From [99mTc]pertechnetate to [99mTc]sestamibi: Dissection of a Complex Reaction Sequence Using Radio-LC-MS

[99mTc]sestamibi ([99mTc][Tc(MIBI)6]+; MIBI = 2-methoxybutylisonitrile) is a clinically established myocardial perfusion SPECT tracer. Its one-pot kit-based synthesis from [99mTc]pertechnetate ([99mTc][TcO4]−) is complex, involving a 6-oxidation state transition (Tc(VII) to Tc(I)) and complete ligand replacement. We aimed to unravel this complex reaction, to inform rational quality control and identify new technetium synthons for molecular imaging. Generator-produced [99mTc]pertechnetate was added to commercial or bespoke clinically used kits, varying the reaction time, temperature, and concentrations of reagents (individually and collectively) and carrier technetium-99. Radioactive products were analysed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) with optical, radiometric, and mass spectrometric (MS-ESI+) detection. At least 11 radioactive intermediates were detected by radio-HPLC. Technetium(V) and technetium(I) intermediates were identified or imputed by radio-HPLC-MS, including [TcVO(cysteinate)2]+, [TcI(MIBI)4L2]+, and [TcI(MIBI)5L1]+ (L = labile monodentate ligand, e.g., H2O). Tc(III) intermediates [TcIII(cysteinate)2(MIBI)]+ and [TcIII(cysteinate)2(MIBI)2]+ were indicated by weak MS-ESI+ ions. We conclude that the reaction proceeds via reduction from [TcVIIO4]− via unknown intermediates to [TcVO(cysteinate)2]+, then via Tc(III) intermediates containing both cysteinate and MIBI ligands (e.g., [TcIII(cysteinate)2(MIBI)2]+), to form Tc(I) without cysteine and with