much of the current success in clinical positron emission tomography ( pet ) can be attributed to the development of 2-(f ) fluoro-2-deoxy - d - glucose ( f18-fdg ) .
it is the most commonly used radiopharmaceutical in the pet , with a half - life of 110 min . before the release of f18-fdg for human use , the quality control tests need to be performed to check the safety of the product .
the bet using gel clot method is a 60-min test and typically performed after the decay of the f18-fdg sample to determine the endotoxin content .
the gel clot is technically the most simple and was the first bet approved by the food and drug administration ( fda ) .
endotoxin is a subset of pyrogens that are strictly of gram - negative origin , a natural complex of lipopolysaccharides occurring in the outer layer of the bilayered gram - negative bacterial cell . from the circulating blood cells of limulus polyphemus , called amebocytes ,
a clear lysate is obtained that forms an opaque gel in the presence of extremely low concentrations of bacterial endotoxins .
pharmacopeia ( usp ) , pharmacopeia , f18-fdg should not contain more than 175/v usp endotoxin units ( eu ) per milliliter of f18-fdg injection , in which v is the maximum administered total dose in milliliters , at the expiration time .
however , no such standards have been recommended by indian pharmacopoeia commission ( ipc ) till date . the ipc is taking measures to include radiopharmaceuticals in the indian pharmacopoeia .
the objective of this study protocol was to set up a standard pyrogen testing facility and to establish documented evidence if the process employed for bet testing of f18-fdg by gel clot method produces the desired results consistently , when performed as per the standard operating procedures .
random samples of the f18-fdg from september 2010 to march 2011 were subjected to the gel clot bet .
f18-fdg was synthesized in the in - house medical cyclotron and radiochemistry lab at the department of nuclear medicine and pet centre , using the tracerlab mxfdg ( ge healthcare , mnster , germany ) .
the assay was performed at four different maximum valid dilutions ( mvds , i.e. 1:10 , 1:100 , 1:350 and 1:700 ) .
the ph of the samples was measured to ensure a ph within the range 6.08.0 for the validity of the gel clot procedure .
the standard gel clot test for assessing the bacterial endotoxin content consisted of four controls : negative water control ( nwc ) , positive water control ( pwc ) , negative product control ( npc ) and positive product control ( ppc ) .
the nwc is the endotoxin - free limulus amaebocyte lysate ( lal ) reagent water .
the matched control standard endotoxin ( cse ) , lal reagent and lal reagent water obtained from charles river laboratories india private limited , bangalore , india , were used in this study .
the sensitivity of the lal reagent contained in each vial was 0.125 eu / ml . the test for confirmation of lysate sensitivity was carried out with every new batch of the lysate as per the usp < 85 > recommendations .
the cse having predetermined amount of endotoxin was prepared according to the directions for use given in the specific certificate of analysis ( coa ) .
the cse stock solution of 20 eu / ml was prepared with endotoxin - free water and vortexed for 5 min .
all the dilutions were made in the depyrogenated 16  125 mm glass tubes using endotoxin - free water and pyrogen - free sterile pipettes tips .
depyrogenations were performed in the dry heat oven at 250c for 1 hour , when the endotoxin - free glass tubes were not obtained commercially .
the bet was performed using 0.1 ml of f18-fdg sample and 0.1 ml of reconstituted lal reagent per tube .
the lal reagent was constituted after the addition of lal reagent water ( endotoxin free ) in the freeze - dried power of lysate .
the pwc and ppc used for these tests were at the endotoxin concentration of 0.5 eu / ml . the four dilutions of the f18-fdg , i.e. 1:10 , 1:100 , 1:350 and 1:700 , were freshly prepared from the batches and subjected to the gel clot assay .
the ppc was separately prepared for all the dilutions at the endotoxin concentration of 0.25 eu / ml . the reaction solution was mixed thoroughly and placed immediately in the incubator at 37c1c for 602 min . at the end of the incubation period ,
if the gel had formed and remained intact in the bottom of the reaction tube after an inversion of 180 , the test was considered positive .
a positive test indicated that the concentration of endotoxin in the tube is greater than or equal to the sensitivity of the lal reagent .
any other state of the reaction mixture constituted a negative test , which indicated an endotoxin concentration less than the lal reagent sensitivity .
the test was considered negative when the tube is inverted and the gel breaks or collapses .
