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Optimization of aqueous acetylation for determination of hydroxy polycyclic aromatic hydrocarbons in water by stir bar sorptive extraction and thermal desorption–gas chromatography–mass spectrometry
Analytica Chimica Acta 535 (2005) 243–250
Optimization of aqueous acetylation for determination of hydroxy polycyclic aromatic hydrocarbons in water by stir bar sorptive extraction and thermal desorption–gas chromatography–mass spectrometry Nobuyasu Itoh, Hiroaki Tao∗ , Takashi Ibusuki Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan Received 14 July 2004; received in revised form 1 December 2004; accepted 1 December 2004 Available online 11 January 2005
Abstract Stir bar sorptive extraction (SBSE) using a 10 mm-long stir bar coated with 24 l of polydimethylsiloxane (PDMS) in combination with the thermal desorption technique and gas chromatography–mass spectrometry was used for determining hydroxy polycyclic aromatic hydrocarbons (OH-PAHs), particularly mono and diOH-PAHs having two- to four-ring structures, in water samples. To improve the recovery for OH-PAHs, in situ derivatization with acetic anhydride was applied prior to SBSE. The optimal conditions for a 10 ml water sample were attained with the addition of 100 mg of sodium hydrogen carbonate, followed by the addition of 20 l of acetic anhydride and extraction for 360 min. In the selected ion monitoring (SIM) mode, the limits of detection were found to range from 0.27 ng l−1 for 2-hydroxynaphthalene (as 2-hydroxynaphthalene acetate) to 25 ng l−1 for 1,4-dihydroxynaphthalene (as 1,4-naphthoquinone) (S/N = 3). The low total recovery (<50%) of the entire procedure was mainly due to the low extraction efficiency of acetyl derivatives from water to PDMS, while the in situ derivatization step achieved more than 88% efficiency. Nine OH-PAHs, such as 1,4-naphthoquinone, 2-hydroxynaphthalene, 2,3-dihydroxynaphthalene, 1,5-dihydroxynaphthalene, 1,3-dihydroxynaphthalene, 1,6-dihydroxynaphthalene, 2,6-dihydroxynaphthalene, 2,7-dihydroxynaphthalene, and 9-hydroxyphenanthrene, were detected in environmental samples under the optimal conditions. The detection of OH-PAHs in environmental water samples has not been reported so far. © 2004 Elsevier B.V. All rights reserved. Keywords: GC–MS; Hydroxy polycyclic aromatic hydrocarbons (OH-PAHs); Stir bar sorptive extraction (SBSE); Thermal desorption (TD); Water sample
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are produced by industrial processes and the incomplete combustion of fossil fuel, as well as by forest fires. The emitted PAHs are transported over long distances in the atmosphere and their deposition in rain/snow is considered to be a significant source of PAHs for surface waters [1,2]. In the atmosphere, the main reaction of PAHs is with OH radicals, converting them into hydroxides (OH-PAHs) [3,4]. This reaction is considered to be the primary step for the degradation and conversion into ∗
Corresponding author. Tel.: +81 29 861 8788; fax: +81 29 861 8308. E-mail address: [email protected] (H. Tao).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.12.002
nitro-PAHs. As their chemical structures resemble those of estrogenic hormones, OH-PAHs are suspected to have endocrine disrupting effects [5]. However, the distribution of OH-PAHs in the environment has not been investigated so far because they are found in trace amounts in the aquatic environment and they have more polar characteristics than PAHs. Recently, OH-PAHs, particularly OH-pyrene and OH-naphthalenes, in biological fluids have been analyzed and used as indicators of PAH exposure [6–12]. Some researchers have used solid phase microextraction (SPME) and stir bar sorptive extraction (SBSE), both of which use polydimethylsiloxane (PDMS) as the adsorbent, for the extraction/preconcentration of OH-PAHs, in combination with the thermal desorption
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(TD) technique and gas chromatography–mass spectrometry (TD–GC–MS) [6,9,10,13]. The performance of the extraction/preconcentration by SPME and SBSE is based on the octanol–water partition coefficient (Ko/w ). As SBSE uses a much larger volume of PDMS (>24 l) than SPME (<0.7 l), SBSE enables more efficient extraction of compounds with relatively low pKo/w [14]. Using SBSE, Desmet et al. [10] showed that the calculated limit of detection (LOD) of OHpyrene is 2 ng l−1 (S/N = 3) in urine samples and Kawaguchi et al. [15] reported that the LODs of 4-nonylphenol and 4-tertoctylphenol in biological fluids are 40 and 4 ng l−1 (S/N = 3), respectively. As for environmental samples, Kawaguchi et al. [16] reported that the LODs of 4-nonylphenol and 4-tertoctylphenol in tap and river water samples are lower than those in biological fluids at 20 and 2 ng l−1 (S/N = 3), respectively. Therefore, this SBSE–TD–GC–MS method is expected to be applicable to the determination of OH-PAHs in environmental samples. To improve extraction efficiency and to facilitate vaporization in GC analysis, derivatizations have been generally applied to polar molecules such as phenols. There are many reports on the in situ derivatization of phenols using benzyl bromide, butyl chloroformate, dimethyl sulfate and acetic anhydride [17]. Among them, derivatization with acetic anhydride to form phenol acetate is one of the simplest methods, and both high derivatization efficiency (>85%) and high recovery (ca. 100%) by the organic phase of the phenol acetate have been reported [18–21]. Thus, this derivatization has been used for such phenols as alkyl-, chloro- and nitro-phenols, and OH-pyrene [10,13,18–26]. Although this method has been in use for more than 20 years [18] and applied to many phenols, optimization of the parameters for the in situ derivatization of phenols especially for OH-PAHs has not been achieved so far. In this study, the parameters for in situ derivatization with acetic anhydride/SBSE of OH-PAHs in artificial seawater are optimized. The recovery at each step and the effect of ionic strength in water are discussed. Then, the optimal analytical conditions are applied to such environmental samples as seawater and puddle water.
