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Quantitative Determination of Nicotine and Cotinine W in Urine and Sputum Using a Combined SPME-GC/MS Method A. E. Witter* and D. M. Klinger Department of Chemistry, Dickinson College, Carlisle, PA 17013; *
[email protected] X. Fan, M. Lam, D. T. Mathers, and S. A. Mabury Department of Chemistry, University of Toronto, Toronto, ON M5S 1A1, Canada
Experiments related to environmental analytical chemistry have increased in this Journal in recent years. Several of these experiments have focused on the analysis of environmental tobacco smoke and its components (1, 2). In the experiment described below, a different aspect of environmental analysis is investigated. Here a naturally formed metabolite that is a biochemical indicator of environmental tobacco smoke exposure is quantified. Background Despite the recent classification of nicotine as a federally regulated, addictive drug, a majority of students observed on our respective campuses continue to smoke. Recent studies indicate that smoking by college students increased 28% between 1993 and 1997 (3). This increase was predictable, according to the authors of the study, because smoking amongst high school students increased by 30% in the mid90s. Environmental tobacco smoke (ETS) is classified by the U.S. EPA as a class A carcinogen. In the U.S., it has been estimated that 50–75% of the nonsmoking population is exposed to ETS either at home or at work (4). Because we believe that students learn best through the idea that “seeing is believing”, we conceived the following experiment which uses the bodies of the students or their smoking peers as the “reaction vessel” in which the following oxidation reaction occurs:
N N
N
CH3 nicotine
P450
N
O
CH3 cotinine
Nicotine is oxidatively metabolized in the liver through the action of microsomal cytochrome P450 enzymes to its primary metabolite, cotinine. Although further metabolic products may form from cotinine oxidation, 80% of the nicotine absorbed by a smoker is metabolized to cotinine (5). Thus, cotinine can be used to assess exposure to environmental tobacco smoke. Solid-phase microextraction (SPME) is a relatively recent sampling technique used in the analysis of volatile and semi-volatile analytes1 that negates the need for extensive sample clean-up (6). Solid phase microextraction can be used to sample either the headspace above an aqueous sample or the aqueous phase itself (7). Equilibria are established between the aqueous phase, the headspace, and the SPME fiber. Be-
cause of its simplicity, SPME is especially amenable to the undergraduate laboratory. Experimental Objectives The objectives of this experiment are to: 1. Use a simple sampling technique (SPME) to concentrate volatile and semi-volatile analytes from urine and sputum for analysis by GC/MS 2. Illustrate how one chooses a suitable internal standard for quantification purposes
Experimental Method The SPME fiber holder was purchased from Supelco (Bellefonte, PA). For nicotine and cotinine analysis, an 85µm polyacrylate fiber was used for analysis (Supelco). Creatinine diagnostic kits were purchased from Sigma–Aldrich (St. Louis, MO). A Hewlett Packard 1800A GC equipped with a Hewlett Packard GCD detector was used for analysis. The column used for analysis was a 30 m × 0.25 mm RTX-5 (Restek Corporation, Bellefonte, PA, film thickness 0.25 mm). The column temperature was set at 60 °C for 0.5 minutes, and then programmed from 60 °C to 260 °C at 25 °C/min. The temperatures of the injection port and ion source were set at 25 °C and 280 °C, respectively. The ionization voltage was 70 eV. Splitless injection mode was used, however the regular inlet liner was replaced with a SPME inlet liner purchased from Supelco. Helium with an inlet pressure of 9 psi was used as the carrier gas in constant flow mode. Two internal standards were used for quantitative purposes: 5-aminoquinoline was used as an internal standard for nicotine, and deuterated cotinine was used as a surrogate for cotinine. Ions used for selective ion monitoring (SIM) were m/z 84 for nicotine, m/z 98 for cotinine, m/z 101 for deuterated cotinine, and m/z 144 for 5-aminoquinoline.
Preparation of Stock Solutions Four stock solutions including nicotine (10 mg/L), 5-aminoquinoline (222 mg/L), cotinine (350 mg/L) and deuterated cotinine (300 mg/L) (Aldrich Chemical Company, Milwaukee, WI) were each prepared separately in water and stored refrigerated when not in use. Serial dilutions of the stock solutions were prepared in urine or sputum. Urine and sputum samples used for analysis were collected from smokers and nonsmokers in urine sampling cups and stored at ᎐20 °C before analysis. If the use of actual urine poses a problem, a synthetic urine matrix can be used.2 Synthetic
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Table 1. Typical Results from Urine and Sputum Analysis of Smokers and Nonsmokers Using the Combined SPME-GC/MS Method Subject ID
Sample ID
Time/ Relative time
Matrix
Concentration in the Sample/ Nicotine
1
1
5 p.m.