random samples of the f18-fdg from september 2010 to march 2011 were subjected to the gel clot bet .
f18-fdg was synthesized in the in - house medical cyclotron and radiochemistry lab at the department of nuclear medicine and pet centre , using the tracerlab mxfdg ( ge healthcare , mnster , germany ) .
the assay was performed at four different maximum valid dilutions ( mvds , i.e. 1:10 , 1:100 , 1:350 and 1:700 ) .
the ph of the samples was measured to ensure a ph within the range 6.08.0 for the validity of the gel clot procedure .
the standard gel clot test for assessing the bacterial endotoxin content consisted of four controls : negative water control ( nwc ) , positive water control ( pwc ) , negative product control ( npc ) and positive product control ( ppc ) .
the nwc is the endotoxin - free limulus amaebocyte lysate ( lal ) reagent water .
the matched control standard endotoxin ( cse ) , lal reagent and lal reagent water obtained from charles river laboratories india private limited , bangalore , india , were used in this study .
the sensitivity of the lal reagent contained in each vial was 0.125 eu / ml . the test for confirmation of lysate sensitivity was carried out with every new batch of the lysate as per the usp < 85 > recommendations .
the cse having predetermined amount of endotoxin was prepared according to the directions for use given in the specific certificate of analysis ( coa ) .
the cse stock solution of 20 eu / ml was prepared with endotoxin - free water and vortexed for 5 min .
all the dilutions were made in the depyrogenated 16  125 mm glass tubes using endotoxin - free water and pyrogen - free sterile pipettes tips .
depyrogenations were performed in the dry heat oven at 250c for 1 hour , when the endotoxin - free glass tubes were not obtained commercially .
the bet was performed using 0.1 ml of f18-fdg sample and 0.1 ml of reconstituted lal reagent per tube .
the lal reagent was constituted after the addition of lal reagent water ( endotoxin free ) in the freeze - dried power of lysate .
the pwc and ppc used for these tests were at the endotoxin concentration of 0.5 eu / ml . the four dilutions of the f18-fdg , i.e. 1:10 , 1:100 , 1:350 and 1:700 , were freshly prepared from the batches and subjected to the gel clot assay .
the ppc was separately prepared for all the dilutions at the endotoxin concentration of 0.25 eu / ml . the reaction solution was mixed thoroughly and placed immediately in the incubator at 37c1c for 602 min . at the end of the incubation period ,
if the gel had formed and remained intact in the bottom of the reaction tube after an inversion of 180 , the test was considered positive .
a positive test indicated that the concentration of endotoxin in the tube is greater than or equal to the sensitivity of the lal reagent .
any other state of the reaction mixture constituted a negative test , which indicated an endotoxin concentration less than the lal reagent sensitivity .
the test was considered negative when the tube is inverted and the gel breaks or collapses .
the mean ph was 7.05 ( 6.57.5 ) . the total numbers of tests performed on the f18-fdg were 100 excluding nwc , pwc and ppc .
table 1 summarizes the results of the undiluted f18-fdg on the positive control solutions at an endotoxin concentration of 0.5 eu / ml ( 4 ) . of the 10 undiluted f18-fdg batches ,
this indicated that all the batches did not contain endotoxin greater than 0.5 eu / ml .
all the 20 positive endotoxin control pwc vials gelled . only 18 out of 20 ppc vials gelled after incubation at 37c for 60 min .
results for undiluted f18-fdg sample table 2 summarizes the results for the 60-min gel clot assay on the four different dilutions of the f18-fdg samples .
all the 20 positive endotoxin control pwc vials gelled . none of the 20 1:10 f18-fdg sample vials gelled after 60-min incubation at 37c . however , 1 out of 20 ppc vials for the 1:10 dilution did not gel probably due to some interference with the gel formation at the lysate sensitivity of 0.125 eu / ml .
all the remaining vials for the 1:100 , 1 : 350 and 1:700 dilutions did not gel .
this indicated that all the vials did not contain endotoxin greater than 0.125 eu / ml . results for the diluted f18-fdg samples
the gel clot method is often considered as the most accurate and sensitive procedure for testing endotoxin content in injectable radiopharmaceutical products because fewer false - positive and false - negative results are observed when this method is used . the total time taken to complete
the lal reaction requires optimal ph range . in the undiluted samples , the measured ph ( 7.05 ) was well within the acceptable range ( i.e. 6.08.0 ) for the gel clot assay .
