A facile direct nucleophilic synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) without HPLC purification

Olga Fedorova • Olga Kuznetsova • Maria Stepanova •

Victor Maleev • Yuri Belokon • Hans-Juergen Wester •

Raisa Krasikova

Received: 13 January 2014

� Akademiai Kiado, Budapest, Hungary 2014

Abstract Due to favourable in vivo characteristics, its

high specificity and the longer half-life of 18F (109.8 min)

allowing for remote-site delivery, O-(2-[18F]fluoroethyl)-

L-tyrosine ([18F]FET) has gained increased importance for

molecular imaging of cerebral tumors. Consequently, the

development of simple and efficient production strategies

for [18F]FET could be an important step to further improve

the cost-effective availability of [18F]FET in the clinical

environment. In the present study [18F]FET was synthe-

sized via direct nucleophilic synthesis using an earlier

developed chiral precursor, the NiII complex of an alkyl-

ated (S)-tyrosine Schiff base, Ni-(S)-BPB-(S)-Tyr-OCH2-

CH2OTs. The purification method has been developed via

solid phase extraction thereby omitting cumbersome HPLC

purification. The suggested SPE purification using combi-

nation of reverse phase and strong cation exchange car-

tridges provided [18F]FET in high chemical, radiochemical

and enantiomeric purity and 35 % radiochemical yield

(decay-corrected, 45 min synthesis time). The method was

successfully automated using a commercially available

synthesis module, Scintomics Hotboxone. Based on the

current results, the proposed production route appears to be

well suited for transfer into an automated cassette-type

radiosynthesizers without using HPLC.

Keywords PET � Fluorine-18 � O-(2-[18F]fluoroethyl)-

L-tyrosine � [18F]FET � SPE purification

Introduction

In recent years positron emission tomography (PET) has

rapidly become a valuable diagnostic tool, particularly with

the development of new targeted 18F-fluorinated PET ra-

diopharmaceuticals ‘‘beyond [18F]FDG’’ with established or

promising oncology application [1]. Among the 18F-labelled

radiopharmaceuticals suitable for brain tumour imaging, O-

(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) is probably the

most frequently used and widely evaluated radiotracer. The

usefulness of [18F]FET in diagnosing intracranial tumors,

identifying the location and extent of the tumors, assessing

for recurrence, guiding biopsies and radiosurgery has been

reported in the literature [2–5]. The added benefit of

[18F]FET is that it can be synthesized in large quantities via

nucleophilic synthesis pathway [2] applicable for PET

satellite concept similarly to [18F]FDG. The [18F]FET was

initially prepared via a two-step procedure, alkylation of the

L-tyrosine di-potassium salt with [18F]fluoroethyltosylate

[2]. Similar to this alkylating agent [6, 7], [18F]fluor-

oethylbromide [7, 8] was employed in routine preparation of

[18F]FET in different PET centers. However, the most suc-

cessful and frequently used synthesis procedure for [18F]FET

O. Fedorova � O. Kuznetsova � M. Stepanova �R. Krasikova (&)

N.P. Bechtereva Institute of the Human Brain of the Russian

Academy of Science (IHB RAS), 9, Pavlova str.,

197376 Saint-Petersburg, Russian Federation

e-mail: raisa@ihb.spb.ru

V. Maleev � Y. Belokon

A.N. Nesmeyanov Institute of Organoelement Compounds of the

Russian Academy of Science (INEOS RAS), B-334, Vavilova

str. 28, 119991 Moscow, Russian Federation

H.-J. Wester

Pharmaceutical Radiochemistry, Technische Universitat

Munchen, Garching, Germany

R. Krasikova

Department of Radiochemistry, St.-Petersburg State University,

7-9, Universitetskaya nab, 199034 Saint-Petersburg, Russian

Federation

123

J Radioanal Nucl Chem

DOI 10.1007/s10967-014-3121-2

is direct nucleophilic fluorination on the protected alkyl

tyrosine derivative, O-(2-tosyloxyethyl)-N-trityl-L-tyrosine

tert.-butylester (1, Table 1) [9]. Typically used synthesis

protocol consists of nucleophilic substitution of tosyl group

in 1 by [18F]fluoride and subsequent acid hydrolysis of the18F-fluorinated intermediate using either TFA/CH2Cl2 [9,

10] or an aqueous HCl [11], while both reactions are carried

out in a single reaction vessel. The suggested method [9]

provides good radiochemical yield and more than 99 %

radiochemical and enantiomeric purity of [18F]FET and

perfectly suits to the automation. Automated production of

[18F]FET using 1 has been established on the multipurpose18F-fluorination module TRACERlab FXFN [11], or cassette-

based apparatuses, such as IBA Synthera [10] (involving

semi-preparative HPLC purification for the isolation of the

product) or SCINTOMICS GRP without HPLC purification.

