Quantitative analysis of despropionyl-bezitramide, the active metabolite of bezitramide (Burgodin) in biological samples by high-performance liquid chromatography with fluorescence detection

A sensitive high-performance liquid chromatographic procedure with fluorescence detection was developed for the quantitative determination of despropionyl-bezitrmidee, the active metabolite of bezitramide (Burgodin), in biological samples. Chromatographic separation was achieved on a Hypersil ODS (C18) 5-mu m column, using a 31:69 (v/v) mixture of 0.1 M ammonium acetate and methanol/acetonitrile (50:50, v/v)-0.1 M ammonium acetate as the eluent. Internal standardization with N-methyldespropionyl-bezitramide was used in order to enhance the precision and the accuracy of the method. For the isolation of the compound from biological samples, a liquid-liquid extraction with n-hexane-isoamyl alcohol (93:7 v/v) was performed. Calibration graphs were prepared for blood and urine, and linearity was achieved over a concentration range 1-50 ng/mL. The quantitation limit for despropionyl-bezitramide in blood and urine was 1 ng/mL. At a 10 ng/mL concentration in blood, coefficients of variation of 3.3 and 6.5% were obtained for within-day and between-day precisions, respectively. For urine, the respective coefficient of variation values of 4.3 and 4.9% were obtained. The selectivity and the accuracy of the method were satisfactory. Samples (blood, urine, stomach contents, bile, liver, kidney) from five fatalities that were due to the combined intake of several drugs, including bezitramide, were analyzed and the results are reported. In addition, one blood sample and 14 urine samples from persons suspected of using bezitramide were analyzed, revealing despropionyl-bezitramide concentrations in urine ranging from 1.3 to 72.3 ng/mL