the f18-fdg produced in the tracerlab mx synthesis module is neutralized in the citrate buffer which contains multivalent ions .
nakao et al . reported the effect of the citrate salt concentration on the endotoxin test for the f18-fdg preparations .
these authors reported that the undiluted sample showed the recovery less than 20% , beyond the acceptance of the usp .
similar results were found in the present study with the undiluted samples as two vials of the ppc ( endotoxin concentration equal to 0.5 eu / ml ) did not gel , indicating the interference with the gel formation . the interference may be due to the presence of the citrate ions . at the expiration time , the maximum administered total dose in milliliters of fdg should contain less than 175 eu .
therefore , the total f18-fdg preparation at any time did not contain more than 8 eu ( 0.5 eu / ml  16 ml ) .
however , the undiluted sample did show the inhibition of the gel formation as shown in table 1 .
therefore , the sample was tested for the various dilutions 1:10 , 1:100 , 1:350 and 1:700 .
the 350 and 700 dilution factors were considered according to the limit of 175 eu / ml ( single patient dose supplied to the other institute or when the final elution is in 23 ml due to some technical errors ) . the rate of inhibition improved with the dilutions in table 2 .
the gel clot assay has a major drawback which limits its use for the short - lived radiopharmaceuticals .
in addition to this drawback , errors made by technical personnel and the misinterpretation of results are also common problems .
the 60-min bet is described in usp<85 > ,  bacterial endotoxins test  , and is also recommended for pyrogenicity testing in the draft guidance of the fda on current good manufacturing practice ( cgmp ) for pet drug products . because the remainder of the required quality assurance ( qa ) testing for f18-fdg injection , with the exception of the sterility test , can be completed in approximately 2030 min , delaying the release of the short - lived f18-fdg injection for an additional 3040 min is not practical and is , in fact , wasteful .
an in - process 20-min bet must be performed before release of the drug product , and a standard 60-min bet must also be completed . some other techniques to determine endotoxins
are also known , such as the chromogenic ( color development ) and the turbidimetric ( turbidity development ) tests , both of which can provide valuable quantitative and qualitative information about the endotoxin concentration in samples . regardless of which method is used , pet laboratories and pharmacies should ensure that their technicians are well trained and educated in the endotoxin testing method of choice .
this study suggests that the bet needs to be performed , standardized and documented in each cyclotron and pet facility .
the dilution of the test sample is the easiest means to resolve the potential product inhibition / enhancement problem during the gel clot testing procedure .
the final f18-fdg preparation at any time point did not contain more than 8 eu , thus making it safe for human administration .
 background : the bacterial endotoxin test ( bet ) performed using gel clot method is a 60-min test and typically performed after the decay of the 2-(18f ) fluoro-2-deoxy - d - glucose ( f18-fdg ) sample to determine the endotoxin content . 
 the objective of this study protocol was to perform bet testing of f18-fdg by gel clot method.materials and methods : ten random decayed samples of the f18-fdg were subjected to the gel clot bet . 
 the assay was performed with undiluted f18-fdg and at four different maximum valid dilutions of 1:10 , 1:100 , 1:350 and 1:700 ( total number of tests = 100 ) . 
 the sensitivity of the lal reagent used was 0.125 eu / ml . endotoxin dilutions were freshly prepared from control standard endotoxin ( cse ) stock solution for each f18-fdg batch testing . if the gel had formed and remained intact in the bottom of the reaction tube after an inversion of 180 , the test was considered positive . 
 any other state of the reaction mixture constituted a negative test.results:in the undiluted samples , the measured ph ( 7.05 ) was well within the acceptable range ( i.e. 6.08.0 ) for the gel clot assay . 
 of the 10 undiluted f18-fdg batches and all the diluted samples , none gelled after 60-min incubation period at 37c . 
 however , the undiluted f18-fdg did inhibit gel formation at the lysate sensitivity of 0.125 eu / ml.conclusion : the total volume of fdg produced was 16 ml in the synthesis module . 
 the total f18-fdg preparation at any time did not contain more than 8 eu ( 0.5 eu / ml  16 ml ) . 
 thus , the product is safe for human administration . 
INTRODUCTION
MATERIALS AND METHODS
F18-FDG sample preparation
Preparation of endotoxin stock solution and endotoxin dilutions
Glassware for BET
Routine test procedure
RESULTS
DISCUSSION
CONCLUSION