Analysis of Radioligand Therapy Components Using Reversed-Phase and HILIC Columns

Importance of the Topic

Radioligand therapy represents a cutting-edge approach to cancer treatment, combining targeting ligands with radioactive isotopes to deliver cytotoxic radiation directly to tumor cells while sparing healthy tissue. Accurate analysis of each molecular component is essential for ensuring product purity, monitoring synthetic steps, and safeguarding patient safety. Analytical methods must accommodate the high polarity and metal-sensitivity of chelator-based compounds under time-critical conditions due to isotope half-life constraints.

Objectives and Study Overview

This study evaluated chromatographic strategies to separate and detect three key components of Lutetium-177 vipivotide tetraxetan (PSMA-617): the main drug, its ligand-linker conjugate, and the DOTA chelator. Goals included comparing reversed-phase and HILIC modes, assessing inert hardware benefits, and establishing a robust method for reaction monitoring and quality control of radioligand precursors.

Instrumentation and Methods Used

Liquid chromatography was performed on an ACQUITY Premier Binary Solvent Manager with PDA detector, interfaced to a Xevo TQ-S Micro mass spectrometer operating in positive ESI. Software control and data processing used MassLynx V4.1. Columns tested included: 1) ACQUITY Premier HSS T3 (RP), 2) Atlantis Premier BEH C18 AX (mixed-mode RP/anion exchange), and 3) Atlantis Premier BEH Z-HILIC (hydrophilic interaction). All columns employed MaxPeak hybrid organic/inorganic surfaces for metal passivation.

Main Results and Discussion

Reversed-phase on HSS T3 achieved good retention of PSMA-617 and the linker conjugate but failed to retain DOTA. The mixed-mode C18 AX column improved resolution of the two larger analytes but still yielded poor, split DOTA peaks. In contrast, HILIC separation on the BEH Z-HILIC column provided baseline retention and sharp peaks for all three compounds, following a 5–50% aqueous gradient with ammonium formate buffer. Comparative runs on stainless-steel hardware showed complete loss of PSMA-617 and DOTA signals due to metal adsorption and iron adduct formation, highlighting inert hardware necessity.

Benefits and Practical Applications

• Consistent separation of highly polar and metal-sensitive analytes in a single HILIC method.
• Enhanced recovery and detection reliability via metal-passivated columns, eliminating the need for mobile phase chelators such as EDTA.
• Applicability for synthesis reaction monitoring, precursor profiling, and DMPK studies of radioligand therapies.

Future Trends and Opportunities

Emerging radioligand compounds will demand further method refinement for higher throughput and multiplexed detection. Advances in stationary phase chemistries and inert surface technologies will extend capability to broader classes of metal-chelator drugs. Integration with automated platforms for on-site synthesis QC and online monitoring will accelerate clinical deployment.

Conclusion

Hydrophilic interaction chromatography on inert BEH Z-HILIC columns offers a robust solution for analyzing PSMA-617, its conjugate, and DOTA chelator. This approach overcomes limitations of reversed-phase and mixed-mode methods, delivering reliable retention, peak shape, and detection essential for quality control in radioligand therapy development.

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