Improved synthesis of 4-[18F]fluoro-m-hydroxyphenethylguanidine using an iodonium ylide precursor

Article abstract of DOI:10.1002/jlcr.3791

Fluorine-18 labeled hydroxyphenethylguanidines were recently developed in our laboratory as a new class of PET radiopharmaceuticals for quantifying regional cardiac sympathetic nerve density in heart disease patients. Studies of 4-[¹⁸F]fluoro-m-hydroxyphenethylguanidine ([¹⁸F]4F-MHPG) and 3-[¹⁸F]fluoro-p-hydroxyphenethylguanidine ([¹⁸F]3F-PHPG) in human subjects have shown that these radiotracers can be used to generate high-resolution maps of regional sympathetic nerve density using the Patlak graphical method. Previously, these compounds were synthesized using iodonium salt precursors, which provided sufficient radiochemical yields for on-site clinical PET studies. However, we were interested in exploring new methods that could offer significantly higher radiochemical yields. Spirocyclic iodonium ylide precursors have recently been established as an attractive new approach to radiofluorination of electron-rich aromatic compounds, offering several advantages over iodonium salt precursors. The goal of this study was to prepare a spirocyclic iodonium ylide precursor for synthesizing [¹⁸F]4F-MHPG and evaluate its efficacy in production of this radiopharmaceutical. Under optimized automated reaction conditions, the iodonium ylide precursor provided radiochemical yields averaging 7.8% ± 1.4% (n = 8, EOS, not decay corrected), around threefold higher than those achieved previously using an iodonium salt precursor. With further optimization and scale-up, this approach could potentially support commercial distribution of [¹⁸F]4F-MHPG to PET centers without on-site radiochemistry facilities.

Full text of DOI:10.1002/jlcr.3791

Raffel David M. (Orcid ID: 0000-0002-7188-9463)
Improved synthesis of 4-[18F]fluoro-m-
hydroxyphenethylguanidine using an iodonium ylide precursor
Running title: Improved synthesis of [18F]4F-MHPG using an iodonium ylide precursor
Yong-Woon Jung, PhD, Guie Gu, MS, David M. Raffel, PhD
Division of Nuclear Medicine, Department of Radiology, University of Michigan Medical
School, Ann Arbor, Michigan, USA
Corresponding Author:
David M. Raffel, Ph.D.
Division of Nuclear Medicine, Department of Radiology
University of Michigan Medical School
2276 Medical Science I, SPC-5610
Ann Arbor, MI 48109 USA
Telephone: (734) 936-0725
FAX:
(734) 764-0288
Email:
raffel@umich.edu
Total Word Count: 4224 (title page, abstract, text, references, tables, and figure legends).
Keywords: iodonium ylides, hypervalent iodine, radiofluorination, cardiac sympathetic
innervation
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been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3791
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Abstract
Fluorine-18 labeled hydroxyphenethylguanidines were recently developed in our laboratory
as a new class of PET radiopharmaceuticals for quantifying regional cardiac sympathetic
nerve
density
in
heart
disease
patients.
Studies
and
of
4-[18F]fluoro-m-
3-[18F]fluoro-p-
hydroxyphenethylguanidine
([18F]4F-MHPG)
hydroxyphenethylguanidine ([18F]3F-PHPG) in human subjects have shown that these
radiotracers can be used to generate high resolution maps of regional sympathetic nerve
density using Patlak graphical analysis. Previously, these compounds were synthesized using
iodonium salt precursors, which provided sufficient radiochemical yields for on-site clinical
PET studies. However, we were interested in exploring new methods that could offer
significantly higher radiochemical yields. Spirocyclic iodonium ylide precursors have
recently been established as an attractive new approach to radiofluorination of electron-rich
aromatic compounds, offering several advantages over iodonium salt precursors. The goal of
this study was to prepare a spirocyclic iodonium ylide precursor for synthesizing [18F]4F-
MHPG and evaluate its efficacy in production of this radiopharmaceutical. Under optimized
automated reaction conditions, the iodonium ylide precursor provided radiochemical yields
averaging 7.8 ± 1.4% (n = 8, EOS, not decay corrected), around three-fold higher than those
achieved previously using an iodonium salt precursor. With further optimization and scale-
up, this approach could potentially support commercial distribution of [18F]4F-MHPG to PET
centers without on-site radiochemistry facilities.
