Validation of a Liquid Chromatography Tandem Mass Spectrometry Method for the Detection of Nicotine Biomarkers in Hair and an Evaluation of Wash Procedures for Removal of Environmental Nicotine Eleanor I. Miller 1, *, Gordon J. Murray 1, Douglas E. Rollins 1, Stephen T. Tiffany 2, and Diana G. Wilkins 1 1 Center for Human Toxicology, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah and 2 University at Buffalo SUNY, Department of Psychology, 228 Park Hall, Buffalo, New York 14260 Abstract The aim of this exploratory study was to develop and validate a liquid chromatography tandem mass spectrometry (LC MS MS) method for the quantification of nicotine, eight nicotine metabolites, and two minor tobacco alkaloids in fortified analytefree hair and subsequently apply this method to hair samples collected from active smokers. An additional aim of the study was to include an evaluation of different wash procedures for the effective removal of environmentally deposited nicotine from tobacco smoke. An apparatus was designed for the purpose of exposing analyte-free hair to environmental tobacco smoke in order to deposit nicotine onto the hair surface. A shampoo/water wash procedure was identified as the most effective means of removing nicotine. This wash procedure was utilized for a comparison of washed and unwashed heavy smoker hair samples. Analytes and corresponding deuterated internal standards were extracted using a cation-exchange solid-phase cartridge. LC MS MS was carried out using an Acquity UPLC system (Waters) and a Quattro Premier XE triple quadrupole MS (Waters) operated in electrospray positive ionization mode, with multiple reaction monitoring data acquisition. The developed method was applied to hair samples collected from heavy smokers (n = 3) and low-level smokers (n = 3) collected through IRB-approved protocols. Nicotine, cotinine, and nornicotine were quantified in both the washed and unwashed hair samples collected from three heavy smokers, whereas 3-hydroxycotinine was quantified in only one unwashed sample and nicotine-1'-oxide in the washed and unwashed hair samples from two heavy smokers. In contrast, nicotine-1'-oxide was quantified in one of the three low-level smoker samples; nicotine was quantified in the other two low-level smoker samples. No other analytes were detected in the hair of the three low-level smokers. * Author to whom correspondence should be addressed. Center for Human Toxicology, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 84108. Email: ei_miller2003@yahoo.co.uk. Introduction Hair testing for biomarkers of tobacco exposure may complement conventional blood or urine testing and provide an alternative means of assessing nicotine and metabolite disposition into the body. It may provide integrative information on nicotine exposure through active smoking, or passive exposure to environmental tobacco smoke (ETS), over extended time periods. Segmental hair analysis may also assist in the interpretation of prior nicotine use. The longer drug detection window offered by hair testing has led to its application in, for example, forensic cases (1,2), workplace testing (3,4), prenatal drug exposure assessment (5,6), drug compliance and abstinence in treatment programs (7,8), and in sports testing (9,10). Additional advantages of hair testing include a non-invasive means of easily supervised sample collection, a reduced risk of sample adulteration, easy sample storage and transportation, reduced risk of exposure to biohazards, and drug stability over long time periods. Segmental hair analysis for nicotine biomarkers enables the determination of drug exposure along the length of the hair shaft and can be used to more accurately assess time of drug exposure. The segmental analysis of hair for nicotine biomarkers could be used to document changes in smoking behavior, to monitor compliance on smoking cessation programs, and to validate self-reports (11). Although some researchers have advocated the use of nicotine hair analysis for monitoring prior exposure, there are controversies over the use of nicotine as an accurate biomarker of tobacco use because it is present in ETS and passive exposure to tobacco smoke can produce positive hair nicotine results (11). Various nicotine cutoff concentrations have been proposed to distinguish smokers and non-smokers; however, none can be applied universally. Previously, one group proposed a cutoff of > 2 ng nicotine/mg hair (12); however, some reported nicotine concentrations for nonsmokers were above this cutoff concentration and also demonstrated an overlap in Reproduction (photocopying) of editorial content of this journal is prohibited without publisher s permission. 321
the hair of infants who were exposed to ETS and parental smoke (13). Another study reported a positive correlation between nicotine concentrations in hair segments and self-reported smoking behavior with and without the use of nicotine gum in a smoking cessation program, and proposed a higher baseline hair nicotine concentration of 5 ng/mg in order to distinguish active smokers from nonsmokers (14). A later study by the same group evaluated nicotine concentrations in hair to assess clinical outcomes of smoking-cessation trials including the use of nicotine gum (15). A pattern of decreased nicotine concentration along 1-cm hair segments was observed in study participants who reported a daily decrease in number of smoked cigarettes and consumed chewing gum. However, the hair nicotine concentrations in the proximal-to-scalp segments in successful smoking cessation subjects were higher than their earlier proposed cutoff of 5 ng/mg, and the authors suggested this might have resulted from a slow dissociation of nicotine from the hair follicle. As a result, the ambiguity with respect to interpretation of hair nicotine concentrations alone, as an accurate marker of tobacco exposure, has been highlighted in these studies, and therefore, it is important to investigate the use of metabolites as an alternative. Nicotine concentrations are higher than cotinine hair concentrations in washed hair from smokers and washed hair collected from nicotine-abstinent individuals exposed to ETS (16). Although cotinine was determined in a greater number of smoker hair samples compared to nonsmoker hair samples, the cotinine concentrations in both populations have been found to overlap (12,13,16). Furthermore, cotinine hair concentrations have been reported to be more than 10 times lower than nicotine hair concentrations (11,17) requiring extremely sensitive detection, particularly in ETS exposure situations (18). The finding that cotinine can be systemically incorporated into hair via ETS exposure suggests that cotinine cannot be used as a marker of tobacco use (19). Therefore the analysis of other nicotine biomarkers and minor tobacco alkaloids in hair may provide additional useful data for interpretation of smoking behavior and also may provide more useful cutoff concentration values to distinguish heavy smokers, low-level smokers, and potentially nonsmokers exposed to ETS and those with no ETS exposure. Drugs can also incorporate into hair by diffusion from sweat and sebum; bathing the growing or mature hair fiber; and by diffusion of external contamination from drugs in smoke or from the environment, such as nicotine, into the mature hair fiber. Previous studies have