Applied Radiation and Isotopes 65 (2007) 676681

The automatic production of 16a-[

18
F]uoroestradiol using a
conventional [
18
F]FDG module with a disposable cassette system
Seung Jun Oh
a,
, Dae Yoon Chi
b
, Christoph Mosdzianowski
c
, Hee Seup Kil
b
,
Jin Sook Ryu
a
, Dae Hyuk Moon
a
a
Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Korea
b
Department of Chemistry, Inha University, Inchon 402-751, Korea
c
GE Healthcare Technologies, Liege, Rue Marie Curie 78, B-4431 Loncin (Liege), Belgium
Received 14 February 2006; received in revised form 17 April 2006; accepted 12 June 2006
Abstract
We have developed a fully automatic method for the synthesis of 16a-[
18
F]uoroestradiol ([
18
F]FES) using a disposable cassette system
and conventional [
18
F]FDG module. [
18
F]FES was synthesized using a GE TracerLab MX module and a modied module control
program. Following [
18
F]uorination, we hydrolyzed the product three times with a mixture of 2 N HCl and CH
3
CN. After HPLC
purication, the decay corrected radiochemical yield of [
18
F]FES was 45.372.8%, which was stable to 98.270.2% at 6 h after synthesis.
This new automated synthesis method provides high and reproducible yields with the advantage of a disposable cassette system.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: [
18
F]Fluoroestradiol; Automatic synthesis; Breast cancer; [
18
F]Fluoride; Disposable cassette system
1. Introduction
16a-[
18
F]Fluoroestradiol ([
18
F]FES) is a radiopharma-
ceutical used in positron emission tomography for imaging
of specic estrogen receptors (Eckelman, 1994). Compared
with 16b-[
18
F]FES, 16a-[
18
F]FES has a 2.5-fold higher
relative binding afnity to these receptors (Katzenellenbogen,
2001). Using this radiopharmaceutical, clear images of
primary and metastatic breast tumors can be obtained.
Tumor uptake of [
18
F]FES has been found to correlate
closely with receptor levels on biopsy samples (Dehdashti
et al., 1995; McGuire et al., 1991). In addition, reduced
tumor uptake of [
18
F]FES has been demonstrated in breast
cancer patients who have started anti-estrogen hormone
therapy (Mortimer et al., 1996, 2001).
[
18
F]FES is manually synthesized from the precursor
3-methoxymethyl-16b,17b-epiestriol-O-cyclic sulfone (1),
resulting in a high radiochemical yield and low by-product
production (Berridge et al., 1990; Lim et al., 1996).
[
18
F]FES has also been automatically synthesized using a
conventional [
18
F]FDG synthetic module, but without a
cassette (Romer et al., 1999). Use of a disposable cassette
would provide stable radiochemical yields without the need
for cleaning procedures. Addition of an automatic rinsing
process, by a modication of the control program, would
reduce the residual activity on the cassette, and would
reduce exposure to radiation during back-to-back runs.
Thus, [
18
F]FES production using a disposable cassette
with conventional [
18
F]FDG module would be more
suitable for routine daily production for clinical purposes.
In the present study, we developed a [
18
F]FES production
method using a conventional [
18
F]FDG module with a
disposable cassette. This method was subjected to quality
control procedures and stability tests for routine clinical
purposes.
2. Materials and methods
2.1. Chemicals
The precursor, 3-methoxymethyl-16b,17b-epiestriol-O-
cyclic sulfone, was obtained from FutureChem (Seoul,
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doi:10.1016/j.apradiso.2006.06.016