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1. Introduction
Noninvasive assessment of the extent and severity of regional cardiac denervation using
radiopharmaceuticals that selectively localize in sympathetic nerve terminals is an emerging
approach to assessing the risk of sudden cardiac death in patients with heart failure.1 Our
18
laboratory has recently developed F-labeled hydroxyphenethylguanidines as a new class of
radiopharmaceuticals for quantifying regional cardiac sympathetic nerve density using PET
and tracer kinetic analysis. Our studies have focused on 4-[18F]fluoro-m-
hydroxyphenethylguanidine ([18F]4F-MHPG) and its structural isomer 3-[18F]fluoro-p-
hydroxyphenethylguanidine ([18F]3F-PHPG) (Figure 1). These radiotracers exhibit
irreversible tissue kinetics through efficient intraneuronal retention in the norepinephrine
storage vesicles localized in cardiac sympathetic nerve terminals. We previously described
the use of iodonium salt precursors with the guanidine group completely protected (tetrakis-
Boc) as an effective method for automated production of [18F]4F-MHPG and [18F]3F-PHPG.2
For example, in production runs of [18F]4F-MHPG (n = 15), this approach provided final
product yields averaging 1.56 ± 0.68 GBq (range 0.61‒2.84 GBq) at end of synthesis (EOS)
with molar activities averaging 58 ± 25 GBq/mol (range 21‒105 GBq/mol), which were
adequate for on-site clinical studies. Under an exploratory Investigational New Drug
clearance from the U.S. Food and Drug Administration, we recently conducted first-in-human
studies of [18F]4F-MHPG and [18F]3F-PHPG in healthy control subjects.3 Encouraged by the
positive results of these studies, we were interested in evaluating alternative approaches to the
production of [18F]fluoro-hydroxyphenethylguanidines with the goal of achieving higher
radiochemical yields for routine clinical production. Among several new methods developed
for radiofluorination of electron-rich arenes, spirocyclic iodonium ylides offer several
advantages, including excellent regioselectivity, high radiochemical yields, and mild, metal-
free reaction conditions.4-7 In practice, the spirocyclic iodonium ylide approach is quite
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similar to our established method using iodonium salt precursors. The goal of this study was
to prepare and evaluate the spirocyclic iodonium ylide analog of the iodonium salt precursor
used previously for [18F]4F-MHPG production to test the general utility of iodonium ylide
method for radiofluorination of 18F-labeled hydroxyphenethylguanidines. The results
demonstrate that this new approach significantly increases the achievable radiochemical
yields of [18F]4F-MHPG, providing yet another example of the effectiveness of the use of
spirocyclic ylide precursors for efficient radiofluorination of electron-rich aromatic
compounds.
2. Results and Discussion
The synthesis of the required tetrakis-Boc protected spirocyclic iodonium ylide precursor 2 is
shown in Scheme 1. Compound 1 (N,N′,N″,N″-tetrakis(tert-butoxycarbonyl)-N-3-benzyloxy-
4-iodophenethylguanidine), previously synthesized for our original approach to [18F]4F-
MHPG,2 was first reacted with dimethyldioxirane (DMDO). The resulting intermediate was
coupled with (1r,3r,5r,7r)-spiro[adamantan-2,2′-[1,3]-dioxane]-4′,6′-dione (SPIAd)5 to give
the spirocyclic iodonium ylide 2.
