Abstract
A method based on liquid chromatography tandem mass spectrometry (LC–MSMS) applying atmospheric pressure chemical ionisation (APCI) in the positive ion mode was developed for the direct determination of nicotine, cotinine, trans-3′-hydroxycotinine, their corresponding glucuronide conjugates as well as cotinine- N-oxide, norcotinine, and nicotine- N′-oxide in the urine of smokers. The assay involves filtration of crude urine, fast liquid chromatography on a reversed-phase column and mass-specific detection using MSMS transitions. Deuterium-labeled nicotine, cotinine, and trans-3′-hydroxycotinine were used as internal standards. Glucuronides used as reference material were either chemically (cotinine- N-glucuronide) or enzymatically synthesized (nicotine- N-glucuronide and trans-3′-hydroxycotinine- O-glucuronide). Precision for the major nicotine analytes at levels observable in urine of smokers was better than 10%. Accuracy expressed in recovery rates in urine matrix for nicotine, cotinine, trans-3′-hydroxycotinine, and cotinine- N-glucuronide ranged from 87 to 113%. Quantitative results for the three glucuronides in urine samples of 15 smokers were compared to an indirect method in which the aglycons were determined with gas chromatography and nitrogen-selective detection (GC–NPD) before and after enzymatic splitting of the conjugates. Good agreement was found for cotinine- N-glucuronide (coefficient of variation, CV: 9%) and trans-3′-hydroxycotinine- O-glucuronide (CV: 20%), whereas the accordance between both methods was moderate for nicotine- N-glucuronide (CV: 33%). The described LC–MSMS method allows the simultaneous determination of nicotine and eight of its major metabolites in urine of smokers with good precision and accuracy. Since the method requires a minimum of sample clean-up and a very short time for chromatography (3 min), it is suitable for determining the nicotine dose in large-scale human biomonitoring studies.
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