these radiopharmaceuticals are formulated from cyclotron - produced or generator - eluted radionuclides and should be subjected to quality control ( qc ) tests to assure their safety and efficacy before injecting to the patient .
fluorodeoxyglucose ( f-18 fdg ) , a glucose analog , is the most popular pet radiopharmaceutical .
o-15 , ga-68 , i-124 , and cu-64 , and so on are also being used for pet imaging .
however , catalysts like kryptofix ( kryptofix 2.2.2 . ) or tetrabutylammonium ( tba ) salts are required for f-18 fdg synthesis .
somatostatin peptide analogues like dotatate , dotanoc , and dotatoc have been labeled with ga for somatostatin receptor imaging .
n-13 is a short - lived pet radionuclide ( t1/2 = 10 min ) and is produced in cyclotron .
the proton irradiation of a natural water target o ( p ,  ) n is an efficient method .
oxo anions of nitrogen n-13 ( n-13-no3 and n-13-no2 ) are produced from radiolytic oxidation . with the use of reducing agents such as devarda 's alloy / sodium hydroxide or titanium - chloride / hydroxide ,
these oxo - anions can be converted to n-13 nh3 qc parameters of n-13 nh3 were studied by various methods .
the prepared radiopharmaceuticals are administered intravenously ; therefore , qc is mandatory , but there is no global consensus in relation to required quality attributes .
the three major standards  ph.int . , ph . eur . , and usp are available for f-18 fdg .
however , there are differences in f-18fdg quality requirements among usp , ep , and int phar . in this study ,
the qc parameters of three pet radiopharmaceuticals , f-18 fdg , n-13 nh3 , and ga-68 dotatate are studied .
pet radiopharmaceuticals , thin layer chromatography ( tlc ) scanner ( ez - scan , usa ) with multimode radiation detector , ( omni - rad ) gas chromatography ( gc ) ( varian 3900 ) with flame ionization detector ( fid ) , multichannel analyzer ( captus 3000 , capintec ) , well counter , dose calibrator ( capintec crc-25pet ) , silica gel chromatography paper , ph paper ( merck ) , lal reagents from charles river laboratories india pvt . ltd .
the f-18 was produced by o ( p , n ) f reaction using 16.5 mev on - site cyclotron ( pet trace-4 , ge healthcare , milwaukee , usa ) by proton irradiation for 45 min at 40a current using o-18 enriched water . at the end of bombardment , the fluoride was transferred directly to the automatic synthesizer module ( tracer lab mxfdg , ge healthcare , milwaukee , usa ) by helium pressure .
( abx , germany ) that contained all chemicals required for the nucleophilic radiosynthesis were used .
the labeled precursor was hydrolyzed under basic medium to eliminate the protecting group and sterilized by 0.22 m millipore filter .
the f-18 fdg ( 16 ml ) was collected in a sterile glass vial and qc was performed .
ga-68 dotatate was prepared using ge-68/ga-68 generator and iqs fluidic labeling module with in - built heater , both from itg germany .
ethanol ( 30% ) was used for elution of prepared peptide ( ga-68 dotatate ) from c-18 cartridge .
eluted product was passed through a 0.22 m low protein binding filter and collected in a sterile vial .
n-13nh3 was produced by o ( p ,  ) n irradiation reaction using on - site cyclotron .
oxoanions of nitrogen ( n-13 no3 , and n-13 no2 ) were produced from radiolytic oxidation .
oxoanions were converted to n-13 nh3 by the use of devarda 's alloy , a reducing agent , in sodium hydroxide .
n-13 nh3 vapors were collected in normal saline in a sterilized vial after passing through 0.22 m filter . a total of 25 samples each of f-18 fdg and ga-68 dotatate and n-13 nh3 were randomly selected .
radionuclide purity was assessed by half - life of radionuclides ( t1/2 ) and was measured by two methods , well counter and formula method .
activity of test samples ( f-18 fdg , ga-68 dotatate ) after every 10 min ( a10 ) was recorded .
t1/2 was calculated from the measured values as per the formula : where , t1/2 and t are in minutes .
