The overall goal of this procedure is to evaluate a new radioactive gallium-68 complex for molecular imaging. This method can help answer key questions in the field of gallium-68 tracer chemistry such as labeling conditions, tracer stability and lipophilicity. The technique of radioactive labeling is straightforward and allows for fast determination of the results while requiring only minimum amounts of material.
To begin this procedure, add three milliliters of 0.25-molar gadoxetic acid injectable solution to a flask. Then add 500 milligrams of oxalic acid to the stirring gadoxetic acid solution. After stirring for one hour, filter the suspension through a frit using reduced pressure.
Wash the residue three times with three milliliters of water. Next, combine the aqueous filtrates in a beaker and place a pH electrode in the solution. Add 12 molar hydrochloric acid to the filtrate until the pH is about 0.1.
After removing the solvent in vacuo, wash the residue thoroughly with hot ethyl acetate to remove the access oxalic acid. Then dry the residue in vacuo. Once the residue is dry, redissolve it in two milliliters of de-ionized water and then cool the solution in an ice bath.
Without removing the ice bath, add 0.5 molar sodium hydroxide drop wise until a colorless, gluey solid is observed. Then remove the water with a pipette. To prepare a 0.11 molar stock solution of gallium trichloride dissolve 1.94 grams of the solid in 100 milliliters of water.
Next, dissolve 80 milligrams of EOB-DTPA in 10 milliliters of water. If necessary, heat the solution to achieve complete dissolution. Add 1.4 milliliters of the gallium trichloride stock solution to the EOB-DTPA solution and place a pH electrobe in the beaker.
Then add diluted aqueous ammonia solution drop wise until the pH of the solution is approximately 4.1. After removing the solvent in vacuo, place the residue in a flask equipped with a steelhead with a central and parallel side neck. Equip the central neck with a cooling finger and the side neck with a vacuum pump outlet.
Heat the residue under reduced pressure. Periodically remove sublimated ammonium chloride from the flask with a slightly wet cloth. Precondition a PSH plus cartridge by flushing it slowly with 1 milliliter of 1 molar hydrochloric acid and subsequently, 5 milliliters of water.
Following this, elute the silica column of the generator with four milliliters of 0.05 molar hydrochloric acid and load the gallium-68 eluate into the PSH plus cartridge. Flush the cartridge with five milliliters of water. After drying with air, elute the gallium-68 from the cartridge with one milliliter of five molar acidified sodium chloride.
Now add 50 microliters of the gallium-68 eluate and 50 microliters of a previously-prepared 19 micromolar EOB-DTPA stock solution to a vial. Add 300 microliters of buffer to raise the pH to 4.0. Shake briefly and incubate the solution at room temperature for five minutes.
Transfer a five to 20 microliter aliquot of the solution to a HPLC vial. Then preform radio HPLC analysis on a reversed phase C18 column. To preform in vitro stability measurements, withdraw samples from the labeling solution containing six to 12 megabecquerel of tracer.
Perform radio TLC analysis on 80 millimeter silica gel-coated aluminum plates using 0.1 molar aqueous sodium citrate as eluent. Analyze the plates with a TLC radioactivity scanner. To determine the stability in phosphate buffered saline, add 150 microliters of 10 millimolar PBS stock solution and 60 microliters of 0.1 molar sodium hydroxide to 65 microliters of labeling solution.
Mix the solution thoroughly. Following this, transfer a one to five microliter aliquot of the solution to a silica plate for TLC analysis. Immediately store the solution in an incubator at 37 degrees Celsius.
Remove aliquots to perform TLC analysis at representative time points over three hours. To determine the stability toward an excess of apotransferrin, add 50 microliters of 10 millimolar PBS stock solution and 430 microliters of 0.1 molar sodium hydroxide to 120 microliters of labeling solution. Add 40 microliters of a 25 milligram per milliliter solution of apotransferrin and thoroughly mix the solution.
Next, transfer a one to five microliter aliquot of the solution to a silica plate for TLC analysis. Immediately store the solution in an incubator at 37 degrees Celsius. Remove aliquots to perform TLC analysis at representative time points over three hours.
To determine the stability in human serum, add 45 microliters of 0.1 molar sodium hydroxide and 500 microliters of human serum to 25 microliters of labeling solution. After thoroughly mixing the solution, transfer a one to five microliter aliquot to a silica plate for TLC analysis. Immediately store the solution in an incubator at 37 degrees Celsius.
Remove aliquots to perform TLC analysis at representative time points over three hours. For determination of the distribution coefficient, add 20 microliters of 10 millimolar PBS stock solution and 170 microliters of 0.1 molar sodium hydroxide to 50 microliters of labeling solution. Transfer 200 microliters of the solution to a plastic V vial and add 200 microliters of n-Octanol.
Close the vial and vortex for two minutes, then centrifuge the sample at 1600 times g for five minutes. Following centrifugation, remove triplicates of 40 microliters from the n-Octanol phase and the aqueous phase and place them in separate plastic V vials. Finally, measure the activity of each sample in a gamma well counter for 30 seconds.
The ligened EOB-DTPA and the non-radioactive gallium three complex were analyzed via proton and carbon NMR spectroscopy, mass spectrometry and elemental analysis. The labeling procedure results in the formation of the desired tracer gallium-68 EOB-DTPA indicated as a radio HPLC peak exhibiting a retention time of 2.8 minutes. Comparison with the retention time of the cold standard at 220 nanometers confirms successful labeling.
The gallium-68 labeling efficiency of EOB-DTPA was investigated by determining the labeling yield as a function of the ligened concentration via HPLC. A representative chromatogram of a TLC plate analyzed with a TLC radioactivity scanner is shown here. To investigate the stability of the tracer, the radiochemical purity over time was determined via TLC.
The standardized percentage of intact tracer is depicted as a function of time here. Activity values and subsequent calculation of the distribution coefficient are listed here. Following this procedure, other methods like in vitro experiments can be performed in order to answer additional questions like biodistribution and uptake dynamics.
After watching this video, you should have a good understanding of the techniques used to evaluate and analyze gallium-68 tracers for molecular imaging. Don't forget that working with radioactive substances can be harmful. It may only be performed by trained personnel.
Safety precautions such as the use of lead shields and tweezers should always be taken while performing this procedure.
