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 me tabolite of nicotine. In an attempt to assess THOC and cotinine (COT) conce ntrations during nicotine transdermal therapy, we developed a new quantitat ive gas chromatography-mass spectrometry (GC-MS) method for simultaneous de termination of total and free THOC and COT in human urine. The method utili zes the following: (a) hydrolysis of conjugated THOC and COT by beta-glucur onidase; (b) basic extraction of THOC and COT with mixed dichloromethane an d n-butyl acetate; (c) derivatization of THOC with bis(trimethylflurosilyl) acetamide; and (d) separation and identification by GC-MS with selective io n monitoring. Lower limits of quantification for the assay were 50 and 20 m u g/L for THOC and GOT, respectively. The intra- and interassay CVs were 4. 4% and 11% for THOC, and 3.9% and 10% for COT at 1000 mu g/L. The results f rom six consecutive 24-h urine collections in 71 subjects administered dail y 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 su bjects. THOC is the major metabolite of nicotine and constitutes 20% of tot al nicotine intake at steady state, whereas urinary nicotine and COT excret ion were 8% and 17%, respectively. The method is useful for simultaneous de termination of free and total THOC and COT and can be used to assess the ur inary excretion of these metabolites during transdermal nicotine therapy.