Despite of the fact that HPLC offers a general purification

method for PET radiotracers, the method is time-consuming

and associated with considerable loss of the product. In

addition, the dedicated HPLC equipment requires additional

space inside the expensive hot cell area and needs regular

qualification service, maintenance and trained operators.

Over the last decade much interest has been devoted to the

development of solid phase extraction (SPE) purification

techniques based on the use of commercially available dis-

posable cartridges. The SPE method is very simple, fast,

reliable and easy adaptable to any automated equipment with

almost no modifications and extra costs. Several successful

applications of the SPE purification technique have been

published, including [18F]FDG [12, 13], 30-deoxy-30-[18F]fluorothymidine ([18F]FLT) [14], 1-H-1-(3-[18F]fluoro-

2-hydroxypropyl)-2-nitroimidazole ([18F]FMISO) [15, 16]

and clinically relevant radiometalated radiotracers, to men-

tion only a few. Consequently, the development of a new,

simple and efficient production method for [18F]FET should

include SPE purification to facilitate its automation and

adaption to modern cassette-based automation platforms.

Although, cartridge based purification processes have

been reported for [18F]FET preparations via 18F-fluorina-

tions of other structural analogues of 1 (2 and 3, Table 1),

these procedures do not exploid precursors for ‘‘pre-

determining’’ the chirality of the product and thus the

enantiomeric purity of produced [18F]FET (which is

essential for amino acids radiotracers). In those cases, an

almost quantitative enantiomeric excess of [18F]FET is

obtained by the adjustment of the reaction conditions and

finally confirmed/quantified by a chiral radio-HPLC quality

control. Nevertheless, racemization which may occur dur-

ing hard fluorination conditions when the precursors 2 and

3 are used, is still inherent. To avoid this problem, bulky

protective groups were introduced for the amino- and

carboxy- functions in the structure of 1, while preventing

protection for racemization using less bulky protective

groups in 2 and 3 was not evident. Recently, we introduced

an alternative synthetic approach to [18F]FET using a chiral

NiII complex of an alkylated (S)-tyrosine Schiff base,

Table 1 Direct nucleophilic synthesis of [18F]FET: labeling precursors, fluorination conditions and purification approaches

No. Structure of labeling precursor PTC and [18F]fluorination

conditions

Enantiomeric

purity (%)

Purification mode Ref.

1a

NH PhPhPh

OTsO

O

O

Bu4N?18F MeCN,

85 �C, 5 min

[98 HPLC [9]

[K/K2.2.2]?18F- MeCN,

100 �C, 10 min

[98 HPLC [11]

2 O

ONH

O

OO

TsO

Bu4N?18F CH3CN,

90 �C, 10 min

Not reported Normal phase SPE purification

of final product

[17]

3 O

OCH3

NHBocO

TsO

[K/K2.2.2]?18F- MeCN,

130 �C, 30 min

Not reported Normal phase SPE purification

of the 18F- fluorinated intermediate

[18]

4 OTsO

HO

O

Ni N

NN

HO

Ph

[K/K2.2.2]?18F- MeCN,

80 �C, 5 min

[95 HPLC [19]

a Precursor 1 is available from ABX, Germany

J Radioanal Nucl Chem

123

Ni-(S)-BPB-(S)-Tyr-OCH2CH2OTs (4, Table 1) as a labeling

precursor [19] (Fig. 1). Nucleophilic substitution of the

tosyl group in 4 with 18F-fluoride in the presence of TBAH/

TBAC or kryptofix 2.2.2/K2CO3 (MeCN, 80 �C, 5 min)

followed by acidic hydrolysis/deprotection (0.5 N HCl,

5 min, 120 �C) yielded [18F]FET in [95 % enantiomeric

purity after final semi-preparative HPLC purification.

