 F-labelled radiopharmaceuticals under conditions of good manufacturing practice (GMP) are described.

Trapping on anion-exchange cartridges
During trapping, the negatively-charged [ 18 F]fluoride ions are attracted to a positivelycharged resin and exchange with the negatively charged ions to adsorb to the surface while the [ 18 O]water continues to the outlet.Depending on the functional groups bound to the resin, the cartridges can be strongly or weakly basic.In addition, the negatively charged ions used in the preconditioning step are a major contributor to the basicity of the reaction mixture after elution.Hence, the type and size of anion-exchange resin as well as the preconditioning agent can substantially influence the work-up procedure of fluorine-18 with regard to azeotropic drying, losses of significant amounts of [ 18 F]F − on the reactor surfaces or the amount of base and phase transfer catalyst needed to elute [ 18 F]F − .

Solid support of the anion-exchange cartridge
Generally, [ 18 F]F − is extracted from the [ 18 O]water on a polymer resin impregnated with quaternary methylammonium salts (QMA).Due to their strong anion exchange capacity, several weaker quaternary and tertiary ammonium salts have been developed to reduce the amount of base and cryptand required to elute the [ 18 F]F − and to perform on-resin radiolabeling reactions.Alternatively, main group elements, such as boranes and phosphazenes, have been studied in the design of cartridge resins for the preparation of dry [ 18 F]fluoride in the absence of an azeotropic drying step.

Quaternary ammonium salts
Resins based on quaternary ammonium salts were investigated with a custom-synthesized 4-(N,N-dialkylamino)-pyridinium functionalized anion-exchange resin for the collection, drying and on-column 18 F-radiolabeling reaction (Mulholland et al. 1989;Toorongian et al. 1990).Problems such as solubility of counter anions and removal of phase transfer catalyst reagents were eliminated, resulting in the radiosynthesis of [ 18 F] FDG with an average AY of 41 ± 15.6% (n = 104), which is comparable to the classical method (44 ± 4% AY, n = 7).In addition, Aerts et al. described the use of long alkyl chain quaternary ammonium salts as resin to quantitatively trap [ 18 F]fluoride and recover > 85% of the trapped activity with 1 mL of a non-protic solvent (Aerts et al. 2010).However, the co-eluted concentration of water is higher than that obtained after classical azeotropic drying of a K 2 CO 3 /K 222 solution, which might cause problems in water-sensitive reactions such as Cu-catalyzed radiofluorinations.Furthermore, the quantitative recovery of [ 18 F]F − (98.5 ± 1.0%, n = 23) with just 2 µmol of K 222 /KHCO 3 in MeOH has been described using a commercially available, mixed-mode quaternary ammonium salt resin (MAX, Oasis) (Fig. 2A) (Iwata et al. 2017).The amount of K 2 CO 3 / K 222 could be even further reduced by a subsequent cation-exchange cartridge (Oasis MCX) (Iwata et al. 2018).This procedure enabled the microscale radiosynthesis several clinically relevant precursors in RCC's comparable to those reported in the literature.
Importantly, since the solvent scale is small, poor reproducibility of the radiosyntheses was obtained.

Tertiary ammonium salts
Instead of quaternary amines, a commercially available piperazine based anion-exchange resin (Oasis WAX) has been studied for trapping of the aqueous [ 18 F]F − and its release by elution with a weak anion-exchanger, in this case pyridinium sulfonate (25 µmol) in DMA (78% elution efficiency) (Fig. 2B) (Antuganov et al. 2019).The reactive [ 18 F]fluoride was then directly used in a Cu-catalyzed radiofluorination reaction obtaining high RCCs for a series of simple arylBPin substrates and a RCY of 35-38% (n = 2) for 4-[ 18 F] FPhe within 90 min.

Phosphonium borane salts
[ 18 F]Fluoride has been prepared according to a simple two-step sequence including trapping of aqueous [ 18 F]F − on a cartridge pre-loaded with the phosphonium borane [(Ph 2 MeP)C 6 H 4 (BMes 2 )] + , followed by quantitative release of [ 18 F]F − from the cartridge using a solution of tetrabutylammonium cyanide (TBACN) (one equivalent with respect to borane) in anhydrous MeCN (Fig. 2C) (Perrio et al. 2017).Loading of the cartridge with borane salt and using equimolar amounts of TBACN to replace [ 18 F]F − on the cartridge as well as to convert the excess borane salt to borane-CN are a prerequisite.Subsequent radiofluorination was successfully applied to model compounds and to the synthesis of [ 18 F]setoperone (25% RCC) (Crouzel et al. 1988).

