6.5 © Springer-Verlag Berlin Heidelberg 2005 II.6.5 Nicotine and cotinine by Mariko Fukumoto Introduction Nicotine is a main water-soluble alkaloid being contained in the tobacco plant ( Nicotiana taba- cum), which acts as an inducing compound for smoking-dependence. Twelve kinds of nicotine metabolites are known in rats [1]. Among them, cotinine is the major metabolite of nicotine; the half-life (T 1/2 ) of cotinine is much longer (10–20 h) than that of nicotine (24–84 min).  e qualitative analysis of cotinine in urine and saliva is being thus carried out as indicators for smoking during its abstinence therapy and for con rming passive smoking.  e cases of ingestion of cigarettes or their butts are household accidents taking place very frequently in Japan.  e nicotine contents being contained in a single cigarette are 7–24 mg [2], which is su cient to kill an infant; but thanks to its vomiting-stimulating action or delayed absorption, the fatalities due to such accidental ingestion are rare. However, a er suicidal in- gestion of nicotine sulfate solution (an insecticide) and of cigarette extract solution obtained by boiling, many people are being brought to critical care medical centers; in such cases, they may be fatal without appropriate and early treatments. For analysis of nicotine and cotinine in human blood and/or urine, methods by GC [3–5], GC/MS [6–8] and HPLC [9–13] were reported. In this chapter, the methods for GC/MS analy- sis of nicotine and cotinine in blood and for HPLC (UV detection) analysis of nicotine in to- bacco extract solutions are presented. GC/MS analysis of nicotine and cotinine in blood and urine Reagents and their preparation • Nicotine and cotinine (Sigma, St. Louis, MO, USA and other manufacturers) are dissolved in the Milli Q water (Millipore, Bedford, MA, USA) to prepare  xed concentrations of their aqueous solutions.  e calibration curves are constructed using human blank sera obtained from healthy nonsmokers; various concentrations of nicotine or cotinine are spiked into the blank sera • 5-Aminoquinoline (internal standard, IS, Aldrich, Milwaukee, WI, USA) is dissolved in the Milli Q water to prepare its 100 ng/mL solution •  e solvent mixture of dichloromethane/isopropyl ether (85:15) is prepared just before use. 500 Nicotine and cotinine GC/MS conditions GC column: a DB-5MS capillary column (30 m × 0.25 mm i. d.,  lm thickness 0.25 µm, J & W Scienti c, Folsom, CA, USA). Instrument: a FinniganMAT GCQ GC/MS system ( ermoFinnigan, San Jose, CA, USA). Column temperature: 40 °C (2.5 min, split mode) → 30 °C/min → 145 °C → 15 °C/min → 220 °C; injection temperature: 250 °C; carrier gas: He; linear velocity: 40 cm/s; ionization: EI; electron energy: 70 eV; detection mode: selected ion monitoring (SIM); fragment ions to be used for SIM and each retention time are shown in > Table 5.1 (total time for the measure- ments, 11 min). Procedure i. A 300-µL aliquot of serum (or urine) is mixed with 20 µL of IS solution and 2 µL of 1 M NaOH solution. ii.  e above mixture is poured into a ChemElut column a (Varian, Harbor City, CA, USA) and le at room temperature for 5 min; the target compounds are eluted with 3.0 mL of dichloromethane/isopropyl ether (85:15). iii.  e eluate is evaporated to dryness under a stream of nitrogen; the residue is dissolved in 30 µL ethyl acetate and 1 µL of it is injected into GC/MS. Assessment and some comments on the method For pretreatments of clinical specimens for analysis of nicotine and cotinine, methods by liquid-liquid extraction [3–5], solid-phase extraction [9], headspace/solid-phase microextrac- tion (SPME) [6] and Extrelut (diatomite) extraction [7, 8, 10–13] were reported. For the liquid-liquid extraction, repeated extraction procedures (3 times on average) are required; recovery rates vary due to emulsion formation, thus resulting in poor reproduc- ibility.  