2238 Paulo Cicero Nascimento1 Luciana Assis Gobo1 Denise Bohrer1 Leandro Machado Carvalho1 Margareth Coutinho Cravo2 Leni Figueiredo Mathias Leite2 1 Department
of Chemistry, Federal University of Santa Maria, 1000, Santa Maria, Brazil 2 CENPES/Petrobras, 950, Cidade ´ Universitaria Rio de Janeiro, Brazil Received November 5, 2014 Revised March 21, 2015 Accepted April 5, 2015
J. Sep. Sci. 2015, 38, 2238–2244
Research Article
Determination of polycyclic aromatic hydrocarbons in fractions in asphalt mixtures using liquid chromatography coupled to mass spectrometry with atmospheric pressure chemical ionization An analytical method using liquid chromatography coupled to mass spectrometry with atmospheric pressure chemical ionization for the determination of polycyclic aromatic hydrocarbons in asphalt fractions has been developed. The 14 compounds determined, characterized by having two or more condensed aromatic rings, are expected to be present in asphalt and are considered carcinogenic and mutagenic. The parameters of the atmospheric pressure chemical ionization interface were optimized to obtain the highest possible sensitivity for all of the compounds. The limits of detection ranged from 0.5 to 346.5 g/L and the limits of quantification ranged from 1.7 to 1550 g/L. The method was validated against a diesel particulate extract standard reference material (NIST SRM 1975), and the obtained concentrations agreed with the certified values. The method was applied to asphalt samples after its fractionation according to ASTM D4124 and the method of Green. The concentrations of the seven polycyclic aromatic hydrocarbons quantified in the sample ranged from 0.86 mg/kg for benzo[ghi]perylene to 98.32 mg/kg for fluorene. Keywords: Asphalt / Atmospheric pressure chemical ionization mass spectrometry / Liquid chromatography / Polycyclic aromatic hydrocarbons DOI 10.1002/jssc.201401231
1 Introduction Polycyclic aromatic hydrocarbons (PAHs) are introduced into the environment by both natural and anthropogenic sources. Natural sources, however, are insignificant compared to anthropogenic sources that are typically attributed to the handling or incomplete combustion of organic matter, especially fossil fuels and their derivatives (pyrogenic processes). Therefore, human exposure to PAHs occurs mainly through environmental contamination [1]. The presence of benzo[a]pyrene is considered by regulatory authorities, such as the World Health Organization, as a marker of the carcinogenic potential of PAHs [2]. Toxicological studies have shown an association between exposure of animals to PAHs and cardiovascular toxicity, bone toxicity, Correspondence: Prof. Dr. Paulo Cicero Nascimento, Department of Chemistry, Federal University of Santa Maria, Av. Roraima, 1000. Santa Maria, Brazil E-mail: [email protected] Fax: +55 55 3220 8870
Abbreviations: ASTM, American Society for Testing and Material; DNA, deoxyribonucleic acid; EPA, Environmental Protection Agency; NIST, National Institute of Standards and Technology; PAH, Polycyclic Aromatic Hydrocarbon; PTFE, Polytetrafluoroethylene; SIM, selective ion monitoring; SRM, Standard Reference Material C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
reproductive toxicity, immune suppression, and cancer [3]. Epidemiological studies have also emphasized that the evidence of cancer, genetic disorders, birth defects, and respiratory and nervous system disorders may be linked to levels of occupational exposure to PAHs [2]. Because of their physicochemical properties, PAHs are persistent in the environment and are able to be transported from one site to another by airborne particles [4]. The presence of PAHs in airborne particles is related to the use of fossil fuels and, because they are compounds with high molecular weight, they are generally present in heavy fractions of petroleum, such as asphalt binder [5]. Asphalt binder is produced from the residue of atmospheric distillation and vacuum distillation of crude petroleum oil and is typically used for paving. Asphalt binder is a mixture of hydrocarbons with molecular weights ranging from 500 to 2000 g/mol with a distillation range of approximately 500⬚C and at least 25 carbons [6]. The major components of asphalt can be separated into asphaltenes and maltenes. The asphaltenes are defined as a fraction insoluble in n-heptane, whereas maltenes are composed of saturated and aromatic compounds and resins that are soluble in n-heptane [7]. To facilitate the handling of paving or roofing (roof waterproofing, for example), the asphalt is heated, which may produce fumes consisting of low molecular weight substances, such as PAHs and other heterocyclic compounds containing www.jss-journal.com
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Table 1. Tested parameters and optimized conditions of the LC–MS system
Parameter
Range of variation tested
Optimized condition
Vaporizer (⬚C) Gas temperature (⬚C) Nebulizer (psi) Gas flow (L/min) Capillary Voltage (V) Corona needle discharge (A)
150, 200, 250 300, 325, 350 20, 30, 60 5, 8, 10, 12 2500, 3000, 3500, 4000 4, 8, 10
250 300 20 8 3000 10
Table 2. Mass spectrometer parameters for PAHs detection
Compound
Fragmentor (V)
[M+H]+
Acenaphthene Acenaphthylene Anthracene Benzo[a]pyrene Benzo[b]fluoranthene Benzo[ghi]perylene Benzo[k]fluoranthene Dibenz[a,h]anthracene Fluoranthene Flurene Indeno[1,2,3-cd]pyrene Naphthalene Phenanthrene Pyrene
111 200 132 143 143 148 128 153 132 210 168 190 142 128
167.1 153.1 155.1 129.1 181.1 179.1 203.1 203.1 253.1 253.1 253.1 277.1 279.1 277.1
will affect air quality [8]. The workers involved in the preparation and application of asphalt are vulnerable to direct exposure to asphalt emissions containing PAHs [9]. Regulatory organizations have warned about the possible carcinogenic effects of asphalt fumes for humans but data are still insufficient. Although there are substantial data showing mutagenic potential and DNA damage caused by exposure to asphalt fumes, research in this area is limited [6]. For the determination of these compounds, GC–MS or LC with fluorescence detection is typically employed [10–13]. Currently, there are official methods for the analysis of PAHs by GC–MS, such as EPA 8100. GC–MS has several advantages in comparison to LC with fluorescence detection, particularly in its resolution capability. GC offers high power chromatographic resolution and MS provides high selectivity and structural information, allowing for the determination of PAHs and their derivatives that exhibit little or no fluorescence [10]. On the other hand, LC–MS for the determination of PAHs and their derivatives is an attractive technique because it does not require derivatization, presents the same resolution capability of GC–MS and, unlike GC, there are no solvent restrictions [14]. However, there are few methods using LC–MS for the determination of PAHs [15]. The objective of this work was to develop a robust and effective method for the determination of 14 PAHs in fractions of asphalt binder. The method was validated against a standard reference material.