2. Experimental 2.1. Materials and reagents The OH-PAHs studied were 1-hydroxynaphthalene (1OHNP), 2-hydroxynaphthalene (2-OHNP), 1,3-dihydroxynaphthalene (1,3-diOHNP), 1,4-dihydroxynaphthalene (1,4diOHNP), 1,5-dihydroxynaphthalene (1,5-diOHNP), 1,6-dihydroxynaphthalene (1,6-diOHNP), 2,3-dihydroxynaphthalene (2,3-diOHNP), 2,6-dihydroxynaphthalene (2,6diOHNP), 2,7-dihydroxynaphthalene (2,7-diOHNP), 9-hydroxyphenanthrene (9-OHPT) and 1-hydroxypyrene (1OHPR). 1-OHNP, 2-OHNP, 1,3-diOHNP, 2,3-diOHNP, and 2,7-diOHNP standards were obtained from Wako
Pure Chemical Industries (Osaka, Japan); 1,4-diOHNP, 1,5-diOHNP, and 1,6-diOHNP standards were from Kanto Chemical Industries (Tokyo, Japan); 2,6-diOHNP standard was from Fluka (Buchs, Germany); and 9-OHPT and 1-OHPR standards were from Aldrich (Steinheim, Germany). The internal standard (I.S.), phenanthrene labeled with 13 C10 (phenanthrene-d10 ; 98% purity), was obtained from Cambridge Isotope Laboratories Inc. (MA, USA). A stock solution containing approximately 1 mg l−1 of each compound was prepared in acetonitrile. Artificial seawater was prepared using artificial salt for marine aquarium (Aquamarine S, YashimaChem, Osaka, Japan). A stir bar coated with 24 l of PDMS was obtained from Gerstel GmbH (M¨ulheim an der Ruhr, Germany) and conditioned by washing (ultrasonic treatment for 5 min) with MeOH/CH2 Cl2 (1:1, v/v) and 100% acetonitrile, followed by placing inside a TDS-2 tube for TD under He flow (50 ml min−1 ) for 20 h at 300 ◦ C. A preheating run was carried out after the conditioning process to ensure that the sorbent would not produce any spurious peaks in the chromatogram. 2.2. In situ derivatization and extraction In situ derivatization of OH-PAHs in water sample (10 ml) was performed using acetic anhydride after the addition of sodium hydrogen carbonate (NaHCO3 ) when necessary. To extract OH-PAH acetates with PDMS, a stir bar coated with PDMS was rotated at 1000 rpm. After extraction, the stir bar was removed with clean tweezers and dried with lint-free tissue. Then, the stir bar was placed inside the TDS-2 tube for TD. 2.3. Thermal desorption and GC–MS measurement Desorption of the extracted OH-PAH acetates was carried out using a TDS-2 TD unit (Gerstel GmbH, M¨ulheim an der Ruhr, Germany) mounted on a CIS-4 PTV injector (Gerstel GmbH, M¨ulheim an der Ruhr, Germany). The temperature of the TDS-2 tube was programmed to increase from 20 to 300 ◦ C at a rate of 60 ◦ C min−1 with a final holding time of 5 min at a flow rate of 50 ml min−1 of He. Desorbed OH-PAHs acetates were cryogenically trapped at the PTV injector, and then transferred into a GC column by heating from −80 to 300 ◦ C at a rate of 12 ◦ C s−1 . GC–MS analysis was carried out with an Agilent 6890/5973N (Agilent Technologies, Palo Alto, CA, USA) equipped with a DB-5MS capillary column (30 m × 0.25 mm i.d.; 0.25 m film thickness) and programmed from 50 (2 min hold) to 300 ◦ C at a heating rate of 5 ◦ C min−1 . Helium was used as the carrier gas at a flow rate of 1.0 ml min−1 in the constant flow mode. The temperature of the MS transfer line was kept at 280 ◦ C. The ionization energy used for electron impact (EI) ionization was 70 eV. The mass range monitored was
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40–550 (m/z) for optimization of the analytical procedures. To determine the LOD and to analyze environmental samples, the selected ion monitoring (SIM) mode was used. Identification of peaks in environmental samples were performed by comparing with both retention time and relative intensities of ion masses in standard solutions. All procedures to obtain the optimal condition and recoveries were performed with three replicates. 2.4. Recovery test at each step To obtain completely acetylated OH-PAHs, acetylation was performed in acetonitrile at 60 ◦ C for 24 h and the absence of non-acetylated OH-PAHs was confirmed. This solution of completely acetylated OH-PAHs was directly injected into the TDS-2 tube filled with glass wool (sample A). Acetonitrile and excess acetic anhydride were removed by N2 stream before analysis. To calculate the extraction efficiency, the solution of completely acetylated OH-PAHs was spiked into artificial seawater and SBSE was carried out for 360 min (sample B). The extraction efficiency was calculated by comparing the peak areas of sample B to those of sample A. Total recovery including the derivatization and the extraction were obtained by adding the solution of non-acetylated OH-PAHs into artificial seawater and in situ derivatization (with 20 l of acetic anhydride and 100 mg of NaHCO3 )/SBSE was carried out for 360 min (sample C). The derivatization efficiency was calculated by comparing the peak areas of sample C to those of sample B. Recovery test was also conducted to the samples such as 35‰ NaClaq (D), Milli-Q water (E) and seawater collected from Kashima Port (F). 2.5. Collection and preparation of environmental samples Seawater samples were collected from Kashima Port near power plant, which faces the Pacific Ocean (Kashima), and a Yacht Harbor in Tokyo Bay; puddle water samples were collected from one of the main streets in Tsukuba City (street A) and two heavily congested traffic streets in Tokyo (streets B and C). The ionic strengths of the puddle water samples were adjusted to that of the seawater sample by adding artificial salt for marine aquarium. All samples were analyzed without any other pre-treatments. 2.6. Calculation of PDMS–water partition coefficient As the distribution of PDMS–water was generally approximated to that of octanol–water, KowWin software (http://esc.syrres.com/interkow/kowdemo.htm) was used for calculating the pKo/w values of OH-PAH acetates. Good agreement between the values obtained with the software and the experimental data has been reported previously [27].
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3. Results and discussion 3.1. Optimization of volume of acetic anhydride The addition of acetic anhydride to form acetate in a water sample has been studied for many phenols, such as alkyl-, chloro-, and nitro-phenols, and OH-pyrene [10,13,18–26]. As the acetylation of phenols enhances their affinity for the organic phase, the recovery of the acetates by organic solvents is high compared to that of intact phenols [18]. Moreover, there are two advantages of the acetylation of phenols prior to GC analysis. First, the acetates would be easier to volatilize than the intact phenols. Second, the acetylation of phenols would reduce peak tailing on the chromatograms due to less interaction with the active sites of liquid phase of the GC column since acetates have less interaction through hydrogen bonding with the active or contamination sites relative to intact phenols [17], and this is important for accurate determination. Although this method has been in use for more than 20 years [18], the volume of acetic anhydride added to 10 ml water samples varies among reports, from less than 0.0125 ml (final concentration: 13.2 mmol l−1 ) [19] to 1.0 ml (final concentration: 1.06 mol l−1 ) [26]. Thus, the optimal volume of acetic anhydride added was determined first. Fig. 1 shows the relationships between the volume of acetic anhydride added and the peak area (A and B) and pH after reaction (C). This experiment was carried out with 1 h extraction of 10 g l−1 OH-PAHs in artificial seawater in the absence of any alkaline compounds described in the next section. No OH-PAH acetates were detected when acetic anhydride was not added. The peak areas of OH-PAH acetates increased with increasing volume of acetic anhydride added up to 20 l (final concentration: 2160 mg l−1 , 21.2 mmol l−1 in artificial seawater), whereas that of 1,4diOHNP acetate was not detected. Instead of 1,4-diOHNP acetate, 1,4-naphthoquinone (1,4-NQ) was detected in all experiments. This is due to the chemical characteristic of 1,4diOHNP, namely, it is easily converted into 1,4-NQ. When more than 20 l of acetic anhydride was added, the peak areas of OH-PAH acetates were markedly decreased whereas 1,4-NQ decreased only slightly and I.S. (phenanthrene-d10 ) slightly increased. As regards the pH, it decreased remarkably from 6.87 to 3.36 by the addition of 10 l of acetic anhydride, whereas it decreased slightly (from 3.14 to 2.20) when the volume was increased from 20 l to 600 l (Fig. 1C). Particularly, when the volume increased from 20 to 100 l, the peak areas of OH-PAH acetates decreased to less than half of those at 20 l addition, although the pH decreased by only 0.47 (from pH 3.14 to 2.67). Since only mono and diOHPAH acetates involving acetylation decreased markedly up to 100 l addition, large volume of acetic anhydride would suppress acetylation rather than extraction into PDMS. Same trend was reported by Bal´ıkov´a and Kohl´ıcˇ ek [22] on other phenols such as phenol, cresols and chlorophenol. They suggested that a large excess of acetic anhydride provides an acidic medium, thereby unfavorably shifting the equilibrium
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Fig. 1. Relationships between volume of acetic anhydride and peak area of various OH-PAHs (A and B), and pH after the reaction (C). Abbreviations: 1-OHNP, 1-hydroxynaphthalene; 2-OHNP, 2-hydroxynaphthalene; 1,4-NQ, 1,4-naphthoquinone; 9-OHPT, 9-hydroxyphenanthrene; 1-OHPR, 1-hydroxypyrene; 2,3-diOHNP, 2,3-dihydroxynaphthalene; 1,5-diOHNP, 1,5-dihydroxynaphthalene; 1,3-diOHNP, 1,3-dihydroxynaphthalene; 1,6diOHNP, 1,6-dihydroxynaphthalene; 2,6-diOHNP, 2,6-dihydroxynaphthalene; 2,7-diOHNP, 2,7-dihydroxynaphthalene.
of the acetylation reaction [22]. Since 20 l addition of acetic anhydride gave the highest amounts for all OH-PAH acetates, the addition volume of acetic anhydride was set at 20 l per 10 ml of artificial seawater, hereafter. 3.2. Optimization of amount of NaHCO3 To enhance the acetylation of phenols, most researchers have used such alkaline compounds as potassium hydrogen carbonate [10,13], sodium hydrogen carbonate (NaHCO3 ) [18–20,23], potassium carbonate [22,24,26] and sodium hydroxide [25] at various concentrations ranging from 0.05 [22] to 0.725 mol l−1 [26]. Thus, the optimal amount of alkaline compound added was determined as the next step. In this experiment, NaHCO3 was selected as the alkaline compound because of its buffer effect, which would prevent the hydrolysis of acetates in the excess acid/base condition. Fig. 2 shows the relationships between the amount of NaHCO3 added and the peak area (A and B) and the change in
Fig. 2. Relationships between amount of NaHCO3 and peak area of various OH-PAHs (A and B), and change in pH (C). Abbreviations are the same as those in Fig. 1.
