The effects of trace metal impurities on Ga-68-radiolabelling with a tris(3-hydroxy-1,6-dimethylpyridin-4-one) (THP) chelator

GMP-grade 68Ge/68Ga generators provide access to positron-emitting 68Ga, enabling preparation of Positron Emission Tomography (PET) tracers and PET imaging at sites that do not have access to cyclotron-produced radionuclides. Radiotracers based on tris(3-hydroxy-1,6-dimethylpyridin-4-one) (THP) chelators enable simple one-step preparations of 68Ga PET radiopharmaceuticals from pre-fabricated kits without pre-processing of generator eluate or post-purification. However, trace metal impurities eluted along with 68Ga could compete for THP and reduce radiochemical yields (RCY). We have quantified trace metal impurities in 68Ga eluate from an Eckert & Ziegler (E&Z) generator using ICP-MS. The metals Al, Fe, natGa, Pb, Ti and natZn were present in generator eluate in significantly higher concentrations than in the starting eluent solution. Concentrations of Fe and natGa in eluate were in the range of 0.01–0.1 μM, Al, Zn and Pb in the range of 0.1–1 μM, and Ti in the range of 0.9–1.5 μM. To assess the ability of THP to chelate 68Ga in the presence of such metal ions, radiolabelling reactions were undertaken in which selected metal ions were added to make them equimolar with THP, or higher. Al3+, Fe3+, natGa3+ and Ti4+ reduced RCY at concentrations equimolar with THP and higher, but at lower concentrations they did not affect RCY. Pb2+, Zn2+, Ni2+ and Cr3+ had no effect on RCY (even under conditions in which each metal ion was present in 100-fold molar excess over THP). The multi-sample ICP-MS analysis reported here is (to date) the most comprehensive and robust quantification of metal impurities in the widely used E&Z 68Ga generator. 68Ga from an E&Z generator enables near-quantitative radiolabelling of THP at chelator concentrations as low as 5 μM (lower than other common gallium chelators) without pre-processing. The combination of Al3+, Fe3+, natGa3+ and Ti4+ in unprocessed 68Ga eluate is likely to decrease RCY of 68Ga radiolabelling if a lower amount of THP chelator is used, and future kit design should take this into account. To increase specific activities by using even lower THP concentrations, purification of 68Ga from trace metal ions will likely be required.


Background
Molecular positron emission tomography (PET) imaging with radiolabelled receptor-targeted peptides and proteins has been transformative in management of cancer patients in clinical centres where it is available. 1,2 Gallium-68 ( 68 Ga) is a metallic radionuclide that emits positrons (t 1/2 ¼ 68 min, b + 90%, E max 1880 keV) suitable for diagnostic imaging with 68 Ga-labelled chelator-peptide conjugates. 3 The short half-life of 68 Ga is compatible with the fast clearance from blood and rapid target localisation of 68 Ga-labelled chelator-peptide conjugates. However, it also necessitates fast radiolabelling approaches.
One-step, kit-based radiolabelling protocols that provide a radiopharmaceutical in quantitative radiochemical yield (RCY), and in pyrogen-free, physiologically-compatible solutions are the ideal for 68 Ga radiopharmaceuticals. 4,5 Such kitbased radiosyntheses reduce the need for expensive infrastructure and highly trained personnel. Kit-based radiosynthesis protocols have been used for formulation of 99m Tc radiopharmaceuticals for almost ve decades. These "one-step" kits consist of a single pre-fabricated vial that contains all nonradioactive components and reagents. For simple kit-based radiosynthesis of 68 Ga radiopharmaceuticals to be realised, the nuclear medicine community needs chelators that bind 68 Ga rapidly (<5 min) and quantitatively (RCY > 95%) at low chelator concentrations (mM range) and near physiological pH (6)(7), preferably avoiding heating or other additional manipulations.