e solid-phase extraction is easier in operationality than liquid-liquid extraction. However, nicotine exists in a liquid form at room temperature; in view of the volatility of nicotine, the solid-phase extraction may not be suitable for nicotine analysis. For every extraction method, as much as 1–5 mL of a specimen is required. For clinical analysis of nicotine and cotinine dealing with many specimens, various technical problems should be overcome. ⊡ Table 5.1 Molecular weights, fragment ions and retention times for nicotine, cotinine and 5-amino- quinoline Molecular weight Fragment ion (m/z) Retention time (min) nicotine 162.23 84, 133, 161 8.0 cotinine 176.22 98, 176 10.4 5-aminoquinoline 144.18 144 10.0 501 In the extraction using a diatomite column, the water (containing target compound(s)) of a specimen adsorbs to the diatomite surface to act as a stationary phase; during the passage of an organic solvent through the diatomite column, the contact between the organic and aqueous phases makes the target compounds liquid-liquid extracted into the organic phase.  ere is no emulsion formation, and excellent cleanup can be realized with high recovery rates. As mentioned above, Extrelut columns were used in many reports; however the author used ChemElut columns made of similar diatomite, which are relatively cheap and easy in handling. In every extraction method, the  nal extracts are diluted more than 10 times; therefore, the  nal solution should be evaporated to dryness, followed by the dissolution of the residue in a small amount of an organic solvent to be injected into GC/MS.  e author tried various or- ganic solvents to test recovery rates of nicotine; it was found that an appreciable amount of nicotine is lost during the condensation (evaporation) step. Especially, when water-soluble or- ganic solvents such as acetone, acetonitrile, methanol and ethanol are used, the loss of nicotine during evaporation is remarkable; the recovery rates also become variable. It is known that the boiling point of nicotine is lowered upon its mixing with acetone, by the action of azeotropic e ects, causing more volatility of nicotine. As extraction solvents for the diatomite columns, dichloromethane, diethyl ether, and di- chloromethane plus isopropyl alcohol or isopropyl ether are being used. In the author’s experi- ence, dichloromethane/isopropyl ether (85:15) gave the best recovery rate. To prevent nicotine from its loss due to evaporation, hydrochloric acid methanolic solution is sometimes added.  e author also tried the addition of various acids including hydrochloric acid, but any acid addition could not improve the recovery rates.  e adsorption of nicotine and cotinine to glassware was tested, because there was a report, in which glassware a er the inactivation treatment (silylation) had been used. However, no adsorption of the compounds was found. SIM chromatogram for nicotine, cotinine and 5-aminoquinoline (IS) extracted from human serum. The peaks of nicotine and cotinine were constructed by combining peak areas at m/z 84, 133 and 161, and at m/z 98 and 176, respectively. The peak of IS is detected with the peak area at m/z 144 only. ⊡ Figure 5.1 GC/MS analysis of nicotine and cotinine in blood and urine 502 Nicotine and cotinine > Figure 5.1 shows an SIM chromatogram for nicotine and cotinine extracted from a clinical specimen.  