In this study we describe our efforts to achieve an HPLC

free radiosynthesis of [18F]FET based on direct nucleo-

philic fluorination of labeling precursor 4 (Fig. 1) and its

automation using the commercially available synthesis

apparatus.

Materials and methods

Materials

Anhydrous acetonitrile (for DNA synthesis, max

10 ppm H2O) was bought from Merck, Germany.

Hydrochloric acid, potassium hydroxide, sodium ace-

tate, anhydrous potassium carbonate, 4,7,13,16,21,24-

hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K2.2.2)

were acquired from Sigma-Aldrich, USA. Chiral pre-

cursor, the NiII complex of an alkylated (S)-tyrosine

Schiff base, Ni-(S)-BPB-(S)-Tyr-OCH2CH2OTs (4) and

reference, O-2-fluoroethyl-L-tyrosine were made avail-

able by the A.N. Nesmeyanov Institute of Organoele-

ment compounds RAS, Moscow. Disposable cartridges

and columns for SPE procedures from different vendors

were used: C18 Sep-Pak plus, tC18 Sep-Pak plus, Silica

Sep-Pak plus (Waters), Lichrolut RP 18E and Lichrolut

SCX (Merck); Maxi-Clean IC-Chelate (Alltech); Su-

pelcleanTM ENVI-Chrom P SPE Tube and Supelclean

LC-SCX resin (Sigma-Aldrich); the resin was packed in

disposable polyethylene SPE tubes of 3.0 mL (Supelco).

The cartridges were conditioned as follows; all reverse

phase cartridges: 10 mL of ethanol following 15 mL of

water; AccellTM Plus QMA light (Waters): 10 mL of

0.5 M K2CO3 and 15 mL of water; SCX and IC che-

late: 5 mL of water; Silica Plus: 5 mL of ether or

CH2Cl2.

Analytical methods

Analytical HPLC system included a Gilson Pump 305, a

Rheodyne type injector fitted with 20 lL loop, Gilson

116 UV absorbance detector in series with a Beckman

170 radiodetector. The HPLC analyses were performed

in two systems with UV detection at 254 nm: System

A: a Nucleosil C18 column, 5 lm, 150 9 4.6 mM

(Supelco), 5 mM sodium acetate buffer (pH 4) con-

taining 5 % of EtOH, flow rate of 1 mL/min; Rt 7.3 min

for [18F]FET; System B: Crownpak (?) (Daicel) col-

umn, an aqueous HClO4 (pH 2)/MeOH (90/10 v/v),

flow rate of 0.8 mL/min, Rt 11.3 and 14.9 min for

D-[18F]FET and L-[18F]FET, correspondingly. TLC

analyses were carried out on the pre-coated plates of

silica gel F254 (Sorbfil, Lenchrom, Russia), radioactivity

distribution was detected by radioTLC scanner (Raytest

GmBH, Germany). TLC system 1: ethyl acetate/

chloroform/acetic acid (4/1/1, v/v/v), Rf of [18F]fluoride

0.05, Rf of [18F]fluorinated intermediate 5—0.70; TLC

system 2: n-butanol/acetic acid/water/ethanol (4/1/1.6/

0.5, v/v/v/v), Rf of [18F]fluoride 0.05, Rf of [18F]FET

0.83. The identity of [18F]FET was confirmed using

system 2 and authentic sample of [19F]FET; the com-

pound spot was visualized by spraying TLC sheet with

ninhydrin solution in ethanol (0.2 %) and heating. The

content of nickel in the final preparation was deter-

mined by the inductively coupled plasma mass spec-

troscopy on ICP-MS PQ-3; Plasma Quad, VG

(England).

