Determination of Strychnine Levels in Serum and Brain via LC-MS/MS in Mice Vaccinated Against Strychnine

Abstract

ID 53762 Poster Board 315Strychnine is an extremely toxic compound that can cause seizures, respiratory failure, and death with no available treatment. To determine if strychnine-specific antibodies could protect against strychnine-related effects and alter strychnine distribution in serum and brain, mice were vaccinated once every two weeks with a strychnine conjugate vaccine (Strychnine-sKLH adsorbed to aluminum) or aluminum alone. One week later, mice received a 0.9 mg/kg IP strychnine dose and blood and brain were collected 30 minutes. To quantitively determine the strychnine in mouse serum and brain, a sensitive method has been developed and validated on liquid chromatography-tandem mass spectrometry (LC-MS/MS) in Electrospray ionization (ESI) inlet with dynamic multiple reaction monitoring (DMRM) mode. The method consists of a protein precipitation spiked with internal standard followed by solid phase extraction (SPE) using a Bond Elut PCX 96 well plate. Serum and brain supernatant samples of 200 mL were pretreated via protein precipitation and internal standard. Chromatographic separation was carried out on a Poroshell EC C18 2.1 x 50mm 2.7u column. The method is linear in the range of 2.5-500 ng/mL and a relative standard error of 4.8%. Maximum detected strychnine in vaccinated mouse serum ranged from 79 to 471 ng/mL, 19 to 124 ng/mL in unvaccinated mice. Strychnine concentrations in vaccinated mouse brains ranged from 10 to 34 ng/gm, 46 to 97 ng/gm in unvaccinated mice. The results of this study showed that strychnine-sKLH reduced brain strychnine concentrations and demonstrate that a strychnine-immunotherapy could be developed to protect against strychnine-related effects.