Chromatographia (2014) 77:1145–1151 DOI 10.1007/s10337-014-2729-2
Original
Determination of Acrylamide and Acrolein in Smoke from Tobacco and E‑Cigarettes Roman Papoušek · Zoltán Pataj · Petra Nováková · Karel Lemr · Petr Barták
Received: 22 November 2013 / Revised: 23 May 2014 / Accepted: 24 June 2014 / Published online: 16 July 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Acrylamide and acrolein are two short-chained hazardous compounds with neurotoxic, carcinogenic, and mutagenic effects. The aim of this paper is to describe a fast and simple procedure for simultaneous determination of both acrylamide and acrolein under standard conditions, suggest a suitable calibration protocol for custom analysis, and demonstrate its applicability to the analysis of gaseous products from, e.g., cigarettes, cigars, or electronic cigarettes. A gas chromatography–mass spectrometry (GC– MS) method was developed to quantify acrylamide and acrolein in smoke vapor from electronic cigarettes, tobacco cigarettes, and cigars. Nonionic and highly polar molecules with a low boiling point and molecular mass need a suitable derivatization method to achieve appropriate retention and selectivity on commonly used relatively nonpolar stationary phases and to enhance the molecular mass for easy MS detection. The derivatization of acrylamide and acrolein was carried out by a bromination method with elemental bromine. The dibromo derivatives were extracted into an organic solvent and following a dehydrobromination procedure the samples were injected into the GC–MS system. Important experimental parameters were varied, after which the bromination time was defined as 30 min, and the injector temperature and the starting temperature of gradient were set at 280 and 50 °C respectively. Acrolein was Published in the special paper collection 9th Balaton Symposium on High-Performance Separations Methods with guest editor Attila Felinger. R. Papoušek (*) · Z. Pataj · P. Nováková · K. Lemr · P. Barták Department of Analytical Chemistry, Faculty of Science, Regional Centre of Advanced Technologies and Materials, Palacký University, 17. listopadu 12, 771 46 Olomouc, Czech Republic e-mail:
[email protected]
found in all tested samples, while acrylamide was detected only in smoke from normal tobacco. Possible mechanisms for the formation of these unsaturated compounds in the samples are discussed. After its validation the newly developed method was successfully and reliably applied to the analysis of both compounds. This short method provides an easy way to determine acrylamide and acrolein in gaseous samples. Keywords Gas chromatography–mass spectrometry · Acrolein · Acrylamide · Bromination · Electronic cigarette · Tobacco smoke
Introduction Indoor air pollution is consistently ranked among the top five environmental risks to public health by the Science Advisory Board (SAB) of the US Environmental Protection Agency (US EPA). Cigarette smoke, which contains several thousand chemical compounds including carcinogens, belongs to the most important pollutants of indoor air. The second-hand smoke is responsible for approximately 3,000 annual lung cancer deaths among non-smokers in the USA alone [1]. Electronic cigarettes (E-cigarettes) represent an alternative method of nicotine consumption, eliminating adverse effects of those substances that result from the combustion and pyrolysis of traditional tobacco cigarettes. The potential risks and benefits of E-cigarettes are uncertain and are still being discussed by researchers and representatives of various health organizations [2]. Acrylamide (CH2=CH–CONH2), the smallest unsaturated primary amide, is the monomer of polyacrylamide used as a flocculant in drinking water treatment and in other industries such as papermaking, textile manufacturing,
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and cosmetics. Polyacrylamide gel is also often applied in analytical biochemistry for electrophoretic separation and protein purification [3, 4]. Acrylamide shows significant neurotoxicity in both laboratory animals and humans and can be dangerous for the immune and blood systems [5, 6]. In rats acrylamide can increase the risk of tumors of mammary glands, central nervous system, thyroid glandfollicular epithelium, uterus, colon, and clitoral gland [7, 8]. The International Agency for Research on Cancer classifies acrylamide as “probably carcinogenic to humans” (group 2A) [5, 9, 10]. Colorless and odorless, it is very soluble in water, ethanol, and acetone [11]. It is generally considered to be formed by thermal treatment of a mixture of amino acids and reducing sugars through the Maillard reaction [12]. As a result of this reaction acrylamide occurs in various cooked starchy foods, e.g., potato chips, French fries, and toasted bread [13]. It has also been detected in cigarette main- and sidestream smoke [14]. Because of its herbicide and biocide effects, acrolein (the simplest unsaturated aldehyde, CH2=CH–CH=O) is mainly used to control algae and submerged and floating weeds in irrigation canals (around 10 ppm concentration) and to treat drilling waters in the oil and gas industry. As a scavenger agent it is also used in the petrochemical industry [15]. Acrolein is an intermediate of industrial production of glycerine, glutaraldehyde, acrylic acid, and other substances [16]. Acrolein is highly toxic via all routes of administration: as a strong irritant it can cause necrosis of skin, eyes, and nasal passages [17–19]. Along with other α,β-unsaturated aldehydes endogenously formed during lipid peroxidation, it has mutagenic effects inducing DNA and protein damage [20]. In 1994, the US EPA classified the effects of acrolein as “suggestive evidence of carcinogenic potential” (group C). The International Agency for Research on Cancer (IARC) revised this classification in 1995, as did the US EPA in 2003. As a result, acrolein is no longer classified as a human carcinogen [21–23]. A colorless and unstable liquid, it is very soluble in water and has a piercing, disagreeable, acrid smell [24]. Acrolein is introduced daily into the environment from many sources such as automobile emissions, cigarette smoke, forest fires, heated cooking oil, and various industrial processes [17, 25]. Another widely used compound, glycerol, thermally decomposes into acrolein at 563 K [24, 26, 27]. A number of methods for determination of either acrylamide or acrolein based on separation methods, namely liquid [28–36] and gas [18, 37–44] chromatography, have been described. Nevertheless, simultaneous determination of both compounds has been described only rarely. Both liquid and gas chromatographic methods usually require derivatization prior to analysis. The formation of suitable oximes [45] or substituted (phenyl)hydrazones [28, 29, 34–36] seems to be the most popular techniques for
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H
NH2
O acrylamide
O acrolein
Fig. 1 Chemical structures of analytes
derivatization of carbonyl compounds, although the geometrical isomerism [42] leading to non-uniform products and an excessive number of chromatographic peaks in the analysis of more complex samples may complicate the evaluation of the analytical results. Derivatization of the second reactive center (i.e., double bond) in acrolein and acrylamide is a suitable alternative, and several methods based on bromination have been described [13, 40, 46–49]. Unfortunately, some described methods differ in bromination reagent, temperature, influence of light and, possibly most importantly, in the recommended time of the reaction. Calibration difficulties related to low stability of the standards of highly reactive compounds have also been reported, especially in the case of acrolein [18, 43]. The aim of this paper is to describe a fast and simple procedure for simultaneous determination of both acrylamide and acrolein (for the relevant structures, see Fig. 1) under standard conditions, suggest a suitable calibration protocol for custom analysis, and demonstrate its applicability to the analysis of gaseous products from both electronic and common tobacco cigarettes and cigars.
Experimental Chemicals and Reagents A standard of acrolein was freshly prepared in our laboratory. A mixture of 100 mL H2O, 5 mL oxidant solution, and 250 μL allyl alcohol (CH2=CH–CH2–OH) was carefully heated in a distillation system. The oxidant solution contained 10 g of sodium dichromate (Na2Cr2O7), 70 mL of H2O, and 25 mL of 96 % sulfuric acid (H2SO4) and was cooled at −5 °C until the time of use. After the collection of 5 mL of distillate, another portion of the oxidant solution (5 mL) and allyl alcohol (250 μL) was added to the distillation system after which another 5 mL of the distillate was collected [50]. The concentration of aqueous solution of acrolein was not known but it was appropriate for identification, and our laboratory-prepared standard helped us avoid the disadvantages of the commercially available acrolein standards and isotopically labeled standards, e.g., expensiveness and stabilizer content against polymerization [51]. In order to conduct the
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validation process properly, a standard of acrolein was purchased as well. Allyl alcohol, H2SO4 (96 %), n-propanol (n-PrOH), and ethyl acetate (EtAc) of analytical grade were purchased from Merck (Darmstadt, Germany). KBrO3, KBr, Na2Cr2O7, Na2S2O3·5H2O, triethylamine (purity 99 %), acrolein (purity 99 %), and acrylamide (purity 99 %) were obtained from Sigma-Aldrich (St. Louis, MO, USA). MilliQ purified water (Merck Millipore, Darmstadt, Germany) was filtrated on a 0.22-μm filter. Apparatus One of the most crucial steps of the analysis was determining the appropriate sample collection that would imitate the real user. Smoking was simulated using our laboratoryprepared apparatus (Fig. 2) connected to a vacuum source and equipped with an air flow controller. This system was meant to simulate those basic variables proposed by the International Organization for Standardization (ISO) [52]. They include procedures to be conducted using a smoking machine that takes 35-mL puffs for the duration of 2 s each minute. Instead of the fiberglass filter and the leak-tight bag which are recommended by ISO for the collection of the particulates in both the smoke and the gas phase [53], the two phases were captured in our system by Milli-Q water, which is a suitable environment for the absorption of polar analytes and their subsequent derivatization [54]. The appropriate vacuum pressure was set in advance by a soap bubble flowmeter, which was used during the experimental work. With this system, not only mainstream smoke (as in the case of E-cigarette) but also sidestream smoke (valve no. 1 was open), which is important in terms of passive
SIDESTREAM SMOKE
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1
VACUUM
MAINSTREAM SMOKE 2 CIGAR
Fig. 2 Apparatus for the capture of tobacco smoke and E-smoke
smoking [1], was absorbed from a cigar. In this case the pressure was readjusted in order to maintain the required flow rates. Gas chromatographic experiments were carried out on an Agilent 7890 series system, coupled with an Agilent 7693 series injector and a mass-selective detector (Agilent 5975 C) and equipped with a capillary column (HP-5 ms, 30 m × 0.25 mm × 0.25 µm) (all Agilent Technologies, Santa Clara, CA, USA). Injection of liquid samples was performed under the following conditions: injection in pulsed splitless mode (20 psi, 0.4 min); injector temperature 280 °C; temperature program 50 °C for 6 min, increased at 10 °C min−1 to 300 °C for 10 min; EI ionization (70 eV). Helium 5.0 was used as carrier gas (0.9 mL min−1). Data were collected in total ion current (TIC) and single ion monitoring (SIM) mode and were evaluated using GC/MSD ChemStation Software (Agilent Technologies, Santa Clara, CA, USA).
