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Applied Radiation and Isotopes 68 (2010) 1937–1943

Contents lists available at ScienceDirect

Applied Radiation and Isotopes

0969-80

doi:10.1

n Corr

E-m

(M.G.R.

journal homepage: www.elsevier.com/locate/apradiso

The possibility of a fully automated procedure for radiosynthesis offluorine-18-labeled fluoromisonidazole using a simplified single,neutral alumina column purification procedure

Saikat Nandy a, M.G.R. Rajan a,n, A. Korde b, N.V. Krishnamurthy c

a Radiation Medicine Centre, Bio-Medical Group, Bhabha Atomic Research Centre, Tata Memorial Hospital Annexe, Parel, Mumbai-400 012, Indiab Radiopharmaceuticals Division, Radiochemistry & Isotope Group, Bhabha Atomic Research Centre, Trombay, Mumbai-400 085, Indiac Medical Cyclotron Facility, Board of Radiation and Isotope Technology, Department of Atomic Energy, Tata Memorial Hospital Annexe, Parel, Mumbai-400 012, India

a r t i c l e i n f o

Article history:

Received 31 March 2009

Received in revised form

23 March 2010

Accepted 1 April 2010

Keywords:

[18F]FMISO

Neutral-alumina purification

Non-radioactive impurities

NITTP

43/$ - see front matter & 2010 Elsevier Ltd. A

016/j.apradiso.2010.04.006

esponding author. Tel.: +91 22 24157098; fa

ail addresses: mgr_rajan@hotmail.com, mgr.ra

Rajan).

a b s t r a c t

A novel fully automated radiosynthesis procedure for [18F]Fluoromisonidazole using a simple alumina

cartridge-column for purification instead of conventionally used semi-preparative HPLC was developed.

[18F]FMISO was prepared via a one-pot, two-step synthesis procedure using a modified nuclear

interface synthesis module. Nucleophilic fluorination of the precursor molecule 1-(20-nitro-10-

imidazolyl)-2-O-tetrahydropyranyl-3-O-toluenesulphonylpropanediol (NITTP) with no-carrier added

[18F]fluoride followed by hydrolysis of the protecting group with 1 M HCl. Purification was carried out

using a single neutral alumina cartridge-column instead of semi-preparative HPLC. The maximum

overall radiochemical yield obtained was 37.4971.68% with 10 mg NITTP (n¼3, without any decay

correction) and the total synthesis time was 4071 min. The radiochemical purity was greater than 95%

and the product was devoid of other chemical impurities including residual aluminum and acetonitrile.

The biodistribution study in fibrosarcoma tumor model showed maximum uptake in tumor, 2 h post

injection. Finally, PET/CT imaging studies in normal healthy rabbit, showed clear uptake in the organs

involved in the metabolic process of MISO. No bone uptake was observed excluding the presence of free

[18F]fluoride. The reported method can be easily adapted in any commercial FDG synthesis module.

& 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The oxygen-dependent covalent binding of the nitroimidazole,misonidazole (1-(2-nitroimidazolyl)-2-hydroxy-3-methoxypropane, MISO) in cells, multicellular spheroids, and tumors hasstimulated interest in using this drug or a congener as an imagingagent for hypoxia in malignant tumors, myocardial infarct, orcerebral ischemia (Rasey et al., 1990; Wiebe, 2004; Hodgkiss,1998; Startford and Workman, 1998; Machulla, 1999). In vivodemonstration of hypoxia requires tissue measurements withoxygen-electrodes, but the invasiveness of this technique haslimited its application. Therefore, non-invasive assessment oftumor hypoxia with a specific radiotracer, prior to radiationtherapy should provide a rational means for selecting patients fortreatment with bioreductive drugs and chemical radiosensitizers.In addition, it is possible to differentiate radiation therapymodalities (neutron versus photon) by correlating results with

ll rights reserved.

x: +91 22 24108625.

