Identification and Quantification of Nicotine Biomarkers in Human Oral ...

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Journal of Analytical Toxicology, Vol. 34, September 2010

Identification and Quantification of Nicotine Biomarkers in Human Oral Fluid from Individuals Receiving Low-Dose Transdermal Nicotine: A Preliminary Study Eleanor I. Miller1,*, Hye-Ryun K. Norris1, Douglas E. Rollins1, Stephen T. Tiffany2, Christine M. Moore3, Michael J. Vincent3, Alpana Agrawal3, and Diana G. Wilkins1 1University

of Utah, Center for Human Toxicology, Department of Pharmacology and Toxicology, Salt Lake City, Utah 84108; at Buffalo SUNY, Department of Psychology, 228 Park Hall, Buffalo, New York 14260; and 3Immunalysis Corporation, 829 Towne Center Drive, Pomona, California 91767 2University

Introduction

Abstract The objective of this preliminary study was to identify and quantify potential nicotine (NIC) biomarkers in post-exposure oral fluid samples collected from 10 NIC-abstinent human participants administered 7 mg transdermal NIC using liquid chromatography– tandem mass spectrometry (LC–MS–MS). Oral fluid samples were collected prior to NIC patch application and at 0.5 and 0.75 h after patch removal using the Quantisal™ oral fluid collection device. The validated LC–MS–MS analyte panel included nicotineN-ββ-D-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1'-N-oxide, cotinine (COT), nornicotine, NIC, anatabine, anabasine, and cotinine-N-ββ-Dglucuronide. Analytes and corresponding deuterated internal standards were extracted by solid-phase extraction. NIC and COT concentrations were quantifiable in oral fluid samples collected from 6 of the 10 participants 0.5 h after patch removal and in oral fluid samples collected from 7 of the 10 participants 0.75 h after patch removal. Based on the mean NIC and COT concentrations in oral fluid and plasma for the participants with both quantifiable NIC and COT at the 0.5 and 0.75 h collection times, the oral fluidplasma ratio was 6.4 for NIC and 3.3 for COT. An ELISA procedure was also validated and successfully applied as a screening tool for these oral fluid samples in conjunction with LC–MS–MS confirmation. An ELISA cut-off concentration of 5.0 ng/mL provided excellent sensitivity for discrimination of COT-positive post-exposure oral fluid samples collected after low-level transdermal NIC exposure and oral fluid samples collected prior to patch application.

* Author to whom correspondence should be addressed: Dr. Eleanor I. Miller, Center for Human Toxicology, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 84108. Email: [email protected].

Medical concerns associated with tobacco use were published as early as the 17th century by the Royal College of Physicians in Edinburgh, Scotland (1). According to the Centers for Disease Control and Prevention (CDC), cigarette smoking is the leading cause of preventable death in the United States, producing an estimated 443,000 deaths each year in 2000–2004 and generating an estimated US$157 billion in annual health-related economic losses (2). A recent national survey by the Substance Abuse and Mental Health Services Administration (SAMHSA) indicated that an estimated 60.1 million people, or 24.2% of the population aged 12 or older, had smoked cigarettes in the past month (3). The nicotine (NIC) metabolic pathway is complex and involves the formation of a number of phase I metabolites through oxidation, hydroxylation, and N-demethylation, as well as numerous phase II metabolites through conjugation with glucuronic acid (4). Microsomal flavin-containing monooxygenase enzyme systems are responsible for the oxidation of NIC to form predominantly the trans-nicotine-1'-oxide isomer (NNO) in humans (5). The identification of the enzyme system(s) responsible for the oxidation of cotinine (COT) to cotinine-N-oxide (CNO) and also for the demethylation of NIC to nornicotine (NNIC) have yet to be identified. Although NNIC has been identified as a metabolite of NIC in humans, it is not unique to metabolism as it is a constituent of the tobacco plant (6). After smoking, NIC itself has a short plasma half-life of 1–2 h (7). In contrast to the short plasma half-life of NIC observed after smoking, the plasma half-life of COT is 6–22 h (8) following intravenous infusion of deuterium-labeled NIC and

