Isolation and structure elucidation of urinary metabolites of mitoxantrone

Three 13C-labeled 1,4-dihydroxy-5,8-bis(2-[(2-hydroxyethyl)amino]-ethyl)amino-9,10- anthracenedione dihydrochloride (mitoxantrone) isotopomers were synthesized to prove the proposed chemical structures of human urinary metabolites by means of nuclear magnetic resonance spectroscopy. After application of labeled mitoxantrone to an anesthetized pig, urine was collected by way of a vesicourethral catheter. The urinary metabolites were isolated by liquid chromatography using a new procedure developed for extraction of mitoxantrone metabolites. Structural elucidation by nuclear magnetic resonance spectroscopy and tandem mass spectrometry confirmed the proposed mono- and dicarboxylic acid structures of the metabolites. High-performance liquid chromatography of native pig urine showed an additional metabolite detected by its UV-visible absorption. The new metabolite was identified as a glucuronic acid conjugate of mitoxantrone by means of nuclear magnetic resonance spectroscopy and tandem mass spectrometry. Incubation with beta-glucuronidase under high-performance liquid chromatography control revealed mitoxantrone as the sole product. Quantitative high-performance liquid chromatography analyses showed that the new urinary metabolite represents the main biotransformational pathway of mitoxantrone in pigs, indicating significant interspecies variation in mitoxantrone biotransformation. Expressed in equivalents of mitoxantrone, the new metabolite amounts to 25% and 31%, respectively, of urinary excreted drug-related material. Extraction of patient urine using the same procedure led to the isolation of pure metabolite B. Tandem mass spectrometric data delivered definitive evidence for the structure of metabolite B as monocarboxylic acid of mitoxantrone.