For initial automated radiofluorination tests with 2, we employed optimized reaction
conditions reported by Rotstein et al.,5 including the use of tetraethylammonium bicarbonate
(TEAB; 4.0 mg) as the fluorine-18 counter ion, anhydrous N,N-dimethylformamide (DMF,
0.5 mL) as the reaction solvent, 5.0 – 6.0 mg of 2, and reaction conditions of 120 °C for 10
min (Scheme 2). The subsequent steps for simultaneous deprotection of the benzyl ether and
the N,N′,N″,N″-tetrakis-Boc groups using 3.0 N HBr followed by HPLC purification of
[18F]4F-MHPG were identical to those used in our original method.2 Radiochemical yields of
these pilot tests (Table 1, Runs 1–3) were dramatically higher than our original approach,
averaging 9.2 ± 0.7% (range 8.4% ‒ 9.7%) at EOS (not decay corrected), more than three-
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fold higher than the average yield of [18F]4F-MHPG achieved with our iodonium salt
precursor approach .2
Encouraged by these trial studies, we sought to optimize reaction conditions through a
series of manual reaction tests. First, we tested the effect of using different fluorine-18
counter ions, including TEA+, K+/Kryptofix 222 and Cs+. The results showed that TEA+ was
the clear choice, as K+/Kryptofix 222 and Cs+ yielded only trace quantities of radiolabeled
intermediate (data not shown). Next, we tested acetonitrile as the reaction solvent, based on a
report suggesting it might provide better yields8, but again low yields of product were
obtained (data not shown). Finally, reaction temperatures and times were optimized using
TEA+ as the fluoride counter ion and DMF as the solvent. The effects of reaction
temperature and reaction time using 4 mg of TEAB in 0.5 mL of DMF solvent are shown in
Figure 2A. The best yields were obtained at 120 °C for reaction times between 3 – 10 min.
Tests of three different concentrations of the fluoride counter ion showed that 4 mg of TEAB
per 0.5 mL of DMF solvent gave the highest yields at a reaction temperature of 120 °C and
reaction times of 5 – 10 min (Figure 2B). The general trend of decreasing radiochemical
yields with longer reactions times is likely due to a combination of thermal decomposition of
the spirocyclic iodonium ylide 2 and the radiolabeled intermediate [18F]3. For example,
melting point tests of 2 showed a broad melting point around 80 °C– 89 °C, followed by
generation of bubbles in the open capillary tube at temperatures between 110 °C – 120 °C.
We hypothesize that the bubbles were caused by generation of CO2 during thermolysis of the
N,N′,N′′,N′′-tetrakis-Boc groups between 110 °C – 120 °C.9,10
The findings of the initial automated tests (Table 1, Runs 1–3) and the manual reaction
tests were confirmed in some additional automated production runs of [18F]4F-MHPG.
Automated syntheses using Cs+ and K+/Kryptofix 222 as fluoride counter ions in DMF
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solvent were again found to provide extremely low yields (Table 1, Runs 4–6). Similarly,
yields were very low using TEA+ as the fluoride counter ion in MeCN solvent (Table 1, Runs
7–8). Using TEA+ in DMF, a short reaction time of 3 min at high temperature (150 °C) gave
a moderate yield of 3.8% (Table 1, Run 9), while a higher yield of 6.2% was achieved for a 5
min reaction time at 120 °C (Table 1, Run 10). Finally, a few runs were performed using
optimized conditions (TEA+ in DMF for 10 min at 120 °C), and the final products were
analyzed with the series of quality control (QC) tests currently used at our institution for
clinical PET studies with this radiopharmaceutical (Table 1, Runs QC1-QC4). The time from
end-of-beam to formulation was 90–95 min, including HPLC purification. An example of the
HPLC data acquired during purification and collection of [18F]4F-MHPG is shown in Figure
3. Good radiochemical yields were achieved (5.8% – 8.3%), consistent with the findings of
the manual reaction tests. Radiochemical purities were high (>99%) and molar activities
ranged from 105.8 to >225 GBq/mmol (Table 1). All other quality control tests (mass
concentration, radiochemical identity, radionuclidic identity, pH, visual inspection for
particulates and color, residual solvents tests, ethanol content, filter membrane integrity, and
sterility) were passed by each batch of [18F]4F-MHPG. Representative data of an HPLC study
to confirm radiochemical identity of the product are shown in Figure 4. Together these results
demonstrate the ability of the spirocyclic iodonium ylide method to reliably produce [18F]4F-
MHPG for clinical PET studies.