2 Materials and methods Figure 1. Chromatogram of the separation of PAHs. (1) 1 mg/L fluorene, 2 mg/L acenaphthylene; (2) 1 mg/L naphthalene, 2 mg/L acenaphthene; (3) 0.4 mg/L phenanthrene; (4) 0.1 mg/L anthracene; (5) 10 mg/L fluoranthene; (6) 0.04 mg/L pyrene; (7) 2 mg/L benz[(b]fluoranthene; (8) 2 mg/L benzo[k]fluoranthene; (9) 0.04 mg/L benzo[a]pyrene; (10) 0.04 mg/L benzo[ghi]perylene, 0.1 mg/L dibenz[a,h]anthracene; and (11) 0.4 mg/L Indeno[1,2,3cd]pyrene.
oxygen, sulfur or nitrogen. These fumes can be inhaled or deposited onto the skin and clothing of workers [6]. Because the asphalt mix is produced and compacted at temperatures higher than 150⬚C, the presence of these gaseous pollutants C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.1 Instrumentation, reagents and solutions An Agilent 1260 Infinity LC–MS chromatograph (Santa Clara, CA, United States) with automatic injection and an Agilent 6430 triple quadrupole mass detector were used. The chromatographic column was a Varian Pursuit 3 PAH 4.6 × 100 mm with 3 m particle size. Acetonitrile and methanol were of Chromasolv LC–MS grade and supplied by Sigma–Aldrich (St. Louis, MO, United States). The PAHs: Acenaphthene, acenaphthylene, anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[ghi]perylene, benzo[k]fluoranthene, dibenz[a,h]anthracene, fluoranthene, www.jss-journal.com
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Figure 2. Extracted chromatogram of the separation of PAHs. (1) 0.1 mg/L dibenz[a,h]anthracene; (2) 0.04 mg/L benzo[ghi]perylene, (3) 0.4 mg/L Indeno[1,2,3-cd]pyrene; (4) 2 mg/L benzo[b]fluoranthene; (5) 2 mg/L benzo[k]fluoranthene; (6) 0.04 mg/L benzo[a]pyrene; (7) 10 mg/L fluoranthene; (8) 0.04 mg/L pyrene; (9) 0.4 mg/L phenanthrene; (10) 0.1 mg/L anthracene; (11) 1 mg/L fluorene; (12) 2 mg/L acenaphthene; (13) 2 mg/L acenaphthylene; (14) 1 mg/L naphthalene. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 3. Analytical parameters of the method
PAH
Intra-day precision (RSD%)
Inter-day precision (RSD%)
Linear range (mg/ L)
Working range (mg/ L)
LOD (mg/ L)
LOQ (mg/ L)
Linear correlation coefficient (r)
Acenaphthene Acenaphthylene Anthracene Benzo[a]Pyrene Benzo[b]fluoranthene Benzo[ghi]perylene Benzo[k]fluoranthene Dibenz[a,h]anthracene Fluoranthene Fluorene Indeno[1,2,3-cd]pyrene Naphthalene Phenanthrene Pyrene
0.47 0.68 0.67 0.60 1.36 0.63 0.92 3.14 2.10 0.49 0.65 1.65 0.58 0.84
2.32 1.32 1.92 2.01 10.36 2.31 3.78 7.02 4.59 1.19 11.22 3.18 3.78 3.24
0.05 – 50 0.005 – 50 0.002 –50 0.002 – 50 0.05 – 50 0.002 – 50 0.05 – 50 0.05 – 50 0.5 – 100 0.05 –50 0.005 –50 0.05 – 50 0.01 –50 0.002 – 50
0.50 – 20.0 0.50 – 20.0 0.025 – 1.00 0.01 – 0.40 0.50 – 20.0 0.01 – 0.40 0.50 – 20.0 0.025 – 1.00 2.50 – 100 0.25 – 10.0 0.10 – 4.00 0.25 – 10.0 0.10 – 4.00 0.01 – 0.40
0.039 0.004 0.002 0.001 0.037 0.005 0.067 0.037 0.346 0.005 0.005 0.010 0.001 0.001
0.129 0.012 0.005 0.002 0.124 0.017 0.222 0.123 1.549 0.016 0.016 0.035 0.004 0.002
0.999 0.990 0.999 0.999 0.999 0.999 0.998 0.995 0.998 0.999 0.998 0.999 0.999 0.999
Table 4. Results of average recovery of PAHs in maltenic fraction (n = 3)
PAH
Recovery in the Recovery in the Recovery in the Average recovery in the acid fraction (%) RSD (%) basic fraction (%) RSD (%) neutral fraction (%) RSD (%) maltenic fraction (%) RSD (%)
Acenaphthene Acenaphthylene Anthracene Benzo[a]pyrene Benzo[b]fluoranthene Benzo[ghi]perylene Benzo[k]fluoranthene Dibenz[a,h]anthracene Fluoranthene Fluorene Indeno[1,2,3-cd]pyrene Naphthalene Phenanthrene Pyrene
97.13 103.63 92.52 97.83 91.75 98.33 94.57 97.29 89.01 105.96 90.32 98.01 105.65 100.48
3.98 0.65 3.13 1.56 7.67 0.60 1.72 3.09 3.15 0.62 6.01 16.00 2.57 14.69
94.75 101.39 91.12 93.14 115.24 96.64 101.55 102.51 91.42 100.79 98.41 109.10 108.09 95.52
fluorene, indeno[1,2,3-cd]pyrene, naphthalene, phenanthrene, and pyrene, were purchased from Fluka and Sigma– Aldrich (St. Louis, MO, United States). Standard solutions of each compound and mixtures were prepared in acetonitrile.
4.67 2.14 1.12 1.37 0.61 0.55 2.84 3.41 9.55 6.45 1.56 11.60 0.45 9.59
99.46 88.50 117.44 91.10 91.68 90.09 80.79 84.42 82.94 83.26 104.44 96.95 84.17 115.04
1.09 1.70 2.87 4.32 9.13 0.60 0.84 2.60 0.85 3.29 1.84 5.19 0.38 12.47
97.11 97.84 100.36 94.02 99.56 95.02 92.31 94.74 87.79 96.67 97.72 101.35 99.30 103.68
2.42 8.34 14.76 3.67 13.64 4.58 11.45 9.83 4.98 12.31 7.25 6.64 13.26 9.79
23 min under a flow of 0.8 mL/min. The injection volume was 20 L. During the run, the column was maintained at 30⬚C. A post-time period of 7 min was adopted to rebalance the system.