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COT. trans-3'-Hydroxycotinine (3HC) also has a longer half-life of 4.6–8.3 h, as observed after intravenous infusion (9). As a result of their longer plasma half-lives, COT and 3HC are often selected as preferred biomarkers of NIC exposure. Although the prototypical cigarette smoker is characterized as someone who consistently smokes at least one pack of cigarettes per day, a large number of current smokers neither smoke daily nor consume one pack of cigarettes on days which they do smoke (10). As a consequence of the inhalation of tobacco smoke, both passive and active smokers maintain an increased health risk including increased rates of coronary heart disease, myocardial infarction, and lung cancer compared to nonsmokers (11,12). In addition, a correlation between higher COT concentrations in serum of nonsmokers and increased risk of coronary heart disease has been demonstrated (13). Furthermore, low-level smoking initiates a transition period that ultimately leads to the development of NIC dependence for many people (10). The serious health implications that result from exposure to tobacco smoke, either through active or passive smoking or both, has led to the need for improved analytical methodologies, which can determine NIC biomarkers in a variety of biological samples in order to assess the extent of tobacco smoke exposure. There have been a limited number of quantitative gas chromatography–mass spectrometry (GC–MS) (14–16) and liquid chromatography–mass spectrometry (LC–MS) (17–19) methods published in recent years, which include the determination of NIC and metabolites in human oral fluid. In these methods, NIC and metabolites were extracted by either mixed mode solid-phase extraction (SPE) or liquid–liquid extraction (LLE) prior to analysis. Kim et al. (14) determined NIC, COT, 3HC, and norcotinine (NCOT) in 33 oral fluid samples collected from a opiate, cocaine, and NIC-addicted pregnant woman enrolled in a methadone maintenance program by SPE and GC–MS with a limit of quantification (LOQ) of 5 ng/mL and linear calibration range of 5–1000 ng/mL. Nicotine concentrations were up to 13 times higher than COT and up to 17 times higher than 3HC concentrations. In another study, Shin et al. (15) utilized an LLE method procedure with diethyl ether and GC–MS for the determination of NIC and COT in oral fluid samples with an LOQ of 1 ng/mL for both analytes and a wide linear calibration range of 1–10,000 ng/mL. In that study, the concentration range of NIC and COT present in oral fluid samples collected from both nonsmokers and smokers ranged from 0 to 207 ng/mL and 0 to 42 ng/mL, respectively. Bentley et al. (17) determined COT and 3HC in oral fluid by automated SPE and LC–MS–MS with sensitive LOQ values of 0.05 and 0.1 ng/mL, respectively, and a linear range of 0.02–10 ng/mL. Oral fluid samples collected from subjects with a range of self-reported recent exposure to environmental tobacco smoke (ETS) produced COT concentrations ranging from 0.025 to 174.5 ng/mL and 3HC concentrations ranging from 0.050 to 44.1 ng/mL. More recently, Kataoka et al. (18) employed on-line intube solid-phase microextraction coupled with LC–MS to quantify NIC, COT, NNIC, and two minor tobacco alkaloids present in oral fluid collected from nonsmokers who chewed NIC gum. Average NIC concentrations in oral fluid reached a maximum of 304 ng/mL after 2 h with a rapid decrease to 0.63 ng/mL by

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4 h, whereas COT concentrations were much lower, averaging 6.2 ng/mL post-administration over a 24-h testing period. Also, Shakleya et al. (19) recently published a validated LC–MS–MS method for the determination of NIC, COT, 3HC, and NCOT in oral fluid collected from “regular tobacco users” using the Quantisal collection device. The LOQ was 1 ng/mL for NIC and NCOT, 0.5 ng/mL for 3HC, and 0.2 ng/mL for COT. NIC, COT, 3HC, and NCOT in oral fluid collected from five “regular tobacco users” ranged from 1.6 to 1440 ng/mL, 3.6 to 320 ng/mL, 5.2 to 53.1 ng/mL, and 0 to 133 ng/mL, respectively. To our knowledge, there are currently no published data that report NIC and NIC metabolite concentrations in oral fluid after administration of a 7-mg transdermal NIC patch for 4 h as a representation of the theoretical NIC dose delivered by smoking a single cigarette. There are a limited number of publications demonstrating the application of enzyme-linked immunosorbent assay (ELISA) (20,21) and enzyme immunoassay (EIA) (22) screening for the semi-quantitative detection of NIC metabolites in oral fluid collected from active and passive smokers, as most tend to report quantitative measurements using GC–MS or LC– MS–MS (14–19). The MS quantitative methods include NIC metabolites that are produced via the CYP 2A6 enzyme system, including COT (14–19), 3HC (14,17,19), and NCOT (14,19). However, some studies have demonstrated that NIC metabolism can be impaired by a genetic polymorphism at the CYP 2A6 gene (23,24). For those individuals who possess this genetic impairment, there may be reduced or even no production of CYP 2A6 metabolites. In such cases, it may be necessary to monitor NIC metabolites that are known to form via other enzyme systems or mechanisms; therefore, we have included metabolites in our method that are not identified products of CYP 2A6 metabolism. The objective of this current study was to identify and quantify potential NIC biomarkers in oral fluid collected from known non-smoking human volunteers following controlled low-dose (7 mg) transdermal NIC exposure for 4 h. Another study objective was to obtain preliminary data on the applicability of a commercially available COT microplate ELISA as a screening tool for the analysis of oral fluid for low-level NIC exposure and to modify our existing LC–MS–MS plasma procedure to accomodate oral fluid.

Experimental Oral fluid for method development

The pooled human oral fluid used in the LC–MS–MS routine analysis was obtained from BioChemed (Winchester, VA). One part pooled blank human oral fluid was mixed with three parts Quantisal buffer (v/v) (Immunalysis, Pomona, CA) to represent the volumes of oral fluid and buffer present in the Quantisal collection device. To confirm that pooled human oral fluid was analyte-free prior to use, the purchased pool was analyzed by LC–MS–MS prior to mixing with the collection device buffer. Also, synthetic oral fluid (Immunalysis) containing 25 mM phosphate buffered saline (pH


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7.0), 30 mM sodium bicarbonate, 0.1% albumin, 0.1% amylase, and 0.1% Proclin 300 preservative was analyzed by ELISA. One part synthetic oral fluid was mixed with three parts Quantisal buffer (v/v) and used in the preparation of ELISA calibrators. Biological samples for clinical study