Automation procedure

The synthesis was performed using modular system

Hotboxone (Scintomics GmbH, Germany), consisted of

the radiodetector system Variodetect D2 with two

gamma sensors, Variodisp8, a high resolution motor

syringe with 8-port PEEK rotor for gas and solvent

transport, serial controlled by Variocontrol software

package and a hardware control interface Vario-

switch32?. The system components (heating block with

reactor vessel, reagents vials, two and three way valves)

OTsO

HO

O

Ni N

NN

HO

Ph

NH2

COOHO

F18

OF18

HO

O

Ni N

NN

HO

Ph0,5M HCl

Ni-BPB-TyrO-CH2CH2OTs

120oC, 5 min

[18F]FET

4 5 Preparative HPLC

K/K2.2.2.+ 18F-

Acetonitrile,80oC, 5 min

Fig. 1 Labeling scheme for O-

(2-[18F]fluoro-ethyl)-L-tyrosine

([18F]FET) using chiral

precursor 4 [19]

J Radioanal Nucl Chem

123

were mounted on Variotecbase modular platform. All the

connections between valves were made by means of the

standard PTFE or PEEK tubing. The reaction vessel was

a conic heat-resistant glass vial which was equipped with

a standard reactor head (PEEK) penetrated with five

lead-through Teflon lines for addition of reagents. The

system assembly is shown on the Fig. 2 with the fol-

lowing reagents setup:

Vial 1: 2 mL of MeCN/water (96/4 v/v) with 9.8 mg of

Kryptofix 2.2.2 and 1.8 mg of K2CO3; Vial 2: 1 mL

MeCN; Vial 3: 0.6 mL MeCN containing 5 ± 0,5 mg of 4;

Vial 4: 0.5 mL of 0.5 M HCl; VARIODISP vial (eluent for

product): 8 mL of CH3COONa 5 vM, pH 4/C2H5OH (95/5

v/v); vessel for dilution: 20 mL of H2O and 2 mL of 0.1 M

NaOH.

Production and separation of [18F]fluoride

No-carrier-added aqueous [18F]fluoride was produced via

the 18O(p,n)18F nuclear reaction by irradiation of an enri-

ched [18O]H2O (95 %, Global Scientific Technologies,

Russia) using a Scanditronix MC17 cyclotron and a low

pressure silver-made water target. The radionuclide was

transferred from the target by means of helium flow and

trapped on a AccellTM Plus QMA light Sep-Pak cartridge

(bicarbonate form) to remove [18O]H2O. [18F]fluoride was

then eluted with 2 mL of solution from vial 1 and collected

in reaction vial. The solvents were evaporated by heating at

120 �C under a stream of nitrogen (100 mL/min). A por-

tion of acetonitrile (1 mL) was added and evaporated

completely resulting in a reactive complex [R/2.2.2]?18F-

as a slightly brownish residue.

Radiosynthesis of [18F]FET

Synthesis was carried out according to earlier described

method [19]. To the dried complex, the precursor 4, Ni-(S)-

BPB-(S)-TyrO-CH2CH2OTs (5 ± 0.5 mg in 600 lL of

anhydrous MeCN) was added and the reaction mixture was

heated at 80 �C for 5 min without mixing. The 18F-incor-

poration rate into 4 was evaluated by radio TLC (system 1).

Decomposition of 18F-fluorinated intermediate 5 and

simultaneous removal of the protecting groups was

achieved through the addition of 0.5 mL of 0.5 M HCl

Waste

H2O Eluent for

product

MeCN

Waste

Waste

N2 input

Waste

Air

18F- Filter

SCX0.5 g

tC18 plus

QMA

V1

2

Vessel for

dilution

Reaction vial

3 4

1

V5

V16

V4

V2 V3

V13

V11V9

V17

V15

V7

V14

V6

V10

Final product

Syringe pump

V12

Fig. 2 Chart flow diagram of the configuration of Scintomics Hotboxone modular system for the radiosynthesis of [18F]FET

J Radioanal Nucl Chem

123

from Vial 4 and heating at 120 �C for 10 min. The per-

formance of hydrolysis was controlled by radio-TLC of an

aliquot of the reaction mixture (system 2).

The SPE purification procedure

In a typical experiment the reaction mixture containing

crude [18F]FET was transferred into Vessel for dilution via

V14 by nitrogen flow supplied from V1 (V5 was off), the

content was agitated by nitrogen flow; the formation of

precipitate (pH 9.0) was observed. The diluted reaction

mixture was transported through a special filter (Millipore

SLGV013NK hydrophilic Millex-GV non-sterile syringe

filter Unit, 0.22 lm, 13 mm) that was inserted into V15 line

to the inlet of tC18 plus Sep-Pak connected via V7 to the

waste position. On this first purification stage the [18F]FET

was kept on tC18 plus, while the precipitate stayed retained

on the filter. The tC18 plus cartridge was rinsed with 5 mL

of water via syringe pump to remove the residual MeCN

and other impurities to the waste. 8 mL of buffer from the

‘‘Eluent for product’’ vessel was taken up by syringe pump

for elution of [18F]FET from tC18 plus. The eluate was

directly passed through a Supelclean LC-SCX resin (0.5 g

packed in 3.0 mL Supelco tube) placed next to tC18 plus

cartridge (via V7 valve in between). The obtained solution

was collected in a sterile product and passed through a

standard 0.22 lm sterile filter (Millipore, Waters).

