Cromatogrfia

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
Allena J. Ji1,2,a, George M. Lawson1, Rodger Anderson1, Lowell C. Dale2, Ivana T. Croghan2and Richard D. Hurt2 

1 Department of Laboratory Medicine and Pathology and 
2 Nicotine Research Center, MayoClinic, Rochester, MN 55905.
a Author for correspondence. Fax 913-268-1497; e-mail ALLENAJI@aol.com.

Abstract
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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 transdermaltherapy, we developed a new quantitative gas chromatography–mass spectrometry (GC–MS) method for simultaneous determination of total andfree THOC and COT in human urine. The method utilizes the following: (a) hydrolysis of conjugated THOC and COT by ß-glucuronidase; (b) basic extraction of THOC and COT with mixed dichloromethane and n-butyl acetate; (c) derivatization of THOC withbis(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 µg/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 µg/L. The results from six consecutive 24-h urine collections in 71 subjects administered daily transdermal nicotinedoses 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 beused to assess the urinaryexcretion of these metabolites during transdermal nicotine therapy.

Introduction
TOP
ABSTRACT
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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trans-3'-Hydroxycotinine (THOC)1 was first recognized as a metabolite of nicotine in the urine of animals (1)(2) and humans (3) 38 years ago. The exact structure of THOC was identified by Dagneet al. (4). THOC was not recognized as the major urinary metabolite of nicotine until 1987 (5)(6)(7)(8) because it is too polar to be extracted from water in a routine nicotine or cotinine (COT) assay. There have been several liquid chromatographic methods for the determination of THOC in serum and urine published in recent years (6)(9)(10)(11)(12)(13). Two gas chromatography–massspectrometry (GC–MS) methods for free THOC, using derivatized or nonderivatized procedures, have been reported (14)(15). These methods did not measure conjugated forms of THOC and COT; both methods had longderivatization procedures or showed peaks of nonderivatized THOC in our GC–MS system. In the present study, we developed a new GC–MS selective ion monitoring assay for the determination of total and free THOCsimultaneously with total and free COT in urine. The assay was applied to 497 urine specimens obtained from 71 smokers admitted to a smoke-free inpatient treatment program at the Mayo Clinic. For 6 days the subjects received transdermal nicotine therapy at doses of 0 (placebo), 11, 22, or 44 mg of nicotine per 24 h. Total 24-h excretion of free nicotine and free COT were determined in a previous report(16) in an attempt to relate the total nicotine "burden" delivered by the transdermal nicotine relative to the total burden while the subject was actively smoking. Because free nicotine and COT account for only about one-fourth of the nicotine administered, we combined urinary free nicotine results, using the method developed in our laboratory (17), with urinary total and free THOC and COT...