Scientific paper Determination of Nicotine and Cotinine in Urine by Headspace Solid Phase Microextraction Gas Chromatography with Mass Spectrometric Detection Irena Br~i} Kara~onji,* Ljiljana Skender, Vi{nja Kara~i} Clinical Toxicological Chemistry Unit, Institute for Medical Research and Occupational Health, Ksaverska c. 2, Post Office Box 291, HR-10001 Zagreb, Croatia. Received: 25-09-2006 Paper based on a presentation at the 12th International Symposium on Separation Sciences, Lipica, Slovenia, September 27-29, 2006. Abstract A simple and rapid method for determination of low concentrations of nicotine and cotinine in urine has been developed. The method is based on headspace solid phase microextraction (HS-SPME) followed by gas chromatographic determination and mass spectrometric detection (GC-MS). The calibration curves were linear (r > 0.9998) over the concentration range tested (1-500 |g L-1), with detection limits of 1.1 |g L-1 and 0.9 |g L-1 for nicotine and cotinine, respectively. The repeatability for nicotine and cotinine (expressed as relative standard deviation) was < 9%. The accuracy of the method ranged from 90 to 99%. The method was applied for the quantitative analysis of nicotine and cotinine in urine samples collected from 30 non-smokers, 15 without any environmental tobacco smoke (ETS) exposure (group I) and 15 who reported exposure to ETS (group II). There were statistically significant differences between groups I and II for nicotine (p < 10-4; ranges 2.1-28.0 and 10.1-499.7 |g L-1) and cotinine (p < 10-5; ranges < 0.9-15.0 and 14.5-200.9 |g L-1). Good sensitivity, short analysis time, small sample volume, no solvent use and sample preparation, make the method convenient for determination of nicotine and cotinine in nonsmokers' urine. Keywords: cotinine, nicotine, nonsmokers, urine, HS-SPME, GC-MS. 1. Introduction Environmental tobacco smoke (ETS) is a widespread pollutant despite the growing awareness of its adverse effects on the health of nonsmokers. Many epide-miological studies reported that ETS can cause lung cancer in adult nonsmokers and that children of parents who smoke have increased frequency of respiratory symptoms and acute lower respiratory tract infections, as well as reduced lung function.1 Recent analyses of a few epidemio-logical and toxicological studies2 suggested that ETS exposure might be a risk factor for cardiovascular disease. The primary components of ETS are sidestream smoke emitted from the smoldering tobacco between puffs and exhaled mainstream smoke from the smoker. ETS, although diluted compared with the smoke inhaled by active smoker, contains many of the same carcinogenic and toxic agents.1 ETS has been classified as a class A carcinogen or human lung carcinogen by U. S. Environmental Protection Agency (US EPA)1 and as carcinogenic to humans by International Agency for Research on Cancer (IARC).3 The biological monitoring of nonsmokers exposed to ETS is the first step toward evaluation of the toxicologi-cal effects of ETS. Nicotine and its main metabolite coti-nine are widely used as biomarkers of ETS exposure and can be measured in blood,4 saliva,4-6 urine,4,6-11 and hair.6,12-14 Concentrations of biomarkers in biological fluids reflect recent exposure, while their concentrations in hair reflect long-term exposure. Only 17% of nicotine is eliminated as cotinine in urine.15 Cotinine is mostly determined in urine, a biological medium easy to obtain. Because of the longer urinary half-life of cotinine compared with nicotine (20 vs. 2 h), cotinine in urine is currently considered to be the biomarker of choice for ETS exposure assessment.16 The half-life of cotinine makes it a good indicator of ETS exposure over the previous day or two. A number of analytical methods have been reported for the analysis of nicotine and cotinine in urine of non-smokers such as gas chromatography using mass spectro-metry (GC/MS)4,6 and nitrogen-phosphorous detector (NPD),10,17 high performance liquid chromatography (HPLC),11,17 radioimmunoassay (RIA),18 and enzyme lin- ked immunosorbent assay (ELISA).1719 Chromatographic