Identification and quantitation of despropionyl-bezitramide in postmortem samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

A sensitive and specific method for the determination and quantitation of ( despropionyl) bezitramide in postmortem samples using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/ MS) is presented. The method is the result from a simple methodological tra nsfer of a liquid chromatographic method with fluorescence detection (LC-FL ) previously developed in our laboratory. A liquid-liquid back-extraction p rocedure using n-hexane isoamyl alcohol (93:7, v/v) as the extraction solve nt is performed for a basic sample cleanup. N-Metethyedespropionyl bezitram ide is used as the internal standard. Chromatographic separation of the ana lytes of interest is achieved on a Hypersil ODS 5-mu m column, using a 80:2 0 (v/v) mixture of 1.0 mM ammonium acetate and methanol/acetonitrile (50: 5 0, v/v) and 1.0 mM ammonium acetate as the mobile phase. To obtain as high a sensitivity and selectivity as possible, a selected reaction-monitoring m ass spectrometric technique is applied. In addition, low-energy collisional -activate dissociation (CAD) product ion spectra are recorded for a few sam ples. Calibration graphs are prepared for blood and urine, and good lineari ty is achieved over a concentration range of 1-150 ng/mL, The intra- and in terassay coefficients of variation (CV%) for the analysis of quality contro l samples at 10 and 50 ng/mL concentration levels do not exceed 10.2%, and percent of targets are within 12.1%, Postmortem samples (blood, urine, stom ach contents, bile, liver, and kidney) from three fatalities, all suspected victims of drug overdoses, are analyzed, and the results are reported. The results obtained with LC-ESI-MS/MS are in close agreement with those obtai ned using the LC-FL method. Moreover, the isolates' identity and structure are confirmed by the CAD product ion spectra, thus allowing to make unequiv ocal conclusions about the prior intake of bezitramide by the three subject s.