While the United States Pharmacopeia (USP) does not cite a release limit for
tetraethylammonium ion (TEA+), Institutional Review Boards may require QC testing of
TEA+ concentrations in the final [18F]4F-MHPG formulation. For example, the European
Pharmacopeia sets a release limit for the closely related tetrabutylammonium ion (TBA+) of
2.6 mg/V (per patient dose), and QC tests have been developed to evaluate TBA+ levels in
PET radiopharmaceutical products.11 Since our optimized iodonium ylide approach to
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[18F]4F-MHPG uses 4.0 mg of TEA+, and the compound is purified using HPLC, it is
unlikely that residual TEA+ levels would preclude use of the final product in human subjects.
Supporting this, a recent report on an improved synthesis of [18F]fluorodopamine for human
studies (which uses HPLC purification of the final product) described a QC testing method
for TEA+ concentrations, which averaged around 50 g/mL when starting with up to 3.5 mg
of tetraethylammonium bicarbonate per reaction.12
The radiochemical yields of [18F]4F-MHPG achieved with the spirocyclic iodonium
ylide 2 under optimized reaction conditions averaged 7.8 ± 1.4% (n = 8, range 5.8% – 9.7%,
Table 1). In comparison, these are lower than the reported yield of 14% achieved with a
closely related spirocyclic iodonium ylide precursor to prepare meta-[18F]fluoro-
benzylguanidine ([18F]mFBG).5 This is not surprising, as the protected m-hydroxyl group in
precursor 2 is electron-rich, which would be expected to cause lower radiochemical yields for
our compound. Nevertheless, the higher and more consistent radiochemical yields of [18F]4F-
MHPG achieved using 2 compared with our earlier results using an iodonium salt precursor
represents a noteworthy improvement in the production of this radiopharmaceutical for
clinical use.
3. Conclusions
The use of a spirocyclic iodonium ylide precursor for automated production of the cardiac
sympathetic innervation radiotracer [18F]4F-MHPG was evaluated and found to consistently
provide higher radiochemical yields than the previous approach using an iodonium salt
precursor. This improved method of [18F]4F-MHPG production is well suited to on-site
production for clinical PET studies, and could possibly be scaled up for distribution of the
radiotracer to stand-alone PET centers without a cyclotron and radiochemistry facility.
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4. Experimental
4.1 General
Reagents and solvents were purchased from commercial sources and used without further
purification unless otherwise noted. N,N′,N″,N″-tetrakis(tert-butoxycarbonyl)-N-3-
benzyloxy-4-iodophenethylguanidine
1,
N,N′,N″,N″-tetrakis(tert-butoxycarbonyl)-N-3-
benzyloxy-4-fluorophenethylguanidine 3 and 4-fluoro-m-hydroxyphenethylguanidine (4F-
MHPG) were previously prepared in our laboratory.2,13,14 Compound 1 was used to
synthesize the spirocyclic iodonium ylide precursor 2. Compound 3 and 4F-MHPG were used
as reference standards for radio-HPLC and radio-TLC analysis. Dimethyldioxirane
(DMDO)15 and (1r,3r,5r,7r)-spiro[adamantan-2,2′-[1,3]-dioxane]-4′,6′-dione (SPIAd)5 were
prepared using previously reported methods.