Corresponding author. Tel.: +82 2 3010 4595; fax: +82 2 3010 4588.
E-mail address: sjoh@amc.seoul.kr (S.J. Oh).
Korea). Solvents and reagents (reagent grade) were
purchased from Sigma-Aldrich (Milwaukee WI, USA),
Lancaster (Lancashire, UK), and Acros Chemicals (Geel,
Belgium), and were used without further purication.
2.2. Chemistry module and synthesis of [
18
F]FES
We used a GE TracerLab MX (Liege, Belgium)
[
18
F]FDG module and made modications of the
[
18
F]FES sequence program and the disposable cassette
used for [
18
F]FDG synthesis. All Sep-Pak cartridges in the
disposable cassette were removed and manifolds 2 and 3
were connected with silicone tubing (Fig. 1).
The design of the disposable cassette is shown in Fig. 1.
This cassette had four reagent supply vials. Acetonitrile
(7 ml) was placed in vial 1 (blue); the precursor (110 mg)
with 2 ml CH
3
CN in vial 2 (red); 5.4 ml of EtOH:H
2
O (7:3)
solution in vial 3 (yellow); and a mixture of 0.69 ml 2 N
HCl and 6.21 ml CH
3
CN in vial 4 (green). Two 30 ml
disposable syringes were installed.
[
18
F]FES was prepared by uorination and hydrolysis
(Fig. 2). [
18
F]Fluoride (3.7 GBq) delivered from the
cyclotron was trapped on a QMA cartridge and eluted to
the reactor with a mixture of 7 mg of K
2
CO
3
in 300 ml H
2
O
and 22 mg of K
222
in 300 ml of CH
3
CN. Following complete
drying, performed under vacuum pressure and nitrogen
purging at 95 1C, we added 110 mg of the precursor,
3-methoxymethyl-16b,17b-epiestriol-O-cyclic sulfone in
2 ml of CH
3
CN. [
18
F]uorination was allowed to proceed
for 8 min at 105 1C and for 2 min at 85 1C. The solvent was
removed at 85 1C for 2 min under vacuum pressure and
nitrogen purging. Hydrolysis was performed by multiple
azeotropic evaporations using a mixture of 2 N HCl and
CH
3
CN. After delivery of 2.2 ml of this mixture to the
reactor, the mixture was heated under nitrogen purging,
while maintaining negative pressure by vacuum, for 2 min
at 85 1C and for 5 min at 95 1C. The hydrolysis procedure
was repeated three times to prevent the production of side
products and to maintain acid concentration (Romer et al.,
1999). After hydrolysis, the reaction mixture was diluted
with the ethanolic solution from the yellow vial, and the
mixture was injected automatically into the HPLC.
Following HPLC purication, using the conditions de-
scribed in Section 2.3, the [
18
F]FES collected was diluted
with 100 ml of H
2
O and trapped on an additional C
18
cartridge to remove excess ethanol. The trapped [
18
F]FES
was eluted with 1 ml of ethanol and diluted with 4 ml of
0.9% saline. Fig. 3
2.3. HPLC Purication
We used a Breeze HPLC pump (Waters, Milford, MA,
USA) with a UV 2457 (Waters) and a NaI PIN Diode
detector system (Bioscan, Washington, DC, USA) for
ARTICLE IN PRESS
18
Finlet
V1 V2 V3 V4
Eluant
Reactor
HPLC
Injector
Sillion tubing Manifold 1 Manifold 2 Manifold 3
O-18 water
Reservoir
Waste
Bottle
Vacuum
Pump
QMA
Cartridge
Waste
Bottle
Product
Vial
100mL
H
2
O
1mL
Ethanol
Syringe
C18
Sep-Pak
Waste
Bottle
Vacuum
Pump
UV
Detector
Radioactive
Detector
Sample
Reservoir
Pneumatic
3-way valve 1
Pneumatic
3-way valve 2
Pneumatic
3-way valve 3
N
2
gas
Venting
needle
Hot-cell
Syringe Pump 2 Syringe Pump 1
HPLC Column
Fig. 1. Diagram of the disposable cassette for the radiochemical synthesis of [
18
F]FES. Silicon tubing connects manifolds 2 and 3. Vials 14 contain
acetonitrile, the precursor, EtOH:H
2
O (7:3), and 0.69 ml 2 N HCl plus 6.21 ml CH
3
CN, respectively. The outlet of manifold 3 is connected to the HPLC
automatic injector.
S.J. Oh et al. / Applied Radiation and Isotopes 65 (2007) 676681 677
HPLC purication. For sample injection from a chemistry
module, we used a 10 ml HPLC loop and an automatic
injector (Rheodyne, Rohnert Park, CA, USA).
For this injection, we used a Rheodyne autoinjector that
has 6-ports in the injector. Each connection was labeled as
follows: port 1, a connection from HPLC pump; port 2,
ARTICLE IN PRESS
O
O
S
O
O
O
O
OSO
3
-
18
F
HO
OH
[
18
F]F
-
0.2 N HCl 18
F
O
O
1 [
18
F]FES
Fig. 2. Automatic synthetic route for [
18
F]FES with a cyclic sulfonate precursor.
Fig. 3. Crude HPLC chromatogram of the automatic synthesis of [
18
F]FES. The upper panel shows a UV chromatogram at 280 nm and the lower panel
shows a chromatogram of radioactivity.
