Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography tandem mass spectrometry

An SPE-LC-MS/MS method was developed, validated and applied to the determination of nicotine and five major metabolites in human urine: cotinine, trans-3'-hydroxycotinine, nicotine-N-glucuronide, cotinine-N-glucuronide and trans-3'-hydroxycoti nine-O-glucuronicle. A 500 mu L urine sample was pH-adjusted with phosphate buffer (1.5 mL) containing nicotineinethyl-d, cotinine-methyl-d, and traiis-3'-hydroxycotinine-methyl-d(3) internal standards. For the unconjugated metabolites, an aliquot (800 mu L) of the buffered solution was applied to a 30 mg Oasis (TM) HLB-SPE column, rinsed with 2% NH(4)OH/H(2)O (3.0 m,L) and H(2)O (3.0 mL) and eluted with methanol (500 mu L). The eluate was analyzed isocratically (100% methanol) by LC-NIS/MS on a diol column (50 x 2.1 mm). For the total metabolites, a beta-glucuronidase/buffer preparation (100 mu L) was added to the remaining buffered solution and incubated at 37 degrees C (20 h). An aliquot (800 mu L) of the enzymatically treated buffered solution was extracted and analyzed in the same manner. The conjugated metabolites were determined indirectly by subtraction. The quantitation range of the method (ng/mL) was 14-10,320 for nicotine, 15-9800 for cotinine and 32-19,220 for trans-3'-hydroxycotinine. The validated method was used to observe diurnal variations from a smoker's spot urine samples, elimination half-lives from a smoker's 24 h urine samples and metabolite distribution profiles in the spot and 24 h urine samples. Copyright (c) 2005 John Wiley & Sons, Ltd.