techniques are preferred over the other types of analyses because they are highly sensitive, specific, and they can analyse both nicotine and cotinine in a single assay.4,6,11 These techniques are often complex and time consuming because they include liquid-liquid 4,6,10,11,17 or solid phase7,20 extraction and preconcentration step prior to analysis. In order to minimize sample preparation and concentrate analytes in a solvent-free manner, simple and rapid headspace solid phase microextraction (HS-SPME) procedure followed by gas chromatographic determination and mass spectrometric detection was developed. SPME is a sampling technique developed by the Pawliszyn research group in the late 1980s,21 in which a fused-silica fibre coated with a thin layer of a selective coating is used to trap and concentrate volatile and semi-volatile analytes directly from the headspace of samples. Although exposure to ETS quite often involves determination of nicotine and cotinine in urine, there are no data on using HS-SPME as sampling technique for assessment of exposure to ETS. The method was applied for the quantitative analysis of nicotine and cotinine in urine samples collected from 30 nonsmokers. 2. Experimental 2.1. Reagents Nicotine, cotinine and diphenylamine (internal standard) were purchased from Sigma (St. Louis, MO, USA). Methanol (gradient grade for liquid chromatography) and potassium carbonate were products of Merck (Darmstadt, Germany). Ultra-pure water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). 2.2. Standard Preparation The stock standard solution for nicotine and cotinine (1 g L-1 of methanol) was prepared as aqueous solution and then diluted with ultra-pure water to working standards. These standards were stored at 4 °C and were stable up to one month. Calibration standards were freshly prepared daily by spiking blank (analyte-free) urine of nonsmoker (no detectable amounts of nicotine and cotinine) with aliquots of working standard solutions of nicotine and cotinine. 10 pL of the internal standard solution of diphenylamine (10 mg L-1) prepared in methanol was added to each calibration level. 2.3. Urine Sample Collection The study was carried out in March 2006 and involved 30 nonsmokers, 15 without any ETS exposure and 15 who reported exposure to ETS because someone in their environment smoked. Median age of subjects was 33 years, 60% were females. All subjects completed a short questionnaire and exposure to ETS, in the 2-day period preceding the sampling, was recorded. The first urine samples were collected in the morning in clean 80-mL bottles and analysed on the day of collection. 2.4. Extraction Procedure One mL of urine sample and 10 pL of diphenylami-ne (10 mg L-1 of methanol) as internal standard for nicotine and cotinine were added in the 6-mL clear glass vial containing 1.3 g of potassium carbonate. The addition of the salt improves extraction efficiency. The vial was sealed with PTFE septum and holed aluminum cap and heated at 80 °C for 60 min. Fused silica fiber 10 mm long with an 85-pm thick polyacrylate coating (PA) was purchased from Supelco (Bellefonte, USA). The SPME needle was pass through the septum, and the extraction fibre was exposed to the headspace above the urine at 80 °C for 15 min. The needle was removed from the vial after retracting the fiber, and inserted into the injection port of GC/MS where analytes were desorbed at 280 °C for 10 min. 2.5. Gas Chromatography-mass Spectrometry The GC-MS analysis of nicotine and cotinine was carried out on Varian 3400 CX gas chromatograph equipped with Saturn ion trap mass spectrometer operating in the electron impact (EI) mode. Compounds were separated on Rtx-5 capillary column (5% diphenyl-95% di-methylpolysiloxane), 30 m x 0.25 mm I. D., 0.25 pm film thickness (Restek, Bellefonte, USA). The oven temperature was kept at 50 °C for 1 min, and then increased at 230 °C at rate of 20 °C min-1. Late-eluating compounds were removed by increasing the temperature to 280 °C at rate of 50 °C min-1. The transfer line temperature was set at 260 °C. The flow rate of the helium was 1 mL min-1. Selected ion monitoring mode was used in the analysis. Monitored ions were m/z 162, 84, 133 (nicotine), m/z 176, 98, 118 (cotinine) and m/z 169, 168 (diphenylamine). Quantification was based on the peak area integration at m/z 162 (nicotine), 176 (cotinine) and 169 (I.S.). 