Clinical samples were collected as part of an Institutional Review Board approved study (IRB #21414, University of Utah) investigating potential NIC biomarkers in NIC-abstinent human study participants (n = 10) following the application of a 7-mg transdermal NIC patch for 4 h to mimic the theoretical NIC dose received through smoking one cigarette (25). A Quantisal collection device (Immunalysis) was used to collect oral fluid samples from clinical study participants prior to NIC patch application and again 0.5 and 0.75 h following removal of the transdermal NIC patch. The samples were stored in a refrigerator at 4°C for approximately 16 h before transportation to the Center for Human Toxicology. Upon laboratory receipt, the oral fluid collection pads were squeezed out into the collection device using serum separator tubes, and the resultant oral fluid/Quantisal buffer mixture was then transferred to clean silanized 16 × 100-mm glass tubes before freezer storage at –20°C. To facilitate comparison, plasma samples from the same clinical study participants were also analyzed prior to NIC patch application and at 0.5 and 0.75 h after patch removal. Reference standards, chemicals, and reagents

(≥ 99% purity). COT microplate ELISA kits were generously provided by Immunalysis. The kits contained a 96-well COT antibody-coated microplate, COT conjugate labeled with horseradish peroxidase, substrate solution containing 3,3',5,5'tetramethylbenzidine (TMB), and stop solution containing 1 N hydrochloric acid. For ELISA analysis, (–)-COT was obtained from Cerilliant. Calibrator and QC solutions

Calibrators and QCs for LC–MS–MS were prepared daily and extracted with each analytical batch. Three calibrator working solutions were prepared in methanol at concentrations of 10, 1, and 0.1 µg/mL for NIC GLUC, CNO, 3HC, NCOT, NNO, NIC hydrogen tartrate salt (weight corrected for NIC), NNIC, COT, AT, and AB (preparation of COT GLUC calibrators involved the two higher concentrations only). The NIC hydrogen tartrate salt was used as it was determined to be more stable than NICfree base. Separate methanolic working solutions were prepared for QC samples at the same concentrations as the calibrator working solutions. Because of the unavailability of different lot numbers for some of these compounds, the same lot numbers were used to prepare both calibrator and QC working solutions; however, they were prepared by two separate analysts. All working solutions were stored in the freezer at –20°C. Table I provides the calibrator and QC concentrations used for each analyte. Also, a deuterated internal standard working solution was prepared in methanol at 1 µg/mL and contained COT GLUC-d3, NIC GLUC-d3, CNO-d3, 3HC-d3, NCOT-d4, NNO-d3, NNIC-d4, NIC-d3, COT-d3, AT-d4, and AB-d4. The working deuterated internal standard solution was stored in the freezer at –20°C. The three calibrators used in the ELISA analysis were prepared by fortifying 100 mL of oral fluid/Quantisal buffer homogenate with the appropriate volume of 10 µg/mL methanolic COT working solution to produce final concentrations of 5, 10, and 20 ng/mL COT per mL of “neat” oral fluid. These calibrators were stored in the refrigerator at 2–8°C until analysis.

The following reference standards and deuterated internal standards were obtained from Toronto Research Chemicals (North York, ON, Canada): cotinine N-β-D-glucuronide (COT GLUC) and cotinine-d3 N-β-D-glucuronide (COT GLUC-d3); nicotine-N-(4-deoxy-4,5-didehydro)-β-D-glucuronide (NIC GLUC) and nicotine-N-(4-deoxy-4,5-didehydro)-β-Dglucuronide-methyl-d3 (NIC GLUC-d3); (S)-CNO and (R,S)CNO-d3; 3HC and 3HC-d3; (R,S)-NCOT and (R,S)-NCOT-d4; (1'S,2'S)-NNO and (1'R,2'S)-NNO mixture and (1'R,2'S)-NNOd3; (R,S)-NNIC and (R,S)-NNIC-d4; (R,S)anatabine (AT) and (R, S)-anatabineTable I. Calibrator and Quality Control Concentrations for LC–MS–MS* 2,4,5,6-d4 (AT-d4); (R,S)-anabasine (AB) and (R, S)-anabasine-2,4,5,6-d4 (AB-d4). Quality Control Concentrations (ng/mL) Calibrators (–)-NIC hydrogen tartrate salt (≥ 98%) Analyte (ng/mL) Low Medium High was obtained from Sigma (St Louis, MO). (–)-COT, (±)-COT-d3, and NIC-d3 were obNIC GLUC 1.0, 2.5, 5.0, 7.5, 10, 25, 50 5.0 25 45 tained from Cerilliant (Austin, TX). SolidCNO 1.0, 2.5, 5.0, 7.5, 10, 25, 50 5.0 25 45 ® phase extraction cartridges [Oasis HLB 3HC 1.0, 2.5, 5, 7.5 10, 25, 50, 75, 100 1.0 10 100 and Oasis MCX (60 mg, 3 mL)] were obNCOT 1.0, 2.5, 5.0, 7.5, 10, 25, 50 5.0 25 45 tained from Waters (Milford, MA). HPLCNNO 1.0, 2.5, 5.0, 7.5, 10, 25, 50, 75, 100 1.0 10 100 grade methanol was obtained from HonCOT 1.0, 2.5, 5, 7.5, 10, 25, 50, 75, 100 1.0 10 100 eywell Burdick & Jackson (Morristown, NNIC 1.0, 2.5, 5.0, 7.5, 10, 25, 50 5.0 25 45 NJ). Ammonium acetate and glacial acetic NIC 1.0, 2.5, 5.0, 7.5, 10, 25, 50 5.0 25 45 acid were obtained from Spectrum (GarAT 1.0, 2.5, 5.0, 7.5, 10, 25, 50, 75, 100 1.0 10 100 dena, CA). Trichloroacetic acid, concenAB 1.0, 2.5, 5.0, 7.5, 10, 25, 50 5.0 25 45 trated formic acid, and concentrated amCOT GLUC 50, 75, 100, 200, 400, 500 75 200 400 monium hydroxide were obtained from Fisher Scientific (Pittsburgh, PA). All * Prepared in 1:3 (v/v) blank oral fluid/Quantisal buffer homogenate. chemicals and reagents were HPLC grade

