SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

M22
SIMULTANEOUS ASSAY FOR NICOTINE AND ITS METABOLITES IN ORAL FLUID BY SPE
AND GCIMS/EI.
Insook Kim*, William D. Darwin, and Marilyn A. Huestis: Chemistry and Drug Metabolism Section, IRP,
NIDA, NIH, 5500 Nathan Shock Drive, Baltimore, MD 21224
Nicotine, the major alkaloid in tobacco, is rapidly and extensively metabolized in humans, but its
metabolism varies among individuals. Nicotine is primarily metabolized by cytochrome P450 (CYP2A6) to
cotinine, which is subsequently hydroxylated to trans-3'-hydroxycotinine. This method was designed to
simultaneous quantifY nicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine in human oral fluid, a
noninvasive biological matrix. An aliquot (0.5 mL) of each oral fluid sample, quality control (QC) sample,
or calibration standard was mixed with 2 mL of 2M sodium acetate buffer (pH 5.5). Deuterated internal
standards (d)-nicotine, d)-cotinine, and d)- trans-3'-hydroxycotinine) were added to each sample. Solid
phase extraction (SPE) columns (200 mg Clean Screen® ZSDAU020, United Chemical Technologies,
Bristol, PA) were preconditioned with elution solvent, methanol, water, and buffer. Each sample was
loaded onto the SPE column and washed with water, 0.2 N HCI, and methanol. Analytes were eluted with
methylene chloride: 2-propanol: ammonium hydroxide (80:20:2 v/v/v), and 100 J.lL of 1% hydrochloric
acid in methanol (v/v) was added prior to evaporation. Extracts were evaporated to dryness under a stream
of nitrogen at 40°C using a Zymark Turbovap LV Evaporator. Extracted residues were reconstituted in
acetonitrile, derivatized with BSTF A (with 1 % TMCS), and analyzed by GCIMSIEI in the selected ion
monitoring (SIM) mode. GCIMS analysis was performed using an HP6890 gas chromatograph interfaced
with HP5973 mass-selective detector, equipped with HP-5MS column (30m x 0.25mm Ld.; 0.25J.1m-fiIm
thickness) with helium gas at 1.0 mLlmin. The instrument was operated in the splitless mode. The initial
column temperature of 70°C was held for 1 min, followed by increases to 190°C at 30°C/min, to 230°C at
5°C/min, to 290°C at 25°C/min. The ions for each analyte were monitored in the following elution order
(quantitative ions are indicated in parenthesis) for the derivatized analytes: d)-nicotine, mlz (87), 165;
nicotine, m/z (84), 162; d)-cotinine, mlz (101), 179; cotinine, m/z (98), 176; norcotinine, m/z (234), 219;
d 3
-trans-3'-hydroxycotinine, mlz (252), 147; and trans-3'-hydroxycotinine, mlz (249), 144. The base peak
and molecular ions of each analyte and internal standards were evaluated by SIM analysis. However, the EI
spectra of these drugs do not possess many fragments that can be used as confirming ions. The mid-mass
range ions for these drugs contain non-specific interferences probably from co-extracted endogenous
material. For this reason, only two ions, the base peak and the molecular ion, were monitored in this assay.
Eight point calibration curves for nicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine were linear
across a concentration range of 2.5 to 500 ng for all compounds/0.5 mL of blank oral fluid. Correlation
coefficients of the calibration curves were >0.99. The limits of determination and quantitation are 2.5
ng/0.5 mL (50 pg on column) for all analytes. Recovery was 90 - Il5% for nicotine, 76 - Il7% for
cotinine, 88 -101% for norcotinine, and 66 -77% for trans-3'-hydroxycotinine at 8, 80, and 400 ng/O.5 mL
(low, mid, and high, respectively), QC sample concentrations. Intra-assay precision (% CV) and accuracy
(percent difference between mean and target concentrations) ranged from 1.6 5.7 and 1.6 - 17.8%,
respectively, at low, mid, and high QC sample concentrations for all analytes. Inter-assay precision and
accuracy for low, mid, and high QC sample concentrations ranged from 4.4 - 8.8 and 0.2 - 12.6%,
respectively, for all analytes. Suitable precision and accuracy were achieved for the simultaneous
determination of nicotine and three metabolites in the oral fluid of smokers. This assay is applicable to
pharmacokinetic studies of nicotine and its metabolites in oral fluid and provides a biomarker for
identification of smoking, which could be helpful in smoking cessation programs.
Keywords: Nicotine, Nicotine Metabolites, Oral Fluid
Page 305