e detection limit by this method was 5 ng/mL (S/N = 3) for both nicotine and conitine. HPLC analysis of nicotine and cotinine in blood and urine Reagents and their preparation •  e standard solutions of nicotine and cotinine, and spiked serum specimens are prepared as described in the GC/MS section. • 5-Aminoquinoline (IS) is dissolved in acetonitrile to prepare 500 ng/mL solution. HPLC conditions HPLC column: Mightysil RP18 (250 × 4.6 mm i. d., particle size 5 µm, Kanto Chemicals, To- kyo, Japan). Instrument: a Hitachi (Tokyo, Japan) HPLC system (pump: Hitachi-7100; data processor: Hitachi D7500; detector: Hitachi L7400 UV detector). Mobile phase: methanol/water/0.1 M acetate bu er solution (pH 4.0)/acetonitrile (13:65:20:2) is adjusted to pH 6.3 with triethylamine. Flow rate: 1.0 mL/min; detector wavelength: 262 nm; analysis time: about 25 min; reten- tion times: nicotine 8.5 min, cotinine 17.8 min and IS 22.3 min. Procedure i. A 100-µL aliquot of serum is mixed well with 100 µL IS solution in a sample tube. ii. A er centrifugation at 10,000 rpm for 10 min, the supernatant fraction is mixed with a saturating amount of sodium carbonate to separate the acetonitrile phase from the aqueous phase. iii. A er centrifugation at 10,000 rpm for 10 min, the upper phase (acetonitrile layer) is trans- ferred to a glass vial containing 50 mg anhydrous sodium sulfate and mixed well for dehy- dration. iv. A 20-µL aliquot of the above supernatant solution and 20 µL mobile phase are drawn into a microsyringe to mix b them in the syringe. An aliquot of the mixture is injected into HPLC. Assessment and some comments on the method In many reports on HPLC analysis of nicotine with a UV detector (UV: 262 or 254 nm), liquid- liquid extraction or diatomite column extraction was used.  ey used as large as 1–5 mL serum for each analysis and  nally condensed the extract solution by evaporation; their methods resulted in the detection limit as low as 5 ng/mL. However, as mentioned above, the loss of 503 nicotine by evaporation is inevitable and the reproducibility is not good by the above methods.  erefore, in this method, the author adopted the deproteinization method with acetonitrile to obtain excellent reproducibility. To the acetonitrile supernatant solution, an excessive amount of sodium carbonate is added to separate the acetonitrile layer from the aqueous one.  e acetonitrile layer can be immediately injected into HPLC; by mixing with the same volume of the mobile phase, the height of acetonitrile peak can be suppressed. As mobile phase, acetate bu er solution and triethylamine were used, because such a vola- tile mobile phase will be able to be used under the same conditions for LC/MS analysis c in the future. > Figure 5.2 shows an HPLC chromatogram for nicotine and cotinine in rat serum a er a single oral dose of cigarette extract solution. HPLC analysis of nicotine in cigarettes On the packages of cigarettes, the amounts of nicotine and tar are described.  e values show the amounts of nicotine and tar being contained in the smoke discharged from a piece of ciga- rettes, measured under a  xed condition using a smoking machine; such a value for nicotine is called “ nicotine yield”. Nowadays, the nicotine and tar yields for Japanese domestic cigarette brands are 0.1–2.4 mg and 1–24 mg, respectively; the cigarettes with low nicotine yields are called “low nicotine (tar) cigarettes”. To minimize the damage to health by smoking, smokers tend to choose low nicotine yield cigarettes. However, actual amounts of nicotine being con- tained in a piece of cigarettes (nicotine content) are 7–24 mg [2]. Upon smoking the low nico- HPLC chromatogram for nicotine, cotinine and IS in rat serum after oral dose of cigarette extract solution. ⊡ Figure 5.2 HPLC analysis of nicotine in tobaccos 504 Nicotine and cotinine tine cigarettes, the smokers unconsciously inhale the smoke to reach the lungs deeply, because such cigarettes give light feeling to them; the deep inhalation results in the absorbed amounts of nicotine to be as large as 2–3 mg per piece, which are similar to those for high nicotine ciga- rettes. Moreover, it is being pointed out by specialists that the number of pieces being consumed tends to be increased for the low nicotine cigarettes, because of the sense of security [14]. In this section, a