1
NMR spectra were obtained on a Varian vnmrs 500 (500.10 MHz for H; 125.70 MHz
13
1
13
for C) spectrometer. H and C NMR chemical shifts () are reported in parts per million
(ppm) relative to internal standard TMS and coupling constants (J) are in Hz. High-
resolution mass spectra were obtained on a VG (Micromass) 70-250S spectrometer using
electrospray ionization (ESI) in positive ion mode at 70 eV. Flash column chromatography
was performed with E. Merck 230-400 mesh silica gel.
4.2 Precursor synthesis
4.2.1
(1r,3r,5r,7r)-spiro[adamantane-2,2′-[1,3]dioxane]-4′,6′-dion-[2-benzyloxy-4-{2′-
(N,N′,N″,N″-tetrakis(tert-butoxycarbonyl)-guanidinyl)ethyl}phenyliodonium] ylide (2).
A solution of dimethyldioxirane (DMDO) in acetone was added dropwise to a cooled (0 °C)
solution
of
N,N′,N″,N″-tetrakis(tert-butoxycarbonyl)-N-3-benzyloxy-4-
iodophenethylguanidine 1 (200 mg, 0.25 mmol) in acetone and acetic acid (4:1, 2.5 mL) until
the starting guanidine compound 1 had disappeared, as assessed by TLC analysis. The
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mixture was stirred at 0 °C for 1 h and then warmed to room temperature. It was then
concentrated under reduced pressure. The residue was diluted with ethanol (5 mL) and treated
by addition of (1r,3r,5r,7r)-spiro[adamantan-2,2′-[1,3]-dioxane]-4′,6′-dione (SPIAd, 70 mg)
in portions. The resulting solution was adjusted to pH 10 by addition of a 10% aqueous
Na2CO3 solution, stirred for 2 h at room temperature, diluted with water (10 mL) and then
extracted with ethyl acetate (350 mL). The combined extracts were washed with brine, dried
over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified
by flash column chromatography (silica gel, 30% – 40% ethyl acetate in hexane) to afford the
product 2 (179 mg, 69%) as a white solid; 1H NMR (500 MHz, CDCl3)  7.44-7.39 (m, 5H),
7.25 (d, J = 7.9 Hz, 1H), 7.04 (s, 1H), 6.99 (d, J = 7.9 Hz, 1H), 5.21 (s, 2H), 3.94 (t, J = 8.0
Hz, 2H), 2.98 (t, J = 8.0 Hz, 2H), 2.52 (br. s, 2H), 2.22 (br. s, 2H), 2.20 (br. s, 2H), 1.88 (br.
13
s, 2H), 1.74 (br. s, 4H), 1.72 (br. s, 2H), 1.52-1.47(m, 36H); C NMR (125 MHz, CDCl3) 
163.80, 157.98, 154.82, 151.38, 147.80, 145.06, 144.12, 135.10, 129.26, 129.21, 129.14,
128.32, 125.59, 114.86, 107.90, 100.19, 84.32, 84.17, 82.53, 72.44, 48.79, 37.53, 36.95,
35.96, 34.08, 33.46, 28.34, 28.27, 28.25, 26.88; HRMS (ESI+) m/z calculated for
C49H64IN3O13 [M+Na]+: 1052.3376, found 1052.3373.
4.3 Radiochemistry
4.3.1 General
Ethanol (200 proof, USP) was purchased from Decon Laboratories, Inc. USP grade sodium
chloride 0.9%, and sterile water for injection, were sourced from Hospira. Millex GV
(#SLGV013SL) and FG (#SLFG025LS) filters were purchased from Millipore. Sep-Pak®
Light QMA cartridges (#WAT023525) were purchased from Waters Corporation. Light
QMA cartridges were conditioned with sequential flushes of 10 mL of ethanol, followed by
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10 mL of sterile water, 10 mL of 0.5M NaHCO3 preconditioning solution and finally 10 mL
of sterile water prior to use.