1
2
8 p.m.
1
3
10 p.m.
1
4
8 a.m.
2
5
5 p.m.
2
6
8 p.m.
Sample Documentation
(mg/L)
Urine
Urine
Cotinine
0.175
nd*
Subject 1: 11-year-old boy
0.163
nd
Parents are heavy smokers:
0.126
nd
subject is assumed to be a
0.238
nd
secondhand smoker.
0.219
nd
Subject 2: 12.5-year-old girl
0.147
nd
Parents are heavy smokers:
2
7
10 p.m.
0.161
nd
subject is assumed to be a
2
8
8 a.m.
0.124
nd
secondhand smoker.
3
9
6:40 p.m.
0.856
0.011
Subject 3: male, regular smoker
3
10
1:00 p.m.
2.48
0.016
4
11
9:00 p.m.
4
12
11:00 p.m.
5
13
9:00 p.m.
5
14
9:30 p.m.
Urine
Urine
Urine
0.007
Subject 4: male, nonsmoker; exposed to
nd
secondhand smoke in bar
0.100
nd
Subject 5: male, nonsmoker
0.077
nd
6
15
8:00 p.m.
0.078
nd
6
16
10:30 p.m.
0.077
nd
6
17
12:30 p.m.
0.085
nd
7
18
8:00 p.m.
7
19
9:00 p.m.
8
20
t = 0 hr
8
21
t = 4 hr
Urine
0.081 0.114
Urine
Urine
0.075
nd
0.080
nd
0.076
nd
0.076
nd
8
22
t = 7 hr
0.071
nd
8
23
t = 11.5 hr
0.071
nd
9
24
t = 0 hr
0.078
nd
9
25
t = 2 hr
0.078
nd
9
26
t = 4 hr
0.085
nd
10
27
t = 0 hr
10
28
t = 47 min
11
29
t = 0 hr
11
30
t = 47 min
12
31
t = 0 hr
12
32
t = 1 hr
13
33
t = 0 hr
13
34
t = 1 hr
Urine
Sputum
Sputum
Sputum
Sputum
0.069
nd
0.087
nd
0.101
nd
0.118
nd
0.082
nd
0.189
nd
0.075
nd
0.104
nd
Subject 6: female, nonsmoker
Subject 7: male, nonsmoker
Subject 8: male, nonsmoker
Subject 9: female, nonsmoker
Subject 10: female, light smoker
Subject 11: male, light smoker
Subject 12: female, nonsmoker
Subject 13 female, nonsmoker
*not detected
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Hazards Because this activity uses real urine samples, the instructor must discuss hazards such as disease transmission associated with biological fluids. Students should work only with their own urine samples and must wear gloves and protective eyewear at all times. Glassware that comes in contact with urine should be rinsed in household bleach following the experiment. Urine spills should be cleaned up with bleach. Because nicotine is highly toxic and can be absorbed through the skin, the instructor should prepare all calibration standards for this experiment. Results and Discussion In this project, we set out to simultaneously monitor nicotine and its primary metabolite, cotinine, in the urine and sputum of humans. For nicotine and cotinine analysis in urine, we found the 85 µm polyacrylate fiber gave consistent, reproducible results when sampling the headspace above alkaline (pH > 10) urine samples.3 Quantitation was achieved using 5-aminoquinoline as a surrogate for nicotine, and deuterated cotinine as a surrogate for cotinine (8). Students were asked to find structures for these molecules, and propose reasons why one standard might be a better choice for an internal standard than the other. Because cotinine and its surrogate deuterated cotinine have similar physical-chemical properties, their behavior during headspace sampling and subsequent ionization is nearly identical, while the behaviors of nicotine and 5-aminoquinoline are not. We also discussed the advantages of SIM for the ions of interest, which allowed us to achieve much lower limits of detection for these analytes than would be possible using full-scan mode. Calibration curves were obtained by plotting the ratio of the nicotine (or cotinine) peak area to its surrogate peak area versus nicotine (or cotinine) concentration (Figure 1). Calibration curves obtained using the internal calibration method showed excellent linearity for the average of six injections. In
2.0
Area Ratio (m /z 84/144)
Sample Extraction Procedures One milliliter of a urine (or sputum) sample was pipetted into a 12-mL glass vial (National Scientific Company, St. Louis, MO) containing 0.7 g of sodium carbonate powder (ACS grade, Fisher Chemical Corp). The vial was fitted with a Teflon-coated silicone septum and a polycarbonate threaded cap. Each septum could be used for a single sample analysis. Ten microliters of deuterated cotinine and 22.5 µL of 5aminoquinoline were added to the urine sample prior to sealing the vial as internal standards for cotinine and nicotine quantification. The vial was heated at 80 °C for 20 minutes in an aluminum block heater (Fisher Chemical Company). The SPME needle was passed through the septum, and the extraction fiber was exposed to the headspace above the urine for exactly 5 minutes. The needle was removed from the vial after retracting the fiber, and inserted into the injection port of the GC/GCD. The analytes were desorbed from the fiber at 250 °C for 9.5 minutes.