2.2 LC–MS conditions 2.3 Sample preparation To develop this method, each PAH was individually injected into the mass spectrometer to obtain its spectrum and also to optimize the fragmentor and the energy of the collision cell. Because PAHs are compounds with low polarity, they were converted into ions by using an APCI ion source. The source and detector conditions were optimized by testing the parameters shown in Table 1. To ensure the best response for the measurement of the analytes, each parameter was varied while the others were maintained at a fixed value. Elution was performed using a gradient that consisted initially of 80% methanol/water for 9 min, 90% methanol/water for 9.5 min, and 100% methanol until the end of the run at C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Asphalt samples were fractionated into asphaltenes and maltenes following the ASTM D4124 method [16]. Briefly, samples were refluxed in isooctane, where the asphaltene fraction, which is composed of polar compounds, precipitated while the maltenic fraction remained soluble. Separation was attained by filtration. The maltenic fraction was separated in their acidic, basic and neutral fractions using the method of Green by non-aqueous preparative ion exchange chromatography [17]. The acidic and basic fractions were diluted to 1:50 in methanol, filtered through a 0.22 m PTFE filter, and injected www.jss-journal.com
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Table 5. Results of the standard reference material NIST 1975 analysis (n = 3)
Compound
Certified/Reference (mg/kg)
Benzo[b]fluoranthene Benzo[ghi]perylenea) Benzo[k]fluoranthene Dibenz[a,h]anthracenea) Fluoranthene Fluorenea) Indeno[1,2,3-cd]pyrenea) Naphthalenea) Pyrenea)
3.20 0.038 0.174 0.079 13.5 0.110 0.12 0.67 0.42
± ± ± ± ± ± ± ± ±
Found (mg/kg) 3.23 ± 0.032 ± < LOQ 0.067 ± 12.93 ± 0.118 ± 0.11 ± 0.62 ± 0.43 ±
0.10 0.006 0.050 0.013 0.6 0.003 0.01 0.01 0.13
0.21 0.0004 0.004 0.24 0.019 0.0007 0.02 0.02
a) Reference values: noncertified values with best estimates of the true value Table 6. PAHs present in the three maltenic fractions of asphalt and the total concentration of each hydrocarbon in this matrix
Fraction(mg/kg) PAH
Acidic
Basic
Neutral
Total(mg/kg)
Anthracene Benzo[a]pyrene Benzo[b]fluoranthene Benzo[ghi]perylene Benzo[k]fluoranthene Fluorene Pyrene
– – – – – 2.48 –
90.32 – – – – – –
1.52 17.81 874.5 1.09 257.1 0.65 3.91
8.97 13.21 67.2 0.86 2.01 98.32 2.97
–: not detected
into the chromatographic system. The neutral fraction was prepared from 2 mL of the fraction that was evaporated to dryness and redissolved in 1 mL of dichloromethane. This new solution from the neutral fraction was diluted 1:10 in methanol, filtered twice through a 0.22 m PTFE filter and injected into the chromatograph. Because SRM 1975 is a liquid extract, an aliquot of 1 g was evaporated to dryness and redissolved in 1 mL of methanol. After this step, this solution was filtered through a 0.22 m PTFE filter and injected into the chromatograph.
3 Results and discussion 3.1 Optimization of the LC–MS method Because the ionization of the analyte in the APCI source occurs in the gas phase, it is important to ensure an efficient drying step (evaporating the solvent) to achieve a better response of the equipment [18]. The best operating conditions obtained after optimization of the source parameters are shown in Table 1. A significant dependence on the gas drying temperature and the vaporizer temperature for the eluent flow from the C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
chromatographic system was observed, which were set at 300 and 250⬚C, respectively. Because the gas flow also aids in the spray drying, a decrease of the signal was observed with an increase of the gas flow and nebulizer pressure; thus, the optimized conditions for these parameters were 8 L/min and 20 psi, respectively. In the case of PAHs, an increase in the corona discharge results in an increase in the ionization level. The ionization was performed with a higher discharge of the corona needle (10 A). The capillary voltage determines the entry of ions into the mass spectrometer, 3000 V was the ideal voltage for these compounds. Although PAHs are very stable condensed aromatic chains in which C–C bonds are hardly broken, it is possible to fragment these compounds. Hutzler et al. [15] monitored them in the selected reaction monitoring mode. However, when this approach was performed in this study, low abundance was observed, therefore the selective ion monitoring (SIM) mode was chosen for this analysis. The mass of the molecular ion of each compound was monitored in the positive polarity. The optimized conditions for the ion determination and the m/z values monitored are shown in Table 2. Figure 1 shows the total ion chromatogram, in which threeco-elutions were observed: fluorene and acenaphthylene (peak 1), naphthalene and acenaphthene (peak 2), and dibenz[a,h]anthracene and benzo[ghi]perylene (peak 9). Although co-eluted compounds have the same retention time, they possess different m/z values. With the detector operating in SIM mode, the monitored m/z values are selected to represent the target compound, i.e., the mass spectrometer detects one m/z at a time [18]. This approach can be seen in Fig. 2, in which the extracted chromatograms for each m/z are displayed. Alternatively, isobaric compounds, such as fluoranthene and pyrene, did not interfere with one another and were chromatographically separated.
3.2 Validation of the method The method was evaluated for inter- and intra-day precision, linearity, detection and quantification limits and accuracy. To perform the precision assay, synthetic samples were prepared by diluting stock solutions in the mobile phase at concentrations in the lower, middle and higher limits of the analytical curve. The intra-day and inter-day precision were evaluated by carrying out three injections of the same standard on the same day and on three consecutive days, respectively. The linearity of the method was checked by linear regression determined by the method of least squares. The LOD and LOQ were calculated from the S/N. S/N = 3 and 10 were used for estimating the LOD and LOQ, respectively [19]. Recovery studies were conducted for all compounds in the three maltenic fractions analyzed. The standard reference material SRM 1975, a diesel particulate extract, was analyzed for method validation. www.jss-journal.com
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Figure 3. Extracted chromatograms of the neutral, basic and acidic fraction for each m/z monitored. (1) benzo[ghi]perylene; (2) benzo[b] fluoranthene; (3) benzo[k]fluoranthene; (4) benzo[a]pyrene; (5) pyrene; (6) anthracene; (7) fluorene.