counts / activity of f-18 fdg sample at 10 s interval was also taken to determine the impurity of n-13 in f-18 fdg . in case of n-13 nh3
a time activity curve was plotted on semilog graph and half - life ( t1/2 ) was taken as the time at which the total activity was decayed to half . for a gamma spectrum of pet radionuclides ,
a repeat spectrum of ga-68 point source was also obtained after 24 and 48 h to determine the ge-68 breakthrough .
radiochemical purity was assessed by silica gel instant thin layer chromatography ( sg - itlc ) . a drop ( 4 - 5 l ) of test sample was placed on a sg - itlc strip ( 10 * 1 cm for f-18 fdg , 8 * 1 for ga-68dotatate and n-13 nh3 ) and air dried .
acetonitrile : water ( 95:5 , v / v ) and sodium citrate was used as mobile phase for f-18 fdg and ga-68 dotatate , respectively .
water : acetone : acetic acid ( 3:2:1 ) saturated with sodium chloride ( nacl ) was used as mobile phase for n-13-nh3 . the radioactivity profile and rf
the amount of organic solvents present in the test samples was estimated by gc using fid detector .
the temperature of detector , oven , and injector were kept constant for all pet radiopharmaceuticals .
hydrogen , air flow , and nitrogen flow rate were also kept constant for all samples .
reference standard of ethanol ( 4000 ppm ) and acetonitrile , ( 400 ppm ) and samples ( 2 - 3 l ) were injected into the gc column and an analysis report was generated .
the concentration of ethanol and acetonitrile in the sample was calculated by the following formula : where c1 and c2 are the concentration of standard and sample .
the chemical purity was calculated for f-18 fdg as kryptofix 2.2.2 ( amino polyether ) may be present in the final product of f-18 fdg .
a total of 4 - 5l of f-18 fdg test sample and the reference standard of kryptofix 2.2.2 ( 0.22 mg / ml ) were developed in the mobile phase composed of methanol : ammonia ( 9:1 , v / v ) .
the developed strips , after drying , were exposed to iodine vapors in a closed container of iodine crystals .
robertson cooked meat medium broth ( rcmb ) was used for cultivation of aerobes and anaerobes , especially pathogenic clostridia .
small inoculation was made near the bottom of the tube and incubated at 37c for 7 days .
the samples were observed every day for 7 days to observe the turbidity or bubble formation by organisms .
the widely used and accepted test for assessing the presence of bacterial endotoxin in a radiopharmaceutical preparation is limulus amebocyte lysate ( lal ) test .
the estimation of bacterial endotoxin test is based on the formation of gel clot in the presence of endotoxin .
this test was performed in randomly selected samples for each pet radiopharmaceutical ( n = 25 ) in a thermally regulated and vibration - free environment .
the permissible limit of endotoxin in injectable formulation is 175 eu / v according to usp and per mlaccording to ph .
the endotoxin concentration in positive water control ( pwc ) was 0.25 and 0.5 eu/ ml .
each sample was diluted at two different maximum valid dilutions ( mvds , i.e. , 350 and 700 times to match with the above - mentioned dilutions of endotoxin .
lal reagent water was used as negative control . after adding reagents in appropriate volume and concentration according to the protocol
, the test vials were incubated for 60 min at 37  1c and were inverted to observe the formation of gel .
a total of 25 samples each of f-18 fdg and ga-68 dotatate and n-13 nh3 were randomly selected .
radionuclide purity was assessed by half - life of radionuclides ( t1/2 ) and was measured by two methods , well counter and formula method .
the initial radioactivity ( a0 ) and activity of test samples ( f-18 fdg , ga-68 dotatate ) after every 10 min ( a10 ) was recorded .
t1/2 was calculated from the measured values as per the formula : where , t1/2 and t are in minutes .
counts / activity of f-18 fdg sample at 10 s interval was also taken to determine the impurity of n-13 in f-18 fdg . in case of n-13 nh3
a time activity curve was plotted on semilog graph and half - life ( t1/2 ) was taken as the time at which the total activity was decayed to half .
for a gamma spectrum of pet radionuclides , a point source was prepared and placed in well counter .
a repeat spectrum of ga-68 point source was also obtained after 24 and 48 h to determine the ge-68 breakthrough .