Similar work-up procedure was applied using other

reverse phase cartridges. Several experiments have been

carried out to purify 18F-fluorinated intermediate 5 using

normal phase Silica Plus cartridges. Reaction mixture

containing crude 5 was mixed with 1.5 mL of CH2Cl2 and

passed through the Silica cartridge in a manual mode; the

aliquot of the eluate was taken and analyzed using radio-

TLC (system 1).

Results and discussion

A detailed outline of the preparation of [18F]FET using

chiral labeling precursor 4 and the activated 18F-complex

with kryptofix 2.2.2 and/or TBAHC in MeCN (80 �C,

5 min) was published previously [19]. Under these condi-

tions the 18F-incorporation rate into 1 was in the range of

60 %. When this reaction was carried out in DMSO under

elevated temperature, 18F-fluorination efficiency was sub-

stantially improved (89 ± 6 %) [20], however this route

did not allow the desired [18F]FET in high and reproducible

enantiomeric purity. Therefore, within this study, the

‘‘classical’’ MeCN/K2.2.2/K2CO3 combination was used

for all the fluorination experiments followed by acid cata-

lyzed hydrlolysis and cleavage of the protecting groups

(Fig. 1). The manufacturing of [18F]FET was accomplished

by use of an automated synthesis module; the setup of the

automated process is outlined on Fig. 2. The performance

of both synthesis steps was evaluated by radio-TLC using

System 1 to assess the effectiveness of 18F-fluorination of 4

and System 2 to control the completeness of hydrolysis and

deprotection. In our previous work the purification of crude

[18F]FET was performed by semi-preparative HPLC puri-

fication using an aqueous buffer/ethanol mixture (0.01 M

CH3COO-NH4?, pH 4/C2H5OH 90/10 (v/v) [19]. The

product fraction was isolated and mixed with PBS buffer to

adjust pH, isotonic formula and ethanol concentration in

the injectable solution, without final re-formulation step.

However, the total purification process took over 20 min

and associated with substantial loss of the product on the

HPLC column. This prompted us to derive HPLC free

strategy to produce [18F]FET with an optimal of product

quality within shorter synthesis time.

In general, it can be summarized that substitution of

HPLC by suitable SPE approaches can be considered as a

milestone for PET radiochemistry. Due to the implemen-

tation of cartridge-based purification techniques the syn-

theses of [18F]FDG, [18F]FMISO, [18F]FLT and other

compounds could be fully automated in a previously

unknown straight forward manner without addition equip-

ment and on the base of modern cassette-based automation

platforms [21]. For the synthesis of [18F]FET via a two step18F-fluoroethylation of L-tyrosine di-sodium salt the SPE

technique was employed for the intermediate purification

of labeling synthon [6] and the final product prior to for-

mulation [6, 8].

In the search for a suitable SPE purification method

for [18F]FET derived from precursor 1, we initially

considered the separation of 18F-fluorinated intermediate

5 (Fig. 1) on the normal phase Silica plus cartridges.

This approach has been found to be useful for the

purification of [18F]FET prepared from labeling precur-

sors 2 and 3 [17, 18], although the enantiomeric purity

of final product was not evaluated (Table 1). In our

experiments the separation of 18F-fluorinated intermediate

5 using Silica Plus SepPak cartridge was efficient; the

content of 5 in the eluate was about 95 %, while all the

labeled by-products retained in the cartridge. However,

the total radioactivity loss was about 60–70 %; additional

amounts of 5 could not be eluted by repeated rinsing

with ether or CH2Cl2.