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Methods Sample preparation and extraction

LC–MS–MS. A 0.5-mL aliquot of oral fluid/Quantisal buffer homogenate sample, calibrator, or QC was fortified with 25 µL of 1 µg/mL deuterated internal standard solution to produce a final concentration of 50 ng/mL. This was equivalent to 0.125 mL “neat” oral fluid. Each tube was acidified (to pH 1) with 0.5 mL of 10% aqueous trichloroacetic acid and subsequently vortex mixed. The acidified oral fluid/Quantisal buffer homogenates were then subjected to SPE according to our previously published procedure for the extraction of these selected analytes from plasma (26). Extracted homogenate residues were reconstituted in 85 µL of initial mobile phase conditions [10 mM ammonium acetate + 0.001 % formic acid (~ pH 4.97)/methanol (85:15, v/v)]. ELISA. Twenty microliters of oral fluid/Quantisal buffer homogenate calibrator, QC, or sample was manually pipetted (Rainin ® , Mettler Toledo, Columbus, OH) into the COT microplate ELISA wells in duplicate. COT enzyme conjugate reagent (100 µL) was subsequently added, and the plate was left in the dark at room temperature for an incubation period of 1 h. Following incubation, the microplate wells were washed with deionized water (6 × 350 µL) with a Columbus™ Strip washer system (Tecan Group, Männedorf, Switzerland) in order to remove any unbound sample and residual enzyme conjugate reagent in the wells. TMB substrate reagent (100 µL) was then added to the wells, and the plate was left to incubate in the dark at room temperature for an additional 30 min. The reaction was stopped after this time by adding 100 µL of 1 N hydrochloric acid (stop reagent). The well contents turned from blue to yellow after addition of the acid to allow detection of the TMB chromophore at a wavelength of 450 nm using a Sunrise™ Microplate Reader (Tecan Group). LC–MS–MS conditions. LC was conducted using an Acquity UPLC® system (Waters). Chromatographic separation was achieved using a Discovery® HS F5 HPLC column (100 mm × 4.0 mm, 3 µm, Supelco®, Bellefonte, PA) with a gradient system consisting of 10 mM ammonium acetate with 0.001% formic acid (pH 4.97)

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Table II. Liquid Chromatography–Electrospray Ionization-Tandem Mass Spectrometry Parameters Analyte

tR (min)

tR SD (min)

Cone Voltage Collision Energy (V) (AU)

COT GLUC

2.01

0.01

20

21

COT GLUC-d3

2.00

0.01

20

21

NIC GLUC

3.36

0.05

15

30

NIC GLUC-d3

3.33

0.06

15

30

CNO

3.68

0.03

25

30

CNO-d3

3.67

0.03

25

30

3-HC

4.91

0.04

25

32

3-HC-d3

4.90

0.04

25

32

NCOT

5.43

0.04

25

35

NCOT-d4

5.41

0.04

25

35

NNO†

7.01

0.17

30

27

NNO-d3

7.03

0.17

30

27

COT

6.50

0.04

25

36

COT-d3

6.59

0.04

25

36

NNIC

6.71

0.51

20

32

NNIC-d

6.69

0.50

20

32

NIC

7.02

0.63

15

30

NIC-d3

7.00

0.61

15

30

AT

7.40

0.68

20

28

AT-d4

7.38

0.68

20

28

AB

8.24

0.83

20

40

AB-d4

8.23

0.81

20

40

* The quantification ion for each analyte is given in the upper row of each box. † t calculated for the second and most abundant diastereomer. R

MRM Transitions* 353.3 → 177.2

356.3 → 180.2

321.2 → 163.0 321.2 → 83.9 324.3 → 166.1 324.3 → 86.9 193.2 → 96.0 193.2 → 98.1

196.4 → 96.0 196.4 → 101.2 193.1 → 79.8 193.1 → 85.9

196.1 → 79.8 196.1 → 88.9 163.0 → 79.8 163.0 → 83.8

167.0 → 83.9†

179.0 →129.9 179.0 → 116.8

182.0 → 129.9 182.0 → 116.8 177.2 → 79.9 177.2 → 97.9

180.1 → 79.9 180.1 → 100.9 149.0 → 79.9 149.0 → 129.9 153.0 → 83.9 153.0 → 134.0 163.2 → 130.0 163.2 → 116.9 166.1 → 129.9 166.1 → 116.9 161.1 → 144.0 161.1 → 116.9 165.1 → 148.0 165.1 → 121.0 163.1 → 130.0 163.1 → 116.9

167.2 → 134.0 167.2 → 122.0


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and methanol at a flow rate of 0.6 mL/min. The initial mobile phase condition was 15% B which was increased linearly to 76% after 11 min, then decreased back to the initial mobile phase condition of 15% B after 11.6 min, and held for 3.4 min to re-equilibrate the LC column (total chromatographic runtime was 15 min). The retention time for each analyte is given in Table II. MS analysis was conducted using a Quattro Premier XE™ triple-quadrupole MS (Waters) with MassLynx™ v. 4.1 software. The MS was operated in electrospray positive mode using multiple reaction monitoring (MRM) acquisition. Two MRM transitions were monitored for each analyte, with the exception of COT GLUC, which produced only one major fragment ion. The following ESI conditions were applied: capillary voltage 3.25 kV; source temperature 100°C; and a desolvation temperature of 350°C. Analyte-specific cone voltages, collision energies, and MRM transitions are provided in Table II. Criteria for identification of “positive” analytes included selected MRM transitions having a signal-to-noise (S/N) ratio of at least 10; the peak-area ratio of analyte quantification ion to deuterated internal standard quantification ion for the samples were within ± 20% of the corresponding calibrator of similar concentration; and the analyte quantification ion-to-analyte qualification ion ratio were within ± 20% of the positive analyte QC, which had more than one fragment ion with ≥ 20% abundance (with the exception of COT GLUC, which produced only one fragment ion). Method development

