Chemosphere 119 (2015) 1200–1207
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Determination of nicotine and cotinine in meconium from Greek neonates and correlation with birth weight and gestational age at birth Nikoleta Tsinisizeli a,b,1, Georgios Sotiroudis b,1, Aristotelis Xenakis b,⇑, Katerina E. Lykeridou a,⇑ a b
Faculty of Health and Caring Professions, Midwifery Department, Technological Educational Institute of Athens, Agiou Spyridonos Str., 12210 Egaleo, Greece Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635 Athens, Greece
h i g h l i g h t s LC–MS/MS quantitation of nicotine and cotinine in meconium from Greek neonates. Concentrations of biomarkers in meconium correlated with mothers’ smoking history. Passive smokers’ concentrations were comparable to those of moderate smokers. Negative correlation between biomarker concentrations and two neonatal outcomes.
a r t i c l e
i n f o
Article history: Received 10 March 2014 Received in revised form 11 September 2014 Accepted 21 September 2014 Available online 1 November 2014 Handling Editor: J. de Boer Keywords: Meconium Tobacco Nicotine Prenatal exposure Neonatal outcomes LC–MS/MS
a b s t r a c t Tobacco exposure during pregnancy is a major factor of morbidity and mortality for both the pregnant woman and the fetus. Several studies in the past have detected and quantified tobacco smoke biomarkers in infant meconium samples. Aim of this study was to measure prenatal exposure to tobacco smoke by detecting nicotine and cotinine in meconium and to try to evaluate the extent of exposure to smoke through passive smoking as well as the relationship between tobacco biomarker meconium concentrations and neonatal outcomes. Tobacco smoke biomarkers nicotine and cotinine were detected and quantitated in meconium from tobacco exposed and non-exposed Greek neonates using liquid chromatography–tandem mass spectrometry. The study included 45 neonates from active, passive and non-smoking women during pregnancy. The results showed significant values of nicotine and cotinine concentration in neonates from both active and passive smokers which reached 125 ng g1 for nicotine and 98.5 ng g1 for cotinine and varied according to the type and level of exposure. In general nicotine and cotinine concentrations correlated with the degree of active smoking by the mother. Similarly, nicotine and cotinine were measured in the meconium of infants of passive smokers at concentrations comparable to those of infants whose mothers were moderate smokers. Our findings show that exposure of the fetus to tobacco biomarkers can be substantial even in passive maternal smoking and there is a statistically significant negative correlation between nicotine or cotinine concentrations in meconium and birth weight or gestational age at birth. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Smoking addiction is one of the most critical problems of public health. The toxic compounds present in cigarettes reach the organism either by active smoking or involuntarily by passive ⇑ Corresponding author at: Institute of Biology, Medicinal Chemistry and Biotechnology (IBMCB), National Hellenic Research Foundation (NHRF), 48 Vassileos Constantinou Ave., 11635 Athens, Greece. Tel.: +30 2107273762; fax: +30 2107273758. E-mail addresses: [email protected] (N. Tsinisizeli), [email protected] (G. Sotiroudis), [email protected] (A. Xenakis), [email protected] (K.E. Lykeridou). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.chemosphere.2014.09.094 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
smoking. In the developing fetus, nicotine crosses both the placental and the blood–brain barrier and its concentration in the fetal compartment is 15% higher than in maternal tissues (Lambers and Clark, 1996). Maternal smoking results in a decreased transfer of nutrients and oxygen to the fetus and has been associated with decreased birth weight, increased risks for premature birth, spontaneous abortion, and infant death from perinatal disorders and from sudden infant death syndrome (SIDS) (Haglund and Cnattingius, 1990; DiFranza and Lew, 1995; Anderson and Cook, 1997; Castles et al., 1999). It may also increase the risk of cognitive and neurodevelopmental delay (Weitzman et al., 2002).