reported the application of hair wash procedures in an attempt to remove externally deposited hair contamination and to prevent the production of falsepositive results (20,21). However, none of the wash procedures tested were 100% effective. The potential influx and efflux of externally deposited smoked drugs such as nicotine and internally bound drugs and metabolites through the application of wash procedures, has still not been established. Consequently, it is essential to identify unique drug metabolites in hair as evidence of active drug use. Several mass spectrometric (MS) methods have been described as distinguishing hair nicotine and cotinine concentrations in smokers and nonsmokers as biomarkers of active smoking and exposure to ETS (12,13,22,23). However, although there are studies that implement hair nicotine and cotinine concentrations as a means of corroborating self-reported smoking, there are currently no studies that report the application of a sensitive analytical methodology specifically for the purpose of validating self-reported measures of heavy or low-level smoking behaviors. Heavy smokers are generally considered to smoke at least one pack of cigarettes per day, however; low-level smokers neither smoke daily nor consume one pack of cigarettes on days on which they do smoke (24). Because of the inhalation of tobacco smoke, both active heavy and low-level smokers, as well as passive smokers, have increased health risks including increased rates of coronary heart disease, myocardial infarction, and lung cancer compared to nonsmokers (25). It is particularly difficult to utilize conventional biomarkers of tobacco smoke or nicotine exposure (e.g., carbon monoxide levels in expired air, nicotine and metabolite concentrations in plasma or saliva) to assess cigarette exposure in low-level smokers, therefore; segmental hair analysis may provide a useful means of assessing this type of smoking behavior. In light of recent proposed U.S. government legislation regarding the provision of health insurance discounts for lowincome individuals or families based on their smoking status, segmental analysis of hair may provide an accurate record of tobacco exposure over the months prior to sample collection. At the time of writing, there are no prospective studies examining the determination of changes in an individual over time, using hair as a biomarker. There are many papers which relate nicotine and cotinine concentrations in serum, urine, and saliva to daily smoking behavior in terms of cigarettes per day (cpd) and a general relationship between cotinine concentrations and cpd in these matrices has routinely been found, although there is some deviation from linearity at the higher smoking frequencies. The window of detection offered through hair analysis is substantially increased compared to these other matrices. This paper is one in a series related to the development and validation of liquid chromatography tandem mass spectrometry (LC MS MS) methods for application in the determination of nicotine biomarkers in plasma, oral fluid, and hair (26,27) following controlled transdermal nicotine delivery and also, ultimately, potential correlation between self-report and low-level smoking through hair analysis. The aim of this exploratory hair study was to develop and validate an LC MS MS method for the quantification of nicotine, eight nicotine metabolites, and two minor tobacco alkaloids in fortified analyte-free hair and subsequently apply this method to hair samples collected from low-level and heavy active smokers. An additional aim of the study was to include an evaluation of different wash procedures for the effective removal of environmentally deposited nicotine from tobacco smoke. Experimental Hair for method development The analyte-free human hair used in the LC MS MS analysis 322
and in the evaluation of wash procedures was obtained from laboratory volunteers within the Center for Human Toxicology at the University of Utah. The hair was determined to be negative for all analytes [below the limit of detection (< LOD)] prior to using it in the preparation of calibrators and QCs. Biological samples for clinical study Clinical hair samples were collected as part of an Institutional Review Board approved study (IRB # PSY0171007A, University at Buffalo SUNY) investigating potential nicotine biomarkers in low-level and heavy smokers. Approximately a pencil thickness of hair was collected from the posterior vertex scalp region of each individual. Samples were wrapped in aluminum foil (which had the root end marked) and stored in the hair collection envelopes (AgriYork, York, U.K.) at room temperature. The scissors were cleaned between participants, using an alcohol swab, and then dried with a paper towel before collecting the sample as close to the scalp as possible. Both groups were comprised of individuals who had naturally colored hair that was at least 1.25-in. long. Smoking-related inclusion criteria for the recruitment of low-level smokers (n = 3) were individuals who had smoked at least 1 cigarette per smoking occasion on 2 29 days over the past 30 days and had smoked at least 25 cigarettes during their lifetime. Heavy smoker recruitment criteria (n = 3) included individuals who smoked at least 20 cigarettes per day, smoked at this rate for at least the last year, and had an expired carbon monoxide level of 15 ppm. Additional criteria for both smoker groups were that participants had made no attempt to quit or reduce smoking over the previous month; had no intention to quit or reduce smoking over the next 2 months; had used only cigarettes as a tobacco source over the past 12 months; and had limited ETS exposure (apart from their own tobacco smoke). The heavy smoker hair strands in the bundle were carefully aligned, and the top 1 cm of hair (proximal segment) was subsequently tied off with string and then cut from the bundle. The proximal 1-cm segment was then cut into 2 3-mm segments using sharp scissors, mixed, and weighed into separate aliquots for the comparison of analyte concentrations detected in washed and unwashed hair samples. Conventionally, a proximal 1-cm segment is considered to reflect approximately one month s tobacco exposure, assuming a typical hair growth rate for scalp hair of 1 cm/month (28). Anatabine and anabasine were important as analytes in the developed method as potential hair biomarkers of tobacco exposure because these compounds are minor tobacco alkaloids that are present in tobacco and should not be present in nicotine-containing medications or therapies; hence, these compounds may be good indicators of unauthorized tobacco use for individuals undergoing treatment programs for addiction. Three low-level smoker hair samples were also similarly prepared and analyzed; however, because of a limited original sample weight, these were not able to be separated into two separate aliquots for comparison of washed and unwashed results. These low-level samples were prepared as 2-mm segments to represent one month s tobacco exposure because we hypothesized that there may be an increased chance of detecting extremely low concentrations in shorter segment lengths, compared to the possibility of perhaps diluting the result with a potentially larger analyte-free hair forming part of a larger segment size. Reference standards, chemicals, and reagents Reference