Forced degradation behavior of epidepride and development of a stability-indicating method based on liquid chromatography-mass spectrometry

A reversed-phase high performance liquid chromatography (RP-HPLC) and LC-tandem mass spectrometry (LC-MS/MS) method was applied to study the forced degradation behavior and stability of epidepride. ¹²³I radioisotope-labeled epidepride, ((-)-N-[[(2S)-1-ethylpyrrolidin-2-yl]methyl]-5-iodo-2,3-dimethoxybenzamide) is a radio-tracer with a very high affinity for dopamine D₂ receptors in the brain and has been used as an imaging agent for single-photon emission computed tomography (SPECT). HPLC studies were performed using ¹²⁷I-epidepride (the non-radioactive compound), instead of ¹²³I-epidepride, with an RP-18 column using a mobile phase consisting of methanol, acetonitrile, and ammonium acetate (pH 7.0, 10 mM). The eluent flow rate and the wavelength for HPLC detection were 0.5 mL/min and 210 nm, respectively. The ligand was exposed to acid (1 N HCl) and alkaline (1 N NaOH) media and was subjected to oxidative decomposition at room temperature using 3% H₂O₂ and to thermal decomposition at 50°C. After various reaction times (0.5, 1, 2, 8, and 24 h), the substances were investigated by HPLC and LC-MS/MS. While no decomposition products were observed after the acidic, alkaline, and thermal treatments, but over 80% of the initial amount of ¹²⁷I-epidepride was oxidized within 24 h in the presence of H₂O₂. Only one major oxidation product with an m/z value of 435 was observed, in addition to the ¹²⁷I-epidepride species (m/z 419). The product was characterized by LC-MS/MS fragmentation; based on the results, the deteriorated type and fragmentation pathways could be proposed for epidepride.