The proposed method exhibits limits of detection and quantification in the range of parts per billion (g/L) (Table 3). Fluoranthene was the only compound that showed higher limits of detection and quantification, most likely because of its difficult ionization and thereby low sensitivity. Good intra-day and inter-day precision were obtained with RSD values within an acceptable range. The results show good linear correlation coefficients (r) and a wide linear range of more than an order of magnitude. The recovery results for evaluating the accuracy of the method were also satisfactory (Table 4) and were within a range of 80 to 110% for the concentration levels studied [19]. The values for the HPAs in the Standard Reference Material (SRM 1975) are certified for benzo[b]fluoranthene, benzo[k]fluoranthene, and fluoranthene, and noncertified (best estimates of the true value) for benzo[ghi]perylene, dibenz[a,h]anthracene, fluorene, indeno[1,2,3-cd]pyrene, naphthalene, and pyrene. The results found using the developed methodology did not differ significantly from the reference according to the Student t-test, at a significance level of 0.05, as shown in Table 5.
3.3 Sample application The asphalt specimen was fractionated into asphaltenes and maltenes, and the maltene fraction was further separated C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
into its acidic, basic, and neutral fractions. Only the maltenic fraction was analyzed because it consists of low-molecularweight hydrocarbons that are soluble in n-heptane, similar to the case of PAHs [20]. The acidic and basic fractions were diluted in methanol, which is the mobile phase of the chromatographic system. The neutral fraction is a more complex matrix that when diluted in methanol forms a precipitate. Thus, the fraction was resuspended in dichloromethane, followed by dilution in methanol. Figure 3 shows the chromatograms obtained for the maltenic acidic, basic, and neutral fractions. The concentration of PAHs in the fractions ranged from 0.053 to 40.24 mg/L with a RSD ranging from 0.55 to 16.07%. Although anthracene and fluorene were the only PAHs found in the basic and acidic fractions, respectively, fluorene, anthracene, pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, and benzo[ghi] perylene were all identified in the neutral fraction. The presence of this elevated number of PAHs in the neutral fraction most likely results from the non-polar character of these compounds. Table 6 presents the concentration of each PAH in each fraction, as well as the calculated concentration of each compound in the asphalt matrix. The overall results show that the developed method was effective for this application and may be used for the determination of PAHs in this type of matrix. www.jss-journal.com
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The Asphalt Institute [21] presented a list of PAHs in bitumes and their respective concentrations. Although the samples are different, the PAHs found in this study were the same as those presented in this publication. Pyrene and benzo[ghi]perylene were within the same concentration ranges found by the cited publication. The other compounds found in this study showed higher values than those reported in the literature, likely as a result of the differences that exist between these complex matrices.
4 Conclusions The developed method exhibits adequate sensitivity, good precision, and accuracy, and a wide linear range for the determination of PAHs. The LOD and LOQ obtained herein indicate that the method allows for the determination of trace amounts of these compounds. Its application in asphalt fractions identified the presence of seven compounds in the samples. As PAHs have a recognized toxicological potential, the development of methods for the determination of these compounds in different matrices is important. The authors acknowledge the financial support provided by CNPq and CAPES. The authors also greatly acknowledge the financial support from PETROBRAS through the Center of Research and Development “Leopoldo Am´erico Miguez de Mello” (CENPES). The authors declare no conflict of interest.
5 References [1] Cavalcante, R. M., Filho, N. S. M., Viana, R. B., Oliveira, I. R. N., Nascimento, R. F., Silveira, E. R., Freire, G. S. S., Quim. Nova. 2007, 3, 560–564. [2] Delgado-Saborit, J. M., Stark, C., Harrison, R. M., Environ. Int. 2011, 37, 383–392. [3] Collins, J. F., Brown, J. P., Alexeeff, G. V., Salmon, A. G., Regul. Toxicol. Pharmacol. 1998, 28, 45–54.
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[4] Wang, Z., Ma, X., Na, G., Lin, Z., Ding, Q., Yao, Z., Environ. Pollut. 2009, 157, 3132–3136. [5] Brandt, H. C. A., De Groot, P.C., Water Research. 2001, 35(17), 4200–4207. [6] Schreiner, C., Regul. Toxicol. Pharmacol. 2011, 59, 270– 284. [7] Browarzik, D., Laux, H., Rahimian, I., Fluid Phase Equilibria. 1999, 154, 285–300. [8] Rubio, M. C., Mart´ınez, G., Baena, L., Moreno, F., J. Clean. Prod. 2012, 24, 76–84. [9] NIOSH, National Institute for Occupational Safety and Health (Ed.), Hazard Review: Health Effects of Occupational Exposure to Asphalt. Hazard Review, DHHS Publication, pp. 2011–2110. ˜ [10] Plaza-Bolanos, P., Garrido Frenich, A., Vidal, J. L. M., J. Chromatogr. A. 2010, 1217, 6303–6326. [11] Ping, C., Hui, Z., Jay, G., Mingxing, S., Guofeng, S., Liang, L., Guoqing, S., J. Sep. Sci. 2015, 38, 864–870. [12] Geng, L., Guibin, L., Yong, C., Hui, Y., Dezhong, D., J. Sep. Sci. 2012, 35, 2796–2804. [13] Fei, Y., Yiming, L., Rui, S., Chunyan, C., Di, P., Qianli, Z., Qingyun, C., Shouzhuo, Y., J. Sep. Sci. 2011, 34, 716–723. [14] Delhomme, O., Millet, M., Herkes, P., Talanta 2008, 74, 703–710. [15] Hutzler, C., Luch, A., Filser, J.G., Anal. Chim. Acta. 2011, 702, 218–224. [16] ASTM, Standard Test Method, Separation of Asphalt into Four Fractions, D4124, 2013. [17] Green, J. B., Yu, S. K. T., Pearson C. D., Reynolds, J. W., Energy Fuels 1993, 7, 119–126. [18] Gross, J. H., Mass Spectrometry – A Text Book, SpringerVerlag, Berlin Heidelberg 2011. [19] Lister, A. S., in: Ahuja, S., Dong, M., (Eds), Handbook of Pharmaceutical Analysis by HPLC, Elsevier, Amsterdam, 2005, pp 191–217. ´ [20] Lins, V. F. C., Araujo, M. F. A. S., Yoshida, M. I., Ferraz, V. P., Andrada, D. M., Lameiras, F. S., Fuel 2008, 87, 3254–3261. [21] Asphalt Institute, Eurobitume, The Bitume Industry – A Global Perspective Production, Chemistry, Use, Specification and Occupational Exposure, Asphalt Institute Inc. and European Bitumen Association, Eurobitume 2011.