standards and deuterated internal standards were obtained from Toronto Research Chemicals (North York, ON, Canada). These included cotinine N-β-D-glucuronide (COT GLUC) and cotinine-d 3 N-β-D-glucuronide (COT GLUC-d 3 ); nicotine-n-(4-deoxy-4,5-didehydro)-β-d-glucuronide (NIC GLUC) and nicotine-n-(4-deoxy-4,5-didehydro)-β-dglucuronide-methyl-d 3 (NIC GLUC-d 3 ); (S)-cotinine N-oxide (CNO) and (R,S)-cotinine-N-oxide-methyl-d 3 (CNO-d 3 ); trans- 3'-hydroxycotinine (3HC) and trans-3'-hydroxycotinine-d 3 (3HC-d 3 ); (R,S)-norcotinine (NCOT) and (R,S)-norcotinine pyridyl-d 4 (NCOT-d 4 ); (1'S, 2'S)-nicotine 1'-oxide and (1'R, 2'S)-nicotine-1'-oxide mixture (NNO) and (±)-trans nicotine- 1'-oxide-methyl-d 3 (NNO-d 3 ); (R,S)-nornicotine (NNIC) and (R,S)-nornicotine-d 4 (NNIC-d 4 ); (R,S)-anatabine (AT) and (R,S)- anatabine-2,4,5,6-d 4 (AT-d 4 ); (R,S)-anabasine (AB) and (R,S)-anabasine-2,4,5,6-d 4 (AB-d 4 ). ( )-Nicotine (NIC) hydrogen tartrate salt ( 98%) was obtained from Sigma (St Louis, MO). ( )-Cotinine (COT), (±)-cotinine-d 3 (COT-d 3 ), and (±)-nicotine-d 3 (NIC-d 3 ) were obtained from Cerilliant (Austin, TX). Solid-phase extraction (SPE) cartridges (Oasis MCX (60 mg, 3 ml) were obtained from Waters (Milford, MA). LC grade methanol, dichloromethane, and isopropanol were obtained from Honeywell Burdick & Jackson (Morristown, NJ). Ammonium acetate and glacial acetic acid were obtained from Spectrum (Gardena, CA). Concentrated formic acid, concentrated hydrochloric acid, and concentrated ammonium hydroxide were obtained from Fisher Scientific (Pittsburgh, PA). Deionized water used in the preparation of reagents, and LC mobile phase buffer was drawn from a Milli-Q filter apparatus (Millipore, Bedford, MA). All chemicals and reagents were HPLC grade ( 99% purity) and were used without further purification. Calibrator and quality control solutions Calibrators and QCs for LC MS MS were prepared daily and extracted with each analytical batch. Four calibrator working solutions, containing all analytes, were prepared in methanol at concentrations of 10, 1, 0.1, and 0.01 µg/ml. The nicotine hydrogen tartrate salt was weight corrected for nicotine at these concentrations (free base equivalents). The NIC hydrogen tartrate salt form was selected as it was determined to be more stable than nicotine free base during initial stability assessments. Separate methanolic working solutions were prepared for QC samples at the same concentrations as the calibrator working solutions. Because of the unavailability of different compound lot numbers for all analytes, the same lot numbers were used to prepare both calibrator and QC working solutions; however, they were prepared by two separate analysts from purified reference material. All working solutions were stored in the freezer at 20 C. Table I provides the calibrator and QC concentration selected for each analyte. A deuterated internal standard working solution was also prepared in methanol at 1 µg/ml and contained COT GLUC-d 3, NIC GLUC-d 3, CNO-d 3, 323
3HC-d 3, NCOT-d 4, NNO-d 3, NNIC-d 4, NIC-d 3, COT-d 3, AT-d 4, and AB-d 4. The working deuterated internal standard solution was also stored in the freezer at 20 C. Methods Sample preparation and extraction Twenty-five microliters of 1 µg/ml deuterated internal standard solution was fortified to 20 ± 0.1 mg analyte-free hair aliquots, to produce a final concentration of 1.25 ng/mg. The specific calibrator and QC concentrations used for heavy and low-level smokers are provided in Table I. A Mettler-Toledo AG104 balance was used to weigh out the hair samples. Next, 1 ml of 1 M aqueous sodium hydroxide solution was added to each test tube and the tubes vortex mixed briefly. The tubes were then placed on a mechanical shaker for 1 h at room temperature. One-hundred microliters of concentrated hydrochloric acid was then added to each test tube, and the tubes centrifuged at 2500 rpm for 5 min. Oasis MCX cartridges (3 cc, 60 mg) were conditioned with 2 ml methanol followed by 2 ml 2% aqueous formic acid, and the acidified supernatants were then loaded onto the cartridges. The cartridges were washed with 1 ml 2% aqueous formic acid and 1 ml methanol. Analytes were eluted with 1.5 ml 5% v/v aqueous ammonium hydroxide in methanol and 1.5 ml dichloromethane/isopropanol/ aqueous ammonium hydroxide (78:20:2, v/v). These eluants were combined, and then 100 µl of 1% hydrochloric acid in methanol was added to the eluants before evaporation at 40 C. The extracts were reconstituted with 130 µl of 10 mm ammonium acetate + 0.001% formic acid/ methanol (85:15, v/v). LC MS MS conditions LC MS MS analysis was conducted using an Acquity UPLC system (Waters, Milford, MA) coupled to a Quattro Premier XE triplequadrupole MS (Waters) with MassLynx v 4.1 software. A Discovery HS F5 HPLC column (100 mm 4.6 mm, 3 µm, Supelco, Bellefonte, PA) was used for chromatographic separation, with a gradient system consisting of 10 mm ammonium acetate with 0.001% formic acid (ph 4.97), and methanol at a flow rate of 0.6 ml/min. Initial mobile phase conditions were 15% B increased linearly to 76% after 11 min, then decreased back to the initial mobile phase condition of 15% B after 11.6 min, and finally held for 3.4 min to re-equilibrate the LC column (total chromatographic run time was 15 min). The MS was operated in electrospray positive ionization mode and acquired multiple reaction monitoring (MRM) data for two MRM transitions per analyte, with the exception of COT GLUC (which produced only one major fragment ion). 324 The ESI conditions were capillary voltage 3.25 kv; source temperature 100 C; and a desolvation temperature of 350 C. The analyte-specific cone voltages, collision energies, and MRM transitions were the same as those previously published in our plasma, urine and oral fluid studies for the same analyte panel (26,27). Quantification criteria of positive analytes included 1. selected MRM transitions had a S/N ratio of at least 10; 2. the peak-area ratio of analyte quantification ion to deuterated internal standard quantification ion for the samples were within ±20% of the corresponding calibrator; and 3. the analyte quantification ion-to-analyte qualification ion ratio were within ±20% of the positive quality control for analytes which had more than one fragment ion with 20% abundance. Evaluation of wash procedures An apparatus was constructed in which an analyte-free bundle of hair was exposed to tobacco smoke, in order to deposit nicotine onto the surface of the hair thus simulating deposition of nicotine via environmental tobacco smoke (Figure 1). The hair bundle was tied securely at both ends using string Table I. Assay Detection and Quantification Limits, Calibrator, and QC Concentrations Calibrator Quality Control Concentrations (ng/mg) LOD LOQ Range Analyte (ng/mg) (ng/mg) (ng/mg) Low Medium High NIC GLUC 0.03 0.05 0.05 2.5 0.08 0.40 1.25 CNO 0.03 0.10 0.10 2.5 0.13 0.40 1.25 3-HC 0.03 0.10 0.10 2.5 0.13 0.40 1.25 NCOT 0.05 0.10 0.10 2.5 0.13 0.40 1.25 NNO 0.01 0.05 0.05 2.5 0.08 0.40 1.25 COT 0.03 0.05 0.05 2.5 0.08 0.40 1.25 NNIC 0.03 0.05 0.05 2.5 0.08 0.40 1.25 NIC 0.05 0.05 0.05 2.5 0.08 0.40 1.25 AT 0.05 0.05 0.05 2.5 0.08 0.40 1.25 AB 0.10 0.10 0.10 2.5 0.13 0.40 1.25 COT GLUC 0.10 0.10 0.10 2.5 0.13 0.40 1.25 Figure 1. Apparatus designed to deposit tobacco smoke components onto the surface of analyte-free hair.