N. Tsinisizeli et al. / Chemosphere 119 (2015) 1200–1207
According to a study in 2002 in the United States, percentages of pregnant women who admitted to using tobacco smoking products the last three months of pregnancy ranged from 6.8% (Utah) to 25.3% (Virginia) (Xia et al., 2011). Prenatal smoking remains one of the most prevalent factors causing infant morbidity and mortality in the United States (Dietz et al., 2010), where smoking during pregnancy accounts for as many as 161 000 perinatal deaths and 4800 infant deaths each year, with more than half of these classified as sudden infant death syndrome (SIDS) (DiFranza and Lew, 1995). Greece, on the other hand, has one of the highest percentages of adult tobacco use worldwide, and the highest adult percentage of current tobacco use among the Organisation for Economic Co-operation and Development (OECD) countries (OECD briefing note for Greece and OECD Health Data; 2013). In a study in the end of 2010 (Filippidis et al., 2013) more than 38% of adult women in Greece admitted to having smoked at least once during the past 30 d. Especially in city areas, such as Athens, smoking rates among women are higher than rural areas (Pitsavos et al., 2003). Those with higher education are actually more likely to smoke and studies have shown that this happens only in Greece and Portugal, among several European countries (Huisman et al., 2005). In a study in Crete (Vardavas et al., 2010), active smoking during pregnancy between pregnant women was reported by 36% of respondents and almost all of the non-smokers (94%) were exposed to passive smoking. In this respect, we were interested in biologically monitoring levels of exposure to tobacco smoke of Greek neonates from mothers of different types of smoking status (heavy smokers, light smokers, passive smokers, non-smokers). Several ways are available for monitoring in utero nicotine exposure, including hair, plasma, urine and meconium. Measuring the nicotine metabolites in urine reveals the most recent tobacco smoke exposure (Köhler et al., 2001). In contrast, nicotine accumulated in fetal hair reflects the smoke exposure over the last three to four months of gestation (Koren, 1995). Meconium, the first neonatal feces, presents several advantages for detecting prenatal exposure to tobacco smoke. Meconium begins to form during the twelfth gestational week, so meconium testing reflects second and mostly third-trimester exposure (Ostrea et al., 1992). Accumulation of meconium over this period of months yields higher tobacco biomarker concentrations than other neonatal matrices. Furthermore, meconium offers the advantage that it can be collected easily and non-invasively in sufficient quantity until about the second postnatal day (Ostrea et al., 2001). In the present study we determined levels of nicotine and cotinine in meconium in both active and passive smokers, as well as in mothers who self-reported no exposure to tobacco smoke. To our knowledge, the biological monitoring of tobacco biomarkers within meconium has never been performed in Greece. The method used was based on the method developed by Gray et al. (2008b). The aims of the study were (a) to compare maternal self-reported tobacco use and meconium analysis results in a group of Greek mothers, (b) compare levels of nicotine and cotinine between active and passive smokers and (c) determine if meconium concentrations can be associated with decreased birth weight and shorter gestation periods, especially in the case of heavy smoking.
2. Materials and methods In total 45 women participated in the study. The specimens of meconium were collected at GAIA maternity hospital of Athens. The material (first day meconium) was collected from the diaper of the neonates using a spatula and pooled into one plastic container per infant (120 mL urine analysis container). Samples were immediately stored at 20 °C until analysis. The estimation of
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the levels of exposure to tobacco smoke was possible through anonymous questionnaires. Each questionnaire consisted of questions asking about the following: age of the women, profession, if they smoked before pregnancy (and number of cigarettes smoked daily), if they smoked during pregnancy (and number of cigarettes smoked daily) and if they stopped smoking during pregnancy and when. Questions about the women’s family (all women were married) were the following: if the husband (or a person they lived with) smoked (and number of cigarettes smoked daily) and if he smoked in the presence of his wife and in their house. Finally, the questionnaire included the following questions about other possible cases of passive smoking: if during pregnancy the women attended closed spaces were people smoked (e.g. work), how often, when was the last time they did that and if they were exposed to second-hand smoke only outdoors or not at all. The questionnaires were completed by the mothers either during the first stage of labor or during the first hour after labor, while meconium specimens were collected during the first day of neonates’ life. Informed consent was obtained from the mother in each case. All specimens and the maternal self-report tobacco surveys were deidentified to protect personal health information. The study protocol and the informed consent document were approved by the Medical School of University of Athens. All the individuals who were classified as smokers smoked at least one cigarette daily. The individuals who had been exposed to second-hand smoke in the past 3 months (even if only outdoors) were classified as passive smokers and those who had never been aware of being exposed to tobacco smoke during the last months of pregnancy were classified as non-smokers. For the analysis approximately 0.5 g of meconium was weighed and mixed with the necessary internal standard solutions followed by a procedure essentially as described by Gray et al. (2008b). 