Development and validation of an LC-MS/MS method for simultaneous quantification of p-Chloroamphetamine in plasma and brain: application to pharmacokinetic and brain distribution studies in mice

A simple, selective, and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of p-Chloroamphetamine (PCA) in mouse plasma and brain tissue. Chromatographic separation was achieved using a Synergi Polar-RP column with gradient elution using 2 mM ammonium formate containing 0.2% formic acid and acetonitrile. Multiple reaction monitoring transitions were m/z 170.0→ 125.0 for PCA and m/z 214.2→ 154.0 for the internal standard, clorprenaline. The method was fully validated according to FDA and ICH guidelines, demonstrating excellent selectivity, linearity (1–1000 ng/mL), accuracy, precision, negligible matrix effects, consistent recovery, and adequate stability in both matrices. A simple methanol-based protein precipitation method enabled efficient extraction with minimal interference. The validated assay was applied to characterize the pharmacokinetics and brain distribution of PCA in male ICR mice following intravenous (1 mg/kg) and oral (10 mg/kg) administration. PCA showed extensive distribution after intravenous dosing, with a large steady-state volume of distribution (5788.42 ± 544.29 mL/kg), moderate clearance (26.92 ± 2.42 mL/min/kg), and a terminal half-life of 149.51 ± 13.98 min. After oral administration, PCA was absorbed with a mean Tmax of 160.0 ± 69.3 min, reaching a Cmax of 0.69 ± 0.39 µg/mL and an apparent oral bioavailability of 104.12%. Following oral dosing, PCA exhibited high total brain penetration, with brain-to-plasma ratios (Kp, brain) of 11.26–16.17 and unbound brain-to-plasma ratios (Kp, uu, brain) of 0.40–0.57. These findings provide the first comprehensive pharmacokinetic and brain distribution profile of PCA and offer quantitative insight into its central serotonergic and neurotoxic actions.