pH (C). This experiment was performed with 1 h extraction of 10 g l−1 OH-PAHs in artificial seawater with the addition of 20 l of acetic anhydride. Regardless of the amount of NaHCO3 , acetylation was markedly (>2 times except for 1,4NQ) enhanced compared to the case where only acetic anhydride was added (Fig. 1A and B). The peak areas of OH-PAH acetates increased with increasing amounts of NaHCO3 up to 100 mg (119 mmol l−1 ), and then slightly decreased thereafter. On the other hand, phenanthrene-d10 and 1,4-NQ, both did not involve acetylation, showed no significant change at any added amounts of NaHCO3 (Fig. 2A). As regards the pH, it dropped to the acidic range (near pH 3) by the addition of acetic anhydride without any addition of NaHCO3 . However, pH was kept in the neutral range after the addition of acetic anhydride when the amount of NaHCO3 exceeded 50 mg (Fig. 2C). The pH change during before and after the reaction was reduced with increasing amounts of NaHCO3 . This is due to the buffer effect of NaHCO3 , as expected, which provides a medium where the acetates are free from hydrolysis. Upon the addition of more than 100 mg of NaHCO3 , calcium carbonate was precipitated during stirring. Since peak area of both phenanthrene-d10 and 1,4-NQ were constant even by the addition of more than 100 mg, excess
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amount of NaHCO3 might suppress the acetylation of OHPAHs rather than cause the adsorbent loss on precipitated calcium carbonate. From the result of this experiment, addition amount of NaHCO3 was set at 100 mg per 10 ml artificial seawater, hereafter. 3.3. Optimization of in situ derivatization/extraction time As OH-PAHs have OH group(s) in their structures, their pKo/w values are lower than those of pesticides and PAHs (Table 1). Thus, their extraction times are expected to be longer than those of other apolar compounds, as reported previously (from 60 to 90 min for PAHs and alkylphenols [15,16,28]). The in situ derivatization/extraction time was studied from 5 to 480 min (Fig. 3). Extracted amounts of all OH-PAH acetates increased with increasing the time. Mono OH-PAHs such as 1- and 2-OHNPs, 9-OHPT and 1-OHPR, reached the steady state at 60–120 min (Fig. 3A), whereas diOHNPs continued to increase up to 360 min (Fig. 3B). Although 1,3- and 2,3-diOHNP acetates have higher pKo/w (3.04) than those of 1- and 2-OHNP acetates (2.77), they took longer time for in situ derivatization/extraction. The longer time for diOHNPs relative to mono OHNPs might depend on their longer acetylation time rather than extraction into PDMS. At least, this result suggests that in situ derivatization/extraction time for each OH-PAHs acetate would depend on the number of OH group(s) in its structure rather than its pKo/w . Since there was no significant increase between 360 and 480 min, the extraction time was set at 360 min, hereafter.
Fig. 3. Relationship between acetylation/extraction time and peak area of various OH-PAHs. Abbreviations are the same as those in Fig. 1.
tion were performed under the optimal conditions discussed above (with 20 l of acetic anhydride, 100 mg of NaHCO3 , and extraction for 360 min). The results are listed in Table 1, together with the monitoring ion masses and the theoretical pKo/w calculated using KowWin software. The lowest LOD (S/N = 3) is 0.27 ng l−1 for 2-OHNP (as 2-OHNP acetate) and the highest LOD (S/N = 3) is 25 ng l−1 for 1,4-diOHNP (as 1,4-NQ). Although the 1,3- and 2,3-diOHNP acetates have the same pKo/w values (Table 1), the LOD of 1,3-diOHNP acetate (11 ng l−1 ) is more than six times higher than that of 2,3-diOHNP acetate (1.6 ng l−1 ). This suggests that the LODs of OH-PAH acetates depend not only on pKo/w but also on other factors, including acetylation efficiency and fragmentation pattern in mass spectrometry. The limits of quantification
3.4. Limits of detection Calibration was performed by extracting artificial seawater samples spiked at seven concentrations of non-acetylated OH-PAHs (0.01, 0.05, 0.10, 0.50, 1.0, 5.0 and 10.0 g l−1 ) and analyzing them in the SIM mode. Acetylation and extrac-
Table 1 Calculated pKo/w of OH-PAH acetates, ion mass and their calibration data under the optimal conditions Compound
pKo/w a
Ion mass (m/z)
Rb
Limit of detection (ng/l) (S/N = 3)
Limit of quantification (ng/l) (S/N = 10)
1-OHNP acetate 2-OHNP acetate 1,4-NQ 2,3-diOHNP acetate 1,5-diOHNP acetate 1,3-diOHNP acetate 1,6-diOHNP acetate 2,6-diOHNP acetate 2,7-diOHNP acetate 9-OHPT acetate 1-OHPR acetate Phenanthrene-d10
2.77 2.77 1.44 3.04 2.36 3.04 2.36 2.36 2.36 3.94 4.53 4.35
144, 115 144, 115 158, 130 160, 202 160, 202 160, 202 160, 202 160, 202 160, 202 194, 165 218, 189 188, 160
1.00 0.999 0.998 1.00 0.996 0.969 0.998 1.00 0.995 0.998 0.997 –
0.35 0.27 25 1.6 3.0 11 1.6 2.7 1.6 2.1 3.2 –
1.2 0.92 75 5.4 10 35 5.4 8.9 5.4 5.2 8.6 –
Abbreviations are the same as in Fig. 1. SBSE was performed with the addition of 100 mg of NaHCO3 , followed by 20 l of acetic anhydride and extraction for 360 min. a Calculated with KowWin software (http://esc.syrres.com/interkow/kowdemo.htm). b Regression coefficients were calculated from seven concentrations (0.01–10 g l−1 ).
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Table 2 Acetylation and extraction efficiencies for OH-PAHs in various samples (%) with theoretical extraction efficiencies for OH-PAH acetates calculated from pKo/w (%) in Table 1 Compound
Acetylation effiiciencya (C/B)
Extraction effiiciencyb (B/A)
Total recoveryc (C/A)
NaClaq/ASW (D/C)
Milli-Q/ASW (E/C)
Kashima/ASW (F/C)
Theoretical extraction efficiency calculated from pKo/w d (%)
1-OHNP acetate 2-OHNP acetate 1,4-NQ 2,3-diOHNP acetate 1,5-diOHNP acetate 1,3-diOHNP acetate 1,6-diOHNP acetate 2,6-diOHNP acetate 2,7-diOHNP acetate 9-OHPT acetate 1-OHPR acetate
90.7 (4.20) 93.4 (4.52) – 99.4 (8.40) 88.2 (7.66) 90.1 (12.5) 89.9 (8.05) 93.4 (9.49) 88.4 (6.78) 96.2 (5.15) 109 (11.6)
51.6 (2.05) 50.1 (2.95) 17.0 (7.55) 24.5 (0.93) 19.0 (0.90) 25.8 (0.99) 22.9 (1.17) 24.0 (1.43) 26.2 (1.37) 47.4 (2.91) 37.1 (5.76)
46.9 (2.62) 46.8 (3.15) 15.4 (7.21) 24.3 (1.87) 16.7 (1.39) 23.3 (3.22) 20.6 (1.97) 22.4 (2.46) 23.2 (2.12) 45.6 (3.68) 40.4 (7.52)
111 (5.85) 107 (4.95) 104 (20.2) 96.3 (7.75) 108 (8.55) 84.7 (12.0) 106 (9.13) 107 (10.7) 108 (8.50) 112 (7.36) 109 (15.0)
92.8 (4.60) 92.1 (4.15) 74.7 (20.9) 80.8 (8.91) 91.7 (11.1) 63.2 (6.32) 91.1 (10.4) 93.1 (11.4) 94.4 (8.68) 109 (7.28) 106 (15.5)
103 (7.88) 99.6 (6.58) 106 (15.0) 96.3 (9.61) 104 (10.1) 42.5 (9.04) 102 (11.5) 102 (12.6) 101 (11.4) 101 (7.66) 94.0 (15.1)
58.6 58.6 6.20 72.4 35.5 72.4 35.5 35.5 35.5 95.4 98.7
Mean values of three replicates (relative standard deviations). ASW, SBSE from artificial seawater; NaClaq, SBSE from 35‰ NaClaq; Kashima, SBSE from seawater collected from Kashima Port. SBSE was performed with the addition of 100 mg of NaHCO3 , followed by 20 l of acetic anhydride and extraction for 360 min. Abbreviations are the same as in Fig. 1. a Acetylation efficiency in artificial seawater. b Extraction efficiency to PDMS for each OH-PAH acetate from artificial seawater. c Total recovery of the entire procedure. d Calculated from KowWin software.