DOTA and HBED-CC chelators are used in the radiopharmaceuticals 68 Ga-DOTA-TATE for imaging neuroendocrine tumours 6 and 68 Ga-HBED-PSMA for imaging prostate tumours, 7 respectively. To date, these chelators have typically required preprocessing of 68 Ga generator eluate to remove competing trace metal impurities and concentrate 68 Ga 3+ , heating to reproducibly incorporate 68 Ga 3+ into the chelator, and post-synthetic purication to remove unreacted 68 Ga 3+ and physiologicallyincompatible buffer components. [8][9][10] Hence, they do not meet the ideal requirements of kit-based radiolabelling.
We have recently developed a THP (tris(3-hydroxy-1,6dimethylpyridin-4-one)) chelator ( Fig. 1) that rapidly and quantitatively incorporates 68 Ga 3+ at room temperature, neutral pH, and low chelator concentrations, giving a single species. [11][12][13] It is consequently well-suited to kit-based 68 Garadiopharmceutical synthesis, unlike other common gallium chelators such as DOTA and HBED-CC. THP has demonstrated superior radiolabelling properties compared to many other chelators, with higher RCYs achievable for THP at very low concentration compared to e.g. DOTA and HBED. 14 THP has been attached to a number of peptides and proteins that target cancer cell surface receptors and the conjugates demonstrate favourable biodistribution and targeting properties in in vivo PET imaging studies. 5,11,[15][16][17][18][19] A THP bioconjugate, THP-PSMA, that targets the prostate specic membrane antigen (PSMA) has been evaluated recently for one-step kit-based radiolabelling with 68 Ga. 5 These resulting GalliProst™ kits can be radiolabelled in 2-5 min, simply by addition of unprocessed 68 Ga generator eluate (typically 5 mL of 0.1 M aqueous HCl containing 170-270 MBq of 68 Ga) to a vial containing THP-PSMA and sodium bicarbonate buffer. 5,16 A new homologue of THP has been developed with further improved radiolabelling properties and affinity and selectivity for Ga 3+ . 13 Currently, 68 Ga is usually produced from pharmaceutical grade 68 Ge/ 68 Ga generators. 20,21 In these generators, the parent radionuclide 68 Ge (t 1/2 ¼ 271 days) is adsorbed on a stationary phase, typically TiO 2 . 68 Ge decay yields 68 Ga, which is normally eluted with an acidic solution (typically aqueous hydrochloric acid) as 68 Ga 3+ . Frequent elutions and continuous use of acid can result in leaching of other metal ions from the generator stationary phase. These can signicantly reduce radiolabelling efficiency due to competition with the radiometal ion for chelator binding. 22 Additionally, the concentration of 68 Ga 3+ is very low: 1 GBq of carrier-free 68 Ga contains only 9.8 pmol of 68 Ga 3+ , equivalent to approx. 2 nM concentration in 5 mL of generator eluate. Most chelators do not exclusively bind a single metal ion, and so both the selectivity of a chelator and the purity of 68 Ga eluate can inuence the RCYs of these reactions. Some 68 Ga chelators, such as HBED, 23 NOTA 24 and TRAP 24 have demonstrated selectivity for Ga 3+ over divalent metal ions.
THP ligands were originally developed as iron-sequestering chelators, 25,26 and so as well as having high affinity for Ga 3+ , THP also has high affinity for Fe 3+ , Al 3+ and other oxiphilic transition metal ions. 12,13,[27][28][29] The highest molar activity that we have achieved for a THP conjugate to date is 80 MBq nmol À1 , 11,19 equating to <0.1% 68 Ga 3+ occupancy of THP in these solutions. This is comparatively low compared to other radiometallated conjugatesfor example, some DTPA-or DOTA-conjugates with 111 In can achieve specic activities up to 1 GBq nmol À1 , 30 and TRAP-conjugates with 68 Ga can achieve over 4.8 GBq nmol À1 , although the latter is obtained under reaction conditions that result in <70% RCY. 31 It is likely that competition from interfering metal ions in 68 Ga generator eluate limits achievement of higher molar activities for THP.
The objectives of this study were to (i) quantify trace metal impurities in 68 Ga eluate from an Eckert & Ziegler generator and (ii) evaluate the competition effects of the most common contaminant metal ions on 68 Ga radiolabelling with THP.