LC–MS–MS. Optimization of MS–MS parameters was conducted by the direct infusion of individual analyte solutions, prepared in methanol at a concentration of 10 µg/mL. Following the optimization of the general capillary voltage (kV), desolvation temperature (°C), desolvation gas, and cone gas flow rates (L/h), the final cone voltages (V) and collision energies (AU) were selected for individual analytes through manual tuning such that the precursor ion response was < 10% abundance in order to attain maximum sensitivity toward the product fragment ions (Table II). The panel of 11 target analytes and their respective deuterated standards were adequately separated within 10 min. Retention time (tR) reproducibility was assessed by calculating tR variability over three interday imprecision batches performed over an approximate four-month period (66–68 injections), as shown in Table II. The number of injections depended on the number of calibrators and QCs (Table I). The % relative standard deviation (% RSD) was ≤ 10% for all analytes and deuterated internal standards tRs. Method linearity was evaluated for each analyte using a simple linear regression data fit and calculation of the coefficient of determination (R2). Calibration curves were produced from peak-area ratios of the quantification ion of target analytes and the quantification ion of the corresponding deuterated internal standards over the concentration ranges shown in Table I. Both weighted 1/x and non-weighted linear regression fits were evaluated with no significant difference in calculated concentration. Therefore, non-weighted fits were selected for routine use. Calibrator and QC concentrations were determined using the calibration curve and were required to be

within 20% of the theoretical target concentration. Method sensitivity was determined by the limit of detection (LOD) and LOQ, which were calculated relative to peak height and, in the case of the LOQ, accuracy and imprecision (within 20%) on three separate days. The LOD was defined as the concentration of analyte that produced an S/N of 3 for selected MRM transitions. The LOQ was the lowest standard in the calibration graph that produced an S/N ratio of ≥ 10 for the selected MRM transitions with acceptable precision and accuracy (within ± 20% of target concentration). The LOD and LOQ parameters were determined empirically as the concentration obtained for a series of decreasing concentrations of analyte fortified in human oral fluid/Quantisal buffer homogenate. The specificity of the method was assessed by the analysis of analyte-free human oral fluid samples collected from six individuals. Each oral fluid sample was extracted and analyzed (n = 3) to determine the presence of any potential interference from endogenous oral fluid matrix components. The total extraction recovery for each analyte was calculated at a low, medium, and high concentration (n = 5 for each level). Oral fluid/Quantisal buffer homogenate 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 also to the unextracted samples. Total extraction recovery (%) was calculated by comparing the average analyte/internal standard peak-area ratio of extracted standards with the average analyte/internal standard peak-area ratio of unextracted standards. LC–MS–MS matrix effects have been evaluated in numerous other publications by comparing the response of extracted analyte-free matrix (from a number of individuals) that had been fortified with analyte and deuterated internal standard after extraction with unextracted standards prepared at the same concentration (19,27). However, this method of calculation does not allow for the inclusion of a matrix effect evaluation relative to the actual quantity of analyte, which is recovered after extraction, and is in most cases most likely to be an underestimate of the actual matrix effect. In an effort to evaluate the effect of matrix on the actual quantity of extracted analyte, the analyte/internal standard peak-area ratio for extracted analyte-free oral fluid samples fortified with analyte and internal standard before extraction from five individuals was compared to the analyte/internal standard peak-area ratio for unextracted standards (n = 5) prepared in initial mobile phase composition at the same concentration. The mean total extraction recovery (%) of the deuterated internal standards was within 10% of the mean total extraction analyte recovery for the three QC concentrations tested, and therefore a comparison of peak-area ratios of extracted samples and unextracted samples was a quantitative assessment of matrix effect. Matrix effect was calculated as a percentage of the mean peak-area ratio of the unextracted samples at low and high QC concentrations. Stability was assessed using oral fluid/Quantisal buffer homogenate QC samples fortified at low and high concentrations over the linear range of the assay. Stability of the analytes following three freeze-thaw cycles of fortified oral fluid was determined as well as short-term stability for fortified human

