No abstract

Recent studies in our laboratories have confirmed that a major unidentified metabolite of nicotine in smokers' urine was susceptible to enzymatic degradation by beta-glucuronidase to afford (S)-(-)-cotinine. In order to establish the identity of this metabolite, the quaternary ammonium conjugate, viz., (S)-(-)-cotinine N-glucuronide, was synthesized. Reaction of methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate with (S)-(-)-cotinine at 60 degrees C for 3 days affords the fully protected conjugate as the bromide salt. Deprotection was accomplished in 1 M NaOH overnight at 25 degrees C. The deprotected inner salt was isolated by Dowex-50W cation-exchange chromatography. Electrospray 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 resulting from loss of water and cleavage of the glycosidic bond. Proton and carbon nuclear magnetic resonance spectra established that the position of glucuronidation was the pyridyl nitrogen. The magnitude of the coupling between H1" and H2" of the sugar ring (8.71 Hz) and nuclear Overhauser enhancements were consistent with the beta-isomer of the glucuronide conjugate. The synthetic (S)-(-)-cotinine N-glucuronide was susceptible to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine. Application of a cation-exchange high-performance liquid chromatographic method enabled the collection of a fraction containing (S)-(-)-cotinine N-glucuronide from a smoker's urine. The electrospray mass spectrum of this fraction contained ions consistent with the presence of (S)-(-)-cotinine N-glucuronide. The concentrated fraction was subjected to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine.(ABSTRACT TRUNCATED AT 250 WORDS)

A thermospray liquid chromatographic/mass spectrometric method has been developed for direct determination of cotinine-N-glucuronide in the urine of smokers. Quantification was performed using methyl-d3-cotinine-N-glucuronide as internal standard and monitoring the protonated aglycons. Using a simple preparation, urine samples from four smokers were analyzed and the results compared favorably with those from a previously reported method that quantifies aglycon release following beta-glucuronidase treatment. Amounts of cotinine-N-glucuronide found in urine from smokers ranged from less than 0.7 to 21 nmol ml-1, indicating wide inter-individual variability in the metabolic production of this metabolite. Cotinine-N-glucuronide was found to be the second most abundant urinary nicotine metabolite. A similar method was developed for trans-3'-hydroxycotinine-N-glucuronide but this compound was not detected in smokers' urine.

No abstract

Eight compounds from a Kentucky 1R4F reference cigarette smoke condensate have been determined by selected ion monitoring-mass spectrometry (SIM-MS) to confirm the validity of multidimensional gas chromatography (MDGC) as a quantitative tool in complex mixture analyses. Four electrostatically precipitated smoke condensate samples of 100 cigarettes each are dissolved individually in 25 mL of 2-propanol. The 2-propanol contains two methyl esters (C8 and C14) and seven deuterium-labeled compounds used as internal standards (IS). Analysis of the compounds of interest, pyridine; acetamide; acrylamide; phenol; o-, m-, and p-cresol; and quinoline, is accomplished by using two heartcuts. Heartcut times of the MDGC analysis are selected such that at least one IS is transferred with each group of compounds being analyzed. This study shows that the MDGC technique previously developed and described can be used for quantitative analyses. A comparison is made between the two types of internal standards. The results obtained for both types of internal standards agree within 20% of each other, on the average, with higher standard deviations for approximately 60% of the compounds where methyl esters are used as internal standards.