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of Chemistry, Federal University of Santa Maria, 1000, Santa Maria, Brazil 2 CENPES/Petrobras, 950, Cidade ´ Universitaria Rio de Janeiro, Brazil Received November 5, 2014 Revised March 21, 2015 Accepted April 5, 2015
J. Sep. Sci. 2015, 38, 2238–2244
Research Article
Determination of polycyclic aromatic hydrocarbons in fractions in asphalt mixtures using liquid chromatography coupled to mass spectrometry with atmospheric pressure chemical ionization An analytical method using liquid chromatography coupled to mass spectrometry with atmospheric pressure chemical ionization for the determination of polycyclic aromatic hydrocarbons in asphalt fractions has been developed. The 14 compounds determined, characterized by having two or more condensed aromatic rings, are expected to be present in asphalt and are considered carcinogenic and mutagenic. The parameters of the atmospheric pressure chemical ionization interface were optimized to obtain the highest possible sensitivity for all of the compounds. The limits of detection ranged from 0.5 to 346.5 g/L and the limits of quantification ranged from 1.7 to 1550 g/L. The method was validated against a diesel particulate extract standard reference material (NIST SRM 1975), and the obtained concentrations agreed with the certified values. The method was applied to asphalt samples after its fractionation according to ASTM D4124 and the method of Green. The concentrations of the seven polycyclic aromatic hydrocarbons quantified in the sample ranged from 0.86 mg/kg for benzo[ghi]perylene to 98.32 mg/kg for fluorene. Keywords: Asphalt / Atmospheric pressure chemical ionization mass spectrometry / Liquid chromatography / Polycyclic aromatic hydrocarbons DOI 10.1002/jssc.201401231
1 Introduction Polycyclic aromatic hydrocarbons (PAHs) are introduced into the environment by both natural and anthropogenic sources. Natural sources, however, are insignificant compared to anthropogenic sources that are typically attributed to the handling or incomplete combustion of organic matter, especially fossil fuels and their derivatives (pyrogenic processes). Therefore, human exposure to PAHs occurs mainly through environmental contamination [1]. The presence of benzo[a]pyrene is considered by regulatory authorities, such as the World Health Organization, as a marker of the carcinogenic potential of PAHs [2]. Toxicological studies have shown an association between exposure of animals to PAHs and cardiovascular toxicity, bone toxicity, Correspondence: Prof. Dr. Paulo Cicero Nascimento, Department of Chemistry, Federal University of Santa Maria, Av. Roraima, 1000. Santa Maria, Brazil E-mail: [email protected] Fax: +55 55 3220 8870
Abbreviations: ASTM, American Society for Testing and Material; DNA, deoxyribonucleic acid; EPA, Environmental Protection Agency; NIST, National Institute of Standards and Technology; PAH, Polycyclic Aromatic Hydrocarbon; PTFE, Polytetrafluoroethylene; SIM, selective ion monitoring; SRM, Standard Reference Material C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
reproductive toxicity, immune suppression, and cancer [3]. Epidemiological studies have also emphasized that the evidence of cancer, genetic disorders, birth defects, and respiratory and nervous system disorders may be linked to levels of occupational exposure to PAHs [2]. Because of their physicochemical properties, PAHs are persistent in the environment and are able to be transported from one site to another by airborne particles [4]. The presence of PAHs in airborne particles is related to the use of fossil fuels and, because they are compounds with high molecular weight, they are generally present in heavy fractions of petroleum, such as asphalt binder [5]. Asphalt binder is produced from the residue of atmospheric distillation and vacuum distillation of crude petroleum oil and is typically used for paving. Asphalt binder is a mixture of hydrocarbons with molecular weights ranging from 500 to 2000 g/mol with a distillation range of approximately 500⬚C and at least 25 carbons [6]. The major components of asphalt can be separated into asphaltenes and maltenes. The asphaltenes are defined as a fraction insoluble in n-heptane, whereas maltenes are composed of saturated and aromatic compounds and resins that are soluble in n-heptane [7]. To facilitate the handling of paving or roofing (roof waterproofing, for example), the asphalt is heated, which may produce fumes consisting of low molecular weight substances, such as PAHs and other heterocyclic compounds containing www.jss-journal.com
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Table 1. Tested parameters and optimized conditions of the LC–MS system
Parameter
Range of variation tested
Optimized condition
Vaporizer (⬚C) Gas temperature (⬚C) Nebulizer (psi) Gas flow (L/min) Capillary Voltage (V) Corona needle discharge (A)
150, 200, 250 300, 325, 350 20, 30, 60 5, 8, 10, 12 2500, 3000, 3500, 4000 4, 8, 10
250 300 20 8 3000 10
Table 2. Mass spectrometer parameters for PAHs detection
Compound
Fragmentor (V)
[M+H]+
Acenaphthene Acenaphthylene Anthracene Benzo[a]pyrene Benzo[b]fluoranthene Benzo[ghi]perylene Benzo[k]fluoranthene Dibenz[a,h]anthracene Fluoranthene Flurene Indeno[1,2,3-cd]pyrene Naphthalene Phenanthrene Pyrene
111 200 132 143 143 148 128 153 132 210 168 190 142 128
167.1 153.1 155.1 129.1 181.1 179.1 203.1 203.1 253.1 253.1 253.1 277.1 279.1 277.1
will affect air quality [8]. The workers involved in the preparation and application of asphalt are vulnerable to direct exposure to asphalt emissions containing PAHs [9]. Regulatory organizations have warned about the possible carcinogenic effects of asphalt fumes for humans but data are still insufficient. Although there are substantial data showing mutagenic potential and DNA damage caused by exposure to asphalt fumes, research in this area is limited [6]. For the determination of these compounds, GC–MS or LC with fluorescence detection is typically employed [10–13]. Currently, there are official methods for the analysis of PAHs by GC–MS, such as EPA 8100. GC–MS has several advantages in comparison to LC with fluorescence detection, particularly in its resolution capability. GC offers high power chromatographic resolution and MS provides high selectivity and structural information, allowing for the determination of PAHs and their derivatives that exhibit little or no fluorescence [10]. On the other hand, LC–MS for the determination of PAHs and their derivatives is an attractive technique because it does not require derivatization, presents the same resolution capability of GC–MS and, unlike GC, there are no solvent restrictions [14]. However, there are few methods using LC–MS for the determination of PAHs [15]. The objective of this work was to develop a robust and effective method for the determination of 14 PAHs in fractions of asphalt binder. The method was validated against a standard reference material.
