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

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.

Authors+Show Affiliations

R. J. Reynolds Tobacco Company, Research and Development, Winston-Salem, NC 27102, USA. heavned@rjrt.comNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Validation Study

Language

eng

PubMed ID

15651085

Citation

Heavner, David L., et al. "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." Biomedical Chromatography : BMC, vol. 19, no. 4, 2005, pp. 312-28.
Heavner DL, Richardson JD, Morgan WT, et al. 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. Biomed Chromatogr. 2005;19(4):312-28.
Heavner, D. L., Richardson, J. D., Morgan, W. T., & Ogden, M. W. (2005). 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. Biomedical Chromatography : BMC, 19(4), 312-28.
Heavner DL, et al. 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. Biomed Chromatogr. 2005;19(4):312-28. PubMed PMID: 15651085.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Heavner,David L, AU - Richardson,Joel D, AU - Morgan,Walter T, AU - Ogden,Michael W, PY - 2005/1/15/pubmed PY - 2005/8/20/medline PY - 2005/1/15/entrez SP - 312 EP - 28 JF - Biomedical chromatography : BMC JO - Biomed Chromatogr VL - 19 IS - 4 N2 - 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. SN - 0269-3879 UR - https://www.unboundmedicine.com/medline/citation/15651085/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_ L2 - https://doi.org/10.1002/bmc.463 DB - PRIME DP - Unbound Medicine ER -