ranged from 0.92 ng l−1 for 2-OHNP (as 2-OHNP acetate) to 75 ng l−1 for 1,4-diOHNP (as 1,4-NQ) as summarized in Table 1. 3.5. Recoveries of OH-PAHs at each step and from spiked samples When using SBSE in combination with in situ derivatization, there are two possible steps in which OH-PAHs are not completely recovered. One is the acetylation step with acetic anhydride and the other is the extraction step from water to PDMS. To identify the recovery at each step, several samples were prepared. Details of the sample preparation and the definitions are described in Section 2. Table 2 summarizes the recoveries for OH-PAHs at each step and the recoveries from the spiked samples, along with the theoretical extraction efficiency calculated from the pKo/w values in Table 1. The acetylation efficiencies are higher than 85% for all OH-PAHs including diOHNPs having two OH groups. On the other hand, the extraction efficiencies are lower than 55% for all OH-PAH acetates, with those for 1,4NQ and 1,5-diOHNP acetate being lower than 20%. These results suggest that the low total recovery (C/A) is due to the low extraction efficiency of PDMS for OH-PAH acetates and 1,4-NQ from water. The effects of ionic strength in water samples were examined using samples D, E and F. The recoveries of OH-PAH acetates except for 1,3-diOHNP acetate from samples D and F were almost the same as those from sample C. As only sample E contained no salt except for NaHCO3 , the low recoveries from sample E were due to its low ionic strength compared to those of the other samples. Therefore, it is necessary to adjust the ionic strength of freshwater samples before in situ derivatization/extraction.
The extraction efficiencies (B/A) of PDMS for all OHPAH acetates were lower than those calculated from pKo/w , whereas that for 1,4-NQ was more than 2 times higher than those calculated (Table 2). Baltussen et al. [14] observed lower extraction efficiencies of apolar compounds and moderate recoveries of polar compounds than those expected from theoretical values, and suspected two reasons for the discrepancy. One was that the distribution of PDMS–water might not always be approximated by pKo/w . The other was that highly apolar solutes might adsorb to glass, causing analyte loss. In our experiments, nonspecific adsorption to glass would be negligible because good linearity was obtained for both 9-OHPT and 1-OHPR acetates even at low concentrations without significant negative y-axis intercepts (Table 1). Less recoveries than those of theoretical values were obtained not only for 9-OHPT and 1-OHPR acetates but also for other OH-PAHs, especially for 1,3- and 2,3-diOHNP acetates. This supports that extraction behavior of PDMS for each compound could not be always approximated only by pKo/w and also affected by other physical and/or chemical characteristics. Recoveries from spiked artificial seawater and environmental samples (>90%) and reproducibility (<20%) were fairly good for all OH-PAH acetates except for the low recovery of 1,3-diOHNP acetate (42.5%). This suggests that the present SBSE–TD–GC–MS method in combination with in situ derivatization is applicable to environmental samples. Low recoveries of 1,3-diOHNP acetate were also observed in samples D and E relative to other OH-PAH acetates, although both acetylation and extraction efficiencies for 1,3-diOHNP acetate in artificial seawater were comparable to those of other diOHNP acetates. This suggests that other factors might affect on this compound.
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Fig. 4. Chromatograms of standard solution containing each OH-PAH at 500 ng l−1 and environmental samples (Kashima, Yacht Harbor; streets A, B and C). For ease of comparison of the chromatograms, the peak intensities of OH-PAH acetates in the environmental samples were magnified 10 times relative to that of I.S. (d). (a) 1,4-NQ; (b) 1-OHNP acetate; (c) 2-OHNP acetate; (d) phenanthrene-d10 ; (e) 2,3-diOHNP acetate; (f) 1,5-diOHNP acetate; (g) 1,3-diOHNP acetate; (h) 1,6-diOHNP acetate; (i) 2,6-diOHNP acetate; (j) 2,7-diOHNP acetate; (k) 9-OHPT acetate; (l) 1-OHPR acetate. (*) Unknown peaks originating from other compounds may overlap.
3.6. Analyses of environmental samples The method optimized in this study was applied to five environmental samples including seawater and puddle water. As the recoveries of samples C, D and F were significantly higher than that of sample E as discussed above (Table 2), the ionic strengths of puddle water were adjusted to that of seawater by adding artificial salt for marine aquarium. Fig. 4 shows the chromatograms of environmental samples (Kashima, Yacht Harbor; streets A, B and C), along with the chromatogram of the standard solution (500 ng l−1 ). For ease of comparison of the chromatograms, the peak intensities of
OH-PAH acetates in the environmental samples were magnified 10 times relative to that of I.S. (d). Detected OH-PAHs in environmental samples were 1,4-NQ, 2-OHNP, 2,3-diOHNP, 1,5-diOHNP, 1,3-diOHNP, 1,6-diOHNP, 2,6-diOHNP, 2,7diOHNP, and 9-OHPT (Table 3). The highest concentration of OH-PAHs was 1.0 g l−1 for 1,3-diOHNP in puddle water collected from street B. Although a large peak at the same retention time of 1,3-diOHNP was observed in puddle water collected from street C, relative intensity of 202 to 160 (m/z) was different from that of 1,3-diOHNP acetates in standard solution. This suggests that an unknown peak originating from other compounds may overlap. This was also
Table 3 Concentrations of OH-PAHs in different water samples (ng l−1 ) Compound
Kashima
Yacht Harbor
Street A
Street B
Street C
1-OHNP acetate 2-OHNP acetate 1,4-NQ 2,3-diOHNP acetate 1,5-diOHNP acetate 1,3-diOHNP acetate 1,6-diOHNP acetate 2,6-diOHNP acetate 2,7-diOHNP acetate 9-OHPT acetate 1-OHPR acetate
n.d. 32 1.6 × 102a 6.3 6.8 1.1 × 102 7.7 n.d. n.d. 16 n.d.
n.d. 15 n.d. 12 12 1.0 × 102 17 n.d. n.d. 23 n.d.
n.d. 47 3.8 × 102 n.d. 15 1.4 × 102 21 23 9.5 73 n.d.
n.d. 14 n.d. n.d. n.d. 1.0 × 103 n.d. n.d. n.d. n.d. n.d.
n.d. 12 n.d. 15 n.d. 1.1 × 103a n.d. 11 n.d. n.d. n.d.
Abbreviations are the same as in Fig. 1. n.d. was used for the less concentration of each OH-PAHs than the limit of quantification in Table 1. a Unknown peaks originating from other compounds may overlap.
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observed for 1,4-NQ in seawater collected from Kashima. To the best of our knowledge, the detection of OH-PAHs in environmental water samples has not been reported so far. Reported concentrations of OH-PAHs in urine samples were ranging 0.3–2500 g l−1 for 1-OHNP [11], 0.3–189 g l−1 for 2-OHNP [11] and 0.80–11.7 g l−1 for 1-OHPR [10]. In all environmental samples, the concentrations of them (1-OHNP, <0.91 ng l−1 ; 2-OHNP, 12–47 ng l−1 ; 1-OHPR, <8.6 ng l−1 ; Table 3) were much lower than those in urine samples. Although the concentration of 1-OHNP in urine samples was reported to be comparable to that of 2-OHNP [11], 1-OHNP was not detected in any environmental samples (Table 3). There was no distinct trend depending on their environmental conditions. To identify the origin and contribution of OH-PAHs in environment, further investigation must be needed.
4. Conclusion Using the SBSE–TD–GC–MS method in combination with in situ derivatization, OH-PAHs were detected at the ppt level in 10 ml water samples. This method showed good recovery of the spiked OH-PAHs from seawater with a small number of treatment steps before analysis. The high sensitivity and the small number of treatment steps before analysis are also suitable for the analysis of other phenols in environmental samples (both seawater and freshwater) and would contribute the automation of the monitoring system.
Acknowledgements This work was supported by a grant-in-aid for the Kanazawa University 21st century COE program from the Ministry of Education, Culture, Sports and Science and Technology of Japan; a grant-in-aid for young scientists from the Kanazawa University 21st-Century COE Program, and the JSPS Science Promotion Fund No. 16915025. We acknowledge two anonymous reviewers for their valuable comments in improving the manuscript.