jan@gmail.com

labeled markers of hypoxic cells with tumor response. Thepotential advantage of neutron over conventional photon radia-tion is the former’s reduced dependence on oxygenation of thetumor and less variability of cell sensitivity to neutrons aroundthe cell cycle (Yang et al., 1995). Consequently, the radiolabeledanalogues of MISO and its derivatives are used as markers ofhypoxic tissues (Jerabek et al., 1986; Rasey et al., 1987, 1996; Kohet al., 1992; Martin et al., 1992; Varagnolo et al., 2000; Gronrosset al., 2001; Lehtio et al., 2003). PET-imaging with [18F]FMISO canhelp to estimate the oxygenation status of tumors in any part ofthe body (Iulina Toma-Dasu et al., 2009). The tracer has also beenused to study the relative hypoxic volume of tumors during thecourse of radiation treatment. Recently, improvement in responseto treatment with new selective experimental chemotherapyagents has been observed by using [18F]FMISO and PET (Eary andKrohn, 2000). Notwithstanding the disadvantages of [18F]FMISO,which, due its lypophilicity, is retained in the brain and is slowlycleared by the hepatobiliary route leading to high backgroundand, hence, requiring delayed imaging; it is still the most usedradiotracer in hypoxia studies in humans and it is in high demandfor PET oncology studies (Koh et al., 1992: Valk et al., 1992;Rajendran et al., 2003, 2006; Couturier et al., 2004), though there

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Scheme 1. Synthesis procedure for FMISO synthesis.

S. Nandy et al. / Applied Radiation and Isotopes 68 (2010) 1937–19431938

are reports of the use of its more hydrophilic derivative,fluoroerythronitroimidazoze, FETNIM (Yang et al., 1995; Gronrosset al., 2001). Additionally, a large number of [18F]-labeledanalogues have been developed such as [18F]FETA (Rasey et al.,1999), [18F]EF1 (Kachur et al., 1999), [18F]EF5 (Couturier et al.,2004; Dolbier et al., 2001; Komar et al., 2008; Mahy et al., 2006)and [18F]FAZA (Kumar et al., 2002) for hypoxia imaging, althoughthe data regarding their use in human studies is limited.

The most commonly used route for the synthesis of[18F]FMISO, to date, is the nucleophilic substitution of thetosylate-leaving group by [18F]fluoride on the tetrahydropyra-nyl-protected precursor 1-(20-nitro-10-imidazolyl)-2-O-tetrahy-dropyranyl-3-O-toluenesulphonylpropanediol (NITTP) followedby the hydrolysis of the protecting group (Kamarainen et al.,2004; Patt et al., 1999). The synthesis procedure is summarized inScheme 1. Recently, a fully automated synthesis of [18F]FMISOusing HPLC purification with a radiochemical yield more than 60%in a synthesis time of approximately 60 min has been reported(Patt et al., 1999). [18F]FMISO radiosynthesis using Sep-Paks

purification with more than 40% radiochemical yield (withoutdecay correction) in about 40 min has also been reported (Tanget al., 2005). Since HPLC purification is quite complicated forroutine operation and significantly adds to the synthesis time, wereport a fully automated radiosynthesis procedure of [18F]FMISOthrough nucleophilic fluorination and employing a neutralalumina cartridge-column purification using a Nuclear Interfacesynthesis module configured for FDG synthesis. A similar studyusing a modified Explora FDG4 module is reported by Wang et al.(2009) with a decay corrected yield of 55% within a total synthesistime of 50 min. Systematic investigation of the dependence ofradiochemical yield on the amount of precursor initially taken hasalso been investigated.

2. Materials and methods

2.1. Reagents and apparatus

NITTP, FMISO reference standard, 75 mM TBAHCO3 solution,molecular-grade acetonitrile, 10% NaCl, 1 M HCl, 1 M NaH2PO4

buffer, sterile and pyrogen-free water for injection, and pharma-ceutical grade ethanol were procured from ABX AdvancedBiochemical Compounds, Germany. Fluorine-18 separation car-tridge, Chromafix 45-PS-HCO3, was obtained from Marcherey-Nagel, Germany. Aluminum oxide active (neutral, Brockmanngrade I–II) for column chromatography was procured from Merck,India. Evacuated 10 mL vials (sterile and pyrogen free) wereobtained from ACILA AG, Germany. Minisart 0.2 m filters werepurchased from Sartorius. Fluid thioglycollate medium andsoyabean casein digest for sterility tests were procured fromHi-Media, Mumbai. Pyrogen levels were tested by LAL method in