2 Materials and methods Figure 1. Chromatogram of the separation of PAHs. (1) 1 mg/L fluorene, 2 mg/L acenaphthylene; (2) 1 mg/L naphthalene, 2 mg/L acenaphthene; (3) 0.4 mg/L phenanthrene; (4) 0.1 mg/L anthracene; (5) 10 mg/L fluoranthene; (6) 0.04 mg/L pyrene; (7) 2 mg/L benz[(b]fluoranthene; (8) 2 mg/L benzo[k]fluoranthene; (9) 0.04 mg/L benzo[a]pyrene; (10) 0.04 mg/L benzo[ghi]perylene, 0.1 mg/L dibenz[a,h]anthracene; and (11) 0.4 mg/L Indeno[1,2,3cd]pyrene.
oxygen, sulfur or nitrogen. These fumes can be inhaled or deposited onto the skin and clothing of workers [6]. Because the asphalt mix is produced and compacted at temperatures higher than 150⬚C, the presence of these gaseous pollutants C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.1 Instrumentation, reagents and solutions An Agilent 1260 Infinity LC–MS chromatograph (Santa Clara, CA, United States) with automatic injection and an Agilent 6430 triple quadrupole mass detector were used. The chromatographic column was a Varian Pursuit 3 PAH 4.6 × 100 mm with 3 m particle size. Acetonitrile and methanol were of Chromasolv LC–MS grade and supplied by Sigma–Aldrich (St. Louis, MO, United States). The PAHs: Acenaphthene, acenaphthylene, anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[ghi]perylene, benzo[k]fluoranthene, dibenz[a,h]anthracene, fluoranthene, www.jss-journal.com
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Figure 2. Extracted chromatogram of the separation of PAHs. (1) 0.1 mg/L dibenz[a,h]anthracene; (2) 0.04 mg/L benzo[ghi]perylene, (3) 0.4 mg/L Indeno[1,2,3-cd]pyrene; (4) 2 mg/L benzo[b]fluoranthene; (5) 2 mg/L benzo[k]fluoranthene; (6) 0.04 mg/L benzo[a]pyrene; (7) 10 mg/L fluoranthene; (8) 0.04 mg/L pyrene; (9) 0.4 mg/L phenanthrene; (10) 0.1 mg/L anthracene; (11) 1 mg/L fluorene; (12) 2 mg/L acenaphthene; (13) 2 mg/L acenaphthylene; (14) 1 mg/L naphthalene. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 3. Analytical parameters of the method
PAH
Intra-day precision (RSD%)
Inter-day precision (RSD%)
Linear range (mg/ L)
Working range (mg/ L)
LOD (mg/ L)
LOQ (mg/ L)
Linear correlation coefficient (r)
Acenaphthene Acenaphthylene Anthracene Benzo[a]Pyrene Benzo[b]fluoranthene Benzo[ghi]perylene Benzo[k]fluoranthene Dibenz[a,h]anthracene Fluoranthene Fluorene Indeno[1,2,3-cd]pyrene Naphthalene Phenanthrene Pyrene
0.47 0.68 0.67 0.60 1.36 0.63 0.92 3.14 2.10 0.49 0.65 1.65 0.58 0.84
2.32 1.32 1.92 2.01 10.36 2.31 3.78 7.02 4.59 1.19 11.22 3.18 3.78 3.24
0.05 – 50 0.005 – 50 0.002 –50 0.002 – 50 0.05 – 50 0.002 – 50 0.05 – 50 0.05 – 50 0.5 – 100 0.05 –50 0.005 –50 0.05 – 50 0.01 –50 0.002 – 50
0.50 – 20.0 0.50 – 20.0 0.025 – 1.00 0.01 – 0.40 0.50 – 20.0 0.01 – 0.40 0.50 – 20.0 0.025 – 1.00 2.50 – 100 0.25 – 10.0 0.10 – 4.00 0.25 – 10.0 0.10 – 4.00 0.01 – 0.40
0.039 0.004 0.002 0.001 0.037 0.005 0.067 0.037 0.346 0.005 0.005 0.010 0.001 0.001
0.129 0.012 0.005 0.002 0.124 0.017 0.222 0.123 1.549 0.016 0.016 0.035 0.004 0.002
0.999 0.990 0.999 0.999 0.999 0.999 0.998 0.995 0.998 0.999 0.998 0.999 0.999 0.999
Table 4. Results of average recovery of PAHs in maltenic fraction (n = 3)
PAH
Recovery in the Recovery in the Recovery in the Average recovery in the acid fraction (%) RSD (%) basic fraction (%) RSD (%) neutral fraction (%) RSD (%) maltenic fraction (%) RSD (%)
Acenaphthene Acenaphthylene Anthracene Benzo[a]pyrene Benzo[b]fluoranthene Benzo[ghi]perylene Benzo[k]fluoranthene Dibenz[a,h]anthracene Fluoranthene Fluorene Indeno[1,2,3-cd]pyrene Naphthalene Phenanthrene Pyrene
97.13 103.63 92.52 97.83 91.75 98.33 94.57 97.29 89.01 105.96 90.32 98.01 105.65 100.48
3.98 0.65 3.13 1.56 7.67 0.60 1.72 3.09 3.15 0.62 6.01 16.00 2.57 14.69
94.75 101.39 91.12 93.14 115.24 96.64 101.55 102.51 91.42 100.79 98.41 109.10 108.09 95.52
fluorene, indeno[1,2,3-cd]pyrene, naphthalene, phenanthrene, and pyrene, were purchased from Fluka and Sigma– Aldrich (St. Louis, MO, United States). Standard solutions of each compound and mixtures were prepared in acetonitrile.
4.67 2.14 1.12 1.37 0.61 0.55 2.84 3.41 9.55 6.45 1.56 11.60 0.45 9.59
99.46 88.50 117.44 91.10 91.68 90.09 80.79 84.42 82.94 83.26 104.44 96.95 84.17 115.04
1.09 1.70 2.87 4.32 9.13 0.60 0.84 2.60 0.85 3.29 1.84 5.19 0.38 12.47
97.11 97.84 100.36 94.02 99.56 95.02 92.31 94.74 87.79 96.67 97.72 101.35 99.30 103.68
2.42 8.34 14.76 3.67 13.64 4.58 11.45 9.83 4.98 12.31 7.25 6.64 13.26 9.79
23 min under a flow of 0.8 mL/min. The injection volume was 20 L. During the run, the column was maintained at 30⬚C. A post-time period of 7 min was adopted to rebalance the system.
