Simple validated method of LC-MS/MS determination of BZ agent in rat plasma samples.

Abstract

Agent BZ (3-quinuclidinyl benzilate) is a centrally acting synthetic anticholinergic agent, considered as a potential military incapacitating chemical warfare agent. Despite its significance as a model compound in pharmacological research and the potential misuse in chemical attacks, few modern analytical method for BZ determination in biological samples has been published. The goal of the presented work is to develop and validate sensitive and rapid LC-MS/MS method for the agent BZ determination in rat plasma. The sample preparation was based on the solid-phase extraction on C-18 cartridges. The reversed phase HPLC coupled with the mass spectrometer with electrospray ionization in the positive ion-selective reaction monitoring mode was employed in the BZ analysis. The atropine was used as an internal standard. The presented method is selective, accurate, precise, linear (r2 = 0.9947) in a concentration range from 0.5 ng/ml to 1 000 ng/mL and sensitive enough (limit of detection 0.2 ng/mL; limit of quantification 0.5 ng/mL) to determine BZ plasma levels in rats exposed to 2mg/kg and 10 mg/kg of BZ. The highest level of BZ in plasma was observed 5 minutes after intramuscular administration (154.6 ± 22.3 ng/mL in rats exposed to 2 mg/kg of BZ and 1024 ± 269 ng/mL in rats exposed to 10 mg/kg). After 48h, no BZ was observed at detectable levels. This new method allows BZ detection and quantification in biological samples after an exposition of an observed organism and it will be further optimized for other tissues to observe BZ distribution in organs.