Using a GE Healthcare PETrace 880 cyclotron, a target containing water enriched with
18
18O was irradiated with 16 MeV protons to yield [18F]fluoride ([18F]F–) via the O(p,n)18F
reaction. [18F]F– was isolated from the enriched water by trapping on a preconditioned Sep-
Pak® Light QMA cartridge. The [18F]fluoride was then used in manual reaction tests or for
automated radiosyntheses of [18F]4F-MHPG using a TRACERLab FXFN synthesis module.
Radiochemical reactions were analyzed by radio-TLC or HPLC. For manual tests, radio-TLC
analysis was performed using a Bioscan AR 2000 Radio-TLC scanner with EMD Millipore
TLC silica gel 60 plates (3.0 cm  6.5 cm). For the automated syntheses, the radiochemical
purity of [18F]4F-MHPG was measured using a Shimadzu LC-2010A HT system equipped
with a Bioscan B-FC-1000 radiation detector under isocratic conditions (Jupiter C18 5
column, 4.6 250 mm, 60 mM NaHPO4 buffer, pH 5.4, with 10% ethanol, flow rate 1.1
mL/min,  = 254 nm, 40 °C oven). A few batches of [18F]4F-MHPG prepared under
optimized automated reaction conditions were tested using the full quality control protocol
developed for human PET studies, including molar activity determination and residual
solvents analyses.
4.3.2 Manual reactions
Tetraethylammonium [18F]fluoride (Et4N[18F]F) was prepared using a TRACERLab FXFN
synthesis module (General Electric, GE). [18F]F– was produced using a short beam time on
the GE PETrace cyclotron (55 μA beam for 30 – 45 s) and trapped on a preconditioned Sep-
Pak® Light QMA cartridge. The trapped [18F]F– was eluted into the reaction vessel with a
mixture of tetraethylammonium bicarbonate (Et4NHCO3, 4.0 mg), H2O (0.3 mL) and MeCN
(0.7 mL). After additional MeCN (0.5 mL) was added to the reaction vessel, the resulting
solution was azeotropically dried under vacuum at 90 °C for 4 min, followed by a nitrogen
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stream and simultaneous vacuum draw at 70 °C for an additional 4 min. Anhydrous N,N-
dimethylformamide (6 mL) was added into the reaction vessel, and the resulting solution was
transferred to a sterile vial to provide ~ 1.5 GBq of anhydrous Et4N[18F]F for manual
radiolabeling tests.
Manual reaction tests for optimizing radiolabeling conditions using the spirocyclic
iodonium ylide precursor 2 were carried out using different reaction temperatures, reaction
times and tetraethylammonium bicarbonate concentrations. For most tests, a stock solution of
Et4NHCO3 was prepared (72.0 mg of dried Et4NHCO3 in 9.0 mL of anhydrous DMF, 41.8
mM), and aliquots of this solution were used for manual reactions. A 0.5 mL aliquot of the
Et4NHCO3 stock solution was added to a 4 mL amber glass vial containing precursor 2 (~
4.0 mg, 3.8 μmol). The reaction vial was sealed with a PTFE/Silicone septum cap and the
solution thoroughly mixed. Using a 100 L glass syringe, a 50 μL aliquot of Et4N[18F]F (7.5-
15.0 MBq, prepared as described above) was added to the reaction vial. The vial was then
heated in an aluminum block at one of four temperatures (105, 120, 135 or 150 °C). After
reaction times of 3, 5, 10, 15 or 20 min, a 10 L aliquot of the reaction mixture was
withdrawn from the reaction vial and analyzed by radio-TLC (EtOAc/hexane, 1:3, v/v) to
determine the radiochemical yield (%) of the radiofluorinated intermediate [18F]3 (see
Scheme 2). Another series of tests evaluated the effect of different amounts of
tetraethylammonium bicarbonate on radiochemical yields. Using a constant temperature of
120 °C, the radiochemical yields of [18F]3 achieved with 2.0, 4.0 and 6.0 mg of Et4NHCO3
dissolved in the 0.5 mL of DMF added to the reaction vial were measured at reaction times of
5, 10, 15 and 20 min.