Reagents
Sterile ultrapure 0.1 M hydrochloric acid aqueous solution for elution of 68 Ga from 68 Ge/ 68 Ga Eckert & Ziegler generator was supplied by ABX GmbH (Radeberg, Germany). One-year old generators (yielding 350-450 MBq) were used in this study. Lead, titanium and zinc standard solutions (1000 mg L À1 ) for AAS were purchased from VWR. Gallium and nickel standards for AAS (TraceCERT TM, 1000 mg L À1 ) were purchased from Sigma-Aldrich. Chromium (as Cr 3+ ) solution for ICP (1000 mg L À1 ) was purchased from Fisher Chemical. Aluminium standard for ICP (1000 mg L À1 ) was purchased from Fluka. Iron AAS standard solution (Specpure®, 1000 mg L À1 ) was from Alfa Aesar. Sodium chloride ultrapure bioreagent was obtained from Fisher Scientic. (+)-Sodium L-ascorbate BioXtra was obtained from Sigma-Aldrich. Hydrochloric acid 20% Primar for trace metal analysis from Fisher Chemical was used for conditioning SCX columns. THP was synthesised according to a previously described procedure. 32 Stock solutions of THP in water (UltraPure™ distilled water from Invitrogen) were stored at À20 C. Instant thin layer chromatography strips impregnated with silica gel (iTLC-SG) were obtained from Agilent Technologies and used to determine RCYs. Ethanol and acetone were of HPLC-grade.
Determination of trace metals in 68 Ga generator eluate 68 Ga 3+ was eluted from an E&Z 68 Ge/ 68 Ga generator with ultrapure HCl (5 mL, 0.1 M) in a single fraction. Eluates were allowed to decay for at least 24 h prior to analysis by ICP-MS. Eluent (ultrapure 0.1 M HCl) was also analysed as a blank. All samples were stored in polypropylene tubes (Elkay, cat. no. 2086) with low trace metal content. The concentration of trace metals was determined on a PerkinElmer NexION 350D inductively coupled plasma mass spectrometer running Syngistix v1.0 soware (London Metallomics Facility, King's College London, UK). The acquisition mode included 5 replicates averaged to give reported values for 27

Competition experiments
To evaluate competition effects of trace metals, THP (5 mM) in sodium bicarbonate buffer was radiolabelled with 68 Ga in the presence of additional Al 3+ , Fe 3+ , nat Ga 3+ , Ti 4+ , Pb 2+ , Zn 2+ , Ni 2+ and Cr 3+ (0.05-500 mM), using the following solutions: THP solutions. An aliquot of THP solution (54 mM, 28 mL) was added to an aliquot of sodium bicarbonate solution (66-88 mL, 0.5 M, pH 8.35) to provide 5 mM concentration of chelator in the nal reaction mixture. As the metal ion standards contained different amounts of acid, the volume of sodium bicarbonate (plus a 10% excess) was separately calculated for each radiolabelling, in order to neutralise 0.1 M HCl (in 68 Ga 3+ eluate) and the additional H + from trace metal stock solutions. Radiolabelling reactions. The solutions containing 68 Ga and added trace metal ion were added to the THP solutions. The reaction mixture was made up to 300 mL total volume (using 50-116 mL of 0.1 M HCl), and contained 1.1 mg (1.5 nmol, 5 mM) of THP. In each reaction solution, the nal pH was 7. The reaction mixture was vortexed and incubated at room temperature for 10 min. Each radiolabelling experiment was replicated three times, with each experiment consisting of 5 technical replicates.
Radiolabelling reactions in the presence of ascorbate. Aqueous sodium L-ascorbate was added to a solution containing 68 Ga 3+ and Fe 3+ , prior to addition of THP. In these reaction solutions, the nal concentration of ascorbate was 166 mM in 300 mL total volume. The pH and the concentrations of THP, 68 Ga 3+ and Fe 3+ were as described above.
