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 .