1–5.6 ppm BD) or 30 μl (11–1220 ppm BD) of DEB-D6 (14.5 μmol/l in acetone). The vial was closed and shaken vigorously. The ice-cold blood samples were immediately centrifuged at 0 °C and plasma was stored at −80 °C until sample preparation for DEB analysis. During the exposures, BD was determined directly from 5-ml gas samples that were collected by means of disposable syringes from the chamber air and immediately injected via a 300 μl sample loop on column in a Shimadzu GC-8A gas chromatograph (GC; Shimadzu, Duisburg, Germany) equipped with a flame ionization detector

using nitrogen with a pressure of 3.75 kg/cm2 as carrier gas. Separation was done on a stainless steel column (3.5 m × 1/8 in. × 2 mm) packed with Tenax TA (60–80 mesh; Chrompack, Frankfurt, Germany). selleck chemical Temperatures of column SB431542 oven and detector were 110 °C and 200 °C, respectively. The combustible gases were hydrogen and synthetic air, each with a pressure of 0.6 kg/cm2. Under these conditions, the retention time of BD was 3.8 min. Chromatograms were recorded and integrated by a CR5A integrator (Shimadzu). Calibration curves were constructed several times by generating BD gas

concentrations ranging from 1 to 2000 ppm in atmospheres of closed chambers. Calibration curves were linear in the whole range. Analysis of linear regression through the origin revealed correlation coefficients (r) of at least 0.997 between peak areas and atmospheric BD concentrations. Each time before starting a BD exposure, a one-point calibration was carried out in the concentration range used in the actual experiment. The limit of detection (three times the background noise) was 0.3 ppm. The coefficient of variation, as a measure for reproducibility, was determined from 6 measurements each carried out at BD concentrations that covered the whole concentration range studied. It was always below ±2.7%. The DEB determination was based on the derivatization with

DTC (according to Munger et al., 1977 and Dupard-Julien et al., 2007). The derivatization procedure was species-specific. Mice: To 0.5 ml of thawed plasma, 1 ml of a DTC solution (0.22 mol/l in a 50 mmol/l phosphate buffer of pH 7.4) was added. After vortexing vigorously, the mixture Rolziracetam stood for 10 min at room temperature, then for 1 h at 50 °C. After cooling to room temperature, the obtained bis(dithiocarbamoyl) esters were extracted twice with 2 ml chloroform each. To the unified organic phase 1 ml of an aqueous sodium chloride solution (10 g/100 ml) was added and the mixture was vortexed thoroughly for 0.5 min. After centrifuging, the organic phase was carefully removed, dried in a gentle stream of nitrogen, resuspended in 50 μl methanol, and transferred in an autosampler vial for LC/MS/MS analysis.