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oral fluid stored for 24 h at 4°C. Autosampler stability was asFor method specificity assessment, there was no signal for sessed for reconstituted extracts stored for 72 h at 4°C. the selected MRM transitions for COT GLUC, CNO, 3HC, NCOT, Intra- and interassay imprecision and accuracy were calcuNNO, AT, or AB. Some of the samples did contain a peak for the lated at low, medium, and high QC concentrations. Intraassay quantification ion transition for NIC GLUC (321.2 → 163.0), imprecision data was calculated from the concentration variCOT (177.2 → 79.9), NNIC (149.0 → 79.9), and NIC (163.2 → ability of the replicate analysis of QCs (n = 5) within an ana130.0) with an S/N of > 10. However the ion ratios did not meet lytical batch. Interassay imprecision data was calculated from the concentration variability of a total of 15 QC samples anTable III. LOD, LOQ, Recovery, and Matrix Effect for Human Oral Fluid/ alyzed in 3 separate analytical batches, Quantisal Buffer Homogenate* which were run over a 4-month period. Recovery (%) Matrix Effect (%) The imprecision is expressed as a stan(n = 5) (n = 5) dard deviation. LOD LOQ The oral fluid LC–MS–MS data was comAnalyte (ng/mL) (ng/mL) Low Medium High Low High pared with the oral fluid ELISA data and the plasma LC–MS–MS data obtained for NIC GLUC 0.50 1.0 94 (3.4) 93 (3.3) 89 (5.3) 109 (8.6) 81 (3.9) 10 clinical study participants at 0.5 and CNO 0.50 1.0 91 (6.8) 104 (6.5) 93 (4.9) 110 (17.6) 99 (7.1) 0.75 h after NIC patch removal. The 3-HC 0.25 1.0 99 (8.0) 105 (8.1) 96 (5.2) 113 (19) 94 (3.2) plasma results were obtained using our NCOT 0.25 1.0 88 (6.2) 94 (7.6) 83 (5.3) 101 (2.9) 88 (5.2) previously published LC–MS–MS proceNNO 0.50 1.0 118 (9.6) 116 (10.3) 117 (6.2) 110 (15) 95 (7.6) dure, which is very similar to this curCOT 0.25 1.0 93 (7.6) 96 (6.5) 94 (5.7) 91 (16) 90 (6.4) rently proposed procedure for oral fluid NNIC 0.25 1.0 97 (10.9) 89 (4.1) 85 (4.1) 109 (6.2) 90 (3.5) NIC 0.50 1.0 107 (5.8) 104 (3.6) 111 (8.1) 120 (6.0) 86 (2.3) testing (26). AT 0.75 1.0 119 (9.2) 106 (12) 111 (6.5) 109 (19) 106 (16) ELISA. An ELISA dose-response curve AB 1.0 1.0 91 (15) 100 (11) 89 (15) 108 (9.9) 82 (4.9) was plotted on a logarithmic scale over COT GLUC 25.0 50.0 101 (9.2) 95 (14) 80 (12) 113 (18) 114 (12) the concentration range 5–100 ng/mL COT by comparing the absorbance value * The number provided in parentheses after the reported % Total Extraction Recovery and % Matrix Effect is the % Relative Standard Deviation. for fortified analyte-free oral fluid/Quantisal buffer homogenate (B) with analytefree oral fluid/Quantisal buffer homogenate (B0). Intraday imprecision within Table IV. Comparison of LC–MS–MS and ELISA Results for Clinical Oral Fluid the same batch (n = 8) and interday imSamples precision for eight different replicates Time ELISA COT Qualitative Qualitative were analyzed in 10 separate batches (n = LC–MS–MS after Patch Semi-Quantitative ELISA Result ELISA Result 80). Cross-reactivity (n = 2) was calcuParticipant Removal NIC COT Equivalents at 10.0 ng/mL at 5.0 ng/mL lated for NIC GLUC, CNO, 3HC, NCOT, Number (h) (ng/mL) (ng/mL) (ng/mL) COT Cut-Off COT Cut-Off NNO, NNIC, NIC, AT, AB, and COT GLUC in the LC–MS–MS method at a concen1 0.5 32 25 > 20 + + tration of 50 ng/mL relative to the COT 0.75 38 27 > 20 + + dose-response curve. 2 0.5 42 12 15 + + 3

Results Method validation

LC–MS–MS. Calibration curves were linear for each analyte over the selected concentration ranges (Table I) with coefficients of determination (R2) values > 0.99. A simple linear regression was fitted to the data points. The LOD and LOQ values (Table III) were derived from the criteria discussed in the Experimental Section. Most analytes were quantifiable down to 1.0 ng/mL of oral fluid/Quantisal buffer homogenate with the exception of COT GLUC, which was 50 ng/mL.

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4 5 6 7 8 9 10

0.75 0.5 0.75 0.5 0.75 0.5 0.75 0.5 0.75 0.5 0.75 0.5 0.75 0.5 0.75 0.5 0.75

45 < LOQ* 13 12 9.1 9.1 12 18 23 0 0 0 0 26 21 26 28

17 < LOQ* 8.2 21 21 0 0 27 28 8.8 8.8 8.0 11 22 23 18 26

15 5.0 6.0 20 20 < 5.0 < 5.0 14 > 20 7.0 6.0 7.0 6.0 15 > 20 > 20 > 20

+ – – + + – – + + – – – – + + + +

* The LOQ for NIC and COT was 1.0 ng/mL oral fluid/Quantisal buffer homogenate (1:3, v/v).

+ + + + + – – + + + + + + + + + +


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ion ratio criteria based on the two selected MRM transitions for establishing the identity of these compounds, and therefore these samples were identified as “negative”. The presence of these very small interfering peaks in the analyte-free oral fluid/Quantisal buffer homogenates provided by nonsmokers could be due to one, or a combination of, passive exposure to ETS (28), NIC intake through dietary sources (5), and endogenous matrix components. The findings of The Third National Health and Nutrition Examination Survey between 1998 and 2001 determined that serum COT concentrations could be utilized to distinguish smokers from nonsmokers, but not nonsmokers with ETS exposure from nonsmokers without ETS exposure (29). The % total extraction recovery was calculated for the low, medium, and high concentrations. This calculation compared the peak-area ratio of analyte quantification ion to deuterated internal standard quantification ion for extracted samples with unextracted samples. The mean % recovery ranged from 80 to 119% (Table III).