Phosphazene bases
Based on the work by Lemaire et al. on  OH − anion-exchange to form the P 2 tBuH + [ 18 F]F − adduct (Fig. 2D) (Lemaire et al. 2010;Mathiessen and Zhuravlev 2013).The resin trapped > 97% of [ 18 F]F − after which the solution of substrate was passed through the column effecting on-column radiofluorination for several model compounds.An advantage of this method compared to the ones mentioned above, is that the column can be reused without any loss in [ 18 F]F − trapping efficiency.However, repeated use is limited to 3 runs.[ 18 F]FDG was produced with a RCY of 41% (n = 1) on a 120 GBq scale with an A m >11 GBq/µmol, which is comparable to the benchmark production of [ 18 F]FDG (44 ± 4% AY, n = 7).

Size of the anion-exchange cartridge
Reducing the size of the anion-exchange cartridge has also been considered as an alternative for reducing water and/or base content.In macro-scale reactions, this method has only been applied once in the radiosynthesis of [ 18 F]fluoroazomycin arabinosid ([ 18 F] FAZA) (Hayashi et al. 2011).A small home-made QMA cartridge containing 25 mg of the QMA resin, compared to the conventional 130 mg, resulted in a high RCY of 52.4 ± 5.3% (n = 8) with just 5.0/6.0 µmol of K 2 CO 3 /K 222 within 50.5 ± 1.5 min.

Miniature anion-exchange cartridges
One of the first reports demonstrated concentration of [ 18 F]fluoride (up to 10 GBq) in 1-5 mL of [ 18 O]H 2 O with a custom-build miniature cartridge (~ 5 µL bed volume) packed with QMA resin (Bejot et al. 2011).Trapping and release of [ 18 F]F − with only 2.1 µmol K 2 CO 3 in H 2 O followed by 4.8 µmol K 222 in MeCN resulted in 95-97% recovery of [ 18 F]F − with a total processing time of ~ 11 min.Since then, commercial micro-cartridges (OptiLynx, 5 µL bed volume) have enabled the concentration of [ 18 F]fluoride (up to 110 GBq) to a final volume of 5-45 µL (Chao et al. 2018;Elizarov et al. 2010;Lebedev et al. 2013).On average, > 99% of [ 18 F]F − is trapped and > 92% is subsequently released with a typical K 2 CO 3 /K 222 mixture (0.50/0.60 µmol) in ~ 3 min.Noteworthy, the Opti-Lynx cartridge filled with Chromabond PS-HCO 3 resin has been successfully used in the preparation of human doses of 5-[ 18 F]fluorouracil (Hoover et al. 2016).Efficient elution of [ 18 F]F − (81%) was obtained with a MeCN/H 2 O mixture of K 3 PO 4 /18-crown-6 (0.5/3.0 µmol).However, a major drawback of these procedures is the inconvenient manual packing of resin in the cartridge and the lack of reproducibility that this operation implies.

Miniature anion-exchange particles
For the integration of anion-exchange resins on the microfluidic device, several techniques have been reported.First, solution suspended anion-exchange beads have been reported by Lee et al. (2005).After trapping of fluorine-18 on a chip (26 MBq in 1 µL [ 18 O]H 2 O), 18 MBq was recovered by elution with 5 nmol of aqueous K 2 CO 3 solution and an azeotropic drying step (69% efficiency).The method provided 4 MBq of [ 18 F]FDG with a RCY of 38% within 14 min.In addition, Leonardis et al. manually loaded recyclable anion-exchange resin particles (Chromabond PS-HCO 3 ) into on-chip cavities (Leonardis et al. 2011).The trapping efficiency of 5-7 GBq [ 18 F]fluoride in 4 mL [ 18 O]H 2 O was > 90% and ≥ 95% of the trapped radioactivity was eluted with 9 µmol K 2 CO 3 in 250 µL MeCN/H 2 O within 6 min.Furthermore, concentration of [ 18 F]fluoride has been demonstrated with 10-15 mg embedded QMA resin (Sep-Pak Accell Plus cartridge) (Salvador et al. 2017).For activities of 19 GBq, trapping of 2 mL of [ 18 F]fluoride in [ 18 O]H 2 O was achieved with 98% efficiency.The trapped activity could then be eluted into a volume of 20 µL K 2 CO 3 (7.2µmol) with > 87% recovery.Instead of filling on-chip cavities, an insert element pre-filled with QMA resin (Sep-Pak ® Light QMA) has been integrated on-chip (Rensch et al. 2014).243-394 MBq in 100-500 µL [ 18 O]H 2 O was concentrated with ~ 90% efficiency, which is comparable to conventional cartridge performance.