method for HPLC analysis of nicotine in cigarettes is described; it cannot be applied to measurements of nicotine in human specimens, because of its low sensitivity as compared with that of GC/MS, but can be applied to measurements of high levels of nicotine in specimens of experimental animals. Reagents and their preparation  e standard solutions of nicotine and cotinine, and spiked serum specimens are prepared as described in the GC/MS section. HPLC conditions HPLC column: Inertsil ODS-3 (150 × 4.6 mm i. d., particle size 5 µm, GL Sciences, Tokyo, Ja- pan). Instrument: a Hitachi HPLC system (the same as above). Mobile phase: 67 mM phosphate bu er solution (pH 7.0)/acetonitrile (88:12) containing 2 mM sodium hexanesulfonate (Sigma, St. Louis, MO, USA and other manufacturers). Flow rate: 1.0 mL/min; detection wavelength: 262 nm; analysis time: about 15 min; reten- tion time of nicotine: 12 min. Procedure i. A  lter and paper are carefully removed, and the chopped tobacco is weighed. ii.  e tobacco is crushed using a blender into powder for 60 s, and a  xed amount of it is suspended in 100 mL of methanol/0.1 M NaOH solution (1:1) and extracted by sonication for 60 min. iii.  e supernatant solution is passed through a  lter paper, diluted 50-fold with 67 mM phosphate bu er solution (pH 7.0) and passed through a Millipore  lter (PTFE, 0.5 µm). A  xed volume of the  ltrate is injected into HPLC. iv. In this method, quantitation is made without IS using the external calibration method. For HPLC analysis of nicotine in serum specimens, solid-phase extraction should be made according to the method described in the section of GC/MS analysis in this chapter. Assessment and some comments on the method  is method can be used for determination of fatal levels (not lower than 0.5 µg/mL) of nico- tine in serum. To obtain more excellent separation ability, the composition ratio of the 67 mM 505 ⊡ Table 5.2 Nicotine contents and nicotine yields for 33 brands of cigarettes being sold in Japan. domestic cigarette brands (n=16) brand names nicotine (1) (mg) contents (%) nicotine yield (mg) tobacco content (g) peace (2) 24.0 2.35 2.4 1.02 peak king size 18.3 2.34 2.4 0.78 hi-light* 14.6 2.02 1.4 0.72 echo 13.7 2.43 1.1 0.57 hope 13.3 1.85 1.2 0.72 caster mild* 13.0 2.00 0.4 0.65 marlboro 12.8 1.61 1.0 0.79 cherry 12.6 1.86 1.2 0.68 mildseven* 12.6 1.87 0.9 0.67 sevenstar* 12.4 1.77 1.3 0.70 cabin mild* 12.0 1.86 0.7 0.65 mildseven super light* 11.8 1.76 0.5 0.67 mi-ne 11.5 1.70 1.0 0.68 mildseven lights 10.9 1.63 0.8 0.67 cabin ultra mild* 8.84 1.55 0.2 0.57 frontier lights 6.94 1.14 0.1 0.61 average 13.1** 1.86 1.04*** 0.70 (±SD) (3.79) (0.33) (0.66) (0.11) phosphate bu er solution/acetonitrile can be changed to 93:7; the concentration of the ion- pairing reagent sodium hexanesulfonate can be increased up to 5 mM. Under these conditions, the analysis time was about 22 min; the retention times of nicotine and cotinine were 20 and 16 min, respectively. > Table 5.2 shows nicotine contents and nicotine yields for 33 brands of cigarettes being sold in Japan. It seems useful for the assessment of toxicity, when a cigarette ingestion incident takes place. Poisoning cases, and toxic and fatal concentrations  e ingestion of 2–5 mg nicotine can provoke vomiting, and the estimated minimal oral lethal dose is 40–60 mg in adults. When amounts of nicotine lager than the toxic dose are ingested, acute symptoms, such as salivation, convulsion and respiratory depression will develop in 1 min–1 h, and the victims may die  nally in severe cases. However, there were survived cases a er ingestion of 1–4 g nicotine. In an autopsy cases, in which victims had ingested 20–25 g of nicotine sulfate and had died within 1 h, the blood nicotine concentrations were 11–63 µg/mL (29 µg/mL on average) [15]. In autopsy cases of nicotine poisoning reported in