Radiolabelling with solutions 68 Ga 3+ pre-processed using cation exchange methodologies Radiolabelling of THP was tested with 68 Ga "pre-processed" on a SCX Bond Elut 100 mg cartridge using methods that use acidied NaCl, 33 ethanol 9 or acetone 10 solutions to elute the trapped 68 Ga 3+ from the cation exchange resin. The amount of NaHCO 3 required to neutralise processed 68 Ga was determined experimentally by titration (in order to account for extra H + released from the SCX column). Processed eluate (50 mL, 7-19 MBq 68 Ga) was added to a solution of THP (5 mM in a nal volume of 300 mL at pH 7). Aer incubation at RT for 10 min, RCY was determined by iTLC.

Trace metal impurities
Using ICP-MS, we determined the concentrations of common trace metal impurities (Al, Cr, Cu, Fe, nat Ga, Mn, Ni, Pb, Sc, Sn, Ti, V, 66 Zn and 68 Zn) in 68 Ga eluate. The concentrations of trace metals are known to correlate with the amount of time between generator elutions. 14,22 To probe this, we obtained generator eluate (5 mL in 0.1 M HCl) either (i) 2 hours aer a previous generator elution (sample set A), or (ii) 1 day aer a previous elution (sample set B). These samples were collected within a period of 20 days, from a generator eluting 330-480 MBq. The mean concentrations of Cr, Cu, Mn, Ni, Sc, Sn and V were all very low (below 10 nM) in both sample sets A and B, and in blank eluent solutions of 0.1 M HCl. We have therefore focused on transition and main group metals Al, Fe, nat Ga, Pb, Ti and Zn (Fig. 2). The mean concentrations of metals Al, Fe, nat Ga, Pb, Ti and nat Zn were all higher in sample sets A and B than in eluent (blank) solution. These concentration differences between eluate and blank solution were statistically signicant in all cases (p < 0.1, Table SI-1 †), with the exception of the difference between mean nat Zn concentrations in sample set A and blank solutions.
The metal present at highest concentrations in generator eluate was Ti. Mean Ti concentration was 1.17 AE 0.37 mM in sample set A, and 0.95 AE 0.53 mM in sample set B. There was no signicant difference in mean Ti concentration between sample sets A and B. The high concentration of Ti in eluate has been previously observed by us and others: it arises from the solid phase titanium dioxide material (contained in a borosilicate glass column) on which 68 Ge 4+ is immobilised. 14,34 The trivalent metal ions Al 3+ and Fe 3+ bind hydroxypyridinones with high affinities, 12,13,[27][28][29]  Many chelators used for 68 Ga radiolabelling, including DOTA, 35 have high affinity for divalent cations such as Zn 2+ and Pb 2+ . Zn impurities arise from the eluent solution, leaching from generator components and decay of 68 Ga. The presence of Zn (occurring as Zn 2+ ) in generator eluate can compromise 68 Ga radiolabelling of DOTA-based radiopharmaceuticals. 21,36,37 In some clinical 68 Ga radiosynthesis protocols, the generator eluate undergoes pre-processing to separate Zn 2+ from 68 Ga prior to biomolecule radiolabelling. 10,38 68 Ga decays exclusively to 68 Zn, and this contributes to the amount of Zn 2+ present in eluate solution. Factors that directly correlate with the amount of 68 Zn in generator eluate include the activity of the generator, and the amount of time between elutions of the generator. Here, we quantied (i) nat Zn concentration (using 66 Zn ICP-MS signal), and (ii) the amount of 68 Zn arising from decay of 68 Ga, " decay68 Zn" (using 68 Zn ICP-MS signal, and subtracting known proportions of nat68 Zn from this measurement).
Mean nat Zn concentration measured 0.21 AE 0.13 mM in sample set A, and 0.30 AE 0.07 mM in sample set B and was higher in both sample sets compared to blank eluent solution (0.15 AE 0.16 mM), although this was only statistically signicant for sample set B (mean difference ¼ 0.15, p ¼ 0.09). Mean decay68 Zn concentration measured 6.1 Â 10 À4 AE 3.4 Â 10 À3 mM in sample set A (in which samples were collected 2 hours aer a previous elution), and 2.9 AE 0.7 Â 10 À2 mM in sample set B (in which samples were collected 1 day aer a previous elution) (mean difference ¼ 2.8 Â 10 À2 , p < 10 À4 ).