Anion-exchange monolith
Integration of the cartridges or resin particles in the chip is typically a challenge.Therefore Ismail et al. used a functionalized polymer monolith (polystyrene imidazolium chloride) instead of packed resin particles (Ismail et al. 2014).Trapping of [ 18 F]fluoride solutions (1.5-7.4MBq) had an efficiency of 97 ± 4% (n = 39) and it was shown theoretically that higher activities could be trapped by extending the length of the monolith.Various eluents were tested, recovering at least > 85% of the activity in a volume of 200 µL.

Preconditioning of the anion-exchange cartridge
Preconditioning of the QMA cartridge has been shown to have a significant effect on the [ 18 F]F − recovery in certain cases.For example, when using an eluent of iodonium salt precursor in MeOH, the presence of HCO 3 − anions on the anion-exchange resin are key.

Oxalate and triflate counteranions (C
For Cu-mediated 18 F-radiolabeling reactions, it has been reported that a combination of appropriate preconditioning and concentration of base in the eluent can improve [ 18 F] F − recovery when eluting with weak bases (pK a < 7) as illustrated by [ 18 F]Cabozantinib (Lien et al. 2018;Mossine et al. 2017).In a full-scale experiment using 15 GBq up to 40% of [ 18 F]F − was lost due to sticking to the reaction vessel or on the anion-exchange column resulting in only 0.7% RCY.Modifying the preconditioning (oxalate solution) and the elution (1.6/6.5/15µmol of K 2 CO 3 /K 2 C 2 O 4 /K 222 ) resulted in only 13% loss of activity, with the RCY increasing to 2.8 ± 0.05% (n = 4) and A m of 17 ± 8 GBq/µmol (Fig. 3A).In general, when using weakly basic eluents such as K 2 CO 3 /K 2 C 2 O 4 /K 222 and K 2 CO 3 /KOTf, the anion-exchange cartridge should be preconditioned with the corresponding weak counteranions, i.e. oxalate and triflate, respectively (Cai et al. 2020;Guibbal et al. 2019;Li et al. 2019a;McCammant et al. 2017;Preshlock et al. 2016a;Wu et al. 2019).Also when using copper complexes as elution agents, preconditioning with aqueous LiOTf solution can result in higher [ 18 F]F − recovery, but strongly depends on the type of QMA cartridge used (Lahdenpohja et al. 2019).

)
In aliphatic radiofluorination reactions, controlling the base amount has been achieved by changing the bicarbonate counteranions on the resin to the inert mesylate anion, using 0.2 M of KOMs solution in the preconditioning step (Lee et al. 2011b(Lee et al. , 2012)).3B).Furthermore, di-and tribasic phosphates have also been reported as counteranions in aliphatic fluorination reactions, but application to a pharmaceutically relevant compound has not been reported (Seo et al. 2011).

Elution of anion-exchange cartridges with non-conventional bases, cryptands, solvents and additives
After trapping, the [ 18 F]fluoride is eluted from the cartridge.Over the years several methods have been examined to optimize, but especially simplify this process.Multiple mixtures of anions and cryptands of both high and low basicity have successfully been applied in the elution of [ 18 F]F − from an anion-exchange cartridge.In addition, solvents ranging from aqueous polar aprotic to polar protic and even charged ones have been used to significantly reduce the time of the subsequent azeotropic drying step.Moreover, instead of bases and cryptands, the elution has been performed with precursors serving as the cation.Finally, ways of making the radiolabeling reactions tolerant to water have been given attention in order to omit a drying step.

Elution with weakly basic potassium salts and cryptand in MeCN/H 2 O
Typically [ 18 F]fluoride is extracted from QMA resins by elution with a ~ 10% aqueous MeCN solution containing relatively large amounts of potassium salts and cryptand, e.g.K 2 CO 3 /K 222 (20:40 µmol).However, also weak bases and neutral phase transfer catalysts can be used for this purpose.So far, these eluents have been tailored to meet the specific needs of a subsequent reaction of interest.
A very straightforward method for decreasing the amount of K 2 CO 3 /K 222 is to omit the trapping/elution step of [ 18 F]F − and instead to directly evaporate the target enriched water.In the preparation of (S)- As an alternative, decreasing the amount of K 2 CO 3 but maintaining the amount of K 222 compared to the classical conditions, sometimes combined with using a larger amount of water, has proven to be successful in the radiolabeling of 2-nitropurinebased nucleosides, e.g.2-[ 18 F]Fludarabine (Guillouet et al. 2014;Marchand et al. 2010Marchand et al. , 2016)).Similarly, the high-base labeling conditions can be adapted by eluting [ 18 F]F − with a small aliquot of the conventional K 2 CO 3 /K 222 solution (Zlatopolskiy et al. 2015).For example, a solution of K 2 CO 3 (0.87 µmol) and K 222 (1.5 µmol) led to efficient elution of [ 18 F]F − from the QMA cartridge (82 ± 2%, n = 27), which resulted in the production of [ 18 F]FAMTO in a RCY of 18 ± 2% with an A m of 105 ± 39 GBq/µmol from EOB within 120 min (Fig. 5A) (Bongarzone et al. 2019).In contrast, the formation of [ 18 F]FAMTO under the classical conditions (K 2 CO 3 /K 222 12/20 µmol) was not observed.