2.1. Reagents and standards Standard solutions of ()-nicotine and ()-cotinine (1.0 mg mL1 in methanol) as well as (±)-nicotine-D4 (0.1 mg mL1 in acetonitrile) and (±)-cotinine-D3 (0.1 mg mL1 in methanol) were obtained from Cerilliant (Round Rock, TX, USA). All reagents were analytical grade or higher. Formic acid, sodium acetate, hydrochloric acid and ammonium hydroxide were purchased from Merck (Darmstadt, Germany). Ammonium acetate was acquired from Sigma Chemicals (St. Louis, MO, USA). All solvents were HPLC grade and obtained from the following suppliers: methanol and isopropanol from Carlo-Erba Reactifs-SDS (Val de Reuil, France) and dichloromethane from Lab-Scan/Poch SA (Gliwice, Poland). Ultrapure water was obtained from a Milli-Q water purification system (Millipore, Billerica, MA, USA). Solid phase extraction columns Discovery DSC-MCAX (100 mg, 3 mL) were purchased from Supelco/Sigma–Aldrich (Bellefonte, PA, USA). 2.2. Instrumentation During sample preparation, sonication was performed by a Raypa UCI-150 ultrasonic cleaner bath (Espinar SL, Barcelona, Spain). MS/MS analysis was performed using an API 3200 QTrapÒ triple quadrupole/linear ion trap mass spectrometer with an APCI source (AB Sciex, Foster City, CA, USA) which was coupled to an Agilent 1200 (Agilent, Waldbronn, Germany) HPLC system (consisting of a G1379B micro vacuum degasser, a G1312A binary pump, a G1329 autosampler and a G1316A column compartment). A nitrogen generator N300DR (Peak Scientific, Renfrewshire, Scotland) supplied auxiliary (nebulising, heater and collision) gases. Analyst software version 1.4.2 (AB Sciex, Foster City, CA, USA) was used for acquisition and data analysis. Statistical evaluations were completed with IBM SPSS 20 for Windows (Chicago, IL, USA).
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2.3. Preparation of standard solutions Standard solutions of nicotine and cotinine in methanol were stored at 20 °C. Dilutions of each standard solution in methanol created 25–10 000 ng mL1 working solutions of nicotine and cotinine. 0.5 g blank meconium were fortified with 25 lL working standards so that final standard concentrations in meconium were 1.25, 2.5, 5, 7.5, 50, 100 and 500 ng g1. Standard solutions of deuterated analogues of nicotine and cotinine were also stored at 20 °C. A 1000 ng mL1 working internal standard solution was prepared by diluting deuterated standard solution with methanol. The final deuterated internal standard concentration after fortifying 0.5 g blank meconium with 25 lL working solution was 50 ng g1. Blank meconium used for the calibration curves was meconium acquired from a woman that had not been exposed to tobacco smoke at all during pregnancy. The blank was analyzed before the analysis of the rest of the samples and showed values smaller than the limits of quantitation determined for this method both for nicotine and cotinine. 2.4. Sample preparation Meconium (0.5 ± 0.01 g) was well mixed and weighed into a 15 mL polypropylene tube. 25 lL of working standard solution (only for the creation of the calibration curve) and 25 lL of internal standard solution were added. Specimens were homogenized (2 mL methanol with 0.01% formic acid (w/v) were added) and then vortexed vigorously and sonicated for 1 h, vortexing every 5 min. Finally they were centrifuged at 8000g for 10 min. The supernatant was transferred to a round bottom screw-top vial and evaporated to dryness under nitrogen at 40 °C. The residue was reconstituted to 2 mL of 2 M sodium acetate buffer, pH 5.5 prior to solid phase extraction. Discovery DSC-MCAX solid phase extraction columns were conditioned with 3 mL methanol, 3 mL water and 2 mL 2 M sodium acetate buffer, pH 5.5. Samples were loaded and allowed to flow by gravity alone. Columns were washed with 2 mL water, dried for 1 min, washed with 1.5 mL 0.2 M aqueous hydrochloric acid, dried for 5 min, washed with 2 1 mL methanol, and finally dried for 5 min. Analytes were eluted with freshly prepared 6 1 mL dichloromethane: 2-propanol: ammonium hydroxide (78:20:2, v/v/v). After the addition of 100 lL 1% hydrochloric acid in methanol (v/v), eluates were dried under nitrogen. Samples were reconstituted in 400 lL methanol with 0.01% formic acid (w/v) and transferred to glass autosampler vials. 2.5. Mass spectrometry Atmospheric pressure chemical ionization (APCI) operating in positive mode was used for all analytes. 100 ng mL1 reference solution in methanol for each standard biomarker was directly infused using a syringe pump in order to optimize the MS/MS parameters for each compound. Source parameters were set to 40 psi curtain gas, 50 psi auxiliary gas, 35 psi nebulizer gas, 6 psi collision gas, 1.0 lA nebulizer current, and 350 °C source temperature after flow injection analysis (FIA) source optimization. Quadrupoles one and three were set to unit resolution. Data acquisition was performed in the multi reaction monitoring (MRM) mode and the following transitions were monitored: (m/z): 163.2 ? 84.2 (nicotine), 167.2 ? 136.0 (nicotine-D4), 177.2 ? 80.0 (cotinine) and 180.2 ? 80.0 (cotinine-D3). Tandem mass spectrometry parameters are presented in the online supplement. 2.6. Liquid chromatography The separation was performed using a Zorbax Eclipse C18 (Agilent, Waldbronn, Germany) column (150 2.0 mm, 4 lm)