and placed inside a glass condenser. A Marlboro Red cigarette was attached to tubing that was attached to the condenser containing the hair bundle. Marlboro Red cigarettes were chosen because they are one of the most preferred brands in the U.S. among adolescent smokers, young adults, and older Table II. Analyte Concentrations in Pooled Unwashed Smokers Hair for Incubation with 1 M Sodium Hydroxide at Different Time Intervals Incubation Time Concentration (ng/mg) ± SD Analyte (h) for n = 3 Hair Aliquots COT 1 0.08 ± 0.00 2 0.08 ± 0.01 4 0.08 ± 0.01 6 0.08 ± 0.00 24 0.09 ± 0.01 NNO 1 0.05 ± 0.02 2 0.04 ± 0.00 4 0.04 ± 0.00 6 0.03 ± 0.00 24 0.04 ± 0.01 NIC 1 0.71 ± 0.09 2 0.73 ± 0.13 4 0.73 ± 0.13 6 0.75 ± 0.03 24 0.87 ± 0.09 Table III. Positive Analyte Concentrations in Unwashed and Washed Analyte-Free Hair Exposed to Cigarette Smoke Unwashed Hair Concentration Washed Hair Concentration (ng/mg, n = 3) Wash (ng/mg, n = 3) Analyte 1 Cigarette 4 Cigarettes Procedure 1 Cigarette 4 Cigarettes NNO < LOQ 0.11 ± 0.00 A negative negative B negative negative C negative 0.06 ± 0.01 D negative negative E negative < LOQ COT < LOQ 0.10 ± 0.00 A negative negative B negative negative C negative < LOQ D negative negative E negative negative NNIC 0.07 ± 0.00 0.21 ± 0.01 A negative negative B negative negative C negative 0.06 ± 0.01 D negative negative E negative negative NIC 1.12 ± 0.05 8.89 ± 0.33 A < LOQ 0.38 ± 0.01 B 0.10 ± 0.00 0.80 ± 0.01 C 0.55 ± 0.07 5.57 ± 0.34 D < LOQ 0.36 ± 0.02 E 0.13 ± 0.05 1.10 ± 0.07 adult smokers (29). The cigarette was then lit, and a vacuum was applied in order to draw the tobacco smoke over the hair bundle. The tubing on the outlet of the condenser was attached to a Dreschel bottle containing a 10 ml scrub solution of 1:1 (v/v) acetone/dichloromethane in order to trap the tobacco smoke that had passed over the hair bundle. Twenty (± 0.2) milligrams of hair (n = 3) was weighed out after exposing the hair bundle to one Marlboro Red cigarette and then three consecutive Marlboro Red cigarettes (tobacco smoke from four cigarettes total). The hair bundle was separated into two halves, by cutting, after exposure to one cigarette and returned to the condenser for exposure to an additional three cigarettes. Upon completion of exposure to smoke, the hair bundle halves were finely cut into 2 3-mm segments and mixed to produce a homogenous sample. The ETSexposed hair aliquots (n = 3) were each subjected to five separate wash procedures to determine if externally deposited nicotine from the tobacco smoke could be effectively removed. Unwashed hair aliquots (n = 3) were also analyzed for comparison with washed hair aliquots. Each wash condition utilized 1 ml of reagent along with ultrasonication at room temperature. The following washes were evaluated: (A) 1 5 min wash with 1:4 (v/v) Herbal Essences Hello Hydration shampoo/milli-q water, followed by 2 5 min Milli-Q water washes; (B) 1 5 min wash with 0.1% aqueous sodium dodecyl sulfate, followed by 1 5 min Milli-Q water wash; (C) 2 10 s dichloromethane washes by vortex mixing; (D) 1 10 min wash with isopropanol, followed by 1 10 min wash with 0.1 M phosphate buffer (ph 6.0); and (E) 1 5 min wash with methanol. Herbal Essences Hello Hydration was selected because it is a readily available and commonly used brand of shampoo in the U.S. Hair wash procedures B and D were slight modifications of previously reported wash procedures (14,24), and wash procedure C was replicated from a previous publication (18). After the washed hair aliquots dried overnight at room temperature, they were extracted and analyzed as described in the Sample preparation and extraction section. Hair incubation optimization To optimize the incubation conditions for digestion of hair, 20 ± 0.2 mg unwashed, pooled, smokers hair (collected from four individuals) was weighed out and incubated in 1 M aqueous sodium hydroxide solution for 1, 2, 4, 6, and 24 h at room temperature with mechanical shaking to determine whether or not the concentration of incorporated nicotine metabolites extracted from smokers hair was dependent on incubation time. The samples were extracted according to the SPE procedure detailed in the Sample preparation and extraction section and 325
analyzed using the calibration ranges reported in Table I. The data were analyzed via a one-way ANOVA and a post-hoc Bonferroni test (p < 0.05). Method validation Linearity was evaluated for each analyte over the concentration range specified in Table I using a simple linear regression data fit and calculation of the coefficient of determination (R 2 ). Calibration curves were generated from peak-area ratios of the quantification ion of target analytes and the quantification ion of the corresponding deuterated internal standards. The mean gradient, intercept, and R 2 values (n = 3) for each analyte are shown in Table I. Calibrator and QC concentrations were calculated using the calibration curve and passed the acceptance criteria if they were found to be within 20% of the theoretical target concentration. The sensitivity of the method was assessed by determining the LOD and the limit of quantification (LOQ). These were calculated relative to peak height, and also in the case of the LOQ, accuracy and imprecision (within 20% of theoretical target concentration) on three separate days. The LOD was defined as the analyte concentration that produced a signal-tonoise ratio (S/N) of 3 for selected MRM transitions. The LOQ was defined as the lowest calibrator concentration standard that produced an S/N ratio of 10 for the selected MRM transitions with acceptable precision and accuracy (within ± 20% of theoretical target concentration). The LOD and LOQ parameters were determined empirically as the concentration obtained for a series of decreasing concentrations of analyte fortified in analyte-free human hair, and subsequently extracted and analyzed. Method specificity was assessed by the analysis of six analytefree human hair samples from different donors. Each hair sample was extracted and analyzed to determine if any potential interference from endogenous hair components was present. The hair samples that were evaluated for specificity were not entirely