Potassium carbonate/potassium oxalate/Kryptofix-222 (K
Soon after the landmark paper by Hamacher et al., a system with potassium oxalate (K 2 C 2 O 4 ) as base and minimal amounts of K 2 CO 3 (≤ 0.36 µmol) was reported for the radiofluorination of butyrophenone neuroleptics (Hamacher and Hamkens 1995;Katsifis et al. 1993).The conventional strong basic K 2 CO 3 /K 222 system led to degradation of the tosylated precursors, but with the alternative system 18 F-incorporation was facilitated.This moderately basic eluent has been used in the nucleophilic substitution of several aromatic scaffolds including those of clinically relevant radiotracers such as (Hamill et al. 2005(Hamill et al. , 2011;;Hostetler et al. 2011a, b;Labas et al. 2011)  In specific cases, not only changing to a mild basic agent is necessary, but also to a neutral phase transfer catalyst.In the radiolabeling reaction of 4-[ 18 F]fluoroglutamine all four possible isomers and a significant amount of elimination byproducts were formed in the presence of K 222 and K 2 CO 3 (Qu et al. 2011).The less basic KHCO 3 /18-Crown-6 (13/28 µmol) catalyst system led to the successful automated preparation of the desired L-glutamine isomer (2 S,4R)-4-[ 18 F]fluoroglutamine (4-[ 18 F]FGln) in 8.4 ± 3.4% AY (n = 10) and high stereoisomeric purity (> 91 ± 8%) (Fig. 5D) (Lieberman et al. 2011).

Elution with tetraalkylammonium salts as base and cryptand in MeCN/H 2 O
Tetraalkylammonium salts have an enhanced solubility in organic solvents and are therefore widely used as an alternative to the K 2 CO 3 /K 222 system.Generally, an ion exchange between the [ 18 F]F − and the counter-anion of the ammonium salt occurs during elution of the activity from the QMA resin with a tetraalkylammonium salts solution.The tetraalkylammonium/[ 18 F]F − complex serves then as the phase transfer catalyst.The different alternatives will hereafter be discussed.

Weakly basic eluents by using polar protic solvents (R-OH)
Limited amounts of base are useful in the radiolabeling of base-sensitive precursors, but they do not always efficiently elute [ 18 F]F − from the anion-exchange cartridge.For example, 3.6/15 µmol K 2 CO 3 /K 222 recovers only 18% of the radioactivity and 26 µmol TBAHCO 3 elutes 74% of the [ 18 F]F − from the QMA resin (Moon et al. 2010).In order to find a proper balance between the selection of base and [ 18 F]F − elution efficiency, polar protic solvents have been studied extensively.

Tetraethylammonium salts in n-butanol (nBuOH)
To obviate an azeotropic drying or solvent-exchange step, the elution of [ 18 F]F − from the QMA cartridge using alcohols other than MeOH or ethanol (EtOH) have been investigated (Zischler et al. 2017).The radiolabeling of boronic and stannyl substrates often use a solution of TEAB (0.7-9.4 µmol) in nBuOH to elute [ 18 F]F − from the anionexchange resin into a vial containing a solution of the precursor and copper catalyst in the desired solvent.Subsequently, the resulting solution is directly heated without a drying step.This general procedure has enabled the preparation of clinically relevant PET tracers including [ 18 F]TRACK, [ 18 F]Triacoxib, [ 18 F]FDOPA and 6-[ 18 F]fluorodopamine ([ 18 F]FDA) (Bailey et al. 2019;Litchfield et al. 2020;Zischler et al. 2017).In the case of [ 18 F]FDOPA, the unoptimized RCY (40 ± 4%, n = 1) was as high as the isolated RCY using the K 2 CO 3 /K 2 C 2 O 4 /K 222 (0.72/6.0/17 µmol) eluent with an azeotropic drying step, showing the potential of this protocol (Fig. 9D) (Preshlock et al. 2016a;Zischler et al. 2017).In contrast, high A m [ 18 F]TRACK (188 GBq/µmol) was obtained using TEAB in nBuOH, but in a AY of only 4% (n = 1), while a AY of 8% (n = 1) was achieved with a solution of TEAB in MeOH (Bailey et al. 2019;Bernard-Gauthier et al. 2018).In accordance with several studies, nBuOH enables the most efficient 18 F-incorporation from the alcohols investigated, however typically radioactivity recovery is around 80% resulting in a decrease in RCY (Zarrad et al. 2017;Zischler et al. 2017;Zlatopolskiy et al. 2018).The addition of more base to the eluent could increase [ 18 F]F − recovery to > 80%, but this can also cause a drop in RCY.

