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 its potential misuse in chemical attacks, few modern analytical methods for BZ determination in biological samples have been published. The goal of the present work is to develop and validate a sensitive and rapid LC–MS/MS method for the determination of agent BZ in rat plasma. The sample preparation was based on 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. Atropine was used as an internal standard. The presented method is selective, accurate, precise, and 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 the BZ plasma levels in rats exposed to 2 mg/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 48 h, no BZ was observed at detectable levels. This new method allows the detection and quantification of BZ in biological samples after exposure of an observed organism and it will be further optimized for other tissues to observe the distribution of BZ in organs.

3‐Quinuclidinyl benzilate (agent BZ) toxicokinetics in rats

“…Preparation of plasma samples was based on the solid‐phase extraction (SPE) method, using Supelco Discovery DSC‐C18 (1 ml, 50 mg) SPE cartridges (Sigma‐Aldrich, St. Louis, MO, USA), previously described by Herman et al 15 Urine and bile samples were diluted 50 times with water and then processed identically as plasma samples.…”

“…After the sample preparation, the concentration of BZ was determined using LC‐MS/MS following the method published by Herman et al 15 The analysis was performed on a Finnigan Surveyor plus system (Thermo Scientific, San Jose, CA, USA). Gemini NX‐C18 column (5 μm, 150 mm × 4.6 mm I.D.)…”

“…BZ determination in plasma was validated in a previous study with linearity ( r 2 = 0.9947) in a concentration range from 0.5 to 1000 ng/ml, LLOQ 0.5 ng/ml, within‐run and between‐run accuracy from 96.2% to 101.9% and within‐run and between‐run precision expressed as the coefficient of variation (CV) less than 10% 15 …”

Trocylation of 3‐quinuclidinol, a key marker for the chemical warfare agent 3‐quinuclidinyl benzilate, for its enhanced detection at low levels in complex soil matrices by electron ionization gas chromatography–mass spectrometry

“…4,5 Analytical methods for its detection exist and involve liquid chromatography-mass spectrometry (LC/MS), relying primarily on BZ's intrinsic UV profile. 6,7 However, due to its fleeting nature as a result of its propensity for hydrolysis in vivo or in the environment, detection of intact BZ in such samples is very unlikely. Now, apart from LC/MS, gas chromatography-mass spectrometry (GC/MS) approaches have been explored for its analysis; however, they are not viable due the compound's lack of significant volatility (m.p.…”