accordance with USP XXV, using Endosafe Reagent Kits fromCharles River Laboratory, USA (US License No: 1197). Radio-activity was measured using an ion chamber (Capintec CRC-15 R).Radiofluorination and conversion of NITTP to [18F]FMISO wascarried out in a Nuclear Interface Module (Munster, Germany)configured for general-purpose fluorination and extensively usedfor [18F]FDG production (Fig. 1). Radio-HPLC analysis was carriedout in a Knauer system (Germany) with a tunable UV absorptiondetector and a radiometric detector system using a C-18 reversephase analytical column (Nucleosil, 5 mM, 250�4 mm dia.). TheUV absorption was monitored at 254 nm. Radio TLC was carriedout on Silica Gel 60, 20�20 and Silica Gel 60 F254, 20�20 (Merck,Germany) and scanned using a RayTest TLC scanner with a BGOscintillation detector and spectrum analyzed with GINAs

software. The residual solvent analysis was carried out using asemi-automatic Gas Chromatograph from Chemito, Indiaequipped with a flame ionization detector (FID) using a PEGcapillary column (BP-20 from SGE, Australia). Alizarin Red S wasprocured from Sigma. Aluminum-Test Kit, Merckoquants (Assayrange: 10–250 mg/L) and Microquants (Assay range: 0.1–6 mg/L)were procured from Merck, India. All other chemicals used wereof either HPLC or analytical grade and procured locally. All animalexperiments were carried out as per guidelines set for animalexperiments and after approval from local animal ethicscommittee. PET-CT imaging of rabbits were done using theDiscovery ST PET-CT scanner (GEMS, GE, USA) available with theBio-imaging Unit, Tata Memorial Hospital, Parel, Mumbai.

2.2. Automated synthesis of [18F]FMISO

The fully automated synthesis of [18F]FMISO consists of threemain steps: (1) nucleophilic fluorination of NITTP, (2) deprotec-tion, and (3) purification through a neutral alumina cartridge-column. [18F]fluoride from the cyclotron is first trapped on aChromafix 45-PS-HCO3 anion exchange cartridge (Fig. 2). This isthen eluted from the column using 75 mM TBAHCO3 (0.4 mL) tothe reaction vessel. The [18F]TBA fluoride was then dried byazeotropic distillation with acetonitrile (0.8 mL). To this, theNITTP precursor (25, 10, or 5 mg, respectively) dissolved inacetonitrile (0.6 mL) was added and SN2 fluorination reactioncarried out at 110 1C for 15 min. The acetonitrile was thenevaporated from the reaction mixture and acid hydrolysis wascarried out with 1 N HCl (1 mL) at 105 1C for 10 min. The reactionmixture was cooled to 50 1C and the reaction mixture is passedthrough the alumina cartridge-column.

2.3. [18F]FMISO purification cartridge-column

The [18F]FMISO purification cartridge-column is ready to use,composed of only neutral alumina, active, Brockmann grade I–IIand packed in our laboratory (Fig. 2). We are not using

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Fig. 1. Schematic representation of general purpose fluorination module.

Fig. 2. Neutral alumina purification column and PS-HCO3 anion exchange column.

S. Nandy et al. / Applied Radiation and Isotopes 68 (2010) 1937–1943 1939

commercially available Sep-Pak neutral alumina cartridges sincewe require more alumina than what is available in one Sep-Pak.Briefly, the cartridge-column is made as follows: 7.7 g of neutralalumina (dry weight) is added to 200 mL of sterile, deionizedwater and allowed to settle leaving the fine particles insuspension, which is decanted off. This is repeated thrice. Thesedimented alumina is kept at 60 1C (overnight) till it dries to afree flowing powder, which is packed inside a polypropylenecartridge (6.5 cm�1.2 cm) taking care to avoid air bubbles. Two20-mm-polyethylene frits are placed, one at the top and other atthe bottom of the cartridge-column to prevent the aluminaparticles entering the product. Larger batches of alumina can bewashed, dried and stored in an air-tight container and used whenrequired. The cartridge-column was washed thoroughly bypassing 20 mL of sterile and bacterial endotoxin free water justbefore use. The reaction mixture from the reactor is loaded to thecolumn and the former rinsed with 1.5 mL of 5% ethanol andadded to the column and the eluate directed to waste and drainedcompletely by pasing Helium gas. Finally, [18F]FMISO is elutedusing 10% ethanol (12 mL) and is collected in the product vialcontaining 10% NaCl (1.7 mL) and 1 M NaH2PO4 (0.7 mL). Fromthe product vial, the [18F]FMISO is transferred to an automated

dispensing unit and aliquoted into different vials in a class 100area through a 0.2 mM filter. The [18F]FMISO was obtained as aclear, colorless solution, free of any suspended particles.