radiochemical purity was assessed by silica gel instant thin layer chromatography ( sg - itlc ) . a drop ( 4 - 5 l ) of test sample was placed on a sg - itlc strip ( 10 * 1 cm for f-18 fdg , 8 * 1 for ga-68dotatate and n-13 nh3 ) and air dried .
acetonitrile : water ( 95:5 , v / v ) and sodium citrate was used as mobile phase for f-18 fdg and ga-68 dotatate , respectively .
water : acetone : acetic acid ( 3:2:1 ) saturated with sodium chloride ( nacl ) was used as mobile phase for n-13-nh3 . the radioactivity profile and rf
the amount of organic solvents present in the test samples was estimated by gc using fid detector .
the temperature of detector , oven , and injector were kept constant for all pet radiopharmaceuticals .
hydrogen , air flow , and nitrogen flow rate were also kept constant for all samples .
reference standard of ethanol ( 4000 ppm ) and acetonitrile , ( 400 ppm ) and samples ( 2 - 3 l ) were injected into the gc column and an analysis report was generated .
the concentration of ethanol and acetonitrile in the sample was calculated by the following formula : where c1 and c2 are the concentration of standard and sample .
the chemical purity was calculated for f-18 fdg as kryptofix 2.2.2 ( amino polyether ) may be present in the final product of f-18 fdg .
the permissible limit for kryptofix 2.2.2 is 0.22 mg / ml . a total of 4 - 5l of f-18 fdg test sample and the reference standard of kryptofix 2.2.2 ( 0.22 mg / ml ) were developed in the mobile phase composed of methanol : ammonia ( 9:1 , v / v ) . the developed strips , after drying , were exposed to iodine vapors in a closed container of iodine crystals .
robertson cooked meat medium broth ( rcmb ) was used for cultivation of aerobes and anaerobes , especially pathogenic clostridia .
small inoculation was made near the bottom of the tube and incubated at 37c for 7 days .
the samples were observed every day for 7 days to observe the turbidity or bubble formation by organisms .
the widely used and accepted test for assessing the presence of bacterial endotoxin in a radiopharmaceutical preparation is limulus amebocyte lysate ( lal ) test .
the estimation of bacterial endotoxin test is based on the formation of gel clot in the presence of endotoxin .
this test was performed in randomly selected samples for each pet radiopharmaceutical ( n = 25 ) in a thermally regulated and vibration - free environment .
the permissible limit of endotoxin in injectable formulation is 175 eu / v according to usp and per mlaccording to ph .
the endotoxin concentration in positive water control ( pwc ) was 0.25 and 0.5 eu/ ml .
each sample was diluted at two different maximum valid dilutions ( mvds , i.e. , 350 and 700 times to match with the above - mentioned dilutions of endotoxin .
lal reagent water was used as negative control . after adding reagents in appropriate volume and concentration according to the protocol
, the test vials were incubated for 60 min at 37  1c and were inverted to observe the formation of gel .
the product of f-18 fdg , ga-68 dotatate , and n-13 nh3 , were clear , colorless , and free from particulate matter .
the ph of all the samples of f-18 fdg , n-13 nh3 , and ga-68 dotatate was in the range of 5.0 - 6.5 .
the half - life of radionuclide of f-18 fdg , n-13 nh3 , and ga-68 dotatate samples with graph were 110 , 10 , and 69 min , respectively [ figures 1a  c ] . the calculated t1/2 ( mean ) from the measured values using the above - mentioned formula was 115.5 , 10.8 , and 67.5 min , respectively .
single peak corresponding to photon energy of 511 kev was observed in the gamma ray spectrum of f-18 fdg and n-13 nh3 [ figures 2a and b ] . in gamma ray spectra of ga-68 ,
one prominent peak corresponding to photon energy of 511 kev and a small sum peak corresponding to 1022 kev were observed [ figure 3a ] .
no peak was observed when the spectrum was recorded with ga-68 point source after 24 and 48 h [ figure 3b ] .
based on half - life measurement and gamma spectra , the radionuclide purity was almost 100% . the retardation factor ( rf ) of f-18 fdg , ga-68 dotatae , and n-13
nh3 were in the range of 0.39 - 0.48 , 0.16 - 0.23 , and 0.64 - 0.73 [ figures 4a  c ] .