More commonly the SPE purification procedures

employed the SPE cartridges or disposable columns packed

with different stationary reverse phases. Typically the

reaction mixture obtained after 18F-fluorination or hydro-

lysis is substantially diluted by water and passed through

the cartridge allowing removal of non-reacted [18F]fluo-

ride, phase-transfer catalysts and fluorination solvent. The

retained 18F-labelled reaction product can be further eluted

J Radioanal Nucl Chem

123

from the cartridge by suitable solvent to proceed with the

next synthesis step. Using chiral metallo complex precur-

sor4 as a labeling precursor, special care must be taken to

ensure the complete removal of nickel. This task was

accomplished via removal of nickel as a precipitate: the

later one was formed when the diluted hydrolysed mixture

was adjusted to pH 9.0 upon controlled addition of 2 mL of

0.5 M NaOH. The obtained turbid solution was directed by

gas flow to C18 cartridge through the Hydrophilic Millex-

GV Non-Sterile Syringe Filter (placed in the tubing)

allowing to retain the precipitate. After rinsing of C18

cartridge by water the [18F]FET was eluted by an aqueous

[18F]FET after SPE purification

[18F]FET before SPE purification

TLC system 2

TLC system 2 HPLC system B

HPLC system B

HPLC system A

HPLC system A

[18F]impurity

[18F]FET

[18F]FET[18F]FET

D-[18F]FET

L-[18F]FET

[18F]FET

[18F]impurity

[18F]impurity

D-[18F]FET

L-[18F]FET

Fig. 3 Radio-TLC and radio-HPLC analysis of [18F]FET before and

after SPE purification; TLC system 2: BuOH/CH3COOH/H2O/EtOH:

4/1/1.6/0.5 (v/v/v/v); HPLC (radiodetection); system A: Nucleosil

C18, 5 lm, CH3COONa 5 vM, pH 4/C2H5OH (95/5 v/v), 1 mL/min;

system B: Crownpak CR (?), HClO4, pH 2/MeOH 90/10; 0.8 mL/

min

Fig. 4 HPLC analysis of the

final formulation of [18F]FET

(system A); overlay of UV at

254 nm and radio detector

J Radioanal Nucl Chem

123

buffer (0.05 M CH3COONa, pH 4, containing 5 % of

EtOH) and transferred to the second purification cartridge

packed with the cation exchange resin, SCX. After passing

through the combination of cartridges, [18F]FET was col-

lected directly into a vented sterile vial by sterile filtration

using a standard 0.22 lm sterilizing filter.

The purification process was optimized with respect to a

choice of reverse phase cartridge, volume and composition

of the eluting buffer. Screening of a series of reverse phase

cartridges (C18 SepPak, tC18 SepPak Plus, Lichrolut RP

18E, ENVI C18) revealed that the tC18 SepPak Plus car-

tridge was the most effective for purification. Using the

combination of this cartridge with a cation exchange resin

SCX the suggested SPE purification procedure afforded

[18F]FET in higher than 99 % radiochemical and higher

than 95 % enantiomeric purity (Fig. 3).

The identity of [18F]FET was confirmed by co-analyzing

the authentic reference both via TLC (system 2) and HPLC

(system A). The chemical purity was assessed by HPLC

using System A with UV detection at 254 nm (Fig. 4). In

addition to a small peak of non-radioactive [19F]FET (Rt

7.3 min), two unidentified peaks (Rt 4.5 and 19.0–20.0 min)

were observed from which the first one could be hydrolyzed

FET, O-(20-hydroxyethyl)tyrosine. Assuming that the all

unidentified impurities had the same absorption and

molecular weight as the product molecule (approximation),

we evaluated their summarized content in the final prepa-

ration of 8 mL volume not more than 180–200 lg. Rec-

ognizing the general limitation by FDA [22] on the

allowable level of up to 120 lg unidentified impurities in a

single injected dose for the application period of 14 days,

the evaluated content of the impurities in [18F]FET ensured

its safe application (when administration is limited to 50 %

of the produced dose). It should be noted that a PET study

usually includes a single (non-repeated) injection of

*185–370 MBq of a radiotracer in an average volume of

3–5 mL; this is only a fraction of the overall activity that

can be produced by means of this method.

To ensure that the final dose is devoid of any nickel

impurities, the nickel concentration of a few batches of

[18F]FET was determined by the inductively coupled

plasma mass spectroscopy and shown to be negligible,

ranging between 0.9 and 1.2 lg/L (Table 2) that was

comparable or less than normal serum concentrations

(1–5 lg/L [23]). Due to suggested removal of nickel in a

precipitated form, the SPE purification provided lower

level of this metal in the final preparations than the pre-

viously reported [19] HPLC purification method (Table 2).

