CHARACTERIZATION OF THE GLUCURONIDE CONJUGATE OF COTININE - A PREVIOUSLY UNIDENTIFIED MAJOR METABOLITE OF NICOTINE IN SMOKERS URINE

Recent studies in our laboratories have confirmed that a major unident ified metabolite of nicotine in smokers' urine was susceptible to enzy matic degradation by beta-glucuronidase to afford (S)-(-)-cotinine. In order to establish the identity of this metabolite, the quaternary am monium conjugate, viz., (S)-(-)-cotinine N-glucuronide, was synthesize d. Reaction of methyl O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranurona te with with (S)-(-)-cotinine at 60-degrees-C for 3 days affords the f ully protected conjugate as the bromide salt. Deprotection was accompl ished in 1 M NaOH overnight at 25-degrees-C. The deprotected inner sal t was isolated by Dowex-50W cation-exchange chromatography. Electrospr ay mass spectra of the inner salt revealed the presence of ions with m /z 353 (M + H)+, 375 (M + Na)+, and 391 (M + K)+ as well as ions resul ting from loss of water and cleavage of the glycosidic bond. Proton an d carbon nuclear magnetic resonance spectra established that the posit ion of glucuronidation was the pyridyl nitrogen. The magnitude of the coupling between H1" and H2" of the sugar ring (8.71 Hz) and and nucle ar Overhauser enhancements were consistent with the beta-isomer of the glucuronide conjugate. The synthetic (S)-(-)-cotinine N-glucuronide w as susceptible to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine. Application of a cation-exchange high-performance l iquid chromatographic method enabled the collection of a fraction cont aining (S)-(-)-cotinine N-glucuronide from a smoker's urine. The elect rospray mass spectrum of this fraction contained ions consistent with the presence of (S)-(-)-cotinine N-glucuronide. The concentrated fract ion was subjected to enzymatic hydrolysis by beta-glucuronidase to aff ord (S)-(-)-cotinine. A thermospray ion-exchange liquid chromatographi c mass spectral method was developed for the direct determination of ( S)-(-)-cotinine N-glucuronide in smokers' urine. The data herein repor ted established the identity of a major urinary metabolite of nicotine as N-beta-D-glucopyranuronosyl-(S)-(-)-cotininium inner salt.