ABSTRACT
[Methyl-d3]-N-1-beta-D-glucopyranosyl-(+/-)-nicotinium inner salt ((+/-)-[methyl-d3]nicotine N-1-glucuronide) was synthesized from (+/-)-[methyl-d3]nicotine via reaction with methyl-2,3,4-tri-O-acetyl-1-bromodeoxy-alpha-D-glucopyranouronate, followed by deprotection with 1 M aqueous NaOH and purification by preparative TLC. Nicotine N-glucuronide was identified and determined directly in smokers' urine. A solid phase extraction method was used to partially isolate the material from urine. Subsequent determination was by thermospray-LC/MS using the synthetic d3-labeled nicotine N-glucuronide as internal standard. The identified urinary component had the same retention time as a synthetic standard and gave the same mass spectrum. The thermospray mass spectrum was characterized from the protonated molecular ion (m/z 339) and the protonated aglycone ion (m/z 163). Quantitative results from this direct method were compared with those from an indirect method, which calculated the nicotine glucuronide in the biological sample from the amount of nicotine released following treatment of the sample with the deconjugating enzyme, beta-glucuronidase. On average, the concentration of nicotine N-glucuronide determined by the direct method was 34% greater than that determined by the indirect method. Concentrations of nicotine N-glucuronide in urine ranged from 2.2 to 7.6 nmol/ml with a limit of detection of 1.3 nmol/ml.
Subject(s)
Glucuronates/urine , Nicotine/analogs & derivatives , Nicotine/urine , Smoking/urine , Adult , Biomarkers/urine , Female , Humans , Male , Mass Spectrometry , Middle Aged , Reproducibility of ResultsABSTRACT
In addition to S(-)-nicotine, several minor tobacco alkaloids ((+/-)-nornicotine, anabaseine, S(-)-anabasine, and S(-)-N-methylanabasine) are present in tobacco smoke. This study demonstrates that these alkaloids increase fractional 3H release in a concentration-dependent manner from rat striatal slices preloaded with [3H]dopamine, with desensitization of this response. The rank order of EC50 values was S(-)-nicotine (3.0 +/- 2.2 microM) > (+/-)-nornicotine (6.7 +/- 2.1 microM) > anabaseine (15.4 +/- 6.1 microM) = S(-)-N-methylanabasine (16.3 +/- 4.7 microM) = S(-)-anabasine (19.3 +/- 3.2 microM). The alkaloids did not modulate fractional 3H release evoked by electrical-field depolarization. Thus, minor tobacco alkaloids may contribute to the apparent neuroprotective effects of smoking in neurodegenerative diseases.
Subject(s)
Anabasine/analogs & derivatives , Anabasine/pharmacology , Corpus Striatum/drug effects , Dopamine/metabolism , Nicotine/analogs & derivatives , Animals , Corpus Striatum/metabolism , Dose-Response Relationship, Drug , In Vitro Techniques , Male , Nicotine/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
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.