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4.3.3 Automated synthesis.
Our manual and automated reaction tests with the spirocyclic iodonium ylide precursor led to
the following improved automated synthesis of [18F]4F-MHPG (Scheme 2). For a 30 min
target irradiation at 55 μA beam current, approximately 65 GBq of [18F]F− was produced and
trapped on the preconditioned Sep-Pak Light QMA cartridge. [18F]F− was eluted from the
cartridge into the reactor vessel of the TRACERlab FXFN system with a solution of
Et4NHCO3 (~4 mg in 0.4 mL of H2O and 1.0 mL of MeCN). The water/acetonitrile mixture
was evaporated under vacuum at 90 °C for 4 min, followed by a nitrogen stream and
simultaneous vacuum draw at 70 °C for an additional 4 min to yield dried Et4N[18F]F. After
cooling to 60 °C, a mixed solution of 0.5 mL of DMF containing 5.5−6.0 mg of the
spirocyclic iodonium ylide precursor 2 was added to the reactor vessel containing Et4N[18F]F.
The mixture was heated in the sealed reactor vessel at 120 °C for 10 min to produce 3-
benzyloxy-4-[18F]fluorophenethyl-N,N′,N″,N″-tetrakis-BOC-guanidine
[18F]3
as
intermediate. After cooling to 70 °C, a solution of 48% HBr (0.5 mL) and MeCN (0.5 mL)
was added to the reaction mixture. The solution was heated at 120 °C for 15 min and then
cooled to 50 °C. Next, a mixture of aqueous NaOH (1.0 mL, 4.0 M in H2O) and buffer
solution (1.5 mL, 5% EtOH in 40 mM NH4OAc) was added into the reactor vessel. This
mixture was injected onto a reverse phase HPLC column (Phenomenex Synergi 10 μm
Hydro-RP 80Å, 250  10 mm, 5% EtOH in 40 mM NH4OAc buffer, flow rate 4.0 mL/min, λ
= 220 nm) and [18F]4F-MHPG was collected at Rt = 27−32 min. The collected fraction was
passed through a 0.22 μm sterilizing filter directly into a septum-sealed, sterile, pyrogen-free
glass vial. An aliquot of the filtered product was analyzed with HPLC to confirm the
radiochemical identity as [18F]4F-MHPG.
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4.3.4 Quality Control Testing.
Molar activity was determined by analyzing an aliquot of [18F]4F-MHPG with known activity
A0 (kBq) using a reverse-phase HPLC system with radiodetection. The area under the UV
absorbance peak associated with the [18F]4F-MHPG radioactivity peak was compared against
a calibration curve to estimate the total mass of 4F-MHPG (g) in the aliquot. The ratio of
sample activity (A0) to the measured total mass (converted from μg to μmol) gave the molar
activity of the sample. Radiochemical purity of the product was measured as the fractional
area under [18F]4F-MHPG peak in the baseline-subtracted curve measured by the
radiodetector. Additional quality control tests included radiochemical identity, radionuclidic
identity, radionuclidic purity, pH, visual inspection for particulates and color, bacterial
endotoxin test, and residual solvents testing.
Acknowledgements
We thank Dr. Allen Brooks for helpful discussions about the TracerLab FXFN system and
Bradley Henderson in the University of Michigan PET Radiopharmaceutical Production
Program for performing quality control testing of several lots of [18F]4F-MHPG.
Disclosure Statement
The authors have no conflicts of interest to disclose.
