LC–MS/MS determination of carbamathione in microdialysis samples from rat brain and plasma

Abstract

A selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of S-(N, N-diethylcarbamoyl) glutathione (carbamathione) in microdialysis samples from rat brain and plasma. S-(N, N-Diethylcarbamoyl) glutathione (carbamathione) is a metabolite of disulfiram. This metabolite may be responsible for disulfiram’s effectiveness in the treatment of cocaine dependence. An analytical method using liquid chromatography–tandem mass spectrometric (LC–MS/MS) was developed to determine carbamathione in vivo using microdialysis sampling from rat brain and plasma. Chromatographic separations were carried out on an Alltech Altima C-18 (50 mm long × 2.1 mm i.d., 3 μm particles) analytical column at a flow rate of 0.3 ml/min. Solvent A consisted of 10 mM ammonium formate, methanol, and formic acid (99:1:0.06, v/v/v). Solvent B consisted of methanol, 10 mM ammonium formate and formic acid (99:1:0.06, v/v/v). A 20 min linear gradient from 95% aqueous to 95% organic was used. Tandem mass spectra were acquired on a Micromass Quattro Ultima “triple” quadrupole mass spectrometer equipped with an ESI interface. Quantitative mass spectrometric analysis was conducted in positive ion mode selected reaction monitoring (SRM) mode looking at the transition of m/z 407–100 and 175 for carbamathione and m/z 392–263 for the internal standard S-hexyl glutathione. The simultaneous collection of microdialysate from blood and brain was used to monitor carbamathione concentrations centrally and peripherally. Good linearity was obtained over a concentration range of 0.25–10,000 nM. The lowest limit of quantification (LLOQ) was determined to be 1 nM and the lowest limit of detection (LLOD) was calculated to be 0.25 nM. Intra- and inter-day accuracy and precision were determined and for all the samples evaluated, the variability was less that 10% (R.S.D.).