our laboratories, students were given the option of collecting authentic urine samples or using synthetic samples prepared by us. Students using authentic samples were asked to design their own hypothesis to test during the 4-hour lab period. Some students chose to examine how the cotinine level varied in active versus passive smokers. Some monitored dermal versus oral exposure to nicotine. Others monitored the concentration of cotinine in cigar versus cigarette smokers. Still others examined whether gender or ethnic differences were discernable in their results. For students who chose to analyze synthetic samples, we suggested a testable hypothesis based on prior knowledge of how the synthetic samples were prepared. Results obtained using SPME (Table 1) detected cotinine in two of our thirteen subjects, although our measured cotinine concentrations overall were lower than cotinine concentrations reported in smoker’s urine by other investigators (9, 10). Cotinine concentrations in smoker’s urine range between 0.12–3.20 mg/L (11). Nonsmoker urine cotinine levels range between 0.005–0.077 mg/L (12). In our experiment, SPME was not as sensitive as the procedures used by these other groups, but our method required less sample handling and urine exposure. Students were asked to consider what some of the limitations of the experimental procedure might be, such as the effect of the urine water volume on the concentration of cotinine detected. One method for “correcting” cotinine concentrations between samples is to measure an additional parameter such as urinary creatinine concentration, and to normalize to this value.4 a 1.5
1.0
0.5
0.0 0.00
0.05
0.10
0.15
0.20
0.25
Nicotine Concentration / (mg/L) 7
Area Ratio (m/z 98/101)
urine containing known amounts of nicotine and cotinine can be prepared ahead of time and used to illustrate the same principles.
6
b
5 4 3 2 1 0 0
2
4
6
8
10
12
Cotinine Concentration / (mg/L) Figure 1. Calibration curves for a) nicotine; and b) cotinine in urine. Conditions are listed in the text.
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Conclusions SPME provides a fast, inexpensive, reproducible way to analyze biological samples for nicotine metabolites in urine and sputum with a minimum of sample exposure. The results demonstrate that the presence of cotinine excreted in urine or sputum can be quantified using headspace analysis. By using a deuterated standard for cotinine quantitation, the effects of extraction and instrumental error are minimized. W
Supplemental Material
Student and teacher instructions are available in this issue of JCE Online. Acknowledgements We gratefully acknowledge the support of Perkin Elmer for their contribution to the ANALEST lab. This work has been financially supported by a Cottrell College Science Award from the Research Corporation awarded to AEW (CC4977), and a Dana Internship awarded through Dickinson College to DMK. Notes 1. Useful information on SPME theory, equipment, optimization, and applications can be downloaded from the Supelco Web site at http://www.supelco.com (accessed May 2002). 2. Synthetic urine can be purchased inexpensively from Ward’s Biology Supply (Rochester, NY). 3. We also used a 100 µm polydimethylsiloxane fiber to sample the headspace, but achieved more consistent results with the polyacrylate fiber. 4. Creatinine, the end product of creatine metabolism, is a normal constituent of urine. Typical urine concentrations are about
1260
25 mg per kg body weight. Inexpensive colorimetric diagnostic kits are available for this purpose from Sigma.
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