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Optimization of aqueous acetylation for determination of hydroxy polycyclic aromatic hydrocarbons in water by stir bar sorptive extraction and thermal desorption–gas chromatography–mass spectrometry Nobuyasu Itoh, Hiroaki Tao∗ , Takashi Ibusuki Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan Received 14 July 2004; received in revised form 1 December 2004; accepted 1 December 2004 Available online 11 January 2005
Abstract Stir bar sorptive extraction (SBSE) using a 10 mm-long stir bar coated with 24 l of polydimethylsiloxane (PDMS) in combination with the thermal desorption technique and gas chromatography–mass spectrometry was used for determining hydroxy polycyclic aromatic hydrocarbons (OH-PAHs), particularly mono and diOH-PAHs having two- to four-ring structures, in water samples. To improve the recovery for OH-PAHs, in situ derivatization with acetic anhydride was applied prior to SBSE. The optimal conditions for a 10 ml water sample were attained with the addition of 100 mg of sodium hydrogen carbonate, followed by the addition of 20 l of acetic anhydride and extraction for 360 min. In the selected ion monitoring (SIM) mode, the limits of detection were found to range from 0.27 ng l−1 for 2-hydroxynaphthalene (as 2-hydroxynaphthalene acetate) to 25 ng l−1 for 1,4-dihydroxynaphthalene (as 1,4-naphthoquinone) (S/N = 3). The low total recovery (<50%) of the entire procedure was mainly due to the low extraction efficiency of acetyl derivatives from water to PDMS, while the in situ derivatization step achieved more than 88% efficiency. Nine OH-PAHs, such as 1,4-naphthoquinone, 2-hydroxynaphthalene, 2,3-dihydroxynaphthalene, 1,5-dihydroxynaphthalene, 1,3-dihydroxynaphthalene, 1,6-dihydroxynaphthalene, 2,6-dihydroxynaphthalene, 2,7-dihydroxynaphthalene, and 9-hydroxyphenanthrene, were detected in environmental samples under the optimal conditions. The detection of OH-PAHs in environmental water samples has not been reported so far. © 2004 Elsevier B.V. All rights reserved. Keywords: GC–MS; Hydroxy polycyclic aromatic hydrocarbons (OH-PAHs); Stir bar sorptive extraction (SBSE); Thermal desorption (TD); Water sample
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are produced by industrial processes and the incomplete combustion of fossil fuel, as well as by forest fires. The emitted PAHs are transported over long distances in the atmosphere and their deposition in rain/snow is considered to be a significant source of PAHs for surface waters [1,2]. In the atmosphere, the main reaction of PAHs is with OH radicals, converting them into hydroxides (OH-PAHs) [3,4]. This reaction is considered to be the primary step for the degradation and conversion into ∗
Corresponding author. Tel.: +81 29 861 8788; fax: +81 29 861 8308. E-mail address: [email protected] (H. Tao).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.12.002
nitro-PAHs. As their chemical structures resemble those of estrogenic hormones, OH-PAHs are suspected to have endocrine disrupting effects [5]. However, the distribution of OH-PAHs in the environment has not been investigated so far because they are found in trace amounts in the aquatic environment and they have more polar characteristics than PAHs. Recently, OH-PAHs, particularly OH-pyrene and OH-naphthalenes, in biological fluids have been analyzed and used as indicators of PAH exposure [6–12]. Some researchers have used solid phase microextraction (SPME) and stir bar sorptive extraction (SBSE), both of which use polydimethylsiloxane (PDMS) as the adsorbent, for the extraction/preconcentration of OH-PAHs, in combination with the thermal desorption
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(TD) technique and gas chromatography–mass spectrometry (TD–GC–MS) [6,9,10,13]. The performance of the extraction/preconcentration by SPME and SBSE is based on the octanol–water partition coefficient (Ko/w ). As SBSE uses a much larger volume of PDMS (>24 l) than SPME (<0.7 l), SBSE enables more efficient extraction of compounds with relatively low pKo/w [14]. Using SBSE, Desmet et al. [10] showed that the calculated limit of detection (LOD) of OHpyrene is 2 ng l−1 (S/N = 3) in urine samples and Kawaguchi et al. [15] reported that the LODs of 4-nonylphenol and 4-tertoctylphenol in biological fluids are 40 and 4 ng l−1 (S/N = 3), respectively. As for environmental samples, Kawaguchi et al. [16] reported that the LODs of 4-nonylphenol and 4-tertoctylphenol in tap and river water samples are lower than those in biological fluids at 20 and 2 ng l−1 (S/N = 3), respectively. Therefore, this SBSE–TD–GC–MS method is expected to be applicable to the determination of OH-PAHs in environmental samples. To improve extraction efficiency and to facilitate vaporization in GC analysis, derivatizations have been generally applied to polar molecules such as phenols. There are many reports on the in situ derivatization of phenols using benzyl bromide, butyl chloroformate, dimethyl sulfate and acetic anhydride [17]. Among them, derivatization with acetic anhydride to form phenol acetate is one of the simplest methods, and both high derivatization efficiency (>85%) and high recovery (ca. 100%) by the organic phase of the phenol acetate have been reported [18–21]. Thus, this derivatization has been used for such phenols as alkyl-, chloro- and nitro-phenols, and OH-pyrene [10,13,18–26]. Although this method has been in use for more than 20 years [18] and applied to many phenols, optimization of the parameters for the in situ derivatization of phenols especially for OH-PAHs has not been achieved so far. In this study, the parameters for in situ derivatization with acetic anhydride/SBSE of OH-PAHs in artificial seawater are optimized. The recovery at each step and the effect of ionic strength in water are discussed. Then, the optimal analytical conditions are applied to such environmental samples as seawater and puddle water.
2. Experimental 2.1. Materials and reagents The OH-PAHs studied were 1-hydroxynaphthalene (1OHNP), 2-hydroxynaphthalene (2-OHNP), 1,3-dihydroxynaphthalene (1,3-diOHNP), 1,4-dihydroxynaphthalene (1,4diOHNP), 1,5-dihydroxynaphthalene (1,5-diOHNP), 1,6-dihydroxynaphthalene (1,6-diOHNP), 2,3-dihydroxynaphthalene (2,3-diOHNP), 2,6-dihydroxynaphthalene (2,6diOHNP), 2,7-dihydroxynaphthalene (2,7-diOHNP), 9-hydroxyphenanthrene (9-OHPT) and 1-hydroxypyrene (1OHPR). 1-OHNP, 2-OHNP, 1,3-diOHNP, 2,3-diOHNP, and 2,7-diOHNP standards were obtained from Wako
Pure Chemical Industries (Osaka, Japan); 1,4-diOHNP, 1,5-diOHNP, and 1,6-diOHNP standards were from Kanto Chemical Industries (Tokyo, Japan); 2,6-diOHNP standard was from Fluka (Buchs, Germany); and 9-OHPT and 1-OHPR standards were from Aldrich (Steinheim, Germany). The internal standard (I.S.), phenanthrene labeled with 13 C10 (phenanthrene-d10 ; 98% purity), was obtained from Cambridge Isotope Laboratories Inc. (MA, USA). A stock solution containing approximately 1 mg l−1 of each compound was prepared in acetonitrile. Artificial seawater was prepared using artificial salt for marine aquarium (Aquamarine S, YashimaChem, Osaka, Japan). A stir bar coated with 24 l of PDMS was obtained from Gerstel GmbH (M¨ulheim an der Ruhr, Germany) and conditioned by washing (ultrasonic treatment for 5 min) with MeOH/CH2 Cl2 (1:1, v/v) and 100% acetonitrile, followed by placing inside a TDS-2 tube for TD under He flow (50 ml min−1 ) for 20 h at 300 ◦ C. A preheating run was carried out after the conditioning process to ensure that the sorbent would not produce any spurious peaks in the chromatogram. 2.2. In situ derivatization and extraction In situ derivatization of OH-PAHs in water sample (10 ml) was performed using acetic anhydride after the addition of sodium hydrogen carbonate (NaHCO3 ) when necessary. To extract OH-PAH acetates with PDMS, a stir bar coated with PDMS was rotated at 1000 rpm. After extraction, the stir bar was removed with clean tweezers and dried with lint-free tissue. Then, the stir bar was placed inside the TDS-2 tube for TD. 2.3. Thermal desorption and GC–MS measurement Desorption of the extracted OH-PAH acetates was carried out using a TDS-2 TD unit (Gerstel GmbH, M¨ulheim an der Ruhr, Germany) mounted on a CIS-4 PTV injector (Gerstel GmbH, M¨ulheim an der Ruhr, Germany). The temperature of the TDS-2 tube was programmed to increase from 20 to 300 ◦ C at a rate of 60 ◦ C min−1 with a final holding time of 5 min at a flow rate of 50 ml min−1 of He. Desorbed OH-PAHs acetates were cryogenically trapped at the PTV injector, and then transferred into a GC column by heating from −80 to 300 ◦ C at a rate of 12 ◦ C s−1 . GC–MS analysis was carried out with an Agilent 6890/5973N (Agilent Technologies, Palo Alto, CA, USA) equipped with a DB-5MS capillary column (30 m × 0.25 mm i.d.; 0.25 m film thickness) and programmed from 50 (2 min hold) to 300 ◦ C at a heating rate of 5 ◦ C min−1 . Helium was used as the carrier gas at a flow rate of 1.0 ml min−1 in the constant flow mode. The temperature of the MS transfer line was kept at 280 ◦ C. The ionization energy used for electron impact (EI) ionization was 70 eV. The mass range monitored was