Japan, the blood concentrations were 64 [16] and 6.3 µg/mL [17]. In animal experiments using male Poisoning cases, and toxic and fatal concentrations 506 Nicotine and cotinine Sprague-Dawley rats with single oral administration each of tobacco exudates or the standard nicotine solutions, their fatal blood nicotine concentrations were 6.29–34.6 and 0.89–57.5 µg/ mL, respectively [18]. A 47-year-old male attempted suicide by ingesting cigarette extract solution obtained by boil- ing (estimated ingested nicotine amount: 500 mg). One hour and 20 minutes later, the stomach contents were removed to some extent by aspiration with a tube; by measuring the concentration of nicotine in the stomach contents, it was estimated that about 250 mg nicotine still remained in the stomach and the intestine. A er gastrolavage and administration of activated charcoal were performed, a blood specimen was sampled for measurement of nicotine concentration about 2 h a er ingestion; the result was 79.5 ng/mL.  e patient showed early poisoning symptoms, such as, hypersalivation, nausea and vomiting, but was discharged 2 days later, because of improvement. In Japan, the incidence of accidental ingestion of cigarettes by small infants is very high. By ingestion of one piece of cigarettes or of more than 3 cigarette butts by infants, there is a high possibility of the appearance of poisoning symptoms [19]. In a report describing the blood ⊡ Table 5.2 (Continued) Imported cigarette brands (n=17) brand names nicotine (1) (mg) contents (%) nicotine yield (mg) tobacco content (g) camel 15.4 2.04 0.9 0.75 dunhill ultimate light 15.0 2.35 0.1 0.64 next ‡ 12.7 1.88 0.1 0.58 parliament 100 ‡ 12.5 1.62 0.8 0.77 lark mild ‡ 11.6 1.59 0.7 0.73 lark 11.3 1.55 0.9 0.73 kent1 ‡ 11.2 1.71 0.1 0.66 vantage 11.1 1.78 0.7 0.62 salem 10.9 1.63 1.0 0.67 virginia slims lights ‡ 10.9 1.63 0.5 0.67 lucky strike ‡ 10.5 1.57 0.8 0.67 island super lights 10.2 1.44 0.6 0.71 kent 9.97 1.44 0.9 0.69 lark super lights ‡ 9.66 1.62 0.4 0.60 philip morris one ‡ 9.53 1.95 0.1 0.49 philip morris super lights ‡ 9.10 1.59 0.4 0.57 salem slim lights 8.03 1.32 0.5 0.61 average 11.2** 1.69 0.56*** 0.66 (±SD) (1.98) (0.26) (0.30) (0.08) (1) the advertised level determined by a smoking machine. (2) non-filtered cigarette. * the best ten brands of domestic cigarettes sold in 1995. ** p < 0.05. *** p < 0.01. ‡ the ten top-selling brands of imported cigarettes in 1995. 507 nicotine concentrations in the cases of accidental cigarette ingestion by infants, the estimated peak blood concentrations for symptomatic and asymptomatic patient groups were 52.9 and 51.0 ng/mL, respectively; there is no signi cant di erence in the concentration between the two groups [20]. In Europe and the United States, even cases of nicotine poisoning, due to ni- cotine chewing gum and nicotine patch, were reported; such a type of acute poisoning cases may appear also in Japan according to widespread use of such smoking-abstinence assisting drugs containing nicotine. Notes a) For the ChemElut columns, various sizes of the columns are available; 0.3–300 mL volumes of sample solution can be applied by selecting each size. Upon elution, the manipulations, such as aspiration under reduced pressure, are not necessary. When a calibration curve with 5 plots is constructed by measuring 10 samples (in duplicate for each plot), the cost for one sample is 570 yen for ChemElut and 1,800 yen for Extrelut. b) To well mix the acetonitrile extract with the same volume of the mobile phase inside a microsyringe, a small amount of air is also aspirated into the syringe to include small bubbles in the solution mixture, followed by its gentle shakings 3–5 times. c)  ere is one report describing LC/MS analysis of nicotine [10], but it is not so practical, because it requires as much as 5 mL volume specimens. References 1) Kyerematen