The suggested synthesis procedure with SPE purification

allowed to obtain [18F]FET in 35 % radiochemical yield

(decay-corrected) in an overall synthesis time of 45 min

with a specific radioactivity at the end of the synthesis

[14 GBq/lmol.

Conclusion

[18F]FET was synthesized via direct nucleophilic synthesis

using earlier developed chiral metallo-complex precursor 4

and purified by means of SPE thereby omitting cumbersome

HPLC purification. The suggested SPE purification using

combination of reverse phase and strong cation exchange

cartridges allowed [18F]FET in high chemical, radiochemical

and enantiomeric purity and 35 % radiochemical yield (decay-

corrected) in 45 min synthesis time. The method was fully

automated using a commercially available synthesis platform

and appeared to be well suited for transfer into an automated

cassette-type radiosynthesizer without using HPLC.

Acknowledgments This work was performed within the CRP pro-

gram of the International Atomic Energy Agency (IAEA), contract

No. 15436.

Conflict of interest We wish to thank Scintomics GmbH, Germany,

for the support of this study. The authors declare that they have no

conflict of interest.

References

1. Coenen HH, Elsinga PH, Iwata R, Kilbourn MR, Pillai MRA,

Rajan MGR, Wagner HN, Zaknun JJ (2010) Fluorine-18 radio-

pharmaceuticals beyond [18F]FDG for use in oncology and neu-

rosciences. Nucl Med Biol 37:727–740

2. Wester H, Herz M, Weber W, Heiss P, Senekovitsch-Schmidtke

R, Schwaiger M, Stocklin G (1999) Synthesis and radiofarma-

cology of O-(2-[18F] fluoroethyl)-L-tyrosine for tumor imaging.

J Nucl Med 40:205–212

3. Popperl G, Kreth FW, Mehrkens JH, Herms J, Seelos K, Koch W,

Gildehaus FJ, Kretzschmar HA, Tonn JC, Tatsch K (2007) FET

PET for the evaluation of untreated gliomas: correlation of FET

uptake and uptake kinetics with tumour grading. Eur J Nucl Med

Mol Imaging 34:1933–1942

4. Galldiks N, Stoffels G, Filss CP, Piroth MD, Sabel M, Ruge MI,

Herzog H, Shah NJ, Fink GR, Coenen HH, Langen KJ (2012)

Role of O-(2-18F-fluoroethyl)-L-tyrosine PET for differentiation

of local recurrent brain metastasis from radiation necrosis. J Nucl

Med 53:1367–1374

Table 2 Nickel concentration in the final formulations of [18F]FET

after HPLC and SPE purifications

HPLC purification Ni content after

SPE purification

(lg/L)Ni content after

HPLC (lg/L)

[19]

Ni content after

HPLC and extra

cartridge

purification

(lg/L)a

Run number 1 2 3 1a 2a 3a 4 5 6

3,5 4,8 4,7 2,0 2,5 2,8 1,2 0,9 1,1

a The product obtained by HPLC purification was additionally passed

through the cartridge IC-Chelate Plus (Alltech)

J Radioanal Nucl Chem

123

5. Dunet V, Rossier C, Buck A, Stupp R, Prior JO (2012) Perfor-

mance of 18F-fluoro-ethyL-tyrosine (18F-FET) PET for the dif-

ferential diagnosis of primary brain tumor: a systematic review

and meta analysis. J Nucl Med 53:207–214

6. Mueller D, Klette I, Kalb F, Baum RP (2011) Synthesis of O-(20-[18F]fluoroethyl)-L-tyrosine based on a cartridge purification

method. Nucl Med Biol 38(5):653–658

7. Gomzina NA, Vassiliev DA, Krasikova RN (2007) The appli-

cation of 2-[18F]fluoroethylbromide in the synthesis of O-(20-[18F]fluoroethyl)-L-tyrosine, radiopharmaceutical for brain

tumors diagnosis by PET. Radiochemistry 49(3):264–269

8. Zuhayra M, Alfteimi A, Von Forstner C, Lutzen U, Meller B,

Henze E (2009) New approach for the synthesis of [18F]fluor-

oethyltyrosine for cancer imaging: simple, fast, and high yielding

automated synthesis. Bioorg Med Chem 17:7441–7448

9. Hamacher K, Coenen HH (2002) Efficient routine production of

the 18F-labelled amino acid O-(2-[18F]fluoroethyl)-L-tyrosine.