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40–550 (m/z) for optimization of the analytical procedures. To determine the LOD and to analyze environmental samples, the selected ion monitoring (SIM) mode was used. Identification of peaks in environmental samples were performed by comparing with both retention time and relative intensities of ion masses in standard solutions. All procedures to obtain the optimal condition and recoveries were performed with three replicates. 2.4. Recovery test at each step To obtain completely acetylated OH-PAHs, acetylation was performed in acetonitrile at 60 ◦ C for 24 h and the absence of non-acetylated OH-PAHs was confirmed. This solution of completely acetylated OH-PAHs was directly injected into the TDS-2 tube filled with glass wool (sample A). Acetonitrile and excess acetic anhydride were removed by N2 stream before analysis. To calculate the extraction efficiency, the solution of completely acetylated OH-PAHs was spiked into artificial seawater and SBSE was carried out for 360 min (sample B). The extraction efficiency was calculated by comparing the peak areas of sample B to those of sample A. Total recovery including the derivatization and the extraction were obtained by adding the solution of non-acetylated OH-PAHs into artificial seawater and in situ derivatization (with 20 l of acetic anhydride and 100 mg of NaHCO3 )/SBSE was carried out for 360 min (sample C). The derivatization efficiency was calculated by comparing the peak areas of sample C to those of sample B. Recovery test was also conducted to the samples such as 35‰ NaClaq (D), Milli-Q water (E) and seawater collected from Kashima Port (F). 2.5. Collection and preparation of environmental samples Seawater samples were collected from Kashima Port near power plant, which faces the Pacific Ocean (Kashima), and a Yacht Harbor in Tokyo Bay; puddle water samples were collected from one of the main streets in Tsukuba City (street A) and two heavily congested traffic streets in Tokyo (streets B and C). The ionic strengths of the puddle water samples were adjusted to that of the seawater sample by adding artificial salt for marine aquarium. All samples were analyzed without any other pre-treatments. 2.6. Calculation of PDMS–water partition coefficient As the distribution of PDMS–water was generally approximated to that of octanol–water, KowWin software (http://esc.syrres.com/interkow/kowdemo.htm) was used for calculating the pKo/w values of OH-PAH acetates. Good agreement between the values obtained with the software and the experimental data has been reported previously [27].
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3. Results and discussion 3.1. Optimization of volume of acetic anhydride The addition of acetic anhydride to form acetate in a water sample has been studied for many phenols, such as alkyl-, chloro-, and nitro-phenols, and OH-pyrene [10,13,18–26]. As the acetylation of phenols enhances their affinity for the organic phase, the recovery of the acetates by organic solvents is high compared to that of intact phenols [18]. Moreover, there are two advantages of the acetylation of phenols prior to GC analysis. First, the acetates would be easier to volatilize than the intact phenols. Second, the acetylation of phenols would reduce peak tailing on the chromatograms due to less interaction with the active sites of liquid phase of the GC column since acetates have less interaction through hydrogen bonding with the active or contamination sites relative to intact phenols [17], and this is important for accurate determination. Although this method has been in use for more than 20 years [18], the volume of acetic anhydride added to 10 ml water samples varies among reports, from less than 0.0125 ml (final concentration: 13.2 mmol l−1 ) [19] to 1.0 ml (final concentration: 1.06 mol l−1 ) [26]. Thus, the optimal volume of acetic anhydride added was determined first. Fig. 1 shows the relationships between the volume of acetic anhydride added and the peak area (A and B) and pH after reaction (C). This experiment was carried out with 1 h extraction of 10 g l−1 OH-PAHs in artificial seawater in the absence of any alkaline compounds described in the next section. No OH-PAH acetates were detected when acetic anhydride was not added. The peak areas of OH-PAH acetates increased with increasing volume of acetic anhydride added up to 20 l (final concentration: 2160 mg l−1 , 21.2 mmol l−1 in artificial seawater), whereas that of 1,4diOHNP acetate was not detected. Instead of 1,4-diOHNP acetate, 1,4-naphthoquinone (1,4-NQ) was detected in all experiments. This is due to the chemical characteristic of 1,4diOHNP, namely, it is easily converted into 1,4-NQ. When more than 20 l of acetic anhydride was added, the peak areas of OH-PAH acetates were markedly decreased whereas 1,4-NQ decreased only slightly and I.S. (phenanthrene-d10 ) slightly increased. As regards the pH, it decreased remarkably from 6.87 to 3.36 by the addition of 10 l of acetic anhydride, whereas it decreased slightly (from 3.14 to 2.20) when the volume was increased from 20 l to 600 l (Fig. 1C). Particularly, when the volume increased from 20 to 100 l, the peak areas of OH-PAH acetates decreased to less than half of those at 20 l addition, although the pH decreased by only 0.47 (from pH 3.14 to 2.67). Since only mono and diOHPAH acetates involving acetylation decreased markedly up to 100 l addition, large volume of acetic anhydride would suppress acetylation rather than extraction into PDMS. Same trend was reported by Bal´ıkov´a and Kohl´ıcˇ ek [22] on other phenols such as phenol, cresols and chlorophenol. They suggested that a large excess of acetic anhydride provides an acidic medium, thereby unfavorably shifting the equilibrium
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Fig. 1. Relationships between volume of acetic anhydride and peak area of various OH-PAHs (A and B), and pH after the reaction (C). Abbreviations: 1-OHNP, 1-hydroxynaphthalene; 2-OHNP, 2-hydroxynaphthalene; 1,4-NQ, 1,4-naphthoquinone; 9-OHPT, 9-hydroxyphenanthrene; 1-OHPR, 1-hydroxypyrene; 2,3-diOHNP, 2,3-dihydroxynaphthalene; 1,5-diOHNP, 1,5-dihydroxynaphthalene; 1,3-diOHNP, 1,3-dihydroxynaphthalene; 1,6diOHNP, 1,6-dihydroxynaphthalene; 2,6-diOHNP, 2,6-dihydroxynaphthalene; 2,7-diOHNP, 2,7-dihydroxynaphthalene.
of the acetylation reaction [22]. Since 20 l addition of acetic anhydride gave the highest amounts for all OH-PAH acetates, the addition volume of acetic anhydride was set at 20 l per 10 ml of artificial seawater, hereafter. 3.2. Optimization of amount of NaHCO3 To enhance the acetylation of phenols, most researchers have used such alkaline compounds as potassium hydrogen carbonate [10,13], sodium hydrogen carbonate (NaHCO3 ) [18–20,23], potassium carbonate [22,24,26] and sodium hydroxide [25] at various concentrations ranging from 0.05 [22] to 0.725 mol l−1 [26]. Thus, the optimal amount of alkaline compound added was determined as the next step. In this experiment, NaHCO3 was selected as the alkaline compound because of its buffer effect, which would prevent the hydrolysis of acetates in the excess acid/base condition. Fig. 2 shows the relationships between the amount of NaHCO3 added and the peak area (A and B) and the change in
Fig. 2. Relationships between amount of NaHCO3 and peak area of various OH-PAHs (A and B), and change in pH (C). Abbreviations are the same as those in Fig. 1.
