Determination of acetylmethadol and metabolites by use of high-performance liquid chromatography

A method is described for the simultaneous determination of l,α-acetylmethadol (LAAM) and five active metabolites — noracetylmethadol, dinoracetylmethadol, methadol, normethadol, and dinormethadol — in biofluids by high-performance liquid chromatography using a normal-phase column and a UV detector at 218 nm. The compounds are recovered from biofluids by a multistep liquid—liquid extraction. The mobile phase is methanol—acetonitrile (70:30, v/v) containing 0.015% ammonium hydroxide as the modifier. Retention times can be varied by adjusting the composition of the mobile phase to maximize peak height for quantitation using l-propranolol as the internal standard or peak separation for the collection of fractions. Using a UV detector the lower limit of sensitivity is 10 ng/ml of biofluid. Using fraction collection of radiolabeled drug and metabolites followed by liquid scintillation counting the lower limit of sensitivity is 1.0 ng/ml. Commonly used or abused narcotics including morphine, heroin, meperidine, methadone and propoxyphene do not interfere with the analysis. The method has been applied to plasma and urine samples from humans, sheep and rats. Extracts of urine from patients receiving maintenance treatment with LAAM contain LAAM and each of the five active metabolites.