Appl Radiat Isot 57:853–856

10. Vaneycken I, Blykers A, Xavier C, Caveliers V (2012) Fully

automated synthesis of 18F-FET using the Synthera Platform.

J Nucl Med 53(1):1476

11. Bourdier T, Greguric I, Roselt P, Jackson T, Faragalla J, Katsifis

A (2011) Fully automated one-pot radiosynthesis of O-(2-[18-

F]fluoroethyl)-L-tyrosine on the TracerLab FXFN module. Nucl

Med Biol 38:645–651

12. Hamacher K, Coenen HH, Stocklin G (1986) Efficient stereo-

specific synthesis of no-carrier-added 2-[18F]fluoro-2-deoxy-D-

glucose using aminopolyether supported nucleophilic substitu-

tion. J Nucl Med 27:235–238

13. Mosdzianowski C, Lemaire C, Lauricella B, Aerts J, Morelle J-L,

Gobert F (1999) Routine and multi-curie level productions of

[18F]FDG using an alkaline hydrolysis on solid support. J Label

Compds Radiopharm 42:515–516

14. Pascali C, Bogni A, Fugazza L, Cucchi C, Crispu O, Laera L,

Iwata R, Maiocchi G, Crippa F, Bombardieri E (2012) Simple

preparation and purification of ethanol-free solutions of 30-deoxy-

30-[(18)F]fluorothymidine by means of disposable solid-phase

extraction cartridges. Nucl Med Biol 39:540–550

15. Wang M, Zhang Y, Zhang Y, Yuan H (2009) Automated synthesis

of hypoxia imaging agent [18F]FMISO based upon a modified

Explora FDG4 module. J Radioanal Nucl Chem 280:149–155

16. Blom E, Koziorowski J (2014) Automated synthesis of [18F]

FMISO on IBA Synthera. J Radioanal Nucl Chem 299:265–270

17. Wang H-E, Wu S-Y, Chang C-W, Liu R-S, Hwang L-C, Lee

T-W, Chen J-C, Hwang J–J (2005) Evaluation of F-18-labeled

amino acid derivatives and [18F]FDG as PET probes in a brain

tumor-bearing animal model. Nucl Med Biol 32:367–375

18. Wang M, Yin D, Li S, Wang Y (2007) Synthesis of O-(2-[18F]

fluoroethyl)-L-tyrosine and its biological evaluation in B16 mel-

anoma-bearing mice as PET tracer for tumor imaging. Sci China

Ser B-Chem 50(2):276–283

19. Krasikova RN, Kuznetsova OF, Fedorova OS, Maleev VI, Sa-

vel’eva TF, Belokon YN (2008) No carrier added synthesis of O-

(20-[18F]fluoroethyl)-L-tyrosine via a novel type of chiral enanti-

omerically pure precursor, NiII complex of a (S)-tyrosine Schiff

base. Bioorg Med Chem 16:4994–5003

20. Krasikova RN, Orlovskaya VV, Stepanova MA, Fedorova OS

(2013) The effect of reaction media and phase transfer catalyst on

the fluorination yield and enantiomeric purity in asymmetric

synthesis of O-(20-[18F]fluoroethyl)-L-tyrosine. Curr Org Chem

17(19):2159–2163

21. Krasikova RN (2013) PET radiochemistry automation: state of

the art and future trends in 18F-nucleophilic fluorination. Curr Org

Chem 17(19):2097–2107

22. Guidance for Industry. Genotoxic and Carcinogenic Impurities in

Drug Substances and Products: Recommended Approaches

(2008) U.S. Department of Health and Human Services Food and

Drug Administration. http://www.fda.gov/downloads/Drugs/.../

Guidances/ucm079235.pdf. Accessed Dec 2008

23. McClatchey KD (2002) Clinical Laboratory Medicine. Lippincott

Williams & Wilkins, Philadelphia, p 1693

J Radioanal Nucl Chem

123