Mean eluate Pb concentrations in both sample set A (0.13 AE 0.16 mM) and sample set B (0.44 AE 0.56 mM) were higher than that observed in blank eluent solution (0.03 AE 0.03 mM), and there was high variability in Pb concentration between individual samples in both sample sets A and B. There was no statistically signicant difference between Pb concentrations in sample sets A and B. It is likely that Pb content arises from lead structures used to the shield the generator. Others have also observed signicant Pb content in generator eluate. 34

Changes in Al, Fe, Ga and Ti concentrations in eluate over six months
To further probe Al, Fe, Ga and Ti concentration in generator eluate, eluate samples were collected six months apart from each other, from a second E&Z generator (Fig. 3). These samples were all obtained 2 hours aer a previous elution. During this six month period, between our two sample collection campaigns, the generator was eluted 130 times.
The most remarkable change was observed for mean Ti breakthrough, which decreased from 1.51 AE 0.09 mM to 0.92 AE 0.05 mM (mean difference ¼ 0.59 mM, p ¼ 1.8 Â 10 À5 ) within six months ( Fig. 3 and Table SI-3 †). Concentrations of Al and nat Ga over this time also decreased. Mean Al concentration decreased from 0.45 AE 0.13 mM to 0.31 AE 0.05 mM (mean difference ¼ 0.14 mM, p ¼ 6.11 Â 10 À2 ) and mean nat Ga concentration decreased from 0.064 AE 0.022 mM to 0.015 AE 0.018 mM (mean difference ¼ 0.049 mM, p ¼ 1.27 Â 10 À2 ). The mean concentration of Fe also decreased from 0.077 AE 0.020 to 0.054 AE 0.020 mM, however in our sample set, this was not statistically signicant.

The effect of metal ion impurities in 68 Ga radiolabelling reactions with THP
To identify the effect of the presence of trace/interfering metal ions on radiolabelling THP with 68 Ga, THP was radiolabelled with 68 Ga in a solution containing a spike of each metal ion. We included the metals identied as being present in generator eluate at concentrations >10 nM (Al, Fe, nat Ga, Pb, Ti and nat Zn), as well as Ni and Cr, which are present in steel needles oen used for preparation of radiopharmaceuticals. In these experiments, we used only a small amount of 68 Ga eluate (2-6 MBq, 70-90 mL) to minimise the amount of interfering metal ions from other sources, such as the generator itself. We have also assumed that the metal ions are present as Al 3+ , Fe 3+ , nat Ga 3+ , Pb 2+ , Ti 4+ , Zn 2+ , Ni 2+ and Cr 3+ .
The results of competition experiments with THP and 68 Ga 3+ in the presence of a single spiked metal ion (at metal ion concentrations of 50 nM to 500 mM, THP concentrations of 5 mM, pH 7 in carbonate solution) are shown in Fig. 4 and summarised in Table SI In many radiopharmaceutical formulations, ascorbic acid or sodium ascorbate is included to minimise radiolysis of the solution. The presence of sodium ascorbate (166 mM, THP concentrations of 5 mM, and 5-7 MBq 68 Ga, pH 7 in bicarbonate solution) in THP radiolabelling reactions did not reduce radiolabelling efficiency of THP with 68 Ga (RCY 96 AE 0.4%, n ¼ 6). Ascorbate can reduce Fe 3+ to Fe 2+ . To assess the practical implications of this, the RCY of [ 68 Ga(THP)] in reactions containing 68 Ga 3+ , THP, a spike of Fe 3+ and ascorbate were compared with the RCY of reactions containing 68 Ga, THP and a spike of Fe 3+ only (Fig. 5) Fig. 3 Concentrations of Al, Ti, Fe and nat Ga in 68 Ga eluate as a function of generator age. Two sets of eluate samples were collected from the same 68 Ga E&Z generator, six months apart from each other. "Blank" eluent (0.1 M HCl solution from ABX Gmb) used in these two sets of samples was also measured. During this period, the generator was eluted 130 times. All eluates were sampled with a pre-elution window of 2 h. Error bars represent standard deviation of concentrations in the samples.

