A new gas chromatography-mass spectrometry method for simultaneous determination of total and free trans-3'-hydroxycotinine and cotinine in the urine of subjects receiving transdermal nicotine.
trans-3'-Hydroxycotinine (THOC) has been recognized as the most abundant metabolite of nicotine. In an attempt to assess THOC and cotinine (COT) concentrations during nicotine transdermal therapy, we developed a new quantitative gas chromatography-mass spectrometry (GC-MS) method for simultaneous determination of total and free THOC and COT in human urine. The method utilizes the following: (a) hydrolysis of conjugated THOC and COT by beta-glucuronidase; (b) basic extraction of THOC and COT with mixed dichloromethane and n-butyl acetate; (c) derivatization of THOC with bis(trimethylflurosilyl)acetamide; and (d) separation and identification by GC-MS with selective ion monitoring. Lower limits of quantification for the assay were 50 and 20 microg/L for THOC and COT, respectively. The intra- and interassay CVs were 4.4% and 11% for THOC, and 3.9% and 10% for COT at 1000 microg/L. The results from six consecutive 24-h urine collections in 71 subjects administered daily transdermal nicotine doses of 11, 22, and 44 mg showed that, on average, free THOC was 76% of total THOC and free COT was 48% of total COT in all subjects. THOC is the major metabolite of nicotine and constitutes 20% of total nicotine intake at steady state, whereas urinary nicotine and COT excretion were 8% and 17%, respectively. The method is useful for simultaneous determination of free and total THOCand COT and can be used to assess the urinary excretion of these metabolites during transdermal nicotine therapy.
Ji
A J
AJ
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA. ALLENAJI@aol.com
Lawson
G M
GM
Anderson
R
R
Dale
L C
LC
Croghan
I T
IT
Hurt
R D
RD
D000279:Administration, Cutaneous; D003367:Cotinine / Q000031:analogs & derivatives* / Q000652:urine; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D009538:Nicotine / Q000008:administration & dosage / Q000378:metabolism* / Q000627:therapeutic use; D018722:Nicotinic Agonists / Q000008:administration & dosage / Q000378:metabolism* / Q000652:urine; D012680:Sensitivity and Specificity; D012907:Smoking / Q000652:urine; D016540:Smoking Cessation
D018722:Nicotinic Agonists; C001381:hydroxycotinine; D009538:Nicotine; D003367:Cotinine