Results and Discussion Derivatization Retention of small, nonionic, and highly polar molecules with a low boiling point on relatively nonpolar stationary phases can be too weak for appropriate separation. Their detection can be negatively influenced by interference with other small molecules or fragments originating from matrix components as well as by a worse response of the massselective detector to low molecular weight compounds. Derivatization of small polar analytes can solve these problems by increasing the molecular weight and decreasing the polarity of the target compounds. The derivatization of acrylamide and acrolein (Fig. 3) was carried out by a modified bromination method reported by Hashimoto [55]. Bromination decreases their polarity, increases extractability, improves selectivity, and increases the molecular mass. The brominated compounds have a specific isotopic pattern and their more symmetrical peaks resulted in better selectivity, higher efficiency, and lower detection limits of the proposed method. In our process, 2 mL of 0.1 M KBrO3 solution and solid KBr in excess were added to 10 mL of water captured smoke (E-smoke or normal tobacco smoke). With the addition of 2 mL of 0.1 M H2SO4 solution, Br2 was created in a redox reaction which was indicated by the yellowish color of the mixture. After 30 min (the effect of the bromination time will be discussed later on) in a cool and dark place, the excess bromine was decomposed by the addition of 0.1 M Na2S2O3 solution (until it became transparent; usually around 1.5 mL). This mixture with the brominated acrylamide and acrolein was then extracted three times with 2.0 mL EtAc. To avoid the reversible nucleophilic
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Fig. 4 Effects of the reaction time on the bromination process of acrylamide (squares) and acrolein (circles)
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To evaluate the effect of the reaction time, six samples were prepared simultaneously. Thus, 15 μL of 1 mg mL−1 acrylamide and 10 μL of acrolein aqueous solution (concentration was unknown) were added to 10 mL of pure water and a different bromination time was used (10, 20, 30, 45, 60, and 90 min). Thirty minutes in a dark and cold place was found to be convenient for both analytes (Fig. 4). Tareke et al. [13] used a similar type of bromination for acrylamide derivatization, but their samples were kept at 4 °C overnight, which proved to be unsuitable for the simultaneous determination of acrylamide and acrolein. Alkene bromination is a two-step electrophilic addition. A bridged bromonium ion is formed in the slower first step. A longer reaction time could be useful, but some undesirable side and subsequent reactions complicate the process. Other brominated derivatives originating from radical and electrophilic substitutions were found in the chromatograms when the reaction conditions were not correctly selected. Dark and cold efficiently eliminate the radical substitution and significantly decelerate the electrophilic one. A reactive carbonyl group can undergo other reactions such as oxidation, electrophilic addition, and nucleophilic substitution [57, 58], depending on the matrix components and primary products of the reaction with excess bromine. As a result of subsequent reactions the yield of dibrominated acrylamide and acrolein decreases after 30 min in a dark refrigerator.
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addition of water to the carbonyl group [56] the EtAc phase was dried over MgSO4. This dried EtAc solution was subsequently preconcentrated to 1.0 mL by blowing a gentle and stream of pure nitrogen onto the surface of the liquid.
Peak area - Acrylamide (counts)
Fig. 3 Bromination and dehydrobromination of acrylamide and acrolein
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As mentioned previously, drying of EtAc extracts eliminated the undesirable reaction between water and reactive analytes. Furthermore, to prevent the reactive carbonyl group from other undesired reactions, 10 μL of n-PrOH was added after the preconcentration. The aldehyde group was protected using n-PrOH, which formed an acetal under acidic conditions [56]. Since the high temperature of the injector caused elimination of the alcohol, the acetal did not appear in the chromatogram and regenerated bromopropenal was quantitatively detected. The temperature of the sample port should be high enough to ensure instantaneous evaporation of the entire sample, which is necessary for satisfactory efficiency of the separation. A common temperature (280 °C) for similar analytes [59, 60] did not ensure quantitative dehydrobromination of acrylamide (Fig. 5) and TEA had to be added before injection of the samples. In the case of acrolein, the applied injection temperature was sufficient and only brominated acrolein appeared in the chromatogram. The conjugation of the non-bonding electron pair of the amino group in acrylamide increases delocalization energy in comparison with acrolein which reduces the negative inductive effect of the bromo groups and results in a more stable derivative [55]. The starting temperature of the column was set at 50 °C, because at a lower temperature the retention time of the earlier eluting brominated acrolein was not reproducible. The reason for that might have been the condensation of EtAc (boiling temperature 77.1 °C) at the beginning of the column, which made the capacity of the stationary phase irreproducible. At temperatures higher than 50 °C, the retention time of brominated acrolein strongly decreased causing difficult determination due to the interference with the solvent.
