Radiosynthesis of 1-(2-[¹⁸F]Fluoroethyl)-L-Tryptophan using a One-pot, Two-step Protocol

The overall goal of this protocol is to radiosynthesize an F18 radiolabeled tryptophan tracer by a one-pot, two-step approach. The radiotracer may find widespread application for imaging tryptophan metabolism. The technique uses the radioisotope with a longer half-life, less reaction precursor, and less reaction volume, and an environmentally benign phase for purification.

The radiotracer may be used for diagnosis of various disorders affecting the brain, including but not limited to epilepsy, neuropsychiatric disorders, and neuro-oncology. This method can be applied to other commercially available radiolabeling modules. Once the F18 fluoride solution is received, begin by surveying the lead box on the surface, and from the distance of one meter, record the maximum radiation exposure rates.

After performing a wipe test to ensure the shipping box is not contaminated, record the F18 fluoride dose and time. Transfer the F18 fluoride solution to the radiochemistry synthesis system. Connect the argon line with a short needle and the F18 fluoride transfer line with a long needle to the F18 fluoride vial.

Then, close the hot cell glass door and lead door. Turn on the argon supply line by clicking Argon Supply. Increase the argon pressure and turn on the F18 fluoride pushing line to push the aqueous F18 fluoride through the QMA light cartridge.

Once all the radioactivity is trapped in the QMA light cartridge, and the radioactivity detector reading is steady, increase the argon pressure and blow argon through the cartridge for another five minutes to remove excess water. After turning off the F18 fluoride pushing line, decrease the argon pressure to zero and switch the six-port two-position valve from the F18 fluoride trapping position to the elution position. Open the reaction vial, turn on the input MVP position one argon line, and push the K222 and potassium carbonate solution into the input MVP position one vial through the QMA light cartridge to elute out the radioactivity into the reaction vial.

Switch the F18 fluoride elution position to the trapping position. Click the Heat button to heat the reactor at 110 degrees Celsius, turn on the output MVP position-four argon line that connects to the reactor, and evaporate the solvent into the output MVP position four vial. Click the Cool button to cool down the reactor to room temperature with compressed carbon dioxide.

Turn off the sweeping line, switch the input MVP position one to position two, and add the anhydrous acetonitrile in the vial two. Two turn on the sweeping line and heater to azeotropically dry F18 fluoride at 110 degrees Celsius. Once the reactor reaches room temperature, turn off the sweeping line, switch the input MVP position two to position three, and add the tosylate radiolabeling precursor in the vial three.

Close the reactor and heat the reaction mixtures at 100 degree Celsius for 10 minutes. To evaporate the reaction solvent, once the reactor cools down, turn on the sweeping line and evaporate the reaction solvent at 100 degree Celsius. Once the reactor cools down, turn off the sweeping line and switch the input MVP position three to position four.

When the hydrochloric acid acetonitrile mixture is added, heat the reaction at 100 degree Celsius for 10 minutes to de-protect the tert-butyl and tert-butyloxycarbonyl protecting groups in the radiolabeling precursor. To neutralize the reaction, when the reactor reaches room temperature, switch the input MVP position four to position five and add two molar sodium hydroxide to the reaction mixture and turn off the input MVP argon line. Next, start transferring the reaction mixture to the intermediate file by clicking the F button in the output MVP to switch the output MVP from the venting position four to position five, and then by clicking the F button in the input MVP to switch the input MVP from position five to position six.

After turning on the output MVP argon line, push the reaction mixture through the stacked neutral aluminum oxide and C8 cartridges to the intermediate vial installed before the HPLC sample loop. For rinsing the reactor, switch the output MVP position five to position six, push the rinse solution in the vial of output MVP position six through the reaction vial, cartridges, and the intermediate vial, successively. Next, begin purification of the mixture by switching the HPLC loop from injection position to load position and turning on the input MVP argon line.

When the mixture is loaded to the five milliliter HPLC loop, switch the Load button to Inject, click Inject HPLC to start the HPLC chromatogram at a flow rate of three milliliters per minute, and then click the HPLC Monitor button to access the real-time HPLC chromatogram. Click the Diversion MVP button to divert the target HPLC fraction to the short C18 column at approximately 12 minutes. After the target radioactivity is collected in the C18 column, switch the four-port two-position diversion valve to the waste collection position, and flush the chiral column at the rate of four milliliters per minute for six minutes to remove the minor D F18 FETrp enantiomer and other ultraviolet impurities.

Click the Diversion MVP button to divert the HPLC mobile phase back to the formulation MVP position one at three milliliters per minute. Observe that the mobile phase passes through the chiral column and C18 column to purify L-F18 FETrp retained in the C18 column. At approximately 32 to 34 minutes, collect the target fraction into the formulation MVP position two vial.

Then push the formulation MVP position two vial solution through the delivery line and sterile filter into the final dose vial. Assay the final dose activity and volume after removing the sterile filter. Flush the system with ultra pure water followed by ethanol at a flow rate of four milliliters per minute for at least 15 minutes per wash.

Turn off the HPLC pump and PLC box, close the program, shut down the compressed airline, argon line, carbon dioxide line, and the main power of the module. Withdraw approximately 0.1 milliliters of the final dose into a 0.2-milliliter insert of a QC vial. Run the hot sample as the partial sequence program is described in the text manuscript.

Analyze the QC data by calculating the chemical and radiochemical purities in enantiomeric excess value and molar activity. After determining the pH value, release the dose if all testing results pass the acceptable range. The representative HPLC chromatograms for QC are shown.

The insignificant peaks between the void volume in 10 minutes of the program were detected in the blank chromatogram. The non-radiolabeled standard reference L-fluoroethyl-trytophan showed the presence of a single isomer separated well from the standard reference of its D counterpart. The final dose of L-F18 tryptophan showed high chemical purity and radiochemical purity.

The stability testing of the final product at the highest dose concentration for up to eight hours showed that L-F18 tryptophan was stable in terms of chemical purity, radiochemical purity, enantiomeric excess, and pH value. The chemical and radiochemical purities greater than 95%were achieved using this protocol. Two columns are used for tracer purification.

Do remember to switch to the C18 column to remove the chemical impurities. We can quickly adapt this method to clinical production of the fluorine-18-labeled tryptophan radiotracer.