An assessment of matrix effects yielded analyte quantification ion to deuterated internal standard quantification ion ratios for extracted samples, at both the low and high QC concentrations, which were within 20% of the ratios determined for unextracted samples at the same concentrations (Table III). Furthermore, the variability between ion ratios between individual samples was ≤ 18%, which also indicated the absence of significant matrix effects. However, the standard deviation of the % matrix effect for low QC concentrations was higher than for high QC concentrations for all but one analyte, indicating that matrix effects are more prevalent at lower analyte concentrations. The mean % observed concentration for analytes that had been fortified in oral fluid/Quantisal buffer homogenate at low and high concentrations (Table I) were calculated to be within ± 18% of the target concentrations, respectively, under all five storage conditions, demonstrating matrix stability for 24 h in the refrigerator at 4°C, 1 week in the freezer at –20°C, and 3 freeze-thaw cycles (–20°C), and also reconstituted extract stability in the LC autosampler for 72 h at 4°C.

Figure 1. Chromatogram of analytes detected in participant’s oral fluid 0.5 h after patch removal (A) and chromatogram of participant’s baseline oral fluid sample (B).

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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 ≤ 15% and ≥ 80%, respectively, over the linear dynamic range of the assay. Interassay imprecision and accuracy, which were calculated from 15 samples fortified at low, medium, and high concentrations over 3 separate analytical batches, were calculated as < 18% and ≥ 81%, respectively. ELISA validation. The ELISA dose-response curve for COT was linear on a logarithmic scale over the concentration range 5–100 ng/mL and produced a coefficient of determination (R2) greater than 0.99. A concentration range of 5.0–20.0 ng/mL COT was selected for analysis of clinical oral fluid samples. Intraday imprecision calculated from the replicate analysis of oral fluid/Quantisal buffer homogenate fortified at 10 ng/mL COT (n = 8) was 3.4%. Interday imprecision for the replicate analysis of oral fluid/Quantisal buffer homogenate at 10 ng/mL COT analyzed on 10 separate days (n = 80) was 3.0%. The analytes which were targeted in the LC–MS–MS method were each tested for cross-reactivity with the COT microplate ELISA (n = 2). All analytes, with the exception of COT and 3HC, demonstrated 0% cross-reactivity with the COT microplate at a concentration of 50 ng/mL compared with the COT doseresponse curve. COT and 3HC were determined to be 100% and 120% cross-reactive, respectively. In addition, NIC GLUC, CNO, NNO, NNIC, NIC, NCOT, AT, AB, and COT GLUC were run at a concentration of 500 and 5000 ng/mL. At the 500 ng/mL concentration, all analytes demonstrated 0% cross-reactivity with respect to COT, and at 5000 ng/mL all immunoassay responses were < 5 ng/mL the lower limit of linearity on the ELISA doseresponse curve.

Discussion Clinical samples: oral fluid

The ELISA assay was included as a screen for oral fluid samples collected from NIC-abstinent human participants receiving low-dose transdermal NIC and to evaluate its potential use as a screening assay for oral fluid samples collected from mimicked “low-level” smokers. Therefore, all ELISA data was regarded as semi-quantitative for screening purposes, while the LC–MS–MS assay was used to quantitatively determine the “positive” analyte concentrations in these samples. The use of an immunoassay screen in combination with LC–MS–MS quantification is the sequence of testing, which is generally recommended for forensic samples by the Society of Forensic Toxicologists (30). The ELISA COT concentrations determined in our study for 12 oral fluid samples following low-level transdermal NIC exposure were in the range of 5.0–20 ng/mL and were > 20 ng/mL for six oral fluid samples (Table IV). Two samples screened as “negative” (< 5.0 ng/mL) for COT. Because no quantifiable 3HC was determined in any of the participant samples by LC–MS–MS, the ELISA concentrations measured in the “positive” samples most likely resulted from COT metabolite in the oral fluid samples. In comparison to the COT con-

