Analysis of Pz-1, a promising therapeutic for organophosphorus poisoning from rodent plasma by liquid chromatography-tandem mass spectrometry

Organophosphorus (OP) pesticides (e.g., parathion) and nerve agents (e.g., soman) can produce acute and long-term neurological problems. Exposure to OP chemicals is responsible for an estimated 200,000 deaths annually. Pz-1 (N-(5-(tert butyl)isoxazol-3-yl)-2-(4-(5-(1-methyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)phenyl)acetamide) is a muscle specific kinase (MuSK) inhibitor which has shown potential as a treatment for OP chemical exposure and as a tyrosine kinase inhibitor to impede the growth of Cancer cells. While development of this treatment requires the availability of a validated analytical method, no method currently exists for analysis of Pz-1 from biological samples. In this study, an analytical method was developed for Pz-1 from rat (and mouse) plasma. Plasma was prepared by precipitating plasma proteins, isolating the supernatant, evaporating to dryness and reconstituting in 1:1 MeOH:water. Prepared samples were analyzed by reversed-phase liquid chromatography tandem mass-spectrometry (LC-MS/MS). The method produced excellent sensitivity, with a limit of detection of 1 nM (455 ng/L). The calibration range was 3-100 nM and the calibration curve produced excellent linear behavior (R² ≥ 0.99 and PRA ≥ 91 %). The method also showed good accuracy and precision. The validated method was used to detect Pz-1 in mouse plasma following intraperitoneal (IP) treatment with 5 mg/kg Pz-1. In summary, this method shows promise as a simple and sensitive method to analyze Pz-1 in rat plasma to facilitate its continued development as a treatment for OP toxicity.