pH (C). This experiment was performed with 1 h extraction of 10 g l−1 OH-PAHs in artificial seawater with the addition of 20 l of acetic anhydride. Regardless of the amount of NaHCO3 , acetylation was markedly (>2 times except for 1,4NQ) enhanced compared to the case where only acetic anhydride was added (Fig. 1A and B). The peak areas of OH-PAH acetates increased with increasing amounts of NaHCO3 up to 100 mg (119 mmol l−1 ), and then slightly decreased thereafter. On the other hand, phenanthrene-d10 and 1,4-NQ, both did not involve acetylation, showed no significant change at any added amounts of NaHCO3 (Fig. 2A). As regards the pH, it dropped to the acidic range (near pH 3) by the addition of acetic anhydride without any addition of NaHCO3 . However, pH was kept in the neutral range after the addition of acetic anhydride when the amount of NaHCO3 exceeded 50 mg (Fig. 2C). The pH change during before and after the reaction was reduced with increasing amounts of NaHCO3 . This is due to the buffer effect of NaHCO3 , as expected, which provides a medium where the acetates are free from hydrolysis. Upon the addition of more than 100 mg of NaHCO3 , calcium carbonate was precipitated during stirring. Since peak area of both phenanthrene-d10 and 1,4-NQ were constant even by the addition of more than 100 mg, excess
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amount of NaHCO3 might suppress the acetylation of OHPAHs rather than cause the adsorbent loss on precipitated calcium carbonate. From the result of this experiment, addition amount of NaHCO3 was set at 100 mg per 10 ml artificial seawater, hereafter. 3.3. Optimization of in situ derivatization/extraction time As OH-PAHs have OH group(s) in their structures, their pKo/w values are lower than those of pesticides and PAHs (Table 1). Thus, their extraction times are expected to be longer than those of other apolar compounds, as reported previously (from 60 to 90 min for PAHs and alkylphenols [15,16,28]). The in situ derivatization/extraction time was studied from 5 to 480 min (Fig. 3). Extracted amounts of all OH-PAH acetates increased with increasing the time. Mono OH-PAHs such as 1- and 2-OHNPs, 9-OHPT and 1-OHPR, reached the steady state at 60–120 min (Fig. 3A), whereas diOHNPs continued to increase up to 360 min (Fig. 3B). Although 1,3- and 2,3-diOHNP acetates have higher pKo/w (3.04) than those of 1- and 2-OHNP acetates (2.77), they took longer time for in situ derivatization/extraction. The longer time for diOHNPs relative to mono OHNPs might depend on their longer acetylation time rather than extraction into PDMS. At least, this result suggests that in situ derivatization/extraction time for each OH-PAHs acetate would depend on the number of OH group(s) in its structure rather than its pKo/w . Since there was no significant increase between 360 and 480 min, the extraction time was set at 360 min, hereafter.
Fig. 3. Relationship between acetylation/extraction time and peak area of various OH-PAHs. Abbreviations are the same as those in Fig. 1.
tion were performed under the optimal conditions discussed above (with 20 l of acetic anhydride, 100 mg of NaHCO3 , and extraction for 360 min). The results are listed in Table 1, together with the monitoring ion masses and the theoretical pKo/w calculated using KowWin software. The lowest LOD (S/N = 3) is 0.27 ng l−1 for 2-OHNP (as 2-OHNP acetate) and the highest LOD (S/N = 3) is 25 ng l−1 for 1,4-diOHNP (as 1,4-NQ). Although the 1,3- and 2,3-diOHNP acetates have the same pKo/w values (Table 1), the LOD of 1,3-diOHNP acetate (11 ng l−1 ) is more than six times higher than that of 2,3-diOHNP acetate (1.6 ng l−1 ). This suggests that the LODs of OH-PAH acetates depend not only on pKo/w but also on other factors, including acetylation efficiency and fragmentation pattern in mass spectrometry. The limits of quantification
3.4. Limits of detection Calibration was performed by extracting artificial seawater samples spiked at seven concentrations of non-acetylated OH-PAHs (0.01, 0.05, 0.10, 0.50, 1.0, 5.0 and 10.0 g l−1 ) and analyzing them in the SIM mode. Acetylation and extrac-
Table 1 Calculated pKo/w of OH-PAH acetates, ion mass and their calibration data under the optimal conditions Compound
pKo/w a
Ion mass (m/z)
Rb
Limit of detection (ng/l) (S/N = 3)
Limit of quantification (ng/l) (S/N = 10)
1-OHNP acetate 2-OHNP acetate 1,4-NQ 2,3-diOHNP acetate 1,5-diOHNP acetate 1,3-diOHNP acetate 1,6-diOHNP acetate 2,6-diOHNP acetate 2,7-diOHNP acetate 9-OHPT acetate 1-OHPR acetate Phenanthrene-d10
2.77 2.77 1.44 3.04 2.36 3.04 2.36 2.36 2.36 3.94 4.53 4.35
144, 115 144, 115 158, 130 160, 202 160, 202 160, 202 160, 202 160, 202 160, 202 194, 165 218, 189 188, 160
1.00 0.999 0.998 1.00 0.996 0.969 0.998 1.00 0.995 0.998 0.997 –
0.35 0.27 25 1.6 3.0 11 1.6 2.7 1.6 2.1 3.2 –
1.2 0.92 75 5.4 10 35 5.4 8.9 5.4 5.2 8.6 –
Abbreviations are the same as in Fig. 1. SBSE was performed with the addition of 100 mg of NaHCO3 , followed by 20 l of acetic anhydride and extraction for 360 min. a Calculated with KowWin software (http://esc.syrres.com/interkow/kowdemo.htm). b Regression coefficients were calculated from seven concentrations (0.01–10 g l−1 ).
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Table 2 Acetylation and extraction efficiencies for OH-PAHs in various samples (%) with theoretical extraction efficiencies for OH-PAH acetates calculated from pKo/w (%) in Table 1 Compound
Acetylation effiiciencya (C/B)
Extraction effiiciencyb (B/A)
Total recoveryc (C/A)
NaClaq/ASW (D/C)
Milli-Q/ASW (E/C)
Kashima/ASW (F/C)
Theoretical extraction efficiency calculated from pKo/w d (%)
1-OHNP acetate 2-OHNP acetate 1,4-NQ 2,3-diOHNP acetate 1,5-diOHNP acetate 1,3-diOHNP acetate 1,6-diOHNP acetate 2,6-diOHNP acetate 2,7-diOHNP acetate 9-OHPT acetate 1-OHPR acetate
90.7 (4.20) 93.4 (4.52) – 99.4 (8.40) 88.2 (7.66) 90.1 (12.5) 89.9 (8.05) 93.4 (9.49) 88.4 (6.78) 96.2 (5.15) 109 (11.6)
51.6 (2.05) 50.1 (2.95) 17.0 (7.55) 24.5 (0.93) 19.0 (0.90) 25.8 (0.99) 22.9 (1.17) 24.0 (1.43) 26.2 (1.37) 47.4 (2.91) 37.1 (5.76)
46.9 (2.62) 46.8 (3.15) 15.4 (7.21) 24.3 (1.87) 16.7 (1.39) 23.3 (3.22) 20.6 (1.97) 22.4 (2.46) 23.2 (2.12) 45.6 (3.68) 40.4 (7.52)
111 (5.85) 107 (4.95) 104 (20.2) 96.3 (7.75) 108 (8.55) 84.7 (12.0) 106 (9.13) 107 (10.7) 108 (8.50) 112 (7.36) 109 (15.0)
92.8 (4.60) 92.1 (4.15) 74.7 (20.9) 80.8 (8.91) 91.7 (11.1) 63.2 (6.32) 91.1 (10.4) 93.1 (11.4) 94.4 (8.68) 109 (7.28) 106 (15.5)
103 (7.88) 99.6 (6.58) 106 (15.0) 96.3 (9.61) 104 (10.1) 42.5 (9.04) 102 (11.5) 102 (12.6) 101 (11.4) 101 (7.66) 94.0 (15.1)
58.6 58.6 6.20 72.4 35.5 72.4 35.5 35.5 35.5 95.4 98.7
Mean values of three replicates (relative standard deviations). ASW, SBSE from artificial seawater; NaClaq, SBSE from 35‰ NaClaq; Kashima, SBSE from seawater collected from Kashima Port. SBSE was performed with the addition of 100 mg of NaHCO3 , followed by 20 l of acetic anhydride and extraction for 360 min. Abbreviations are the same as in Fig. 1. a Acetylation efficiency in artificial seawater. b Extraction efficiency to PDMS for each OH-PAH acetate from artificial seawater. c Total recovery of the entire procedure. d Calculated from KowWin software.
