Table 2.
Troubleshooting table.
| step(s) | problem | solution |
|---|---|---|
| Steps 7 and 8 | Unchelated 68Ga is discovered in the solution | Take a sample and measure its pH. The final pH during the reaction should be between 3 and 4. If the pH is <3, add sodium acetate buffer (Reagent Setup) to the reaction mixture stepwise in 100-ml aliquots until the pH is between 3 and 4. If the pH is higher than 4, adjust the pH with diluted HCl or use a lower amount of sodium acetate buffer for the next labeling so that the pH of the reaction mixture is between 3 and 4 After re-adjusting the pH of the reaction mixture, incubate the reaction solution for an additional 7 min and analyze an aliquot by radio-iTLC and/or radio-HPLC. |
| Unchelated 68Ga is still present in the solution after control of the pH of the reaction mixture | Incubate the reaction solution for an additional 5 min and analyze another aliquot by radio-iTLC and/or radio-HPLC | |
| Unchelated 68Ga is still present in the solution after the additional incubation | Add 5–7 nmol DOTA-like conjugated peptide (if specific activity is not a main focus) and incubate for an additional 5 min. Analyze another aliquot by radio-iTLC and/or radio-HPLC | |
| Steps 7–9 | Unchelated 68Ga is still present in the solution after incubation with additional peptide | Apply the reaction mixture to an activated SPE (e.g., Sep-Pak) cartridgea. Wash the cartridge with 5 ml of water (for injection) and elute dropwise the 68Ga-labeled peptide with 0.5 ml of a 1:1 ethanol:saline solution |
| Steps 7 and 8 | Unchelated 68Ga is present in the solution and you suspect that this is due to iron contamination of the reaction mixture | Check all used reagents for iron contamination (including the generator eluate and other components (e.g., reaction vial)) using a colorimetric test (Box 1) or an atomic absorption spectrometer. Replace the contaminated reagent or component. If iron is detectable on the preconditioned SCX cartridge, rinse the SCX cartridges before the labeling with 1 ml of HCl (5.5 M), wait 2 min, and rinse again with 1 ml of HCl (5.5 M) and then with 10 ml of water. If iron is detectable in the reaction vial, rinse the vial before the labeling with 1 ml of HCl (5.5 M) and then with 10–20 ml of water |
| Steps 7–10 | Multiple radioactive peaks are observed from a sample that is known to be analytically pure and free of isomers | Radiolysis may be occurring. Add ∼5 mg of a radical scavenger to the original reaction buffer and start the labeling procedure again. Possible radical scavengers include ascorbic acid, ethanol, gentisic acid and methionine |
Sep-Pak purification of the labeled peptide: If a Sep-Pak C-18 cartridge is required to remove unreacted Ga3+, it should be activated by preconditioning with ethanol. For this purpose, rinse the cartridge with 5 ml of ethanol and then with 5 ml of water (‘for injection’ quality). Transfer the reaction mixture through the C-18 cartridge and rinse the cartridge again with 2 ml of water (‘for injection’ quality). After elution of the 68Ga-labeled peptide with ethanol, it is essential to either remove the organic solvent from the resulting solution or to dilute the solution with isotonic saline solution before using the labeled compound as a radiopharmaceutical for animal studies or patient administration. The solvent can be evaporated by allowing a gentle stream of inert gas (helium, argon or nitrogen) to pass over the solution. To avoid adherence of the labeled peptide onto the inner surface of the reaction vial, the solution should not be concentrated to dryness.