Elution with positively charged precursor molecules in alcoholic solvents: minimalist approach
Besides potassium and tetraalkylammonium salts, labeling precursors bearing a positively charged nitrogen, iodine, sulfur or oxygen functionality (onium salts) have been investigated for the quantitative recovery of [ 18 F]F − from an anion-exchange resin (Feni et al. 2017;Richarz et al. 2014).Solutions of onium salts in especially MeOH can directly extract [ 18 F]F − from a QMA resin in excellent yields of > 95%.Low-boiling methanol can subsequently be removed completely at 70-80 °C within 2-3 min without the need for azeotropic drying.The resulting onium [ 18 F]fluoride salts are directly converted into 18 F-labeled compounds without addition of a base or any other additives under so-called minimalist conditions.

Onium salts in alcoholic polar aprotic solvent
The minimalist procedure has also been successfully adapted to the radiofluorination of onium salts in the absence of a solvent exchange step.In the case of uronium salts, the [ 18 F]fluoride work-up procedure includes elution of [ 18 F]F − from the QMA with an uronium salt (11-21 µmol) in a mixture of EtOH and DMSO (Neumann et al. 2016).The elution procedure obviates the need for a solvent exchange step and subsequent heating of the resulting solution directly provides the product.However, the elution efficiency can be as low as 50% due to the low amounts of base and EtOH used.Addition of weak bases such as TBAOTf and TEAB to the eluent increased the elution efficiency, but the gain was offset by a decrease in RCY (Beyzavi et al. 2017).Under these adapted minimalist conditions, [ 18 F]Bavarostat was produced under cGMP in a RCY of 14 ± 4% (n = 23) and an average A m of 204 ± 75 GBq/µmol (Fig. 11A) (Celen et al. 2020;Strebl et al. 2017).Recently, [ 18 F]Atorvastatin has been prepared using the uronium precursor in MeOH/ DMSO (1/3 v/v), which resulted in 88 ± 5% elution efficiency, a RCY of 19 ± 6% (n = 10) and an A m of 65 ± 32 GBq/µmol (Clemente et al. 2020).In addition, a balance between [ 18 F]F − recovery and 18 F-incorporation has been achieved in the alcohol-enhanced Cu-mediated radiofluorination of (aryl)(mesityl)iodonium salts with a solution of the radiolabeling precursor (21 µmol) in 20% MeOH in DMF (Fig. 11B) (Orlovskaya et al. 2019b).A further increase of the MeOH content was detrimental and caused a rapid decrease of the 18 F-incorporation yield due to the formation of 18 F -(MeOH) n clusters which substantially decrease the nucleophilicity of fluoride.Finally, 18 F-labeled pyridines with electron-withdrawing substituent(s) has been prepared by slow of [ 18 F]F - from the QMA cartridge with a solution of a quaternary ammonium triflate precursor in MeCN/tert-butanol (tBuOH) (1/4 v/v) (Basuli et al. 2018).observed with fractions of the eluate of [K 222 ]OH -, using the entire eluate resulted in only incorporation (10-20%) caused by degradation of the precursors in the presence of large amounts of OH − .This could be compensated by increasing the amount of precursor, but several alternative procedures have been developed using the OH − /[ 18 F]F − exchange principle.These procedures use basic ligands or other non-ionic weak bases with a high conjugate pK a in the presence of trace amounts of water to generate OH − in situ (Mossine et al. 2017).