protected by a guard column of the same packing material. The mobile phase consisted of phase A as 10 mM ammonium acetate (pH 6.8) and phase B as acetonitrile with 0.01% formic acid (w/v). The flow rate was set at 1.0 mL min1. In order to resolve the analytes, a gradient system was applied which comprised of 20% phase B for 3 min increased to 95% over 2 min, held at 95% for 1 min and decreased to 20% over 2 min where it was re-equilibrated for 3 min. The total analysis time was 11 min. The system was operated at an ambient temperature (23–25 °C). The column outlet was directly connected to the APCI probe with PEEK tubing. Injection volume was 10 lL. 2.7. Data analysis Limits of detection (LODs) for nicotine and cotinine were 1.25 ng g1 and limits of quantification (LOQs) were 5.03 ng g1 and 1.25 ng g1, respectively (empirically determined as the lowest concentration with a signal to noise ratio of at least 3:1 for detection and 10:1 for quantification) (Shabir, 2003). Samples were analyzed using multiple reaction monitoring and the concentrations were calculated from the ratios of native and labeled ions compared to a calibration curve for nicotine and cotinine respectively. Peak area ratios of target analytes and respective internal standards were calculated at each concentration. Calibration by deuterated internal standardization was performed using simple least squares regression with 1/x weighting. 2.8. Statistical analysis Statistical evaluations were completed with IBM SPSS 20 for Windows (Chicago, IL, USA). The level of statistical significance was set at p < 0.05. In the correlation analysis the level of significance was determined using Spearman’s coefficient. Mann–Whitney tests were used for the comparison of the median values. In all statistical evaluations, values below the LOQ were excluded, with the exception of the median values comparison. 3. Results Of the 45 women who participated in the study 22 (49%) were active smokers, 17 (38%) were passive smokers and 6 women had not been exposed to tobacco (13%) during their pregnancy. Concentrations of nicotine and cotinine in meconium are presented in Table 1 (sorted and numbered by decreasing concentrations of nicotine for each category of women). Information about the participants’ smoking habits and exposure to tobacco smoke, as well as birth weight and gestational age at birth for each corresponding neonate are also presented in this table. Nicotine and cotinine concentrations for each sample were highly correlated with each other (rs = 0.674, p < 0.001) (Fig. 1) but cotinine was detected in meconium generally at lower concentrations than observed for nicotine in this matrix. The maximum concentration of nicotine measured in the smokers’ group was 125 ng g1 (respectively maximum concentration of cotinine 98.5 ng g1) while the maximum concentration of nicotine in the group of passive smokers was 59.4 ng g1 (respectively maximum concentration of cotinine 76.5 ng g1), which is comparable to values of samples from women who smoked about 5–7 cigarettes per day. It is noteworthy that passive smokers with husbands not smoking more than 10 cigarettes per day showed a trend of much smaller cotinine: nicotine concentration ratios compared to active smokers ratios (Table 1). In general, concentrations of nicotine and cotinine in meconium correlated with the mothers’ smoking history. More specifically for active smokers Spearman correlation values were rs = 0.618, p = 0.004 (nicotine) and rs = 0.538, p = 0.010 (cotinine) while for
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Table 1 Nicotine and cotinine values (ng g1) in meconium from infants of active smokers, passive smokers and non-exposed women, smoking status of each mother, neonatal weight and gestational age at birth. All concentrations are listed to three significant figures. Sample number
a
Nicotine (ng g1)
Cotinine (ng g1)
Number of cigarettes smoked daily (mother)
Husband smoking/number of cigarettes smoked daily
Relatives smoking
Exposure at other places
Birth weight (g)
Gestation age (weeks+days)
Active smokers 1 125 2 108 3 92.1 4 83.7 5 57.1 6 55.5 7 53.0 8 51.9 9 49.3 10 44.7 11 42.4 12 38.6 13 38.2 14 33.6 15 31.4 16 26.6 17 25.7 18 22.3 19 17.6 20 7.09 21
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Determination of nicotine and cotinine in meconium from Greek neonates and correlation with birth weight and gestational age at birth Nikoleta Tsinisizeli a,b,1, Georgios Sotiroudis b,1, Aristotelis Xenakis b,⇑, Katerina E. Lykeridou a,⇑ a b
Faculty of Health and Caring Professions, Midwifery Department, Technological Educational Institute of Athens, Agiou Spyridonos Str., 12210 Egaleo, Greece Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635 Athens, Greece
h i g h l i g h t s LC–MS/MS quantitation of nicotine and cotinine in meconium from Greek neonates. Concentrations of biomarkers in meconium correlated with mothers’ smoking history. Passive smokers’ concentrations were comparable to those of moderate smokers. Negative correlation between biomarker concentrations and two neonatal outcomes.
