Researchers have demonstrated a novel analytical technique that allows for the real-time detection of radioactive daughter products within actinium-225 radiopharmaceuticals. Published in the journal EJNMMI Radiopharmacy and Chemistry, the study presents an inline high-performance liquid chromatography setup that combines liquid scintillation detection with LaBr3-based gamma-spectroscopy. This approach addresses a significant limitation in current quality control methods, which rely on slow offline fraction collection and delayed gamma-counting to identify the distribution of daughter radionuclides. The new method enables a faster and more informative analysis of the complex decay chain associated with actinium-225, a radionuclide increasingly used in targeted alpha therapy for cancers such as prostate and neuroendocrine tumors.
The study, led by Guilhem Claude and colleagues, focuses on the analytical challenges posed by the decay of actinium-225. The decay chain comprises seven radionuclides that emit alpha, beta, and gamma radiation, with half-lives ranging from milliseconds to hours. In the specific inline detection setup described, the researchers were able to detect four distinct species: francium-221, bismuth-213, actinium-225, and lead-209. Short-lived daughters such as astatine-217 and polonium-213 decay completely within the residence time in front of the detector due to the cell size and HPLC flow rate, making them indistinguishable from their parent isotopes in this configuration. Thallium-209 is generated in only about 2 percent of bismuth-213 decays and consequently contributes negligibly to the recorded signal.
Traditional analytical methods for actinium-225 labeled radiopharmaceuticals typically use thin-layer chromatography and high-performance liquid chromatography followed by offline fraction collection. These procedures require waiting for secular equilibrium with gamma-emitting daughters before counting can occur, a process that is slow and does not depict the distribution of daughter radionuclides in real time. The proposed inline setup aims to overcome these limitations by providing continuous, real-time radionuclide speciation without interrupting the chromatographic process for fraction collection. This is particularly important because the decay of alpha emitters is considerably more complex than that of lutetium-177, a commonly used therapeutic radionuclide.
The experimental design involved synthesizing [225Ac]Ac-PSMA I&T, a radiopharmaceutical targeting the prostate-specific membrane antigen. The actinium-225 used in the study had been separated from thorium-229 at least one day before the complexation reaction, meaning it already contained daughter radionuclides at the time of radiolabeling. HPLC measurements were typically performed approximately four to five hours after the completion of radiolabeling. The HPLC system used an Agilent 1260 Quaternary pump with a UV detector set at 200 nm. An Elysia-Raytest Gabi Nova detector was used for gamma-spectroscopy, and a Ramona LS-Pump was used to pump Gold Star liquid scintillator at a rate of 3.6 mL/min, corresponding to a 1:3 ratio of HPLC eluate to scintillation cocktail.
Chromatograms obtained from the gamma and scintillation channels showed complementary profiles. Three peaks were observed in the gamma-trace, whereas four peaks were visible in the scintillation trace. The peak with the longest retention time was attributed to [209Pb]Pb-PSMA I&T, as it was detected only in the scintillation trace and was absent in the gamma-chromatogram. This assignment was confirmed by fraction collection followed by liquid scintillation counting, which showed the characteristic beta-emission of lead-209. The gamma-emitting isotopes were identified directly using inline gamma-spectrometric measurement. Peak 1 contained predominantly uncomplexed francium-221 together with at most a minor contribution of free bismuth-213. Peak 2 was assigned to [213Bi]Bi-PSMA I&T and peak 3 to [225Ac]Ac-PSMA I&T based on their characteristic gamma-spectra recorded inline.
To confirm the peak assignments, non-radioactive standards of lanthanum-PSMA I&T, bismuth-PSMA I&T, and lead-PSMA I&T were analyzed by UV detection. Lanthanum was used as a non-radioactive surrogate for actinium. The retention times of these standards corresponded to those observed in the radioactive chromatograms. Small delays between UV and radio peaks resulted from the dead volume between detectors and were consistent for each compound. The UV trace shown in the study corresponds to these non-radioactive reference compounds used for retention-time assignment, whereas radionuclide assignment in the radioactive sample was based on the combination of inline gamma-spectroscopy, liquid scintillation detection, and confirmatory fraction analysis.
