The development of peptide-based radiopharmaceuticals for the detection of amyloid fibrils associated with Alzheimer’s disease requires the formation of stable complexes with high radiochemical yield and purity. Specific fragments of the amyloid-ß peptide, when conjugated to chelating agents such as DOTA and radiolabeled with metallic radionuclides, represent a promising strategy for the development of molecular imaging tracers for nuclear medicine applications.

The aim of this study was to synthesize an amyloid-ß peptide fragment, conjugate it to the DOTA chelator, and assess the radiochemical yield, purification efficiency, radiochemical purity, and stability of gallium-68 labeling.

The amyloid-ß peptide central fragment Aß16-20 (KLVFF) was synthesized and conjugated to the DOTA chelator via solid-phase peptide synthesis. The DOTA-KLVFF conjugate was cleaved and analyzed by high-performance liquid chromatography (HPLC) to assess its chromatographic profile prior to radiolabeling. Radiolabeling was performed manually with [68Ga]GaCl3 eluted from a 68Ge–68Ga generator with 6 mL of 0.1 M HCl (∼12.21 mCi/mL) and purified in a cationic filter. Approximately 500 µL of eluate was added to 40 µL of peptide (830 µg/mL), solubilized in 1 mL of 0.2 M sodium acetate buffer (pH 4.0), followed by heating at 95°C for 15 minutes. The radiolabeled peptide was purified using a Sep-Pak C18 cartridge with 50% ethanol. The chromatographic behavior and radiochemical purity of [Ga]Ga-DOTA-KLVFF were evaluated by instant thin-layer chromatography (iTLC), before and after purification, respectively using ammonium acetate/methanol (AcONH4:MeOH, 1:1) as mobile phase. Radiochemical stability was qualitatively assessed by iTLC at 0, 30 min, 1h, and 1.5h.

The DOTA-KLVFF peptide was successfully synthesized with a yield of 80%. After purification, preliminary radiolabeling with 68Ga achieved a radiochemical yield of approximately 89%. The purified product, [68Ga]Ga-DOTA-KLVFF, exhibited a radiochemical purity of 99%. iTLC analysis showed a retention factor (Rf) of 0.9–1.0 for the radiolabeled peptide, while free [68Ga]GaCl3 presented an Rf of 0.0–0.1, indicating effective separation between the desired product and radiochemical impurities. A preliminary stability study demonstrated maintenance of radiochemical purity over time, with values of 94.3 ± 2.6% at time zero, 91.0 ± 0.6% after 30 min, 87.4 ± 0.4% after 1h and 94.4 ± 3.6% after 1.5h. These results indicate efficient complex formation and satisfactory short-term stability of [Ga]Ga-DOTA conjugate under the evaluated conditions.

The central amyloid-ß peptide fragment Aß16–20 (KLVFF) conjugated to DOTA exhibited efficient gallium-68 radiolabeling, high radiochemical yield, effective purification, and excellent radiochemical purity, along with qualitative stability over the evaluated period. These findings demonstrate the robustness of the experimental protocol and support the potential of this conjugate as a platform for the development of radiopharmaceuticals targeting amyloid fibrils in future studies.