Strong bases
The first report on the in situ OH − production described the strong organic base P 2 Et (45 µmol, pK a =32.9) in aqueous MeCN, which resulted in nearly quantitative elution of [ 18 F]F − (Fig. 12A) (Lemaire et al. 2010).The elution volume was smaller with eluents containing larger amounts of water or bases with higher pK a values.Importantly, pK a values of around 30 were required for quantitative elution of the [ 18 F]F − from the cartridge.It still needs to be clarified whether a base strength of this magnitude is compatible with common precursors for nucleophilic 18 F-fluorination.

Metal catalysts
[ 18 F]Fluoride can also be eluted from the anion-exchange cartridge using an organic, aqueous solution of metal catalysts.In case of Cu-mediated radiofluorination reactions, Cu(OTf ) 2 (48 µmol), and to a lesser extent Cu(OTf ) 2 (py) 4 , in DMA have been shown to be a suitable elution agent for [ 18 F]F − (79% elution efficiency) (Lahdenpohja et al. 2019;Mossine et al. 2017).Even though higher amounts of copper complex slightly improved the [ 18 F]F − recovery, large amounts of copper in the reaction solution complicated the purification of the final products on preparative scale.An example of this method has been given by Preshlock et al. in the automated production of [ 18 F]FMZ with a AY of 16% (n = 1) using Cu(OTf ) 2 (py) 4 (40 µmol) in DMA as eluent (Fig. 12D) (Preshlock et al. 2016a).Also a solution of manganese catalysts (4-30 µmol) in pure acetone or a mixture with MeCN can directly elute [ 18 F]F − from an anion-exchange cartridge with > 90% efficiency (Huang et al. 2014;Liu et al. 2018).The eluate has successfully been used in the late-stage 18 F-labeling of several unactivated aliphatic C − H bonds.

Direct addition of [ 18 F]fluoride in [ 18 O]H 2 O to reaction medium with additives
An alternative to the radiofluorination on a solid-phase without a need of azeotropic drying is the use of additives in the reaction medium.These additives allow the radiolabeling reaction to take place in the presence of a significant amount of water.To this end, highly viscous solvents, metal catalysts and enzymes have been studied as additives.

Ionic liquids
The rapid and convenient synthesis of 18 F-fluoroalkanes has been enabled by the use of ionic liquids as reaction media, allowing the direct addition of the [ 18 O]H 2 O target water containing [ 18 F]fluoride and obviating the time-consuming azeotropic drying step (Kim et al. 2003(Kim et al. , 2004)).Generally, ionic liquids contain a lipophilic cation moiety based on imidazolium salts e.g.[bmim] and a counter anion that does not possess an exchangeable fluorine to prevent exchange with the [ 18 F]fluoride ion.However, large amounts of starting activity are not possible, because amounts of H 2 O in the reaction mixture reduce the yield of the 18 F-fluorinated product.Besides a successful application in the synthesis of [ 18 F]FDG, RCY of 59.1 ± 5.1% (dc, n = 3) compared to 44 ± 4% AY (n = 7) with the conventional method, no clinically relevant compounds or drug scaffolds have been shown (Fig. 13A) (Hamacher et al. 1986;Kim et al. 2003Kim et al. , 2004)).A severe disadvantage of this approach is the high viscosity of these solvents, which limits their wider application.

Catalysts
Sergeev et al. reported the radiofluorination in up to 25 vol% of water by using titanium dioxide (TiO 2 ) nanoparticles as catalyst (Sergeev et al. 2015).The TiO 2 catalyst coordinates both aqueous [ 18 F]fluoride and tosyl precursors to its surface via hydrogen bonding and thereby facilitates the reaction.In the presence of TBAHCO 3 (0.36 µmol) as a phase-transfer agent, efficient radiolabeling of aromatic, aliphatic, and cycloaliphatic tosylated precursors has been performed without the need for a drying step.For example, the RCY and A m for TiO 2 catalyzed synthesis of [ 18 F]Fallypride were found to be 71.0 ± 2.3% (n = 3) and 185 ± 74 GBq/µmol, respectively (Fig. 13B).This is similar to those typically obtained with conventional procedures, e.g.66 ± 8% RCY (n = 8) and A m of 15-78 GBq/µmol with 15 µmol TBAHCO 3 and azeotropic drying (Lazari et al. 2014).However, increasing the amount of radioactivity in the reaction also increases the volume of water, which consequently requires a [ 18 F]fluoride concentration step.In addition, triflate, nosylate and precursors with additional oxo moieties are not compatible with this method.Finally, enzymatic approaches offer a unique, mild, and selective method for the incorporation of fluorine-18 into substrates modified both on the adenine base and on the ribose ring by direct addition of [ 18 F]F − in [ 18 O]H 2 O.The fluorinase enzyme from Streptomyces Cattleya has been successfully applied as catalyst in the 18 F-labeling of nucleosides, e.g.[ 18 F]-5′-fluoro-5′-deoxyadenosine ([ 18 F]-5′-FDA), [ 18 F]-5′-fluoro-5′-deoxyinosine and [ 18 F]-5-fluoro-5-deoxyribose (Deng et al. 2006;Martarello et al. 2003;Winkler et al. 2008).However, the fluorinase is a relatively specific enzyme, it can never be a general fluorination reagent, and large amounts of the enzyme are required which makes purification of the product rather difficult.