ORCID
David M. Raffel http://orcid.org/0000-0002-7188-9463
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FIGURE 1. The structures of [18F]4F-MHPG and [18F]3F-PHPG
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SCHEME 1. The synthesis of the spirocyclic iodonium ylide precursor 2
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SCHEME 2. The two-step reaction yielding [18F]4F-MHPG from precursor 2
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20
18
16
14
12
10
8
A
T = 105 °C
T = 120 °C
T = 135 °C
T = 150 °C
6
4
2
0
0
5
10
15
20
25
Time (min)
20
18
16
14
12
10
8
B
TEAB: 2 mg
TEAB: 4 mg
TEAB: 6 mg
6
4
2
0
0
5
10
15
20
25
Time (min)
FIGURE 2 Radiochemical yields of the radiolabeled intermediate [18F]3 as a function of
reaction time for: (A) different reaction temperatures, and (B) different amounts of
tetraethylammonium bicarbonate (TEAB)
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A
B
30000
25000
20000
15000
10000
5000
0
Gamma Detector
Cut
Peak
0
10
20
30
40
50
1200
1000
800
600
400
200
0
UV Detector
0
10
20
30
40
50
Time (min)
FIGURE 3 HPLC purification of [18F]4F-MHPG; (A) radiation detector chromatogram,
showing collection of [18F]4F-MHPG from 27 to 32 min; and (B) the corresponding UV
detector data at  = 220 nm.
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A
B
400
350
300
250
200
150
100
50
9.8
|
Gamma Detector
5.4
|
0
0
2
4
6
8
10 12 14 16
70
60
50
40
30
20
10
0
UV Detector
9.1
|
0
2
4
6
8
10 12 14 16
Time (min)
FIGURE 4. Coinjection study to confirm radiochemical identity. An aliquot of [18F]4F-
MHPG product was spiked with cold standard [19F]4F-MHPG and analyzed using HPLC.
The relative retention times of the peaks in the UV chromatogram (Rt = 9.1 min) and the
radiation detector chromatogram (Rt = 9.8 min) is consistent with an established delay time of
0.7 min between the UV and radiation detectors. A small amount of free [18F]F– (~ 2% of the
total activity) appears at Rt = 5.4 min in the radiation detector trace. HPLC conditions:
Synergi 10 Hydro-RP column, 4.6  250 mm, 60 mM sodium phosphate buffer, pH 5.4 with
10% ethanol, flow rate 1.0 mL/min, UV absorbance at 220 nm.
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Table 1. Automated tests of [18F]4F-MHPG production with spirocyclic iodonium ylide
precursor 2.
Molar
Reaction
Time
(min)
Run
#
X+
Temperature
(°C)
Radiochemical
Purity (%)
Radiochemical
Yield (%)
Solvent
Activity
(GBq/mol)
[18F]F–
1
2
TEA+
TEA+
TEA+
Cs+
DMF
DMF
DMF
DMF
DMF
DMF
MeCN
MeCN
DMF
DMF
DMF
DMF
DMF
DMF
DMF
120
120
120
120
120
120
120
120
150
120
120
120
120
120
120
10
10
10
10
10
10
10
10
3
> 98
> 98
> 99
—
8.4
9.4
9.7
—
—
—
3
—
4
—
5
K+/K222
K+/K222
TEA+
TEA+
TEA+
TEA+
TEA+
TEA+
TEA+
TEA+
TEA+
> 89
> 96
> 75
> 95
> 99
> 96
> 98
> 99
> 99
> 99
> 99
0.3
1.1
0.4
1.0
3.8
6.2
8.3
6.9
5.8
6.7
7.0
—
6
—
7
—
8
—
9
—
10
11
QC1
QC2
QC3
QC4
5
—
10
10
10
10
10
—
130.5
105.8
137.9
>225
Reactions were performed in 0.5 mL of solvent using 5.0–6.6 mg precursor and 3.5–5.4 mg of fluoride counter
ion.
Symbol '—' denotes that the value was not determined. K222 = Kryptofix 222.
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GRAPHICAL TABLE OF CONTENTS
The use of a spirocyclic iodonium ylide precursor for preparation of the cardiac sympathetic
innervation radiotracer 4-[18F]fluoro-m-hydroxyphenethylguanidine has been shown to
provide significantly higher radiochemical yields than were previously obtained with a
structurally related iodonium salt precursor.
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