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

Abstract For the investigations of alpidem overdose cases in humans, an HPLC method with fluorescence detection was developed. The method was based on 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 urinary sample, after deconjugation with β-glucuronidase/ arylsulfatase enzyme and addition of internal standard, was simply diluted (1/1) with pure water and injected onto the chromatographic system connected to spectrofluorimetric detector (λ1 = 255 nm, λ2= 423 nm). The analytical column was C8 type material specially deactivated for basic compounds, the eluent mixture was constituted by a phosphate solution 0.025M (pH=4.6), acetonitrile, methanol, 45/30/15 (v/v/v), supplied at 1.5 ml.min−1 flow rate. The determination limit of the method, in human urine, was 20 ng.ml−1; the method was linear in the range 20–1000 ng.ml−1 in urine. The method showed a good ...