representative of the hair samples collected from the study subjects, and ideally, a wider range of hair colors, races, and also cosmetically treated hair should be analyzed and evaluated for potential endogenous interferences and matrix effects. Total extraction recovery was calculated for each analyte at the low, medium, and high QC concentration (n = 5). Specifically total extraction recovery is defined here as the % of ana- Table IV. Assay Imprecision, Total Extraction Recovery, and Matrix Effect Intraassay Imprecision (n = 5) ± Interassay Imprecision (n = 20) Matrix Effect (%) Standard Deviation ng/mg Standard Deviation ng/mg Recovery (%) (%RSD) (n = 5) (%RSD) (n = 5) Analyte Low Medium High Low Medium High Low Medium High Low High NIC GLUC 0.07 ± 0.00 0.37 ± 0.01 1.04 ± 0.04 0.07 ± 0.00 0.44 ± 0.05 1.19 ± 0.15 89.5 85.2 87.4 109.4 112.6 (5.8) (8.3) (2.4) (6.3) (2.6) CNO 0.12 ± 0.00 0.35 ± 0.02 1.18 ± 0.01 0.11 ± 0.01 0.36 ± 0.05 1.17 ± 0.04 98.2 78.1 86.3 108.4 108.0 (3.1) (4.0) (4.4) (5.0) (3.1) 3HC 0.11 ± 0.00 0.39 ± 0.03 1.07 ± 0.04 0.11 ± 0.01 0.37 ± 0.03 1.08 ± 0.05 77.9 62.9 79.7 116.0 118.7 (6.7) (7.9) (3.6) (7.6) (7.2) NCOT 0.11 ± 0.01 0.40 ± 0.04 1.23 ± 0.06 0.12 ± 0.01 0.39 ± 0.04 1.22 ± 0.04 87.4 63.4 80.7 113.5 115.6 (6.6) (3.7) (2.3) (3.7) (4.0) NNO 0.07 ± 0.00 0.37 ± 0.02 1.07 ± 0.02 0.07 ± 0.00 0.38 ± 0.04 1.09 ± 0.05 85.4 75.3 85.5 99.3 88.7 (2.8) (3.0) (3.6) (8.1) (2.7) COT 0.07 ± 0.01 0.40 ± 0.03 1.11 ± 0.05 0.07 ± 0.01 0.39 ± 0.05 1.10 ± 0.04 89.3 74.1 88.3 115.9 103.8 (5.3) (2.9) (3.0) (3.1) (1.9) NNIC 0.07 ± 0.00 0.37 ± 0.05 1.26 ± 0.03 0.07 ± 0.01 0.39 ± 0.05 1.23 ± 0.04 85.5 74.4 84.8 115.7 107.3 (4.9) (1.6) (4.3) (1.8) (3.3) NIC 0.07 ± 0.01 0.40 ± 0.06 1.45 ± 0.04 0.07 ± 0.01 0.39 ± 0.05 1.20 ± 0.17 117.8 101.8 93.7 120.1 109.5 (7.5) (4.1) (2.0) (22.8) (5.3) AT 0.07 ± 0.00 0.33 ± 0.01 1.10 ± 0.01 0.07 ± 0.01 0.37 ± 0.05 1.09 ± 0.04 93.6 92.4 81.6 112.1 101.3 (6.7) (11.4) (4.2) (6.1) (3.2) AB 0.11 ± 0.01 0.38 ± 0.05 1.33 ± 0.04 0.12 ± 0.01 0.39 ± 0.05 1.33 ± 0.05 97.0 85.8 90.1 117.1 110.2 (2.5) (2.7) (2.7) (4.5) (5.9) COT GLUC* 0.12 ± 0.00 0.37 ± 0.03 1.45 ± 0.05 0.13 ± 0.01 0.35 ± 0.02 1.41 ± 0.10 14.7 13.5 23.1 118.9 117.6 (13.4) (19.8) (11.6) (5.9) (3.9) * COT GLUC interassay imprecision was for n = 15. 326
lyte recovered following fortification, incubation, and extraction. Analyte-free hair was fortified with analyte before SPE, and unextracted samples were prepared at identical analyte concentrations. Deuterated internal standard solution was added to the SPE eluant before evaporation and to the unextracted samples. Total extraction recovery (%) was calculated from the comparison of the average analyte peak-area ratio of extracted standards with the average analyte peak-area ratio of unextracted standards. LC MS MS matrix effects were calculated by the comparison of the analyte peak-area ratio for extracted analyte-free hair samples from five individuals fortified with analyte and internal standard before extraction with the analyte peak-area ratio for five unextracted standards prepared in initial mobile phase composition at the same concentration. Matrix effects were calculated as a percentage of the mean peak-area ratio of the unextracted samples at low and high QC concentrations. Autosampler stability was assessed for reconstituted hair extracts (n = 4), fortified at low and high QC concentrations, which were stored for 72 h at 4 C. These QCs had been analyzed and met our acceptance criteria. They were re-analyzed 72 h later, along with freshly prepared QCs fortified at the same concentrations. Intra- and interassay imprecision and accuracy was calculated at low, medium, and high QC concentrations (Table II). The intraassay imprecision was calculated from the concentration variability of the replicate analysis of QCs (n = 5) within an analytical batch. The interassay imprecision was calculated from the concentration variability of a total of 20 QC samples analyzed in four separate analytical batches. The imprecision is expressed as a standard deviation. Results Hair incubation optimization There was no significant difference between incubation times for cotinine and nicotine-1'-oxide; however, there was significantly higher nicotine determined after 24 h incubation compared to the other incubation times (Table III). However, since the determination of unique nicotine metabolites in hair may be a more useful means of determining tobacco smoke exposure than the determination of nicotine itself due to problems of external deposition of nicotine on the surface of the hair shaft, a 1 h incubation time with sodium hydroxide was selected for a faster but comparably efficient extraction of metabolites relative to other incubation times. trans-3-hydroxycotinine and nornicotine were detected in all three hair aliquots at each incubation time, although the concentrations were not quantifiable (< LOQ of 0.03 ng/mg). Depending on the analyte to be determined, the extraction kinetics may be very different for washed hair compared to unwashed hair. The incubation optimization experiments were initially conducted on unwashed hair because the focus of the experiment was to investigate primarily metabolite concentrations in smokers hair. In addition, experiments conducted after the incubation experiments have determined that washing one of the heavy smoker samples resulted in loss of COT to below the LOQ, whereas the unwashed hair has a quantifiable amount. Decreases in 3HC, NNO, and NNIC were also observed for washed hair compared to unwashed hair. Furthermore, COT was not detected in any of the unwashed low-level smoker samples. NNO was quantified in one sample and only detected in one sample. For this reason, these data suggest that it would Table V. Heavy Smoker and Low-Level Smoker Hair Concentrations Sample ID Analyte Concentration (ng/mg) Average Segment Weight cpd Length (mg) 3-HC COT NNO NNIC NIC AT Heavy Smoker 1 30 1 cm 48.1 < LOQ < LOQ < LOQ 0.05 1.18 negative Washed Heavy Smoker 1 30 1 cm 47.5 < LOQ 0.06 < LOQ 0.07 1.78 negative Unwashed Heavy Smoker 2 22 1 cm 11.7 < LOQ 0.19 0.44 0.24 9.83 negative Washed Heavy Smoker 2 22 1 cm 11.7 < LOQ 0.25 0.59 0.26 12.65 negative Unwashed Heavy Smoker 3 25 1 cm 14.9 < LOQ 1.52 0.46 0.33 17.88 0.08 Washed Heavy Smoker 3 25 1 cm 15.0 0.17 2.46 0.98 0.40 19.09 0.10 Unwashed Low Level 2 5 2 mm 1.4 1.9 negative negative 0.35 negative negative negative Smoker 1 Low Level 0 5 2 mm 10.4 11.4 negative negative < LOQ negative 0.43 negative Smoker 2 327
