Quantitative Analysis of Muscarinic Antagonist Atropine and Tiotropium in Microvolume Plasma Using Liquid Chromatography-Mass Spectrometry: Application for Pharmacokinetic Studies.

Despite the availability of current resources, the development of medical countermeasures remains crucial in combatting the threat posed by chemical warfare agents, such as organophosphorus compounds (OPs), which are toxic nerve agents requiring rapid medical intervention. Within the available therapeutic arsenal, muscarinic antagonists such as atropine are administered to mitigate the effects of excessive cholinergic system stimulation, which leads to respiratory tract obstruction due to hypersecretions and bronchoconstriction. Tiotropium, an FDA-approved bronchodilator, acts as a muscarinic receptor antagonist and could, therefore, serve as a potential alternative. To assess its potential efficacy in attenuating OP-induced respiratory effects in a murine intoxication model, it was necessary to first characterize its pharmacokinetic properties. A liquid chromatography-mass spectrometry method was developed and validated following ICH M10 guidelines for the quantification of atropine and tiotropium in 10 μL of plasma. The sample pretreatment procedure involved solid-phase extraction. Chromatographic separation was achieved using a fully porous sub 2 μm C18 column. The analysis was completed in just 4 min, with analytes identified and quantified using two selected reaction monitoring transitions. The mean extraction recoveries exceeded 90% for both drugs, and no matrix effect was observed. The lower limits of quantification were 0.5 ng/mL for tiotropium and 1.0 ng/mL for atropine, with an upper limit of 1000 ng/mL. The signal-to-concentration ratio demonstrated recoveries of back-calculated concentrations ranging from 94% to 108% (relative standard deviation (RSD) < 9.0%). Within-run and between-run precisions were both below 8% with accuracies ranging from 87% to 110%. This highly specific and sensitive method has proven useful for analyzing samples from pharmacokinetic studies conducted in mice. Following intraperitoneal administration, the AUC for tiotropium was approximately twice that of atropine, while its Tmax was half as long (4.9 vs. 8.2 min). The terminal half-lives were approximately 7.5 min for tiotropium and 9.8 min for atropine.