2.4. Quality control and stability

The pH of [18F]FMISO was checked with pH test paper strip.The radiochemical purity was first checked by radio-TLC using asilica gel 60-coated plate developed in methanol/ammonia (95:5,v/v) solvent system. TLC of the reference standard FMISO was alsodeveloped in the same solvent and stained with iodine vapor forcomparison. Additionally the chemical and radiochemical puritywere also analyzed using an analytical HPLC by monitoring the UVabsorbance (l¼254 nm) as well as the radioactivity profile. Themobile phase consisted of 70% MeOH and 30% water and HPLCcarried out in an isocratic system at a flow rate of 0.5 mL/min. Thepresence of non-radioactive impurities was also verified undersimilar HPLC conditions by analyzing pure NITTP, reference-standard FMISO and a mixture of the two. Finally, the identity ofthe [18F]FMISO prepared was confirmed by co-eluting peaks(radioactive as well as UV) in HPLC analysis and comparing the UVpeak of [18F]FMISO with that of cold reference standard of FMISO.Radiochemical stability was checked with a 10 mL solution [9 mLsaline+1 mL [18F]FMISO (10% ethanol)] using radio TLC andanalytical HPLC up to 8 h post synthesis.

2.5. Gas chromatograph analysis

Sealed samples from each synthesis of [18F]FMISO were storedat room temperature and analyzed later, for the residual solvents,mainly, the presence of toxic acetonitrile, using gas chromato-graphy (Channing et al., 2001). The GC column was maintained ata temperature of 50 1C during the operation. An aqueous solutioncontaining 200 ppm each of ethanol and acetonitrile was used asstandard solutions. One mL of [18F]FMISO sample, after radioactivedecay, was injected and compared against the calibration dataobtained from 1 mL injection of the standard solutions.

2.6. Test for aluminum ions

The test for aluminum ions (Al3 +) in the product wasperformed using the following two methods: (1) The AlizarinRed S method (Nakao et al., 2005): In a test tube containing 50 ml

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of the sample, 50 ml of a 10% ammonia solution and then 50 mL ofan aqueous solution of Alizarin Red S were added. The colour(wine red) developed in the presence of Al3 + , stable after adding

Table 1Dependence of radiochemical yield on amount of precursor (reaction conditions:

radiofluorination at 110 1C, 15 min and acid hydrolysis at 105 1C, 10 min).

S. no Amount ofprecursor,NITTP (mg)

Radiochemical yielda

(%) (individualsynthesis)

RadiochemicalYielda (%)(average)

1 5 24.0 23.4070.6522.723.5

2 10 39.2 37.4971.6835.837.5

3 25 34.3 34.1073.2030.631.538.435.6

a Without decay correction.

Fig. 3. (a) TLC of radiofluorinated NITTP and (b) radio-

500 ml of 1 N acetic acid, was compared visually with that ofstandard Al3 + aqueous solutions (0, 5, 10, and 20 mg/mL Al3 +)treated similarly. Standard solutions were tested. (2) The sampleswere also analyzed by Microquants Al test kit as per standardprotocol provided with the kit.

2.7. Sterility and bacterial endotoxin analysis

Sterility tests were performed according to the IndianPharmacopoeia (IP 1996 and IP addendum 2005) protocol. In thistest, 1 mL of the [18F]FMISO sample after radioactive decay, wasinoculated in fluid thioglycollate medium and incubated at 37 1Cfor 14 days to observe the growth of aerobic and anaerobicbacteria. Similarly, 1 mL of the decayed [18F]FMISO sample wasalso inoculated in soyabean casein digest medium and maintainedat 22–25 1C for 14 days to detect fungal growth. The bacterialendotoxin test was performed in accordance with USP XXV. Thetest was based on the formation of gel clot in the sample byLimulus Amoebocyte Lysate (sensitivity: 0.125 EU/mL) reagent.

TLC of the reaction mixture after acid hydrolysis.