be most useful to analyze and compare measured concentrations of nicotine and metabolites in both unwashed and washed hair, when sufficient hair is available, in order to ensure the Figure 2A. Positive analytes for heavy smoker sample (chromatograms correspond to analytes > LOQ for heavy smoker sample). 328 collection of complete metabolite information and enhance interpretation of hair data. Method validation Calibration curves were determined to be linear for each analyte over the selected concentration range (Table I); each graph had a coefficient of determination (R 2 ) values > 0.99. Weighted 1/x and nonweighted linear regression fits were evaluated with no significant difference in calculated concentration (paired t-test); therefore, non-weighted fits were selected for routine use. The LOD and LOQ values (Table I) were derived from the criteria stated in the Methods section. Based on a 20-mg hair weight, five of the analytes were quantifiable at 0.1 ng/mg and seven analytes at 0.05 ng/mg. In the method specificity evaluation, there was no signal for selected MRM transitions for any of the analytes at their established analyte retention times; therefore, there was not an effect on the signal from endogenous hair components from the six hair samples tested. The average percent total extraction recovery was calculated for the low, medium, and high concentrations. With the exception of COT GLUC, these ranged from 62.9 to 117.8%, with the majority of observed recoveries being > 80%. The lower COT GLUC recovery is likely due to the use of an MCX cation exchange cartridge in the extraction procedure. The amide moiety present in COT GLUC is not protonated at ph 1 (loading ph) and so it cannot participate in the cation exchange mechanism; therefore, it only interacts with the SPE cartridge through the hydrophilic-lipophilic backbone of the sorbent. In addition, the aqueous and methanolic wash steps employed in the SPE extraction, in order to clean up the hair matrix resulted in the loss of this extremely polar compound. However, the quantification and detection limits were still acceptable considering the loss of this compound during the extraction process. LC MS MS matrix effect assessment demonstrated analyte quantification ion to deuterated internal standard quantification ion ratios for extracted samples, at both the low and high QC concentrations, which were generally < 20% of the ratios determined for unextracted samples at the same concentrations (Table II). Further-
more, the variability between ion ratios between individual samples was generally < 9%, which also indicated the absence of significant matrix effects. The variability between ion ratios for NIC at the low QC level was higher than for other analytes (22.8%); however, this result was acceptable. The % CV of the % matrix effect for low QC concentrations was generally higher than for high QC concentrations, indicating that matrix effects are more prevalent at lower analyte concentrations. The mean % observed concentration for analytes in the autosampler stability evaluation for reconstituted hair extracts stored at 4 C for 72 h were within 16% of the theoretical target concentration with % CV values which were 10%. Intraassay imprecision and accuracy, which were calculated from replicate (n = 5) analysis of QC samples within a batch, fortified at low, medium, and high concentrations were 17% over the linear dynamic range of the assay. Interassay imprecision and accuracy, which were calculated from 20 samples fortified at low, medium, and high concentrations over 4 separate analytical batches, were calculated as 19% (Table II). Figure 2B. Blank hair with internal standard (chromatograms for analytes < LOD not shown). Hair wash and extract procedures The concentrations determined in hair exposed to the tobacco smoke from one cigarette in the smoking apparatus were 1.07 1.17 ng/mg NIC; 0.07 ng/mg NNIC; and detectable COT and NNO (Table IV). The concentrations determined in hair exposed to the tobacco smoke from four consecutively lit cigarettes were 8.56 9.22 ng/mg NIC; 0.20 0.22 ng/mg NNIC; 0.10 COT; and 0.11 NNO. It is conceivable that the presence of very low levels of COT and NNO in the hair bundles exposed to tobacco smoke can be attributed to the oxidation of NIC to COT and NNO during the combustion of tobacco in the lit cigarette. The results show that the COT and NNO levels determined in hair exposed to the tobacco smoke from one cigarette within the confines of a condenser tube would not affect the quantification of actual metabolites in this assay. However, the results for the concentrations of COT and NNO determined in hair exposed to four cigarettes are clearly quantifiable and would certainly contribute to the metabolite concentrations that would be reported for these particular samples, where no actual metabolites were present, only artifacts of the oxidation of tobacco that had been externally deposited onto the hair shaft. 329
Figure 2C. Low quality control (chromatograms correspond to analytes > LOQ for heavy smoker sample). 