S.J. Oh et al. / Applied Radiation and Isotopes 65 (2007) 676681 678
10 mL loop; port 3, sample reservoir; port 4, waste port;
port 5, 10 mL loop; port 6, to HPLC column. We have a
10 mL sample reservoir at the injector port of 3 and the
waste port of the autoinjector was connected to a vacuum
pump. We also have a 0.22 mm vented sterile lter (Millex-
GS, Millipore, Billerica, MA, USA) between the sample
reservoir and injector port 3 to remove insoluble com-
pounds and air from the reaction mixture (Gee and Bender,
1997). By vacuum from the pump, the reaction mixtures
moved to the sample loop from the sample reservoir; they
were automatically injected to the HPLC column by
rotation of the injector after complete moving of sample
to loop.
A Nucleosil 100-7 C
18
(Machery-Nagel, 10 mm, 10
250 mm) HPLC column was used; this was eluted with
ethanol:water (65:35) at 3.5 ml/min, and monitored at
280 nm with a UV detector.
We used three pneumatic 3-way valves (Alltech, USA),
operated by compressed air. The valve position was in
Fig. 1. Valve 1 was used to collect puried [
18
F]FES from
HPLC column and valves 2 and 3 were used to remove
EtOH and concentrate [
18
F]FES on solid phase extraction
cartridge. The puried [
18
F]FES was diluted with 100 ml of
H
2
O in the 200 ml collection bottle and passed through a
C
18
Sep-Pak Plus (360 mg sorbent) or an Oasis HLB (30
and 200 mg of sorbent) cartridge (Waters, USA) to remove
excess EtOH and to trap the [
18
F]FES. Before synthesis, we
added 100 mL of H
2
O to this 200 mL bottle and made a
connection of 1 mL of EtOH syringe to 3-way valve 2 from
outside of the hot-cell. We describe this connection in
Fig. 1. The trapped [
18
F]FES was eluted to the vial which
has 20 mg/mL ascorbic acid with 1 ml EtOH passed via a
sterile 0.22 mm lter.
2.4. Evaluation of automatic synthesis with high
radioactivity
We used 37 GBq/1 ml and 74 GBq/2 ml of [
18
F]uoride
for high radioactivity [
18
F]FES synthesis and reproduci-
bility tests (n 10 and 5, respectively) with 2 mg precursor
for these syntheses.
All radiochemical yields were decay-corrected and end-
of-synthesis (EOS) yields were calculated.
2.5. Quality control and stability checking
Following synthesis of [
18
F]FES from 37 GBq/mL
[
18
F]uoride (n 10) or from 74 GBq/mL [
18
F]uoride
(n 5), we measured radiochemical purity by radio TLC
(developing solvent: acetone; Merck 60 F254, Germany)
and analytical HPLC (Thermo Separation Products,
USA). The residual organic solvents in the nal product
were analyzed by gas chromatography (Acme-2000,
YoungLin Instrument, Korea). Supelcowax-10 (Sigma-
Aldrich) fused silica capillary columns (60 m0.25 mm
0.25 mm lm thickness) were used for analysis. The
temperatures of the oven, injector and detector were 75,
250 and 250 1C, respectively. We used a ame ionization
detector system with a ow rate of 105 ml/min. Gas
chromatography data were analyzed with an Autochro-
2000 (Younglin Instrument, Korea). Tests for pyrogen,
Kryptox
2.2.2
and sterility were performed as described
previously (Hung, 2002). The pH of the nal solution was
measured using a pH meter (Corning, USA).
Long-term radiochemical stability for up to 6 h at room
temperature was determined on each 10 ml solution (9 ml
saline+370 MBq/mL [
18
F]FES solution) using a radioTLC
and an analytical HPLC (n 3), with acceleration stability
tests performed at pH values of 1, 4, 7, 10, and 13.
3. Results
Radiochemical yields depended on the amount of
precursor (Table 1). Starting with 2 mg precursor, the
HPLC puried radiochemical yield after [
18
F]uorination
for 8 min at 105 1C and for 2 min at 85 1C was 45.372.8%.
Although the radiochemical yield increased as the amount
of precursor increased (68.475.2% from 10 mg precursor),
we could not completely separate the organic impurities
from [
18
F]FES when we started with higher amounts of
precursor. We could not analyze them and only considered
that they were cold [
18
F]FES and hydrolyzed precursor
ARTICLE IN PRESS
Table 1
Yield and specic activity of synthesized [
18
F]Fluoroestradiol (n 3)
Precursor amount (mg) 1 2 5 10
Yield of radiochemical (%) 20.473.7 45.372.8 55.778.4 68.475.2
Measured specic activity (GBq/mmol) 57.971.7 57.773.8 32.979.4 20.271.8
*Reaction conditions:
1. Precursor: 3-methoxymethyl-16b,17b-epiestriol-O-cyclic sulfone.
2. 3.7 GBq/mL [
18
F]F