single peaks were observed in each sample of all three pet radiopharmaceuticals used which demonstrated the radiochemical purity more than 99%
. graphical demonstration of time - activity curve on semilogthermic graph for determination of t1/2 of ( a ) f-18 , ( b ) n-13 , and ( c ) ga-68 spectra obtained by using mca ( a ) f-18 fdg ( b ) n-13 nh3 spectra of ga-68 obtained by using mca ( a ) after elution , ( b ) 24 h after elution instant thin layer chromatograms of ( a ) fluorine-18 fluorodeoxyglucose , ( b ) ga-68 dotatate , and ( c ) n-13 nh3 the concentration of ethanol and acetonitrile of f-18 fdg sample was in the range of 20 - 1695ppm and 3 - 57 ppm , respectively [ figure 5a , b ] .
however , the concentration of ethanol in ga-68 dotatate was in the range of 294 - 1147 ppm [ figure 6a , b ] .
gc was not performed for n-13 nh3 samples as organic solvents were neither used nor formed during the synthesis of n-13 nh3 .
the violet - brown color was developed in a standard kryptofix strip when exposed to iodine vapors .
however , no color band was visible when f-18 fdg sample strips were exposed to iodine vapors [ figure 7 ] .
gas chromatograms of ( a ) ethanol and acetonitrile standard and ( b ) fluorine-18 fluorodeoxyglucosesample gas chromatograms of ( a ) ethanol standard and ( b ) ga-68 dotatate sample showing chromatograms of ( a ) kryptofix standard ( 0.22 mg / ml ) , ( b ) f-18 fdg samples , developed in methanol : ammonia ( 9:1 , v / v ) and exposed to iodine vapors ; ( c ) iodine chamber no microbial growth was observed in the samples of f-18 fdg , n-13 nh3 , and ga-68 dotatate .
gel formation was not observed in the samples of f-18 fdg , n-13 nh3 , ga-68 dotatate , and in negative water control .
however , gel was formed in pwc and positive product control at an endotoxin concentration of 0.25 and 0.50 eu / ml . all the results are summarized in table 1 .
the visual inspection per batch of in - house synthesized pet radiopharmaceutical is important and mandatory prior to use in the patient and is also a measure of process performance and validation .
presence of particulate matter in a test sample indicates possible failure at some stage of radiopharmaceutical synthesis .
this may include the failure of the purification , sterilizing filtration , and inadequate environmental control during assembly of reagents in the preinstallation stage .
however , a slight yellow color in the f-18 fdg preparation is acceptable according to ph .
a precisely measured count at two points within a 10 min interval is sufficient to determine the physical half - life of f-18 and ga-68 .
however , the counts were also taken at 10 min interval up to 2 h and half - life was calculated at which the activity / counts were reduced to half . in our tests , the gamma spectrum of a test sample demonstrated a major peak at 511 kev and a sum peak at 1022 kev .
but the presence of a 511or 1022 kev peak in the g ray spectrum is not sufficient to determine radionuclidic identity .
impurities such as n ( arising from o impurity in the o - water ) or other positron emitters may not be detected , as gamma peaks with an energy of 511 and 1022 kev sum peak are a common feature of positron emitters .
a combination of gamma spectrum and the half - life measurement provides the best assurance of the identity and purity of the radionuclide
. the measured half - life will be lower if n-13 impurity is present in fdg .
as the half - life of n-13 is only 10 min therefore , activity as a measure of counts at 2 min interval is important .
the ga-68 sample prepared for gamma spectra was kept for 48 h in order to decay all ga-68 present to a level that permits the detection of long lived ge-68 impurity .
the gamma photons resulting from the decay of ge-68 will have energy of 511 kev and a sum peak of 1022 kev may also be observed . in our study , we could not find any energy peak at 24 and 48 h. these results demonstrated that ge-68 is not present at detectable range .
the availability of nonmetallic silica based column generator eliminates the additional purification step for removal of metallic ions by cation exchange column .
only single peak was observed in tlc scans of f-18 fdg , ga-68 dotatate , and n-13 nh3 .
the rf of the principal peak corresponded with the studied radiopharmaceuticals [ table 1 ] .
radio - tlc provides an easy and reliable means to determine radiochemical identity and purity of radiopharmaceuticals .
