SIMULTANEOUS QUANTITATION OF AMPHETAMINE AND 4'-HYDROXYAMPHETAMINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A relatively simple HPLC procedure used to detect and quantify amphetamine and 4'-hydroxyamphetamine in rat urine has been developed. Following Bond Elut(R) solid phase extraction, the two analytes - amphetamine and 4'-hydroxyamphetamine, and the two internal standards - methamphetamine and 4'-hydroxymethamphetamine were separated by HPLC using a phenyl column and detected by UV at 215 nm. The limit of quantitation for amphetamine and 4'-hydroxyamphetamine was 0.92 and 0.81 mu g/ml, respectively. Based on replicated analysis of controls at 1.6, 6.5, and 16.0 mu g/ml, the method is accurate (94 - 103% of target), and precise (% CVs of 1.3 - 5.2). The linear range of the assay is suitable for the quantitation of both analytes at urine concentrations that commonly result from administration of a single dose of amphetamine to the rat.