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 flu orescence detection was developed for the quantitative determination o f despropionyl-bezitrmidee, the active metabolite of bezitramide (Burg odin), 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 am monium acetate as the eluent. Internal standardization with N-methylde spropionyl-bezitramide was used in order to enhance the precision and the accuracy of the method. For the isolation of the compound from bio logical samples, a liquid-liquid extraction with n-hexane-isoamyl alco hol (93:7 v/v) was performed. Calibration graphs were prepared for blo od 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, coeffici ents of variation of 3.3 and 6.5% were obtained for within-day and bet ween-day precisions, respectively. For urine, the respective coefficie nt of variation values of 4.3 and 4.9% were obtained. The selectivity and the accuracy of the method were satisfactory. Samples (blood, urin e, stomach contents, bile, liver, kidney) from five fatalities that we re due to the combined intake of several drugs, including bezitramide, were analyzed and the results are reported. In addition, one blood sa mple and 14 urine samples from persons suspected of using bezitramide were analyzed, revealing despropionyl-bezitramide concentrations in ur ine ranging from 1.3 to 72.3 ng/mL.