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2.8. Biodistribution

Biodistribution of [18F]FMISO was performed in Swiss miceweighing about 25–30 g, bearing fibrosarcoma tumors (developedby subcutaneous injection of 106 cells per animal). After 14 daysof the injection of the cells, a tumor diameter of 1–2 cm wasobserved. [18F]FMISO in isotonic 10% ethanolic water wasadministered to three groups of mice with four animals pergroup. Considering the small weight of the mice used, only100 KBq in 0.3 mL per animal was administered, intravenouslyinjected through the tail-vein. The animals were sacrificed at 30,120, and 240 min and the various organs and blood wereremoved. These were weighed and counted for radioactivity.The radioactivity in each organ was measured and expressed asthe percentage of injected dose present per gram of the organ. Theresults are summarized in Table 2.

2.9. PET-CT imaging

The normal biodistribution of [18F]FMISO was studied by PET/CT imaging of normal healthy rabbit (�2.5 kg, maintained in ouranimal house) after overnight fasting. [18F]FMISO (111 MBq/1.0 mL) was injected intravenously through the ear vein. Imageswere recorded after 1 and 4 h post injection after standardanesthesia administration.

Fig. 5. HPLC chromatogram of [18F]FMISO. The radioactive and UV detectors are

connected in series.

3. Results and discussion

3.1. Synthesis and quality control

Starting from the precursor, NITTP, [18F]FMISO was prepared ina synthesis module configured for [18F]FDG synthesis. Purificationwas achieved using a single neutral alumina cartridge-column.The radiochemical yield expressed as the percentage of radio-activity finally obtained as [18F]FMISO compared with the starting18F activity, without decay correction, was 23.4070.65% (n¼3),37.4971.68% (n¼3), and 34.173.2 (n¼5) with 5, 10, and 25 mgof NITTP, respectively (Table 1). Hence, the highest yield wasobtained with 10 mg of precursor in our study with three

Fig. 4. (a) TLC of [18F]FMISO and (b

quantities of precursor. The overall yield did not seem toimprove by using more than 10 mg of NITTP .

The final [18F]FMISO obtained was clear, colourless, and free ofany turbidity or suspended particles and the pH was observed tobe in the range of 6.5–7.0. The entire synthesis was monitored byTLC at the three important steps, viz., (1) after radiofluorination ofthe NITTP, (2) after acid hydrolysis of the 18F-substituted NITTP,and finally (3) after purification through the single neutralalumina cartridge-column and elution with 10% ethanolic water.The TLC of the radiofluorinated NITTP showed the presence of free[18F]fluoride with a Rf of 0.04 whereas the radiofluorinated NITTPwas observed as a broad peak with a Rf of 0.85 as shown inFig. 3(a). The TLC pattern of acid hydrolyzed radiofluorinatedNITTP was also of similar nature showing two peaks onecorresponding to free fluoride and another to [18F]FMISO(Fig. 3(b)). The TLC of the final purified [18F]FMISO showed onlyone prominent peak with a Rf of 0.69 as shown in Fig. 4(a). Thedifference in the Rf value of the THP-protected reaction product,[18F]NITTP and [18F]FMISO were in accordance with their polarity.The Rf value of [18F]FMISO was further verified by TLC of the

) TLC of iodine stained FMISO

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reference standard FMISO in the same solvent system and thenstaining with iodine vapor (Fig. 4(b)). The Rf value of [18F]FMISOexactly matched with that of reference standard FMISO afteriodine staining. The HPLC chromatogram of [18F]FMISO is shownin Fig. 5. From the chromatogram it is seen that the retention timeof [18F]FMISO is 7.9 min and the corresponding retention timefrom the UV chromatogram is 10.04 min. The difference in theretention times of radioactivity and UV peak is attributed to thedead volume of the 1 m long tubing between the radioactivitydetector followed by the UV detector as configured in our HPLCsystem. This always gives a difference in the retention times of theradioactivity peak and the UV peak. The retention time of

Fig. 7. PET/CT scan of rabbit following [18F]FMISO

Table 2Biodistribution of [18F]FMISO in fibrosarcoma tumor model.

Organ % Injected radioactivity per gram tissue30 min (n¼4) 120 min (n¼3) 240 min (n¼4)

Blood 4.4572.22 1.4270.19 1.7071.07

Bone 1.8470.18 2.2971.06 2.90771.07

Muscle 2.1870.16 1.4571.00 1.4470.88

Liver 4.5970.24 5.3471.60 1.9270.34

Intestine+gall bladder 4.5871.86 5.0471.51 11.3474.72

Fibrosarcoma tumor 2.1970.48 7.7772.66 4.4870.89

Fig. 6. HPLC Chromatogram of reference standard FMISO (only UV).