Drying of reactive [ 18 F]fluoride ion from [ 18 O]H 2 O
A complete methodological alternative to avoid a solvent evaporation step or the drying on a stationary phase is the conversion of aqueous [ 18 F]fluoride into gaseous 18 F-synthons or deposing it in an electric field.In both methods the radionuclide can be easily dried, either via distillation and trapping in a solvent or on an activated resin or via releasing under a low electric field.Both methods make an azeotropic drying process obsolete.F]HF by an argon flow and subsequent trapping on solid bound phosphazene base P 2 Et (Fig. 14A) (Mathiessen et al. 2011).Under optimal conditions, the [ 18 F]HF transfer yield was 16-82%.Mannose triflate (52 µmol) was converted into [ 18 F]FDG at 120 °C for 20 min without any loss of stereochemical integrity and a RCY of 82 ± 5% (n = 3).However, this method appears technologically demanding for automation.[ 18 F]AcF as novel synthon for the transfer of [ 18 F]fluoride through gas lines in an anhydrous form was produced by trapping [ 18 F]F − on an anion-exchange column (MP-64) at 60 °C followed by the reaction with acetic anhydride (Fig. 14B) (Jiang et al. 2015).The resultant gaseous [ 18 F]AcF was efficiently trapped in anhydrous apolar protic solvents after purification through a column containing Porapak Q resin and sodium sulfate at 30 °C in 87 ± 3% RCY (n = 10) in 15 min.The purified [ 18 F]AcF has also been trapped on solid-phase extraction cartridges such as Oasis WAX, but limited information is available to date.Following dissolution of [ 18 F]AcF in MeCN containing TEAB (52 µmol),   O]H 2 O by applying a potential and then release the activity into 275 µL of eluent with an overall efficiency of ~ 60% (Saiki et al. 2010;Wong et al. 2012).An alternative platform makes use electrowetting-on-dielectric (EWOD) (Chen et al. 2014;Keng et al. 2012).An EWOD microfluidic chip typically consists of two parallel plates, one patterned with electrodes and one coated with a conductor to act as a ground electrode.An electrical potential is then applied to the desired electrodes to achieve operations such as droplet transport or heating.For example, a 200 µL droplet of [ 18 F]fluoride in [ 18 O]H 2 O with base and phase transfer catalyst is loaded and its volume reduced by heating the nearby electrodes.An electrical potential is then applied to the electrodes to move the droplet to a heating site.Here, the target water is further removed by evaporation resulting in a residue of 5 µL in 10 min.[ 18 F]FLT and [ 18 F]Fallypride have been successfully synthesized on an EWOD device in 63 ± 5% RCY (n = 5) and 65 ± 6% RCY (n = 7), respectively (Javed et al. 2014a, b).These yields are comparable to, or greater than, those obtained by conventional approaches e.g.40% RCY (n = 1) for [ 18 F]FLT and 66 ± 8% RCY (n = 8) for [ 18 F]Fallypride (Lazari et al. 2014;Suehiro et al. 2007).In addition, the A m of [ 18 F]Fallypride (730 GBq/µmol) was two times higher on the EWOD device than synthesized in a macroscale radiosynthesizer (330 GBq/µmol), despite starting with significantly less radioactivity (270 MBq and 7400 MBq, respectively).Although this approach is suitable for modest starting volumes, impractically large chips and many time-consuming sequential 200 µL evaporations are required to handle volumes of ~ 2 mL of [ 18 F]fluoride in enriched water that are generated in most cyclotrons.

Magnetic droplet microfluidics (MDM)
Finally, Fiel et al. described the use of magnetic particles to manipulate [ 18 O]H 2 O droplets in the [ 18 F]fluoride preconcentration step (Fiel et al. 2015).A magnet is moved to manipulate the magnetic particles which hold the small target water droplet by surface tension, but also possess an anion-exchange property that is capable of capturing and releasing the [ 18 F]F − .The capture efficiency was 79% using low initial activities (~ 74 MBq) and 25 mg of magnetic particles.After evaporation of the solvent, [ 18 F]F − is released with 50 µL of K 2 CO 3 (7.3µmol) in 91-95% efficiency.The preconcentration step took approximately 5 min and the [ 18 F]fluoride solution was preconcentrated by 15-fold.However, it was found that when the initial 18 F-activity was increased to ~ 300 MBq, the capture efficiency already decreased to ca. 59%, which makes clinical application questionable.