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centration range observed in our present study, previous studies utilizing EIA determined COT concentrations in 49 smokers (314.0 ± 171.9 ng/mL) and 31 nonsmokers (1.6 ± 1.2 ng/mL) (22). Another study measured ELISA COT concentrations ranging from 0.87 to 14.9 ng/mL in oral fluid samples collected from 67 nonsmokers with different levels of ETS exposure and from 2.7 to 37 ng/mL for 27 smokers (21). These results indicate an observed overlap in COT concentrations between nonsmokers and smokers. The average semi-quantitative ELISA COT concentration determined in oral fluid samples collected for our study was higher than the average EIA COT concentrations (22) and ELISA concentrations (20,21) determined for nonsmokers with various degrees of ETS exposure. The LC–MS–MS quantitative results for each clinical study participant are provided in Table IV. An oral fluid sample collected from a participant 0.5 h after patch removal is shown in Figure 1A, and an oral fluid sample collected from the same participant before patch application is shown in Figure 1B (chromatograms are for positive analytes only). Oral fluid concentrations in Figure 1A were 12 ng/mL NIC and 21 ng/mL COT at 0.5 h after patch removal, as determined by LC–MS– MS. The NIC concentration in the sample collected 0.75 h after patch removal decreased to 9.1 ng/mL, and the COT concentration remained constant at 21 ng/mL. NIC and COT concentrations were quantifiable in oral fluid samples collected from 6 of the 10 participants 0.5 h after patch removal and in oral fluid samples collected from 7 of the 10 participants 0.75 h after patch removal. Average NIC and COT concentrations (n = 13) at these two collection times were 25 ± 12 ng/mL and 21 ± 6.0 ng/mL, respectively. NIC was quantified in both post-exposure oral fluid samples collected from 1 of the 10 participants; however, no COT was detectable. COT was quantified in both post-exposure oral fluid samples collected from 2 of the 10 participants in which no NIC was detectable. Based on the average NIC and COT concentrations in oral fluid and plasma for the participants with both quantifiable NIC and COT at the 0.5 and 0.75 h collection times, the oral fluid/plasma ratio was 6.4 for NIC and 3.3 for COT. As expected, the COT values measured in our current study are lower than previously reported values for regular tobacco users (14,16,17). The LC–MS–MS COT concentration range observed for 13 post-exposure samples in our study (8.2–28 ng/mL) containing both quantifiable NIC and COT is similar to the COT range (2.8–23 ng/mL) previously reported for oral fluid collected from six occasional smokers (17). Although the route of NIC administration is different between these two studies and the frequency and time of smoking is not reported for the occasional smokers, it is apparent that the COT concentrations measured in our study are either within the range (n = 8) observed for occasional smokers or slightly higher than the reported range (n = 5). There is a general lack of consensus regarding the definition of an optimal cut-off concentration for COT for the discrimination of smokers from nonsmokers in oral fluid. However the optimal COT cut-off concentration in plasma for the general U.S. population has recently been reported as 3 ng/mL (31). In 1983, Benowitz (32) documented that a blood COT concentration > 10 ng/mL had never been measured in a nonsmoker in their studies. Considering the recent introduction of clean air


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government policies and reduced smoking practices compared to 25 years ago, it is probable that this cut-off concentration established in 1983 for plasma is presently too high. Various researchers have applied COT cut-off concentrations in the range of 5–100 ng/mL to distinguish smokers and nonsmokers (33,34). Because oral fluid to plasma concentration ratios are known to range from 1.1 to 1.4 for COT and are therefore regarded as interchangeable, earlier work recommended a 10 ng/mL COT oral fluid cut-off to distinguish nonsmokers from smokers (32,35). Our preliminary LC–MS–MS and ELISA data have demonstrated that NIC-abstinent participants have COT oral fluid concentrations undetectable by LC–MS–MS or ELISA before transdermal NIC patch application. After removal of the patch, the COT oral fluid concenrations ranged from 8.2 to 28 ng/mL by LC–MS–MS and 5.0 to > 20 ng/mL by ELISA for 9 out of 10 participants (Table IV). There was no COT detected by LC– MS–MS or ELISA in post-exposure oral fluid samples collected from one participant; however, NIC was quantified in both of these samples. Plasma COT and NIC concentrations for this participant 0.5 and 0.75 h after transdermal patch removal were detectable but < LOQ. Clinical samples: plasma

The mean NIC and COT concentrations in the plasma samples collected from the 10 participants in our study were 2.8 ± 2.2 ng/mL and 5.5 ± 3.9 ng/mL, respectively, 0.5 h after patch removal. These mean concentrations were almost identical for NIC (2.9 ± 2.2 ng/mL) and slightly increased to 6.1 ± 4.1 ng/mL for COT 0.75 h after patch removal. Therefore, an average oral fluid/plasma ratio of 6.4 for NIC was determined in our study for the two post-patch collection points combined, which is similar to the ratio determined in another study investigating plasma and oral fluid NIC concentrations following the administration of transdermal NIC (36). However, despite the similarity in oral fluid/plasma NIC ratio between transdermal NIC studies, a prior intravenous NIC infusion study indicated that there was no correlation between plasma and oral fluid NIC concentrations (37). An average oral fluid/plasma ratio of 2.7 was determined for COT in our study for the two post-patch collection points combined. This is a higher ratio than previously reported studies in which a ratio range of 1.2– 1.4 was observed following intravenous COT infusion (38) and in an NIC transdermal study (39). It is possible that the difference in the observed oral fluid/plasma ratios between transdermal NIC studies is due to variables such as the patch dose, time of patch application, stimulation of oral fluid production, and oral fluid collection protocol.

Conclusions An SPE-LC–MS–MS procedure for the accurate and precise determination of fortified NIC, eight NIC metabolites, and two minor tobacco alkaloids from human oral fluid/Quantisal buffer homogenate has been successfully developed and validated. Furthermore, this procedure was applied in the sensitive quantification of NIC and/or COT in oral fluid collected from 10

NIC-abstinent clinical study participants 0.5 and 0.75 h after removal of a 7-mg transdermal NIC patch, which had been worn for 4 h. An ELISA method was also successfully applied as a screening tool for these samples in conjunction with LC–MS– MS confirmation. An ELISA cut-off concentration of 5.0 ng/mL provided excellent sensitivity for discrimination of COT “positive” post-exposure oral fluid samples collected after low-level transdermal NIC exposure. The findings of this study have shown that both LC–MS–MS and ELISA methods can be complementary, demonstrating excellent sensitivity and specificity for oral fluid samples collected from this particular population as part of a clinical study investigating NIC pharmacokinetics and potential biomarkers of NIC exposure in plasma and oral fluid of low-level smokers.

Acknowledgments This research was supported by the National Cancer Institute Grant Number: R01 CA12040412. The authors would like to thank the Center for Clinical and Translational Science (CCTS) at the University of Utah Health Sciences Center for financial support (#ULIRR025764) and the CCTS staff for help with the clinical study.

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Manuscript received January 26, 2010; revision received April 12, 2010.