[18F]FMISO was validated using a reference FMISO standard(Fig. 6). The presence of UV active non-radioactive impuritieswas also examined. The precursor, NITTP shows a retention timeof 15.13 min. When a mixture of NITTP and reference standardFMISO were analyzed two well resolved peaks were obtained at9.13 min (FMISO) and 15.80 min (NITTP), respectively. The HPLCanalysis of the radiofluorinated NITTP precursor showed two UVpeaks at 10.13, and 16.2 min, respectively, but a single radioactivepeak at 10.40 min. By comparing this with the retention times ofFMISO and NITTP standards, it is apparent that the first UV peakcould be that of [18F]NITTP whereas the second one that ofunreacted NITTP, which is comparatively more non-polar due tothe presence of THP moiety. Free 18F� did not elute out under theHPLC condition and this was verified by carrying out HPLC of free18F� . The HPLC of the [18F]FMISO showed only one UV peak andone radioactive peak. Hence, it could be concluded that the final[18F]FMISO was free from any kind of non-radioactive impurity.The formation of MISO, which is reported to show someneurotoxicity at therapeutic dose, is virtually impossible underthe experimental conditions employed. Since it has to beproduced by the hydrolysis of the precursor NITTP followed bythe etherification of the –OH generated from the tosyl group.

GC analysis showed no traces of acetonitrile in the final[18F]FMISO. However, ethanol is present as expected since theproduct is eluted with 10% ethanol in water. The permissible levelof Al3 + in the product is 5 mg/mL. The Alizarin Red S methoddetects Al3 + on the basis of colour development reactionsbetween aluminum hydroxide (formed from Al3 + in the presenceof ammonium base) and Alizarin Red S with a visual detectionlimit of 5 mg/mL (Nakao et al., 2005). We found no colourformation with our [18F]FMISO product, tested post radioactivedecay, indicating that the Al3 + ion concentration is below theallowed limit of 5 mg/mL. Further analysis with Microquants Al3 +

test kit (sensitivity 0.1–6 mg/mL) based on Chromazurol S methodconfirmed that Al3 + in the final [18F]FMISO was in the range of3–4 mg/mL. The biodistribution study in fibrosarcoma tumorbearing mice showed maximum uptake in tumor 2 h postinjection (% injected dose/g: 7.872.7) (Table 2) but decreasedafter 4 h post injection (% injected dose/g¼4.4870.89). Boneuptake did not alter significantly. The biodistribution result was inaccordance with the findings by Yang et al. (1995). PET/CT imagesof the rabbit were recorded 1 h and 4 h post injection and areshown in Fig. 7(a) and (b). There is significant uptake in organsinvolved in the metabolic pathway of MISO like liver, GI tract, etc.Uptake in brain is also observed as expected.

injection (a) 1 h and (b) 4 h post injection.

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4. Conclusion

We have developed a novel, fully automated radiosynthesisprocedure for [18F]FMISO achieving satisfactory chemical andradiochemical purity using single neutral alumina cartridge-column for purification instead of semi-preparative HPLC. Further,we could do this in a general purpose fluorination moduleconfigured for FDG synthesis with moderately good yield37.4971.68% with 10 mg NITTP, without decay correction, in4071 min. The synthesis procedure is fast, reliable, and verysimilar to [18F]FDG synthesis procedure with consistent radio-chemical yield, and gives a product that fulfills the criteria ofradiopharmaceutical quality. This new procedure can easily beused for the routine production of [18F]FMISO using conventionalcommercially available FDG synthesis module.

Acknowledgements

We would like to thank Dr. P.S.Soni, Head, Medical CyclotronFacility, Board of Radiation and Isotope Technology (BRIT),Department of Atomic Energy, Mumbai for help related to thecyclotron production of 18F. The support from Dr. V. Rangarajanand technologists from Bio-imaging Unit, Tata Memorial Hospital,Mumbai, India for PET/CT imaging of rabbits is sincerely acknowl-edged. Help from Quality Control and Microbiology Staff of BRIT indetermining the Al3 + ion concentration and analyzing thesamples for BET and sterility test is sincerely acknowledged.

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