330 These artifacts were removed through washing with the optimized wash procedure (Table IV). It is possible that the low levels of surface COT and NNO created through passive exposure would be removed with everyday hygiene procedures. The analyte-free bundle of hair that had been exposed to tobacco smoke from both one cigarette and four consecutive cigarettes in the apparatus yielded negative (< LOD) results for wash procedures C E (the organic wash was evaporated and analyzed). The aqueous washes in procedures A and B were not analyzed. The washed hair aliquots were subsequently extracted and also yielded negative results. Wash procedure A was selected as the most effective means of removing externally deposited nicotine in this experiment because the NIC concentration determined in the extracted hair aliquot exposed to the tobacco smoke from one cigarette was < LOQ and after exposure to tobacco smoke from four consecutive cigarettes was the lowest concentration of all the wash procedures tested (in addition to wash procedure D). Because wash procedure A required less time than wash procedure D, with comparable results, it was selected as the optimum wash procedure. It is apparent, however, that this optimum wash procedure results in some influx of externally deposited NIC into the hair shaft because quantifiable NIC was determined in the washed then extracted hair aliquot exposed to tobacco smoke from four consecutive cigarettes. The issue of producing false-positive results through washing of hair samples has been raised in previous publications (21,22). The results from our wash study suggest that this might be a concern for NIC because although the optimized wash procedure does remove externally deposited NIC, it also produced a false-positive NIC result in analyte-free hair exposed to tobacco smoke after washing and extracting. Furthermore, the wash experiments applied to the heavy smoker samples determined that there was a decrease in metabolite concentrations in washed hair aliquots compared with unwashed hair aliquots (detailed in the Application to clinical samples section). Wash procedure B, a similar wash procedure to wash procedure A, was identified as the next optimum for removal of externally deposited NIC. Negative extracted hair results were obtained for COT, NNIC, and NNO after application of this wash procedure. However, the NIC concentrations determined in analyte-free hair exposed to tobacco smoke from both
one and four cigarettes were higher (almost double for hair exposed to smoke from four cigarettes) compared to wash procedure A. Detectable COT and quantifiable NNO and NNIC were determined in the extracted hair aliquots which had been exposed tobacco smoke from four cigarettes and then washed according to procedure C. These observed COT levels were < LOQ, whereas the COT levels determined in hair collected from lowlevel smokers were not detectable (Table V). There was, however, no COT, NNO, or NNIC detected in hair aliquots that had been exposed to tobacco smoke from one cigarette and then washed according to procedure C. The NIC concentrations determined in hair exposed to the tobacco smoke from both one and four cigarettes then washed according to procedure C was more than 5 times higher than the NIC concentrations determined in hair washed by procedures A and B. This is most likely due to the less polar nature of the dichloromethane wash solvent compared with the polar aqueous wash procedures. Wash procedure E was also effective in the removal of externally deposited nicotine onto hair exposed to tobacco smoke from both one and four cigarettes. Although this procedure removed an approximately 88% of externally bound NIC in both passive exposure scenarios, it was not as effective as wash procedures A, B, or D. Application to clinical samples NIC, COT, and NNIC were quantified in the washed and unwashed hair samples collected from heavy smokers, ranging from 1.18 to 19.09, 0.06 to 2.46, and 0.05 to 0.40 NNIC, respectively. 3HC was quantified in only one, unwashed sample (0.17 ng/mg), where all other washed or unwashed samples contained detectable but not quantifiable levels. NNO was quantified in both washed and unwashed samples (0.44 0.98 ng/mg) for two individuals and detected in washed and unwashed samples for one individual. AT was quantified in both washed and unwashed samples from one individual (0.08 and 0.10 ng/mg, respectively). NIC and COT concentrations in the hair of 42 active smokers have been reported to range from 0.9 to 33.9 ng/mg and 0.09 to 4.99 ng/mg, respectively, compared to 0.54 to 1.82 ng/mg NIC and 0.01 to 0.13 ng/mg COT in nonsmokers (12). The NIC and COT concentrations determined in our study are comparable with these reported ranges for active smokers. At the time of writing, there are no reported hair concentrations for the other analytes in our test panel. Positive analyte chromatograms for heavy smoker sample number 3 are provided in Figure 2A along with a blank with internal standard (Figure 2B) and a low QC chromatogram (Figure 2C). The optimized wash procedure, which demonstrated the effective removal of externally deposited NIC onto hair, resulted in an average concentration decrease (n = 3) of 20.7% NIC, 31.8% COT, 39.4% NNO, 17.9% NNIC, and 22.5% AT for the washed and unwashed heavy smoker hair samples with quantifiable concentrations. This observed decrease in concentration may be a result of nicotine removal by sweat, or the removal of drug from within the hair shaft; however, it may also be a combination of drug influx and efflux into and out of the hair shaft instigated by hair swelling during washing. NIC was quantified in two low-level smoker samples (0.43 and 0.41 ng/mg); and NNO was detected in one sample and, interestingly, quantified in one sample at 0.35 ng/mg in which no other analytes were detected. The NIC concentrations observed in the two samples could be a result of passive exposure to ETS or a result of smoking. These preliminary data suggest that the quantification of 3HC, NNIC, and AT in some or all of the heavy smokers might present a means of differentiation from low-level smokers in which these analytes were not detected. NNO was also quantified in all of the heavy smoker samples, where it was only quantified at a lower concentration in one low-level smoker sample. The n number is presently too small to accurately evaluate whether or not the presence and quantity of these biomarkers can differentiate heavy and low-level smokers, and further studies are warranted. Conclusions An LC MS MS procedure for the accurate and precise determination of nicotine, eight metabolites, and two minor tobacco alkaloids from fortified human hair has been successfully developed and validated. Furthermore this procedure was successfully applied to three heavy smoker hair samples in the sensitive quantification of NIC, COT, and NNIC in all three samples; NNO in two samples and detection in one sample; quantification of 3HC in one sample and detection in two samples; and AT quantification in one sample. The