a r t i c l e
i n f o
Article history: Received 10 March 2014 Received in revised form 11 September 2014 Accepted 21 September 2014 Available online 1 November 2014 Handling Editor: J. de Boer Keywords: Meconium Tobacco Nicotine Prenatal exposure Neonatal outcomes LC–MS/MS
a b s t r a c t Tobacco exposure during pregnancy is a major factor of morbidity and mortality for both the pregnant woman and the fetus. Several studies in the past have detected and quantified tobacco smoke biomarkers in infant meconium samples. Aim of this study was to measure prenatal exposure to tobacco smoke by detecting nicotine and cotinine in meconium and to try to evaluate the extent of exposure to smoke through passive smoking as well as the relationship between tobacco biomarker meconium concentrations and neonatal outcomes. Tobacco smoke biomarkers nicotine and cotinine were detected and quantitated in meconium from tobacco exposed and non-exposed Greek neonates using liquid chromatography–tandem mass spectrometry. The study included 45 neonates from active, passive and non-smoking women during pregnancy. The results showed significant values of nicotine and cotinine concentration in neonates from both active and passive smokers which reached 125 ng g1 for nicotine and 98.5 ng g1 for cotinine and varied according to the type and level of exposure. In general nicotine and cotinine concentrations correlated with the degree of active smoking by the mother. Similarly, nicotine and cotinine were measured in the meconium of infants of passive smokers at concentrations comparable to those of infants whose mothers were moderate smokers. Our findings show that exposure of the fetus to tobacco biomarkers can be substantial even in passive maternal smoking and there is a statistically significant negative correlation between nicotine or cotinine concentrations in meconium and birth weight or gestational age at birth. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Smoking addiction is one of the most critical problems of public health. The toxic compounds present in cigarettes reach the organism either by active smoking or involuntarily by passive ⇑ Corresponding author at: Institute of Biology, Medicinal Chemistry and Biotechnology (IBMCB), National Hellenic Research Foundation (NHRF), 48 Vassileos Constantinou Ave., 11635 Athens, Greece. Tel.: +30 2107273762; fax: +30 2107273758. E-mail addresses: [email protected] (N. Tsinisizeli), [email protected] (G. Sotiroudis), [email protected] (A. Xenakis), [email protected] (K.E. Lykeridou). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.chemosphere.2014.09.094 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
smoking. In the developing fetus, nicotine crosses both the placental and the blood–brain barrier and its concentration in the fetal compartment is 15% higher than in maternal tissues (Lambers and Clark, 1996). Maternal smoking results in a decreased transfer of nutrients and oxygen to the fetus and has been associated with decreased birth weight, increased risks for premature birth, spontaneous abortion, and infant death from perinatal disorders and from sudden infant death syndrome (SIDS) (Haglund and Cnattingius, 1990; DiFranza and Lew, 1995; Anderson and Cook, 1997; Castles et al., 1999). It may also increase the risk of cognitive and neurodevelopmental delay (Weitzman et al., 2002).