2.6. Method Validation To construct 5- point calibration curves, blank urine sample spiked with nicotine and cotinine at concentration ranging from 1 to 500 pg L-1 was prepared and analysed using the above procedure. Repeatability and accuracy were evaluated by analysing blank urine samples spiked with two different concentrations of nicotine and cotinine (20 and 100 pg L-1) in six replicates. Detection limits (DL) were calculated by a signal-to-noise ratio of 3. 2.7. Statistics Because the data did not follow a normal distribution, the results within groups were presented as median and range, and the significance of the difference between groups was calculated by using the Mann-Whitney U-test. The test was considered statistically significant when p < 0.05. Undetectable concentrations were set at 0.5xDL. We performed all statistical analyses using Statistica for Windows, release 5.5 (StatSoft Inc., Tulsa, Oklahoma, USA). 3. Results and Discussion The described method permits the separation, sensitive, fast and reliable determination of low ^g L-1 nicotine and cotinine levels in urine. Calibration curves were linear over the concentration range 1-500 ^g L-1 with correlation coefficients > 0.9998. Relative standard deviations (RSD) and accuracy of the method ranged between 5-9% and 90-99%, respectively. Analytical parameters of the method are presented in Table 1. They are comparable with repeatability and accuracy reported by authors using the liquid-liquid (LL) 4,6 or solid phase (SP) 20 extraction methods. Detection limits were 1.1 ^g L-1 and 0.9 ^g L-1 for nicotine and cotinine, respectively. Shin et al.4, Torano et al.6 and James et al.20 reported lower detection limits for nicotine and cotinine in urine using LL and SP extraction methods. However, our results suggest that the proposed HS-SPME extraction method is sensitive enough to distinguish passive smokers from those who reported no ETS exposure. Figure 1 shows total and selected ion chromato-grams of nicotine and cotinine in the urine of nonsmoker exposed to ETS. No interfering peaks were observed in the urine samples studied. Urine is usually a sample of choice for determining exposure to ETS since collection procedure is non-invasive. Our method was applied for determination of nicotine and cotinine in urine of 15 nonsmokers without any ETS exposure and 15 who reported exposure to ETS. Concentrations of nicotine and cotinine in the urine of these subjects are presented in Table 2. Table 1: Repeatability and accuracy of the method (N = 6). Compound Concentration Repeatability Accuracy Compound (^g/L-1) (RSD %) (%) Nicotine 2G 8.5 9l.5 1GG 5.4 98.8 Cotinine 2G 6.5 9G.1 Nicotine 1GG 5.9 9l.2 Cotinine Table2: Mass concentration of nicotine and cotinine in nonsmokers' urine. Mass concentration (^g/L-1) Nonsmokers without ETS exposure (N = 15) Nonsmokers with ETS exposure (N = 15) Median (Range) 9.9 (2.1-28.G) 6.2 ( 0,9998) v konsentracijskem intervalu, ki smo ga preverjali (1-500 |g L-1) z mejo določljivosti 1,1 |g L-1 za nikotin in 0,9 |g L-1 za kotinin. Ponovljivost, izražena kot standardni odmik, je pod 9 %. Točnost je med 90 in 99 %. Metodo smo uporabili za kvantitativno določitev nikotina in kotinina v vzorcih urina, ki smo jih zbrali pri 30 nekadilcih, od tega pri 15 takih, ki niso bili izpostavljeni okoljskemu tobačnemu dimu (skupina I), in 15 takih, ki so javili tako izpostavljenost (skupina II). Ugotovili smo statistično značilne razlike med skupinama I in II pri vsebnosti nikotina (p < 10-4; obseg koncentracij 2,1-28,0 in 10,1-499,7 |g L-1) in kotinina (p < 10-5; obseg koncentracij <0,9-15,0 in 14,5-200,9 |g L-1). Občutljivost, kratek čas analize, majhen vzorčni volumen, odsotnost topil in kakršnekoli priprave vzorca so dobre lastnosti predlagane metode za določanje nikotina in kotinina v urinu nekadilcev.