Radiolabelling THP with cation-exchange processed 68 Ga
Numerous clinical 68 Ga-radiolabelling protocols include eluate pre-processing steps in which 68 Ga is isolated on a cation exchange cartridge, followed by a washing step and subsequent elution of 68 Ga from the cartridge. These methods concentrate 68 Ga and isolate 68 Ga from (i) non-radioactive metal ion impurities (such as Fe, Ti and Zn) and (ii) 68 Ge that is oen present, albeit in very low amounts, in generator eluate containing high concentrations of HCl. There are three well-used methods that employ solutions of different composition to wash and to release 68 Ga from the cartridge: the "NaCl method", in which aqueous 68 Ga solutions contain 5 M NaCl and 0.13 M HCl, 33,38 the "ethanol method", in which 68 Ga solutions contain 90% ethanol and 0.9 M HCl, 9 and the "acetone method", in which 68 Ga solutions contain 97.6% acetone and 0.05 M HCl. 10 The reactivity and speciation of 68 Ga in these mixtures with organic solvents could have effects on radiolabelling efficiency. To assess the tolerance of THP radiolabelling with preprocessed 68 Ga in the presence of other components in solution such as NaCl, ethanol and acetone, we reacted THP with solutions of 68 Ga processed according to these three methods. In these aqueous reaction solutions (nal volume 300 mL) containing pre-processed 68 Ga eluate (7-19 MBq, 50 mL, containing either ethanol, acetone or NaCl eluate solutions), the nal THP concentration was 5 mM or 0.5 mM, and pH ¼ 7.
We compared the RCY of these reactions with the RCY of reactions using unprocessed 68 Ga (eluate straight from the generator). At 5 mM THP concentration, the RCY of [ 68 Ga(THP)] was unaffected by using 68 Ga eluent processed using either the NaCl method or the ethanol method, with RCY >96% (Table 1). At 0.5 mM THP concentration, the RCY was unaffected for reactions containing 68 Ga eluent processed using the ethanol method, and slightly improved for reactions containing 68 Ga eluent processed using the NaCl method (Table 1). In contrast, in reactions containing 68 Ga eluent processed using the acetone method, the RCY of [ 68 Ga(THP)] was signicantly decreased at both THP concentrations of 5 mM and 0.5 mM.

Discussion
Previous studies 12,13,[27][28][29] have shown that THP and hydroxypyridinones have particularly high affinity for hard, oxiphilic cations such as Al 3+ , Fe 3+ and Ga 3+ , with markedly lower affinity for divalent metal ions such as Zn 2+ and Cu 2+ . Although THP has shown remarkably high affinity for Ga 3+ (log K 1 ¼ 35.0, and pGa ¼ 30.0 at pH 7.4) and quantitative RCYs when reacted with 68 Ga 3+ at chelator concentrations as low as 0.5 mM, trace metal impurities, particularly hard, oxiphilic metal ions, have a potential to compete with 68 Ga 3+ for complexation to THP. 12,13,19 For example, for [Fe(THP)], log K 1 ¼ 34.2, and p M ¼ 29.1 at pH 7.4. 13 Our competition experiments showed that the presence of Al 3+ , Fe 3+ , nat Ga 3+ and Ti 4+ reduces RCY of [ 68 Ga(THP)] at concentrations equimolar with THP and higher. Analytical ICP-MS studies indicate that all of these metals are present in generator eluate, and that the amounts can vary signicantly with (i) the time between generator elutions and (ii) the age of the generator. These ndings have implications for design of kits that contain THP chelator bioconjugates. At present, the GalliProst™ kit contains 40 mg of THP-PSMA, and when reconstituted by simple addition of unprocessed 68 Ga generator eluate, THP-PSMA is at a concentration of 5 mM. Here, the range of concentrations of Ti (1.17-1.51 mM), Fe (0.07-0.09 mM), nat Ga (0.04-0.21 mM) and Al (0.61-0.91 mM) observed in eluate are all <5 mM. Our experience to date 5,16 shows that the presence of these metals does not affect 68 Ga radiolabelling using GalliProst™ kits. However, if lower amounts/concentrations of THP bioconjugates were to be used, the combination of Al, Fe, Ga and Ti in (unprocessed) 68 Ga eluate could decrease RCY of 68 Ga radiolabelling in such kits. 68 Ga radiolabelling of THP is less susceptible to the presence of some metal ions (Zn 2+ , Ni 2+ , Pb 2+ ) compared to DOTA. Prior work has shown that the presence of a range of metal ions signicantly decrease RCY of 68 Ga-DOTA-based derivatives. 