N. Tsinisizeli et al. / Chemosphere 119 (2015) 1200–1207
According to a study in 2002 in the United States, percentages of pregnant women who admitted to using tobacco smoking products the last three months of pregnancy ranged from 6.8% (Utah) to 25.3% (Virginia) (Xia et al., 2011). Prenatal smoking remains one of the most prevalent factors causing infant morbidity and mortality in the United States (Dietz et al., 2010), where smoking during pregnancy accounts for as many as 161 000 perinatal deaths and 4800 infant deaths each year, with more than half of these classified as sudden infant death syndrome (SIDS) (DiFranza and Lew, 1995). Greece, on the other hand, has one of the highest percentages of adult tobacco use worldwide, and the highest adult percentage of current tobacco use among the Organisation for Economic Co-operation and Development (OECD) countries (OECD briefing note for Greece and OECD Health Data; 2013). In a study in the end of 2010 (Filippidis et al., 2013) more than 38% of adult women in Greece admitted to having smoked at least once during the past 30 d. Especially in city areas, such as Athens, smoking rates among women are higher than rural areas (Pitsavos et al., 2003). Those with higher education are actually more likely to smoke and studies have shown that this happens only in Greece and Portugal, among several European countries (Huisman et al., 2005). In a study in Crete (Vardavas et al., 2010), active smoking during pregnancy between pregnant women was reported by 36% of respondents and almost all of the non-smokers (94%) were exposed to passive smoking. In this respect, we were interested in biologically monitoring levels of exposure to tobacco smoke of Greek neonates from mothers of different types of smoking status (heavy smokers, light smokers, passive smokers, non-smokers). Several ways are available for monitoring in utero nicotine exposure, including hair, plasma, urine and meconium. Measuring the nicotine metabolites in urine reveals the most recent tobacco smoke exposure (Köhler et al., 2001). In contrast, nicotine accumulated in fetal hair reflects the smoke exposure over the last three to four months of gestation (Koren, 1995). Meconium, the first neonatal feces, presents several advantages for detecting prenatal exposure to tobacco smoke. Meconium begins to form during the twelfth gestational week, so meconium testing reflects second and mostly third-trimester exposure (Ostrea et al., 1992). Accumulation of meconium over this period of months yields higher tobacco biomarker concentrations than other neonatal matrices. Furthermore, meconium offers the advantage that it can be collected easily and non-invasively in sufficient quantity until about the second postnatal day (Ostrea et al., 2001). In the present study we determined levels of nicotine and cotinine in meconium in both active and passive smokers, as well as in mothers who self-reported no exposure to tobacco smoke. To our knowledge, the biological monitoring of tobacco biomarkers within meconium has never been performed in Greece. The method used was based on the method developed by Gray et al. (2008b). The aims of the study were (a) to compare maternal self-reported tobacco use and meconium analysis results in a group of Greek mothers, (b) compare levels of nicotine and cotinine between active and passive smokers and (c) determine if meconium concentrations can be associated with decreased birth weight and shorter gestation periods, especially in the case of heavy smoking.
2. Materials and methods In total 45 women participated in the study. The specimens of meconium were collected at GAIA maternity hospital of Athens. The material (first day meconium) was collected from the diaper of the neonates using a spatula and pooled into one plastic container per infant (120 mL urine analysis container). Samples were immediately stored at 20 °C until analysis. The estimation of
1201
the levels of exposure to tobacco smoke was possible through anonymous questionnaires. Each questionnaire consisted of questions asking about the following: age of the women, profession, if they smoked before pregnancy (and number of cigarettes smoked daily), if they smoked during pregnancy (and number of cigarettes smoked daily) and if they stopped smoking during pregnancy and when. Questions about the women’s family (all women were married) were the following: if the husband (or a person they lived with) smoked (and number of cigarettes smoked daily) and if he smoked in the presence of his wife and in their house. Finally, the questionnaire included the following questions about other possible cases of passive smoking: if during pregnancy the women attended closed spaces were people smoked (e.g. work), how often, when was the last time they did that and if they were exposed to second-hand smoke only outdoors or not at all. The questionnaires were completed by the mothers either during the first stage of labor or during the first hour after labor, while meconium specimens were collected during the first day of neonates’ life. Informed consent was obtained from the mother in each case. All specimens and the maternal self-report tobacco surveys were deidentified to protect personal health information. The study protocol and the informed consent document were approved by the Medical School of University of Athens. All the individuals who were classified as smokers smoked at least one cigarette daily. The individuals who had been exposed to second-hand smoke in the past 3 months (even if only outdoors) were classified as passive smokers and those who had never been aware of being exposed to tobacco smoke during the last months of pregnancy were classified as non-smokers. For the analysis approximately 0.5 g of meconium was weighed and mixed with the necessary internal standard solutions followed by a procedure essentially as described by Gray et al. (2008b). 