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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

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

David L Heavner, Joel D Richardson, Walter T Morgan, Michael W Ogden

Biomed Chromatogr. 2005 May;19(4):312-28.

PMID: 15651085

Abstract:

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'-hydroxycotinine-O-glucuronide. A 500 microL urine sample was pH-adjusted with phosphate buffer (1.5 mL) containing nicotine-methyl-d3, cotinine-methyl-d3 and trans-3'-hydroxycotinine-methyl-d3 internal standards. For the unconjugated metabolites, an aliquot (800 microL) of the buffered solution was applied to a 30 mg Oasis HLB-SPE column, rinsed with 2% NH4OH/H2O (3.0 mL) and H2O (3.0 mL) and eluted with methanol (500 microL). The eluate was analyzed isocratically (100% methanol) by LC-MS/MS on a diol column (50 x 2.1 mm). For the total metabolites, a beta-glucuronidase/buffer preparation (100 microL) was added to the remaining buffered solution and incubated at 37 degrees C (20 h). An aliquot (800 microL) 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	CAS Number	Price
AP110952700-B	(±)-Cotinine-(methyl-d3)	110952-70-0	Price
AP143827207	Creatinine-(methyl-d3)	143827-20-7	Price
AP202656153	(±)-Cotinine-(methyl-d3) solution	202656-15-3	Price
AP34834678	trans-3'-Hydroxycotinine solution	34834-67-8	Price

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