Laboratory of Toxicology, University of Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium
* Nucleoside Research & Mass Spectrometry Unit, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium

A liquid-liquid back extraction procedure using n-hexane-isoamyl alcohol (93:7, v/v) as the extraction solvent was performed for a basic sample clean-up. N-methyldespropionyl-bezitramide was used as the internal standard. Chromatographic separation was achieved on a Hypersil ODS (C18) 5-mm column, using a 80:20 (v,v) mixture of 1.0 mM ammonium acetate and methanol/ acetonitrile (50:50, v/v)-1.0 mM ammonium acetate as the mobile phase. To obtain as high a sensitivity and selectivity as possible, a selected reaction monitoring mass spectrometric technique in the daughter ion mode was applied. In addition low energy collision-activated dissociation (CAD) product ion spectra were recorded for a few samples.

Calibration graphs were prepared for blood and urine and good linearity is achieved over a concentration range of 1 to 150 ng/mL (r > 0.9994). The intra- and interassay coefficients of variation (CV%) for the analysis of quality control samples were: within-day CV% 6.4 and 1.6%, between-day CV% 10.2 and 7.4% for the 10 and 50 ng/ml levels in blood, respectively. At the same concentration levels percent of targets are within 12.1% and 9.2%.

Postmortem samples (blood, urine, stomach contents, bile, liver and kidney) from three fatalities, all suspected victims from a drug overdose, were analyzed. The following results were found for fatality 1,2 and 3 respectively: blood 106 ng/mL, 6 ng/mL, NA (matrix not available); urine 2.5 ng/mL, < LOD of 0.7 ng/mL, NA; stomach contents 5.1 µg/g, 3.5 µg/g, NA; bile 621 ng/g, NA, NA; liver 2 µg/g, 0.3 µg/g, 2.5 µg/g; kidney 1.7 µg/g, 0.2 µg/g, 0.3 µg/g. The results obtained with LC-ESI-MS/MS are in close agreement with those obtained using the LC-FL method. Moreover the isolates' identity and structure is fully confirmed by the CAD product ion spectra, thus allowing unequivocal conclusions about the prior intake of bezitramide by the three subjects.