The study also investigated how the addition of diethylenetriaminepentaacetic acid (DTPA) to the reaction mixture altered the observed daughter distribution. DTPA is commonly included in radioligand therapy formulations to capture free radiometals and reduce off-target distribution. In the present experiments, the presence of DTPA reduced the formation of [213Bi]Bi-PSMA I&T and [209Pb]Pb-PSMA I&T complexes. This is consistent with the rapid complexation of recoil-released daughter radionuclides by DTPA. DTPA recomplexed the recoiled daughters, which then did not form a new DOTAGA complex. Partial transchelation from the DOTAGA chelator to DTPA may also occur, as previously reported for bismuth complexes. Owing to their high hydrophilicity, these DTPA complexes are not retained on the reversed-phase column and co-elute with the uncomplexed nuclides near the void volume.
The absence of a detectable [221Fr]Fr-PSMA I&T complex suggests that the daughter complexes arise from recomplexation processes occurring after recoil release of daughter radionuclides. Francium possesses alkali-metal character with a large ionic radius, and its coordination chemistry remains largely unexplored due to the absence of stable isotopes. To the authors’ knowledge, coordination of francium by DOTA-type chelators has not been reported. Consequently, recoil-released francium is expected to remain uncomplexed in solution, whereas downstream daughters such as bismuth and lead can recomplex with ligands present in excess. Recombination is facilitated by the large excess of ligand present in the reaction mixture, with a metal-to-ligand ratio of approximately 1:1500 depending on the starting activity.
An elevated baseline preceding the elution of [225Ac]Ac-PSMA I&T was observed in both detectors and returned to its initial level after the complex had eluted. This effect is most likely due to recoiled francium-221 generated continuously from the [225Ac]Ac-PSMA I&T complex during the chromatographic run. This recoiled francium elutes over a broader time window than the parent complex itself. This interpretation is supported by the inline gamma-spectroscopic data, in which this region showed essentially only the signal attributable to francium-221. The effect was observed in both the gamma and liquid scintillation channels, although the absolute signal intensity differs because of the different detector responses.
The authors note that because isotope-specific detector response factors have not yet been established, no rigorous absolute quantification of the minor bismuth-213 fraction in peak 1 is reported. A quantification of the amount of free bismuth-213 relative to the amount of [213Bi]Bi-PSMA I&T is possible by using a narrow energy range centered around the gamma line of bismuth-213, which does not overlap with any nuclide of the decay chain. However, a comparison of sensitivity, limit of quantification, and detector-specific response factors for each radionuclide was not established systematically in the present study. This will require further studies, especially due to the contribution of recoiled francium-221 to adjacent signal regions. The developed HPLC method provides a useful analytical tool for investigating the redistribution and recomplexation behavior of radionuclides within the actinium-225 decay chain.
The study concludes that the combined detection setup enables rapid observation of isotope distribution and daughter behavior during chromatographic analysis. It avoids fraction handling by operating as a closed system, thereby reducing contamination risk and radiation exposure. This approach may contribute to improved quality control procedures for actinium-225 radiopharmaceuticals and other alpha-emitting radionuclides. However, at the present stage, the method should be regarded as a qualitative to semi-quantitative proof-of-principle. Formal quantification and validation will require dedicated studies of specificity, precision, robustness, and sensitivity. Work is in progress to apply the same configuration to additional ligands and radionuclides to evaluate the general applicability of this dual-detection approach.
Subject of Research: Analytical chemistry of actinium-225 radiopharmaceuticals
Article Title: Inline gamma spectroscopy and liquid scintillation HPLC reveal daughters in 225Ac-radiopharmaceutical
Article References: Claude, G., Balzer, M., Brenner, W., Bruchertseifer, F., Morgenstern, A., Thonon, D., & Spreckelmeyer, S. (2026). Inline gamma spectroscopy and liquid scintillation HPLC reveal daughters in 225Ac-radiopharmaceutical. EJNMMI Radiopharmacy and Chemistry, 11(1), Article 49. https://doi.org/10.1186/s41181-026-00476-6
Image Credits: AI Generated
DOI: 10.1186/s41181-026-00476-6
Keywords: actinium-225, HPLC, gamma-spectroscopy, liquid scintillation, radiopharmaceuticals, quality control, daughter radionuclides, prostate cancer, alpha therapy, PSMA, Inline, gamma
