Levomethorphan (dissolved in a mixed solution of 5% Emulphor™ EL-620/5% ethanol/90% isotonic sodium chloride solution, 2.5 mg/mL, rat 4–6) was administered to male DA pigmented rats, which were 5 weeks old and around 90 g mean weight. The drugs were given once daily at 5 mg/kg by intraperitoneal injection for ten successive days.

Liver microsomes from individual male dark agouti (DA) rats (n=4, 6 weeks old, around 125 g mean weight) were prepared by ultracentrifugation. For the in vitro experiments with rat and human liver microsomes, the reaction mixture consisted of 0.1 M potassium phosphate buffer (pH 7.4) with an NADPH generating system (1.3 mM NADP, 3.3 mM G-6-P, 0.4 U/ mL G-6-PDH, 3.3 mM MgCl2), 50 μM substrate (dextromethorphan or levomethorphan), and 0.5 mg protein/mL microsomes (rat or human liver microsomes) in a final volume of 200 μL. Dextromethorphan and levomethorphan were dissolved in methanol, and the final concentration of the organic solvent was 0.1%. The incubation was started by adding the microsomal fraction and then continued for 0, 5, 10, or 20 min. The reaction was terminated by adding an equal volume of a mixed organic solution of 50% acetonitrile and 50% methanol, including 10 μM levallorphan (IS), and vigorous shaking.