2.1. Reagents and standards Standard solutions of ()-nicotine and ()-cotinine (1.0 mg mL1 in methanol) as well as (±)-nicotine-D4 (0.1 mg mL1 in acetonitrile) and (±)-cotinine-D3 (0.1 mg mL1 in methanol) were obtained from Cerilliant (Round Rock, TX, USA). All reagents were analytical grade or higher. Formic acid, sodium acetate, hydrochloric acid and ammonium hydroxide were purchased from Merck (Darmstadt, Germany). Ammonium acetate was acquired from Sigma Chemicals (St. Louis, MO, USA). All solvents were HPLC grade and obtained from the following suppliers: methanol and isopropanol from Carlo-Erba Reactifs-SDS (Val de Reuil, France) and dichloromethane from Lab-Scan/Poch SA (Gliwice, Poland). Ultrapure water was obtained from a Milli-Q water purification system (Millipore, Billerica, MA, USA). Solid phase extraction columns Discovery DSC-MCAX (100 mg, 3 mL) were purchased from Supelco/Sigma–Aldrich (Bellefonte, PA, USA). 2.2. Instrumentation During sample preparation, sonication was performed by a Raypa UCI-150 ultrasonic cleaner bath (Espinar SL, Barcelona, Spain). MS/MS analysis was performed using an API 3200 QTrapÒ triple quadrupole/linear ion trap mass spectrometer with an APCI source (AB Sciex, Foster City, CA, USA) which was coupled to an Agilent 1200 (Agilent, Waldbronn, Germany) HPLC system (consisting of a G1379B micro vacuum degasser, a G1312A binary pump, a G1329 autosampler and a G1316A column compartment). A nitrogen generator N300DR (Peak Scientific, Renfrewshire, Scotland) supplied auxiliary (nebulising, heater and collision) gases. Analyst software version 1.4.2 (AB Sciex, Foster City, CA, USA) was used for acquisition and data analysis. Statistical evaluations were completed with IBM SPSS 20 for Windows (Chicago, IL, USA).
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2.3. Preparation of standard solutions Standard solutions of nicotine and cotinine in methanol were stored at 20 °C. Dilutions of each standard solution in methanol created 25–10 000 ng mL1 working solutions of nicotine and cotinine. 0.5 g blank meconium were fortified with 25 lL working standards so that final standard concentrations in meconium were 1.25, 2.5, 5, 7.5, 50, 100 and 500 ng g1. Standard solutions of deuterated analogues of nicotine and cotinine were also stored at 20 °C. A 1000 ng mL1 working internal standard solution was prepared by diluting deuterated standard solution with methanol. The final deuterated internal standard concentration after fortifying 0.5 g blank meconium with 25 lL working solution was 50 ng g1. Blank meconium used for the calibration curves was meconium acquired from a woman that had not been exposed to tobacco smoke at all during pregnancy. The blank was analyzed before the analysis of the rest of the samples and showed values smaller than the limits of quantitation determined for this method both for nicotine and cotinine. 2.4. Sample preparation Meconium (0.5 ± 0.01 g) was well mixed and weighed into a 15 mL polypropylene tube. 25 lL of working standard solution (only for the creation of the calibration curve) and 25 lL of internal standard solution were added. Specimens were homogenized (2 mL methanol with 0.01% formic acid (w/v) were added) and then vortexed vigorously and sonicated for 1 h, vortexing every 5 min. Finally they were centrifuged at 8000g for 10 min. The supernatant was transferred to a round bottom screw-top vial and evaporated to dryness under nitrogen at 40 °C. The residue was reconstituted to 2 mL of 2 M sodium acetate buffer, pH 5.5 prior to solid phase extraction. Discovery DSC-MCAX solid phase extraction columns were conditioned with 3 mL methanol, 3 mL water and 2 mL 2 M sodium acetate buffer, pH 5.5. Samples were loaded and allowed to flow by gravity alone. Columns were washed with 2 mL water, dried for 1 min, washed with 1.5 mL 0.2 M aqueous hydrochloric acid, dried for 5 min, washed with 2 1 mL methanol, and finally dried for 5 min. Analytes were eluted with freshly prepared 6 1 mL dichloromethane: 2-propanol: ammonium hydroxide (78:20:2, v/v/v). After the addition of 100 lL 1% hydrochloric acid in methanol (v/v), eluates were dried under nitrogen. Samples were reconstituted in 400 lL methanol with 0.01% formic acid (w/v) and transferred to glass autosampler vials. 2.5. Mass spectrometry Atmospheric pressure chemical ionization (APCI) operating in positive mode was used for all analytes. 