GA, Taylor LH, DeBethizy JD et al. (1988) Pharmacokinetics of nicotine and 12 metabolites in the rat. Drug Metab Dispos 16:125–129 2) Fukumoto M, Kubo H, Ogamo A (1997) Determination of nicotine content of popular cigarettes. Vet Hum Toxicol 39:225–227 3) Hengen N, Hengen M (1978) Gas liquid chromatographic determination of nicotine and cotinine in plasma. Clin Chem 24:50–53 4) Feyerabend C, Russel MAH (1979) Improved gas chromatographic method and microextraction technique for the measurement of nicotine in biological fluids. J Pharm Pharmacol 31:73–76 5) Kogan MJ, Verebey K, Jafee JH et al. (1981) Simultaneous determination of nicotine and cotinine in human plasma by nitrogen detection gas-liquid chromatography. J Forensic Sci 26:6–11 6) Yashiki M, Nagasawa N, Kojima T et al. (1995) Rapid analysis of nicotine and cotinine in urine using headspace- solid phase microextraction and selected ion monitoring. Jpn J Forensic Toxicol 13:17–24 7) Azuma E, Sashikuma F, Itani S et al. (1986) Simultaneous determination of nicotine and cotinine in urine by gas chromatography. Eisei Kagaku 32:276–280 (in Japanese with an English abstract) 8) Mizunuma H, Hirayama Y, Sakurai S et al. (1982) Sensitive determination of nicotine in urine and blood. Eisei Kagaku 28:13–17 (in Japanese with an English abstract) 9) Kyerematen GA, Damiano MD, Dvorchik BH et al. (1982) Smoking-induced changes in nicotine disposition: application of a new HPLC assay for nicotine and its metabolites. Clin Pharmacol Ther 32:769–780 10) Pacifici R, Pichini S, Altieri I et al. (1993) Determination of nicotine and two major metabolites in serum by solid- phase extraction and high-performance liquid chromatography, and high-performance liquid chromatogra- phy-particle beam mass spectrometry. J Chromatogr 612:209–213 11) Zuccaro P, Altieri I, Rosa M et al. (1995) Solid-phase extraction of nicotine and its metabolites for high-perfor- mance liquid chromatographic determination in urine. J Chromatogr B 668:187–188 12) Zuccaro P, Altieri I, Rosa M et al. (1993) Determination of nicotine and four metabolites in the serum of smokers by high-performance liquid chromatography with ultraviolet detection. J Chromatogr 621:257–261 Poisoning cases, and toxic and fatal concentrations 508 Nicotine and cotinine 13) Pichini R, Altieri I, Pacifici R et al. (1992) Simultaneous determination of cotinine and trans-3′-hydroxycotinine in human serum by high-performance liquid chromatography. J Chromatogr 577:358–361 14) U.S. Department of Health and Human Services (2000) Reducing tobacco use: a report of the Surgeon General, Atlanta, Georgia: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health 15) Baselt RC, Cravey RH (1977) A compendium of therapeutic and toxic concentrations of toxicologically signifi- cant drugs in human biofluids. J Anal Toxicol 1:81–103 16) Fukui M, Takinomi T (1970) A case report of nicotine poisoning. Jpn J Legal Med 24:83 (in Japanese) 17) Takayasu T, Ohshima T, Lin Z et al. (1992) An autopsy case of fatal nicotine poisoning. Jpn J Legal Med 46:327– 332 (in Japanese with an English abstract) 18) Fukumoto M, Shirai M, Akahori F et al. (2000) Acute toxicity of tobacco in rats. J Toxicol Clin Toxicol 38:517– 518 19) Smolinske SC, Spoerke SK, Spiller SK et al. (1988) Cigarette and nicotine chewing gum toxicity in children. Hum Toxicol 7:27–31 20) Hoshino K, Matsuura H, Arimoto K et al. (1996) Blood concentrations of nicotine and cotinine after accidental tobacco ingestion. Jpn J Pediatr 100:1387–1391 (in Japanese with an English abstract) . conitine. HPLC analysis of nicotine and cotinine in blood and urine Reagents and their preparation •  e standard solutions of nicotine and cotinine, and. (24–84 min).  e qualitative analysis of cotinine in urine and saliva is being thus carried out as indicators for smoking during its abstinence therapy and