2.2 LC–MS conditions 2.3 Sample preparation To develop this method, each PAH was individually injected into the mass spectrometer to obtain its spectrum and also to optimize the fragmentor and the energy of the collision cell. Because PAHs are compounds with low polarity, they were converted into ions by using an APCI ion source. The source and detector conditions were optimized by testing the parameters shown in Table 1. To ensure the best response for the measurement of the analytes, each parameter was varied while the others were maintained at a fixed value. Elution was performed using a gradient that consisted initially of 80% methanol/water for 9 min, 90% methanol/water for 9.5 min, and 100% methanol until the end of the run at C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Asphalt samples were fractionated into asphaltenes and maltenes following the ASTM D4124 method [16]. Briefly, samples were refluxed in isooctane, where the asphaltene fraction, which is composed of polar compounds, precipitated while the maltenic fraction remained soluble. Separation was attained by filtration. The maltenic fraction was separated in their acidic, basic and neutral fractions using the method of Green by non-aqueous preparative ion exchange chromatography [17]. The acidic and basic fractions were diluted to 1:50 in methanol, filtered through a 0.22 m PTFE filter, and injected www.jss-journal.com
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Table 5. Results of the standard reference material NIST 1975 analysis (n = 3)
Compound
Certified/Reference (mg/kg)
Benzo[b]fluoranthene Benzo[ghi]perylenea) Benzo[k]fluoranthene Dibenz[a,h]anthracenea) Fluoranthene Fluorenea) Indeno[1,2,3-cd]pyrenea) Naphthalenea) Pyrenea)
3.20 0.038 0.174 0.079 13.5 0.110 0.12 0.67 0.42
± ± ± ± ± ± ± ± ±
Found (mg/kg) 3.23 ± 0.032 ± < LOQ 0.067 ± 12.93 ± 0.118 ± 0.11 ± 0.62 ± 0.43 ±
0.10 0.006 0.050 0.013 0.6 0.003 0.01 0.01 0.13
0.21 0.0004 0.004 0.24 0.019 0.0007 0.02 0.02
a) Reference values: noncertified values with best estimates of the true value Table 6. PAHs present in the three maltenic fractions of asphalt and the total concentration of each hydrocarbon in this matrix
Fraction(mg/kg) PAH
Acidic
Basic
Neutral
Total(mg/kg)
Anthracene Benzo[a]pyrene Benzo[b]fluoranthene Benzo[ghi]perylene Benzo[k]fluoranthene Fluorene Pyrene
– – – – – 2.48 –
90.32 – – – – – –
1.52 17.81 874.5 1.09 257.1 0.65 3.91
8.97 13.21 67.2 0.86 2.01 98.32 2.97
–: not detected
into the chromatographic system. The neutral fraction was prepared from 2 mL of the fraction that was evaporated to dryness and redissolved in 1 mL of dichloromethane. This new solution from the neutral fraction was diluted 1:10 in methanol, filtered twice through a 0.22 m PTFE filter and injected into the chromatograph. Because SRM 1975 is a liquid extract, an aliquot of 1 g was evaporated to dryness and redissolved in 1 mL of methanol. After this step, this solution was filtered through a 0.22 m PTFE filter and injected into the chromatograph.
3 Results and discussion 3.1 Optimization of the LC–MS method Because the ionization of the analyte in the APCI source occurs in the gas phase, it is important to ensure an efficient drying step (evaporating the solvent) to achieve a better response of the equipment [18]. The best operating conditions obtained after optimization of the source parameters are shown in Table 1. A significant dependence on the gas drying temperature and the vaporizer temperature for the eluent flow from the C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
chromatographic system was observed, which were set at 300 and 250⬚C, respectively. Because the gas flow also aids in the spray drying, a decrease of the signal was observed with an increase of the gas flow and nebulizer pressure; thus, the optimized conditions for these parameters were 8 L/min and 20 psi, respectively. In the case of PAHs, an increase in the corona discharge results in an increase in the ionization level. The ionization was performed with a higher discharge of the corona needle (10 A). The capillary voltage determines the entry of ions into the mass spectrometer, 3000 V was the ideal voltage for these compounds. Although PAHs are very stable condensed aromatic chains in which C–C bonds are hardly broken, it is possible to fragment these compounds. Hutzler et al. [15] monitored them in the selected reaction monitoring mode. However, when this approach was performed in this study, low abundance was observed, therefore the selective ion monitoring (SIM) mode was chosen for this analysis. The mass of the molecular ion of each compound was monitored in the positive polarity. The optimized conditions for the ion determination and the m/z values monitored are shown in Table 2. Figure 1 shows the total ion chromatogram, in which threeco-elutions were observed: fluorene and acenaphthylene (peak 1), naphthalene and acenaphthene (peak 2), and dibenz[a,h]anthracene and benzo[ghi]perylene (peak 9). Although co-eluted compounds have the same retention time, they possess different m/z values. With the detector operating in SIM mode, the monitored m/z values are selected to represent the target compound, i.e., the mass spectrometer detects one m/z at a time [18]. This approach can be seen in Fig. 2, in which the extracted chromatograms for each m/z are displayed. Alternatively, isobaric compounds, such as fluoranthene and pyrene, did not interfere with one another and were chromatographically separated.
3.2 Validation of the method The method was evaluated for inter- and intra-day precision, linearity, detection and quantification limits and accuracy. To perform the precision assay, synthetic samples were prepared by diluting stock solutions in the mobile phase at concentrations in the lower, middle and higher limits of the analytical curve. The intra-day and inter-day precision were evaluated by carrying out three injections of the same standard on the same day and on three consecutive days, respectively. The linearity of the method was checked by linear regression determined by the method of least squares. The LOD and LOQ were calculated from the S/N. S/N = 3 and 10 were used for estimating the LOD and LOQ, respectively [19]. Recovery studies were conducted for all compounds in the three maltenic fractions analyzed. The standard reference material SRM 1975, a diesel particulate extract, was analyzed for method validation. www.jss-journal.com
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Figure 3. Extracted chromatograms of the neutral, basic and acidic fraction for each m/z monitored. (1) benzo[ghi]perylene; (2) benzo[b] fluoranthene; (3) benzo[k]fluoranthene; (4) benzo[a]pyrene; (5) pyrene; (6) anthracene; (7) fluorene.
