Sensitive fluorescence assay for d,l-methadone

d,l-Methadone forms a fluorophore when reacted with paraformaldehyde in concentrated sulfuric acid. Based on this reaction, a fluorescence assay suitable for quantitative d,l-methadone analysis from plasma and other tissues was developed. d,l-Methadone was extracted at pH 9.2 from the deproteinized filtrate of plasma or of aqueous tissue homogenate into an organic phase of 25% isobutanol in ethylene dichloride. After an aliquot of the organic phase was evaporated to dryness at 50--55 degrees with an air jet, paraformaldehyde (0.1%, in concentrated sulfuric acid) was added, and fluorescence was read at 450 nm with excitation at 275 nm. By this method, d,l-methadone can be assayed in the presence of its metabolites, morphine, diacetylmorphine (heroin), codeine, and cocaine; however, amphetamine, meperidine, and quinine interfere.