in the Urine of Subjects Receiving

trans-3*-Hydroxycotinine (THOC) has been recognized as the most abundant metabolite of nicotine. In an attempt to assess THOC and cotinine (COT) concentra-tions during nicotine transdermal therapy, we devel-oped a new quantitative gas chromatography–mass spectrometry (GC–MS) method for simultaneous deter-mination of total and free THOC and COT in human urine. The method utilizes the following: (a) hydrolysis of conjugated THOC and COT by b-glucuronidase; (b) basic extraction of THOC and COT with mixed dichlo-romethane 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 mg/L for THOC and COT, respec