as starting radioactivity.
3. [
18
F]uorination for 8 min at 105 1C and for 2 min at 85 1C.
4. 3 azeotropic hydrolysis with 2 N HCl and CH
3
CN.
5. HPLC purication.
6. Radiochemical yield: decay-corrected radiochemical yield after HPLC purication.
S.J. Oh et al. / Applied Radiation and Isotopes 65 (2007) 676681 679
impurities. The measured specic activity of the product
was 57.773.8 GBq/mmol when we started with 2 mg of
precursor, but only 20.271.8 GBq/mmol when we started
with 10mg of precursor. (n 3 for each condition) Starting
with 37GBq of [
18
F]F

(n 10), the radiochemical yield

was 48.372.7%, whereas starting with 74 GBq of [
18
F]F

(n 5), the yield was 45.273.8%.

Total synthesis time, including HPLC purication and
formulation, was 75.875.2 min. After concentrating
[
18
F]FES on the C
18
cartridge, we obtained an injectable
solution containing 10% ethanol. The C
18
plus cartridge
showed a trapping efciency 495%, whereas the 30 mg
sorbent HLB Oasis cartridge had a trapping efciency of
6070%. Although the 200 mg sorbent HLB cartridge
showed 495% of trapping efciency, we only obtained
5060% [
18
F]FES from total trapped radioactivity with
1 mL of EtOH elution.
At the time of synthesis, the purity of the radiochemical
was 98.770.8%. The results of quality control procedures
showed that the synthesized [
18
F]FES was suitable for
routine clinical use. Peak retention times of cold FES and
[
18
F]FES were the same in the co-injection HPLC
procedures. Radiochemical purity was 98.270.2%
(n 3) after 6 h, and remained 495% after several
acceleration tests.
4. Discussion
Starting with 74 GBq of
18
F and using a conventional
[
18
F]FDG synthetic module with a disposable cassette, we
obtained a high radiochemical yield of [
18
F]FES
(45.273.8%). In this procedure, we modied only
sequence software and the disposable cassette, without
any hardware changes.
We used a two-stage heating protocol for [
18
F]uorina-
tion. A reaction temperature of 105 1C was not high and
there was a low possibility of the reaction vessel cap
rupture due to high internal pressure generated by the high
temperature. However, because our TracerLab MX mod-
ule did not have a cooling system comparable to that
reported previously (Romer et al., 1999), it was necessary
to reduce the reaction temperature in order to decrease the
internal pressure in the reactor. Without this temperature
reduction, we may have lost much of the radioactivity
when we opened the valve connected to the reactor.
Therefore, after 8 min of heating at 105 1C, we reduced the
[
18
F]uorination temperature to 85 1C for 2 min to reduce
the internal pressure of the reactor.
We also subjected the reaction product to repeated
azeotropic hydrolysis. Although this procedure requires a
long preparation time, it has several advantages, including
low contamination with side-products and efcient re-
moval of unreacted [
18
F]F

(Lim et al., 1996; Romer et al.,

1999). The hydrolysis step included nitrogen purging and
application of negative pressure. Under these conditions,
and in the presence of 0.2 N HCl, the unreacted [
18
F]F

changed to H[
18
F]F, which could then be evaporated to the
waste bottle. As a result, our radioactive HPLC chromato-
gram was very clean.
After HPLC purication, the nal product had a high
concentration of ethanol. Previously, the nal product was
diluted with saline or evaporated to remove excess ethanol
(Lim et al., 1996; Romer et al., 1999). We used a solid-
phase extraction method to reduce the quantity of ethanol
in the nal [
18
F]FES injectable solution, obviating the
need for cumbersome procedures such as heating for
evaporation.
In this study, we used a commercial disposable cassette
system. This cassette could be replaced for each run,
ensuring clean and reproducible conditions for each
production, preventing cross-contamination, and resulting
in high, reproducible yields. Although cassette replacement
entails higher costs, the addition of an automatic rinsing
process with a modication of the control program may
reduce the residual activity on the cassette, as well as
reducing radiation exposure in cases of back-to-back runs
and lowering replacement costs.
5. Conclusion
We have developed an automated method of [
18
F]FES
production using a commercial FDG module and a
modied disposable cassette system, resulting in consis-
tently high radiochemical yields. Automated synthesis of
[
18
F]FES may enhance the clinical applications of this
radiochemical.
Acknowledgment
This study was supported by the Korean Ministry of
Science & Technology (MOST), through its real-time
molecular imaging research program.
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