22,36,37 For example, in 68 Ga-radiolabelling reactions of DOTA-TATE, a 2 : 1 Pb 2+ : DOTA-TATE molar ratio decreases RCY of 68 Ga-DOTA-TATE to less than 80% (compared to near quantitative RCY in the   22,34,39 The amounts of metal ion impurities in our multi-sample ICP-MS analyses are of a similar magnitude to previously reported data for eluate deriving from generators with a titanium dioxide solid phase. 14,22,34 Our data on Al, Ti, Fe and Ga concentrations in eluates obtained six months apart from each other suggest that the concentration of these metal ions decreases over time/number of elutions. We assessed whether the inclusion of other chemical components, such as ascorbate or organic solvents commonly used in 68 Ga generator eluate pre-processing affect RCY of [ 68 Ga(THP)]. Addition of ascorbate to 68 Ga radiolabelling reactions did not result in decreased RCYs of [ 68 Ga(THP)], and in this respect, it is a suitable radiolytic stabiliser for radiopharmaceuticals containing THP chelators. At Fe concentrations of 50 and 500 mM, the presence of ascorbate improved RCY of [ 68 Ga(THP)], compared to reactions undertaken with a spike of Fe, but without ascorbate. We attribute this to Fe 3+ reduction to Fe 2+ . Fe 2+ has signicantly lower affinity for hydroxypyridinones compared to Fe 3+ . Others' experimental data show that the rate of ascorbate reduction of Fe 3+ to Fe 2+ is pH dependent, and can decrease by an order of magnitude as the pH in aqueous media increases from 5 to 6. 40 Over the timeframe of THP radiolabelling reactions (<5 min), and at Fe concentrations in generator eluate (<5 mM), ascorbate reduction of Fe 3+ is unlikely to be of practical importance.
In some radiopharmacies, it has become standard practice to process 68 Ga generator eluate, in order to separate 68 Ga from 68 Ge breakthrough. The presence of sodium chloride or ethanol in solutions of processed 68 Ga eluate does not adversely affect RCY of [ 68 Ga(THP)] compared to radiolabelling reactions in which unprocessed eluate is used, but the use of processed eluate containing acetone does result in a slight decrease in RCY and should be avoided in the context of THP-based radiopharmaceuticals.

Concluding remarks
Metal impurities commonly present in 68 Ga generator eluate can deleteriously affect chelator 68 Ga radiolabelling of various bifunctional chelator-derived radiopharmaceuticals. 14,22,36, 37 We have provided experimental evidence that 68 Ga radiolabelling of THP is less susceptible to the presence of some of these metal ions (Zn 2+ , Cr 3+ , Ni 2+ , Pb 2+ ) compared to other clinically used chelators such as DOTA.
Other metal ions (Fe 3+ , Ti 4+ , Al 3+ , nat Ga 3+ ) can interfere with THP radiolabelling if present at sufficient concentration. However, the low concentrations at which these metals occur in generator eluate, combined with the amount of THP-based conjugate currently used in prefabricated kits, results in quantitative RCY of these 68 Ga-THP conjugates. If lower amounts/ concentrations of THP bioconjugates were used, the combination of Al 3+ , Fe 3+ , nat Ga 3+ and Ti 4+ in unprocessed 68 Ga eluate could decrease RCY of 68 Ga radiolabelling in such kits, and kit design should take this into account.

Conflicts of interest
There are no conicts of interest to declare.