Liquid chromatography-tandem mass spectrometry-based pharmacokinetic and metabolic analysis of 4-bromo-2,5-dimethoxyphenethylamine and its metabolites in human plasma.

4-Bromo-2,5-dimethoxyphenethylamine (2C-B) is widely used recreationally and has recently gained interest as a treatment for mental health disorders. In this study, a liquid chromatography-tandem mass spectrometry method to quantify 2C-B and its metabolites 4-bromo-2,5-dimethoxyphenylacetic acid (BDMPAA) and 4-bromo-2-hydroxy-5-methoxyphenylacetic acid (B-2-HMPAA) in human plasma was developed and validated. Moreover, pharmacokinetic analysis was performed on samples from clinical study participants who received 30 mg of 2C-B. The metabolic degradation of 2C-B and its metabolites via monoamine oxidases (MAOs), cytosolic enzymes, and cytochrome P450 enzymes was assessed and their activation potencies at the serotonin 2A receptor were investigated. Optimal chromatographic separation was achieved using a Kinetex 2.6 μm XB-C18 analytical column and a mobile phase gradient of water and acetonitrile supplemented with 0.1% formic acid. Using electrospray ionization, a linear range of 0.5-100 ng/mL for 2C-B, 2.5-1000 ng/mL for BDMPAA, and 0.5-1000 ng/mL for B-2-HMPAA was achieved. The method demonstrated high accuracy, precision, and extraction recovery with minimal matrix effects. MAO-A, MAO-B, cytosolic enzymes, and CYP2D6 were identified as key enzymes involved in the metabolic degradation of 2C-B. Unlike 2C-B, BDMPAA and B-2-HMPAA did not activate the human serotonin 2A receptor, suggesting that these metabolites do not contribute to the psychedelic effect. This study provides insights into the pharmacokinetics, metabolism, and pharmacological activity of 2C-B and its metabolites. The validated liquid chromatography-tandem mass spectrometry method offers a reliable tool for future clinical studies investigating the therapeutic potential and metabolism of 2C-B. SIGNIFICANCE STATEMENT: A rapid and nonlaborious liquid chromatography-tandem mass spectrometry method was developed and validated for pharmacokinetic analysis of 4-bromo-2,5-dimethoxyphenethylamine (2C-B) and its metabolites 4-bromo-2,5-dimethoxyphenylacetic acid and 4-bromo-2-hydroxy-5-methoxyphenylacetic acid in human plasma. To assess the metabolites' relevance in psychedelic drug action, serotonin 2A receptor activity was studied. Unlike 2C-B, the metabolites failed to activate the receptor. Monoamine oxidase A and B, and cytosolic enzymes were confirmed in 4-bromo-2,5-dimethoxyphenylacetic acid formation, whereas CYP2D6 was found to metabolize 2C-B through a minor pathway.