The proposed method exhibits limits of detection and quantification in the range of parts per billion (g/L) (Table 3). Fluoranthene was the only compound that showed higher limits of detection and quantification, most likely because of its difficult ionization and thereby low sensitivity. Good intra-day and inter-day precision were obtained with RSD values within an acceptable range. The results show good linear correlation coefficients (r) and a wide linear range of more than an order of magnitude. The recovery results for evaluating the accuracy of the method were also satisfactory (Table 4) and were within a range of 80 to 110% for the concentration levels studied [19]. The values for the HPAs in the Standard Reference Material (SRM 1975) are certified for benzo[b]fluoranthene, benzo[k]fluoranthene, and fluoranthene, and noncertified (best estimates of the true value) for benzo[ghi]perylene, dibenz[a,h]anthracene, fluorene, indeno[1,2,3-cd]pyrene, naphthalene, and pyrene. The results found using the developed methodology did not differ significantly from the reference according to the Student t-test, at a significance level of 0.05, as shown in Table 5.
3.3 Sample application The asphalt specimen was fractionated into asphaltenes and maltenes, and the maltene fraction was further separated C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
into its acidic, basic, and neutral fractions. Only the maltenic fraction was analyzed because it consists of low-molecularweight hydrocarbons that are soluble in n-heptane, similar to the case of PAHs [20]. The acidic and basic fractions were diluted in methanol, which is the mobile phase of the chromatographic system. The neutral fraction is a more complex matrix that when diluted in methanol forms a precipitate. Thus, the fraction was resuspended in dichloromethane, followed by dilution in methanol. Figure 3 shows the chromatograms obtained for the maltenic acidic, basic, and neutral fractions. The concentration of PAHs in the fractions ranged from 0.053 to 40.24 mg/L with a RSD ranging from 0.55 to 16.07%. Although anthracene and fluorene were the only PAHs found in the basic and acidic fractions, respectively, fluorene, anthracene, pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, and benzo[ghi] perylene were all identified in the neutral fraction. The presence of this elevated number of PAHs in the neutral fraction most likely results from the non-polar character of these compounds. Table 6 presents the concentration of each PAH in each fraction, as well as the calculated concentration of each compound in the asphalt matrix. The overall results show that the developed method was effective for this application and may be used for the determination of PAHs in this type of matrix. www.jss-journal.com
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The Asphalt Institute [21] presented a list of PAHs in bitumes and their respective concentrations. Although the samples are different, the PAHs found in this study were the same as those presented in this publication. Pyrene and benzo[ghi]perylene were within the same concentration ranges found by the cited publication. The other compounds found in this study showed higher values than those reported in the literature, likely as a result of the differences that exist between these complex matrices.
4 Conclusions The developed method exhibits adequate sensitivity, good precision, and accuracy, and a wide linear range for the determination of PAHs. The LOD and LOQ obtained herein indicate that the method allows for the determination of trace amounts of these compounds. Its application in asphalt fractions identified the presence of seven compounds in the samples. As PAHs have a recognized toxicological potential, the development of methods for the determination of these compounds in different matrices is important. The authors acknowledge the financial support provided by CNPq and CAPES. The authors also greatly acknowledge the financial support from PETROBRAS through the Center of Research and Development “Leopoldo Am´erico Miguez de Mello” (CENPES). The authors declare no conflict of interest.
5 References [1] Cavalcante, R. M., Filho, N. S. M., Viana, R. B., Oliveira, I. R. N., Nascimento, R. F., Silveira, E. R., Freire, G. S. S., Quim. Nova. 2007, 3, 560–564. [2] Delgado-Saborit, J. M., Stark, C., Harrison, R. M., Environ. Int. 2011, 37, 383–392. [3] Collins, J. F., Brown, J. P., Alexeeff, G. V., Salmon, A. G., Regul. Toxicol. Pharmacol. 1998, 28, 45–54.
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[4] Wang, Z., Ma, X., Na, G., Lin, Z., Ding, Q., Yao, Z., Environ. Pollut. 2009, 157, 3132–3136. [5] Brandt, H. C. A., De Groot, P.C., Water Research. 2001, 35(17), 4200–4207. [6] Schreiner, C., Regul. Toxicol. Pharmacol. 2011, 59, 270– 284. [7] Browarzik, D., Laux, H., Rahimian, I., Fluid Phase Equilibria. 1999, 154, 285–300. [8] Rubio, M. C., Mart´ınez, G., Baena, L., Moreno, F., J. Clean. Prod. 2012, 24, 76–84. [9] NIOSH, National Institute for Occupational Safety and Health (Ed.), Hazard Review: Health Effects of Occupational Exposure to Asphalt. Hazard Review, DHHS Publication, pp. 2011–2110. ˜ [10] Plaza-Bolanos, P., Garrido Frenich, A., Vidal, J. L. M., J. Chromatogr. A. 2010, 1217, 6303–6326. [11] Ping, C., Hui, Z., Jay, G., Mingxing, S., Guofeng, S., Liang, L., Guoqing, S., J. Sep. Sci. 2015, 38, 864–870. [12] Geng, L., Guibin, L., Yong, C., Hui, Y., Dezhong, D., J. Sep. Sci. 2012, 35, 2796–2804. [13] Fei, Y., Yiming, L., Rui, S., Chunyan, C., Di, P., Qianli, Z., Qingyun, C., Shouzhuo, Y., J. Sep. Sci. 2011, 34, 716–723. [14] Delhomme, O., Millet, M., Herkes, P., Talanta 2008, 74, 703–710. [15] Hutzler, C., Luch, A., Filser, J.G., Anal. Chim. Acta. 2011, 702, 218–224. [16] ASTM, Standard Test Method, Separation of Asphalt into Four Fractions, D4124, 2013. [17] Green, J. B., Yu, S. K. T., Pearson C. D., Reynolds, J. W., Energy Fuels 1993, 7, 119–126. [18] Gross, J. H., Mass Spectrometry – A Text Book, SpringerVerlag, Berlin Heidelberg 2011. [19] Lister, A. S., in: Ahuja, S., Dong, M., (Eds), Handbook of Pharmaceutical Analysis by HPLC, Elsevier, Amsterdam, 2005, pp 191–217. ´ [20] Lins, V. F. C., Araujo, M. F. A. S., Yoshida, M. I., Ferraz, V. P., Andrada, D. M., Lameiras, F. S., Fuel 2008, 87, 3254–3261. [21] Asphalt Institute, Eurobitume, The Bitume Industry – A Global Perspective Production, Chemistry, Use, Specification and Occupational Exposure, Asphalt Institute Inc. and European Bitumen Association, Eurobitume 2011.
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