ranged from 0.92 ng l−1 for 2-OHNP (as 2-OHNP acetate) to 75 ng l−1 for 1,4-diOHNP (as 1,4-NQ) as summarized in Table 1. 3.5. Recoveries of OH-PAHs at each step and from spiked samples When using SBSE in combination with in situ derivatization, there are two possible steps in which OH-PAHs are not completely recovered. One is the acetylation step with acetic anhydride and the other is the extraction step from water to PDMS. To identify the recovery at each step, several samples were prepared. Details of the sample preparation and the definitions are described in Section 2. Table 2 summarizes the recoveries for OH-PAHs at each step and the recoveries from the spiked samples, along with the theoretical extraction efficiency calculated from the pKo/w values in Table 1. The acetylation efficiencies are higher than 85% for all OH-PAHs including diOHNPs having two OH groups. On the other hand, the extraction efficiencies are lower than 55% for all OH-PAH acetates, with those for 1,4NQ and 1,5-diOHNP acetate being lower than 20%. These results suggest that the low total recovery (C/A) is due to the low extraction efficiency of PDMS for OH-PAH acetates and 1,4-NQ from water. The effects of ionic strength in water samples were examined using samples D, E and F. The recoveries of OH-PAH acetates except for 1,3-diOHNP acetate from samples D and F were almost the same as those from sample C. As only sample E contained no salt except for NaHCO3 , the low recoveries from sample E were due to its low ionic strength compared to those of the other samples. Therefore, it is necessary to adjust the ionic strength of freshwater samples before in situ derivatization/extraction.
The extraction efficiencies (B/A) of PDMS for all OHPAH acetates were lower than those calculated from pKo/w , whereas that for 1,4-NQ was more than 2 times higher than those calculated (Table 2). Baltussen et al. [14] observed lower extraction efficiencies of apolar compounds and moderate recoveries of polar compounds than those expected from theoretical values, and suspected two reasons for the discrepancy. One was that the distribution of PDMS–water might not always be approximated by pKo/w . The other was that highly apolar solutes might adsorb to glass, causing analyte loss. In our experiments, nonspecific adsorption to glass would be negligible because good linearity was obtained for both 9-OHPT and 1-OHPR acetates even at low concentrations without significant negative y-axis intercepts (Table 1). Less recoveries than those of theoretical values were obtained not only for 9-OHPT and 1-OHPR acetates but also for other OH-PAHs, especially for 1,3- and 2,3-diOHNP acetates. This supports that extraction behavior of PDMS for each compound could not be always approximated only by pKo/w and also affected by other physical and/or chemical characteristics. Recoveries from spiked artificial seawater and environmental samples (>90%) and reproducibility (<20%) were fairly good for all OH-PAH acetates except for the low recovery of 1,3-diOHNP acetate (42.5%). This suggests that the present SBSE–TD–GC–MS method in combination with in situ derivatization is applicable to environmental samples. Low recoveries of 1,3-diOHNP acetate were also observed in samples D and E relative to other OH-PAH acetates, although both acetylation and extraction efficiencies for 1,3-diOHNP acetate in artificial seawater were comparable to those of other diOHNP acetates. This suggests that other factors might affect on this compound.
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Fig. 4. Chromatograms of standard solution containing each OH-PAH at 500 ng l−1 and environmental samples (Kashima, Yacht Harbor; streets A, B and C). For ease of comparison of the chromatograms, the peak intensities of OH-PAH acetates in the environmental samples were magnified 10 times relative to that of I.S. (d). (a) 1,4-NQ; (b) 1-OHNP acetate; (c) 2-OHNP acetate; (d) phenanthrene-d10 ; (e) 2,3-diOHNP acetate; (f) 1,5-diOHNP acetate; (g) 1,3-diOHNP acetate; (h) 1,6-diOHNP acetate; (i) 2,6-diOHNP acetate; (j) 2,7-diOHNP acetate; (k) 9-OHPT acetate; (l) 1-OHPR acetate. (*) Unknown peaks originating from other compounds may overlap.
3.6. Analyses of environmental samples The method optimized in this study was applied to five environmental samples including seawater and puddle water. As the recoveries of samples C, D and F were significantly higher than that of sample E as discussed above (Table 2), the ionic strengths of puddle water were adjusted to that of seawater by adding artificial salt for marine aquarium. Fig. 4 shows the chromatograms of environmental samples (Kashima, Yacht Harbor; streets A, B and C), along with the chromatogram of the standard solution (500 ng l−1 ). For ease of comparison of the chromatograms, the peak intensities of
OH-PAH acetates in the environmental samples were magnified 10 times relative to that of I.S. (d). Detected OH-PAHs in environmental samples were 1,4-NQ, 2-OHNP, 2,3-diOHNP, 1,5-diOHNP, 1,3-diOHNP, 1,6-diOHNP, 2,6-diOHNP, 2,7diOHNP, and 9-OHPT (Table 3). The highest concentration of OH-PAHs was 1.0 g l−1 for 1,3-diOHNP in puddle water collected from street B. Although a large peak at the same retention time of 1,3-diOHNP was observed in puddle water collected from street C, relative intensity of 202 to 160 (m/z) was different from that of 1,3-diOHNP acetates in standard solution. This suggests that an unknown peak originating from other compounds may overlap. This was also
Table 3 Concentrations of OH-PAHs in different water samples (ng l−1 ) Compound
Kashima
Yacht Harbor
Street A
Street B
Street C
1-OHNP acetate 2-OHNP acetate 1,4-NQ 2,3-diOHNP acetate 1,5-diOHNP acetate 1,3-diOHNP acetate 1,6-diOHNP acetate 2,6-diOHNP acetate 2,7-diOHNP acetate 9-OHPT acetate 1-OHPR acetate
n.d. 32 1.6 × 102a 6.3 6.8 1.1 × 102 7.7 n.d. n.d. 16 n.d.
n.d. 15 n.d. 12 12 1.0 × 102 17 n.d. n.d. 23 n.d.
n.d. 47 3.8 × 102 n.d. 15 1.4 × 102 21 23 9.5 73 n.d.
n.d. 14 n.d. n.d. n.d. 1.0 × 103 n.d. n.d. n.d. n.d. n.d.
n.d. 12 n.d. 15 n.d. 1.1 × 103a n.d. 11 n.d. n.d. n.d.
Abbreviations are the same as in Fig. 1. n.d. was used for the less concentration of each OH-PAHs than the limit of quantification in Table 1. a Unknown peaks originating from other compounds may overlap.
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observed for 1,4-NQ in seawater collected from Kashima. To the best of our knowledge, the detection of OH-PAHs in environmental water samples has not been reported so far. Reported concentrations of OH-PAHs in urine samples were ranging 0.3–2500 g l−1 for 1-OHNP [11], 0.3–189 g l−1 for 2-OHNP [11] and 0.80–11.7 g l−1 for 1-OHPR [10]. In all environmental samples, the concentrations of them (1-OHNP, <0.91 ng l−1 ; 2-OHNP, 12–47 ng l−1 ; 1-OHPR, <8.6 ng l−1 ; Table 3) were much lower than those in urine samples. Although the concentration of 1-OHNP in urine samples was reported to be comparable to that of 2-OHNP [11], 1-OHNP was not detected in any environmental samples (Table 3). There was no distinct trend depending on their environmental conditions. To identify the origin and contribution of OH-PAHs in environment, further investigation must be needed.
4. Conclusion Using the SBSE–TD–GC–MS method in combination with in situ derivatization, OH-PAHs were detected at the ppt level in 10 ml water samples. This method showed good recovery of the spiked OH-PAHs from seawater with a small number of treatment steps before analysis. The high sensitivity and the small number of treatment steps before analysis are also suitable for the analysis of other phenols in environmental samples (both seawater and freshwater) and would contribute the automation of the monitoring system.
Acknowledgements This work was supported by a grant-in-aid for the Kanazawa University 21st century COE program from the Ministry of Education, Culture, Sports and Science and Technology of Japan; a grant-in-aid for young scientists from the Kanazawa University 21st-Century COE Program, and the JSPS Science Promotion Fund No. 16915025. We acknowledge two anonymous reviewers for their valuable comments in improving the manuscript.
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