SIMPLE, RAPID AND SPECIFIC IDENTIFICATION AND QUANTIFICATION OF A METABOLITE OF ALPIDEM, A NEW IMIDAZOPYRIDINE ANXIOLYTIC, IN HUMAN URINE, BY DIRECT-INJECTION INTO HPLC COLUMN WITH FLUORESCENCE DETECTION

For the investigations of alpidem overdose cases in humans, an HPLC me thod with fluorescence detection was developed. The method was based o n the identification and quantification of an alpidem metabolite (the product of oxidation of the propyl group) in urine, since no unchanged alpidem was found in urine of subjects dosed with the drug. The urina ry sample, after deconjugation with beta-glucuronidase/arylsulfatase e nzyme and addition of internal standard, was simply diluted (1/1) with pure water and injected onto the chromatographic system connected to spectrofluorimetric detector (lambda(1) = 255 nm, lambda(2) = 423 nm). The analytical column was C-8 type material specially deactivated for basic compounds, the eluent mixture was constituted by a phosphate so lution 0.025M (pH = 4.6), acetonitrile, methanol, 45/30/15 (v/v/v), su pplied at 1.5 ml.min(-1) flow rate. The determination limit of the met hod, in human urine, was 20 ng.ml(-1); the method was linear in the ra nge 20-1000 ng.ml(-1) in urine. The method showed a good selectivity i n respect to endogenous compounds and to commonly prescribed psychotro pic drugs. The method, validated over a 4 day period, by two analysts, was demonstrated to be precise and accurate.