method was also successfully applied to three low weight low-level smoker hair samples: NIC was quantified in two samples; and NNO was detected in one sample and interestingly quantified in one sample where no other analytes were detected. These preliminary data would suggest that the presence of quantifiable 3HC, NNIC, and AT and higher NNO concentrations could distinguish heavy and low-level smokers; however, the n number should be increased to more accurately evaluate this conclusion and will be reported in future work. The data from this study form part of a larger clinical study investigating nicotine pharmacokinetics and potential biomarkers of nicotine use in low-level smokers and heavy smokers. Acknowledgments This research was supported by the National Cancer Institute Grant Number R01 CA12040412. The authors also gratefully acknowledge Mr Austin Parker and Ms Amanda Reiser for preparing the hair samples for the manuscript. References 1. M.R. Moeller, P. Fey, and H. Sachs. Hair analysis as evidence in forensic cases. Forensic Sci. Int. 63: 43 53 (1993). 2. P. Kintz, A. Tracqui, and P. Mangin. Detection of drugs in human 331
hair for clinical and forensic applications. Int. J. Leg. Med. 105: 1 4 (1992). 3. T. Cairns, V. Hill, M. Schaffer, and W. Thistle. Amphetamines in washed hair of demonstrated users and workplace subjects. Forensic Sci. Int. 145: 137 142 (2004). 4. T. Cairns, V. Hill, M. Schaffer, and W. Thistle. Levels of cocaine and its metabolites in washed hair of demonstrated cocaine users and workplace subjects. Forensic Sci. Int. 145: 175 181 (2004). 5. E. Vinner, J. Vignau, T. Thibault, X. Codaccioni, C. Brassart, L. Humbert, and M. Lhermitte. Hair analysis of opiates in mothers and newborns for evaluating opiate exposure during pregnancy. Forensic Sci. Int. 133: 57 62 (2003). 6. J. Klein, T. Karaskov, and G. Koren. Clinical applications of hair testing for drugs of abuse: the Canadian experience. Forensic Sci. Int. 107: 281 288 (2000). 7. C. Brewer. Hair analysis as a tool for monitoring and managing patients on methadone maintenance. A discussion. Forensic Sci. Int. 63: 277 283 (1993). 8. P. Kintz. Determination of buprenorphine and its dealkylated metabolite in human hair. J. Anal. Toxicol. 17(7): 443 444 (1993). 9. Y. Gaillard, F. Vayssette, and G. Pepin. Compared interest between hair analysis and urinalysis for doping controls. Results for amphetamines, corticosteroids and anabolic steroids in racing cyclists. Forensic Sci. Int. 107: 361 379 (2000). 10. D. Thieme, J. Gross, H. Sachs, and R.K. Mueller. Analytical strategy for detecting doping agents in hair. Forensic Sci. Int. 107: 335 345 (2000). 11. C. Eliopoulos, J. Klein, and G. Koren. Validation of self-reported smoking by analysis of hair for nicotine and cotinine. Ther. Drug Monit. 18: 532 536 (1996). 12. P. Kintz, B. Ludes, and P. Mangin. Evaluation of nicotine and cotinine in human hair. J. Forensic Sci. 37: 72 76 (1992). 13. S. Pichini. The analysis of nicotine in infants hair for measuring exposure to environmental tobacco smoke. Forensic Sci. Int. 17: 253 258 (1997). 14. T. Uematsu, A. Mizuno, S. Nagashima, A. Oshima, and M. Nakamura. The axial distribution of nicotine content along hair shaft as an indicator of changes in smoking behaviour: evaluation in a smoking-cessation programme with or without the aid of nicotine chewing gum. Br. J. Clin. Pharmacol. 39: 665 669 (1995). 15. A. Mizuno, T. Uematsu, T. Ishikawa, N. Yoshimine, and M. Nakashima. Clinical outcome of smoking-cessation trial of nicotine chewing gum evaluated by analysis of nicotine in hair. Ther. Drug Monit. 19: 407 412 (1997). 16. N.J. Haley and D. Hoffman. Analysis for nicotine and cotinine in hair to determine cigarette smoker status. Clin. Chem. 31(10): 1598 1600 (1985). 17. C. Eliopoulos, J. Klein, M.K. Phan, B. Knie, M. Greenwald, D. Chitayat, and G. Koren. Hair concentration of nicotine and cotinine in women and their newborn infants. J. Am. Med. Assoc. 271: 621 623 (1994). 18. W. Al-Delaimy. Hair as a biomarker for exposure to tobacco smoke. Tob. Control 11: 176 182 (2002). 19. F. Pragst and M.A. Balikova. State of the art in hair analysis for detection of drug and alcohol abuse. Clin. Chem. Acta 370(1-2): 17 49 (2006). 20. G. Romano, N. Barbera, and I. Lombardo. Hair testing for drugs of abuse: evaluation of external cocaine contamination and risk of false positives. Forensic Sci. Int. 123: 119 129 (2001). 21. T. Cairns, V. Hill, M. Schaffer, and W. Thistle. Removing and identifying drug contamination in the analysis of human hair. Forensic Sci. Int. 147: 97 108 (2004). 22. T. Chetiyanukornkul, A. Toriba, R. Kizu, K. Kimura, and K. Hayakawa. Hair analysis of nicotine and cotinine for evaluating tobacco smoke exposure by liquid chromatography mass spectrometry. Biomed. Chromatogr. 18(9): 655 661 (2004). 23. J. Ryu, M.W. Seong, and M.H. Nam. Simultaneous and sensitive measurement of nicotine and cotinine in small amounts of human hair using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 20(18): 2781 2782 (2006). 24. S. Shiffman. Light and intermediate smokers: background and perspective. Nicotine Tob. Res. 11: 122 125 (2009). 25. A. Rosengren, L. Wilhelmsen, and H. Wedel. Coronary heart disease, cancer and mortality in male middle-aged light smokers. J. Intern. Med. 231: 357 362 (1992). 26. E.I. Miller, H.R. Norris, D.E. Rollins, S.T. Tiffany, and D.G. Wilkins. A novel validated procedure for the determination of nicotine, eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography electrospray ionization-tandem mass spectrometry. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 878(9 10): 725 737 (2009). 27. E.I. Miller, H.R. Norris, D.E. Rollins, S.T. Tiffany, C.M. Moore, M.J. Vincent, A. Agrawal, and D.G. Wilkins. Identification and quantification of nicotine biomarkers in human oral fluid from individuals receiving low-dose transdermal nicotine: a preliminary study. J. Anal. Toxicol. 34(7): 357 366 (2010). 28. Society of Hair Testing. Recommendations for hair testing in forensic cases. Forensic Sci. Int. 145(2): 83 84 (2004). 29. http://www.oas.samhsa.gov/nhsda/tobacco/chapter7.htm Manuscript received October 6, 2010; revision received January 7, 2010. 332