narrow range of rf values of the principal spot in the test samples confirmed the radiochemical identity of f-18 fdg , ga-68 dotatate , and n-13 nh3 .
radioactivity was measured in a dose calibrator after calibration with an appropriate reference standard , cs-137 ( 662 kev ) .
the total radioactivity , volume ( mbq / ml or mci / ml ) and reference time of radiopharmaceuticals were specified on product vial label . on the basis of this information , radioactivity per patient was dispensed and exposure to the worker was also reduced .
the ph of f-18 fdg , ga-68 dotatate , and n-13 nh3 fall within the range , that is , 4.5 - 8.5 .
the allowed range of acceptable ph for radiopharmaceuticals is broad ; hence , ph meter was not used .
a narrow range ph strip was used and the results were within the reference ph range .
the ph check of n-13 nh3 is very important as the radio - labeled nitrates may be carried over during the reduction and distillation process .
acetonitrile / ethanol is used during synthesis for reagent preparations and conditioning of the purification cartridges .
traces of these organic solvents may potentially contaminate f-18 fdg and ga-68 dotatate and therefore should be controlled .
the permissible limit of acetonitrile is 400 ( 0.04% ) and of ethanol is 4000 ppm ( 0.4% ) .
ga-68 dotatate was eluted in ethanol , but the concentration of ethanol in random samples was within the permissible limit [ table 1 ] .
organic solvent was not used in the preparation protocol of n-13 nh3 synthesis and thus there was no requirement of gc .
no color band / spot was visualized in the chromatogram of f-18 fdg when exposed to iodine vapors after developed in methanol : ammonia ( 1:1 ) .
these results demonstrated that kryptofix was not present at detectable range in f-18 fdg samples .
kryptofix , a cyclic crown ether , acts as catalyst and binds the potassium ion to prevent the formation of f-18 kf , therefore , enhances the reactivity of ( f-18 ) ion .
kryptofix may cause apnea and convulsions ; therefore , it must be within appropriate limits ( 0.22 mg / ml ) and conformance with these limits is to be demonstrated .
currently available automatic synthesis modules have multiple removal steps to reduce the amount of kryptofix to negligible level .
we have used rcmb as it has the ability to initiate growth of bacteria from very small inocula and also maintain the viability of cultures for longer duration .
moreover , rcmb is routinely used in the department of microbiology , so there was no need for growing reference bacteria .
the gel was not formed in any of the samples indicated the absence of endotoxin in f-18 fdg , ga-68 dotatate , and n-13 nh3 test samples .
all positive controls demonstrated the gel formation even with the endotoxin concentration of 87.5eu / ml . however , negative control and samples did not show any gel formation .
f-18 fdg , ga-68 dotatate , and n-13 nh3 comply with requirements for intravenous administrations .
f-18 fdg , ga-68 dotatate , and n-13 nh3 conformed to various quality attributes of purity ; efficacy and safety in the samples included in this study and comply with all requirements for injectable radiopharmaceutical products .
 purpose of the study : to study quality control parameters of routinely prepared positron emission tomography ( pet ) radiopharmaceuticals.materials and methods : three pet radiopharmaceuticals fluorine-18 fluorodeoxyglucose ( f-18 fdg ) , n-13 ammonia ( n-13 nh3 ) , and ga-68 dotatate ( n = 25 each ) , prepared by standardized protocols were used . the radionuclide purity , radiochemical purity , residual solvents , ph , endotoxins , and sterility of these radiopharmaceuticals were determined.results:the physical half - life of radionuclide in radiopharmaceuticals , determined by both graphical and formula method , demonstrated purity of radionuclides used . 
 ph of all pet radiopharmaceuticals used was in the range of 5 - 6.5 . 
 no microbial growth was observed in radiopharmaceutical preparations . 
 the residual solvents , chemical impurity , and pyrogens were within the permissible limits.conclusions:all three pet radiopharmaceuticals were safe for intravenous administration . 
INTRODUCTION
MATERIALS AND METHODS
pH
Radionuclide purity
Gamma ray spectra
Radiochemical purity
Residual solvent
Chemical purity
Sterility
Bacterial endotoxin test
RESULTS
DISCUSSION
CONCLUSION