Conclusions
The large number of publications on the preparation of reactive [ 18 F]fluoride from oxygen-18 enriched water over the last decade reflects the growing importance of the complex activation step in 18 F-fluorination chemistry.Success and progress have been achieved in base-sensitive nucleophilic 18 F-fluorination reactions, providing improved, modified or even completely new fluorine-18 work-up procedures.The currently most used elution system still comprises the conventional K 2 CO 3 /K 222 eluent, but the mild base tetraethylammonium bicarbonate (TEAB) as phase-transfer catalysts has been shown to be a useful alternative in the 18 F-labeling of iodonium(III) containing precursors, e.g.[ 18 F]UCB-J.In addition, the moderately basic K 2 CO 3 /K 2 C 2 O 4 /K 222 eluent as well as positively charged precursors in alcoholic medium, i.e. the minimalist approach, in especially Cu-mediated nucleophilic 18 F-fluorination reactions have found widespread use.
It should be mentioned that many developments listed in this review only comprise small adaptations compared to the conventional methodology and are focused on a specific reaction or substrate.Hence, several of these methods have already proven their advantage and suitability for 18 F-labeled tracer synthesis, but more comparative evaluations are desirable.In contrast, new approaches have been reported and hold promise, but often only concern specific reactions with primarily simple substrates as application.From these methods, the recent [ 18 F]triflyl fluoride procedure has to be mentioned as a successful example of a new [ 18 F]fluoride work-up methodology because of its use in clinical PET tracer production.
Despite the large variety of new activation procedures, none has completely replaced the conventional K 2 CO 3 /K 222 elution method and further expansion of miniaturization and kit-like procedures is an area of research with room for improvement.Automated cassettes and technologies like microfluidic devices to produce reactive [ 18 F]fluoride will increasingly be used in PET chemistry by providing safe, standardized and automated GMP production tools.Considering the many promising developments going on, there is great confidence in further inventions and the establishment of a few standardized drying methodologies for the routine production of a broad scale of PET tracers.

Fig. 7 A
Fig. 7 A Radiosynthesis of [ 18 F]DCFPyL after elution of [ 18 F]fluoride with a TBAHCO 3 solution.B Radiosynthesis of [ 18 F]FDPA after elution of [ 18 F]fluoride with a very mild basic TBAOMs solution.C Radiosynthesis of [ 18 F]FDOPA after elution of [ 18 F]fluoride with a weakly basic TBAOTf solution.D Radiosynthesis of [ 18 F]Uracil after elution of [ 18 F]fluoride with a TEAB solution

Fig. 10
Fig. 10 Radiosynthesis of [ 18 F]FDOPA after elution of [ 18 F]fluoride with the diaryliodonium precursor in an alcoholic solution (A) and the conventional K 2 CO 3 /K 222 solution (B) Fig. 11 A Radiosynthesis of [ 18 F]Bavarostat after elution of [ 18 F]fluoride with the uronium precursor in an alcoholic solution.B Radiosynthesis of 4-[ 18 F]FPhe after elution of [ 18 F]fluoride with the iodonium precursor in alcoholic polar aprotic solution

Fig. 12
Fig. 12 Radiosynthesis of [ 18 F]FDG after elution of [ 18 F]fluoride with the strong basic P 2 Et in aqueous MeCN (A) and the weak basic K 2 HPO 4 in aqueous tBuOH (b); radiosynthesis of [ 18 F]FMZ after elution of [ 18 F]fluoride with the weak basic DMAP•OTf in aqueous DMF (C) and the metal catalyst Cu(OTf ) 2 (py) 4 in aqueous MeCN (D); n.m.: not mentioned 18 F]fluoride species via 18 F-gaseous synthons -[ 18 F]fluoride relay A relatively new concept for the synthesis of reactive [ 18 F]fluoride species is [ 18 F]fluoride relay.In [ 18 F]fluoride relay, fluorine-18 is first transferred from aqueous [ 18 F]F − to an

18 F
-fluorination of the mannose triflate provided [ 18 F]FDG in > 90% incorporation rate.Except for a few model radiofluorination reactions, the scope of [ 18 F]AcF has not been investigated to date.