100 ng mL1 reference solution in methanol for each standard biomarker was directly infused using a syringe pump in order to optimize the MS/MS parameters for each compound. Source parameters were set to 40 psi curtain gas, 50 psi auxiliary gas, 35 psi nebulizer gas, 6 psi collision gas, 1.0 lA nebulizer current, and 350 °C source temperature after flow injection analysis (FIA) source optimization. Quadrupoles one and three were set to unit resolution. Data acquisition was performed in the multi reaction monitoring (MRM) mode and the following transitions were monitored: (m/z): 163.2 ? 84.2 (nicotine), 167.2 ? 136.0 (nicotine-D4), 177.2 ? 80.0 (cotinine) and 180.2 ? 80.0 (cotinine-D3). Tandem mass spectrometry parameters are presented in the online supplement. 2.6. Liquid chromatography The separation was performed using a Zorbax Eclipse C18 (Agilent, Waldbronn, Germany) column (150 2.0 mm, 4 lm)
protected by a guard column of the same packing material. The mobile phase consisted of phase A as 10 mM ammonium acetate (pH 6.8) and phase B as acetonitrile with 0.01% formic acid (w/v). The flow rate was set at 1.0 mL min1. In order to resolve the analytes, a gradient system was applied which comprised of 20% phase B for 3 min increased to 95% over 2 min, held at 95% for 1 min and decreased to 20% over 2 min where it was re-equilibrated for 3 min. The total analysis time was 11 min. The system was operated at an ambient temperature (23–25 °C). The column outlet was directly connected to the APCI probe with PEEK tubing. Injection volume was 10 lL. 2.7. Data analysis Limits of detection (LODs) for nicotine and cotinine were 1.25 ng g1 and limits of quantification (LOQs) were 5.03 ng g1 and 1.25 ng g1, respectively (empirically determined as the lowest concentration with a signal to noise ratio of at least 3:1 for detection and 10:1 for quantification) (Shabir, 2003). Samples were analyzed using multiple reaction monitoring and the concentrations were calculated from the ratios of native and labeled ions compared to a calibration curve for nicotine and cotinine respectively. Peak area ratios of target analytes and respective internal standards were calculated at each concentration. Calibration by deuterated internal standardization was performed using simple least squares regression with 1/x weighting. 2.8. Statistical analysis Statistical evaluations were completed with IBM SPSS 20 for Windows (Chicago, IL, USA). The level of statistical significance was set at p < 0.05. In the correlation analysis the level of significance was determined using Spearman’s coefficient. Mann–Whitney tests were used for the comparison of the median values. In all statistical evaluations, values below the LOQ were excluded, with the exception of the median values comparison. 3. Results Of the 45 women who participated in the study 22 (49%) were active smokers, 17 (38%) were passive smokers and 6 women had not been exposed to tobacco (13%) during their pregnancy. Concentrations of nicotine and cotinine in meconium are presented in Table 1 (sorted and numbered by decreasing concentrations of nicotine for each category of women). Information about the participants’ smoking habits and exposure to tobacco smoke, as well as birth weight and gestational age at birth for each corresponding neonate are also presented in this table. Nicotine and cotinine concentrations for each sample were highly correlated with each other (rs = 0.674, p < 0.001) (Fig. 1) but cotinine was detected in meconium generally at lower concentrations than observed for nicotine in this matrix. The maximum concentration of nicotine measured in the smokers’ group was 125 ng g1 (respectively maximum concentration of cotinine 98.5 ng g1) while the maximum concentration of nicotine in the group of passive smokers was 59.4 ng g1 (respectively maximum concentration of cotinine 76.5 ng g1), which is comparable to values of samples from women who smoked about 5–7 cigarettes per day. It is noteworthy that passive smokers with husbands not smoking more than 10 cigarettes per day showed a trend of much smaller cotinine: nicotine concentration ratios compared to active smokers ratios (Table 1). In general, concentrations of nicotine and cotinine in meconium correlated with the mothers’ smoking history. More specifically for active smokers Spearman correlation values were rs = 0.618, p = 0.004 (nicotine) and rs = 0.538, p = 0.010 (cotinine) while for
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Table 1 Nicotine and cotinine values (ng g1) in meconium from infants of active smokers, passive smokers and non-exposed women, smoking status of each mother, neonatal weight and gestational age at birth. All concentrations are listed to three significant figures. Sample number
a
Nicotine (ng g1)
Cotinine (ng g1)
Number of cigarettes smoked daily (mother)
Husband smoking/number of cigarettes smoked daily
Relatives smoking
Exposure at other places
Birth weight (g)
Gestation age (weeks+days)
Active smokers 1 125 2 108 3 92.1 4 83.7 5 57.1 6 55.5 7 53.0 8 51.9 9 49.3 10 44.7 11 42.4 12 38.6 13 38.2 14 33.6 15 31.4 16 26.6 17 25.7 18 22.3 19 17.6 20 7.09 21
