A reversed-phase high-performance liquid chromatography (HPLC) and liquid chromatography-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 radiotracer with a high affinity for dopamine D2 receptors in the brain and has been used as an imaging agent for single-photon emission computed tomography. HPLC studies were performed using 127I-epidepride (the nonradioactive 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 (30 minutes, 1 hour, 2 hours, 8 hours, and 24 hours), the substances were investigated by HPLC and LC-MS/MS. Although no decomposition products were observed after the acidic, alkaline, and thermal treatments, >80% of the initial amount of 127I-epidepride was oxidized within 24 hours 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, and the deteriorated type and fragmentation pathways were proposed for epidepride

Studies of the neurobiological causes of anxiety disorders have suggested that the γ-aminobutyric acid (GABA) system increases synaptic concentrations and enhances the affinity of GABA_A (type A) receptors for benzodiazepine ligands. Flumazenil antagonizes the benzodiazepine-binding site of the GABA/benzodiazepine receptor (BZR) complex in the central nervous system (CNS). The investigation of flumazenil metabolites using liquid chromatography (LC)-tandem mass spectrometry will provide a complete understanding of the in vivo metabolism of flumazenil and accelerate radiopharmaceutical inspection and registration. The main goal of this study was to investigate the use of reversed-phase high performance liquid chromatography (PR-HPLC), coupled with electrospray ionization triple-quadrupole tandem mass spectrometry (ESI-QqQ MS), to identify flumazenil and its metabolites in the hepatic matrix. Carrier-free nucleophilic fluorination with an automatic synthesizer for [¹⁸F]flumazenil, combined with nano-positron emission tomography (NanoPET)/computed tomography (CT) imaging, was used to predict the biodistribution in normal rats. The study showed that 50% of the flumazenil was biotransformed by the rat liver homogenate in 60 min, whereas one metabolite (M1) was a methyl transesterification product of flumazenil. In the rat liver microsomal system, two metabolites were identified (M2 and M3), as their carboxylic acid and hydroxylated ethyl ester forms between 10 and 120 min, respectively. A total of 10–30 min post-injection of [¹⁸F]flumazenil showed an immediate decreased in the distribution ratio observed in the plasma. Nevertheless, a higher ratio of the complete [¹⁸F]flumazenil compound could be used for subsequent animal studies. [¹⁸F] According to in vivo nanoPET/CT imaging and ex vivo biodistribution assays, flumazenil also showed significant effects on GABA_A receptor availability in the amygdala, prefrontal cortex, cortex, and hippocampus in the rat brain, indicating the formation of metabolites. We reported the completion of the biotransformation of flumazenil by the hepatic system, as well as [¹⁸F]flumazenil’s potential as an ideal ligand and PET agent for the determination of the GABA_A/BZR complex for multiplex neurological syndromes at the clinical stage.

Studies on the neurobiological causes of anxiety disorders suggest that the GABA system in-creases synaptic concentration and enhances the affinity of GABAA (type A) receptors for ben-zo-diazepine ligands. Flumazenil antagonizes the benzodiazepine binding site of the GABA (γ-aminobutyric acid) type /benzodiazepine receptor (BZR) complex in the central nervous sys-tem (CNS). The integration of the metabolites of flumazenil by LC-tandem mass spectrometry will complete understanding of the in vivo metabolism of flumazenil and accelerate radio-pharmaceutical inspection and registration. The main goal of our study was to investigate using reversed-phase HPLC (PR-HPLC) coupled with electrospray ionization triple quadrupole tan-dem mass spectrometry (ESI-QqQ MS) for the identification of flumazenil and its metabolites in a hepatic matrix. And a carrier-free nucleophilic fluorination with automatic synthesizer for [18F]flumazenil which applied to in vivo nano-positron emission tomography (Nano-PET)/computed tomography (CT) imaging and ex vivo bio-distribution used to analyze in normal rats. The study showed that 50% of the flumazenil was bio-transformed at 60 min by the rat liver homogenate, while one metabolite (M1) was a methyl transesterification product of flumazenil. In a rat liver microsomes system, two metabolites were identified (M2 and M3) as its carboxylic acid and hydroxylated ethylester forms, respectively. [18F]flumazenil in vivo nanoPET/CT imag-ing and ex vivo bio-distribution assay also showed significant effects on GABAA receptor availa-bility in the amygdala, prefrontal cortex, cortex, and hippocampus in the rat brain, worriless about the formation of metabolites. We showed completion of the bio-transformed course of flumazenil by the hepatic system; and [18F]flumazenil can be a good ligand and serve as a PET agent for determination of GABAA/BZR complex for multiplex neurological syndromes in the clinical stage.

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