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Simultaneous determination of trace benzotriazoles and benzothiazoles in water by large-volume injection/gas chromatography–mass spectrometry Weihai Xu a,b,∗ , Wen Yan a , Tobias Licha b a b
CAS Key Laboratory of Marginal Sea Geology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China Department Applied Geology, Geoscience Centre of the University of Göttingen, Göttingen 37077, Germany
a r t i c l e
i n f o
Article history: Received 15 July 2015 Received in revised form 8 October 2015 Accepted 8 October 2015 Available online xxx Keywords: Benzotriazole Benzothiazole Large-volume injection Silylation GC–MS
a b s t r a c t Benzotriazole (BTR) and benzothiazole (BTH) derivatives have been acknowledged as emerging pollutants due to their widespread contamination in the environment and their adverse effects on aquatic organisms. A rapid and reliable analytical method, based on solid phase extraction (SPE) and large-volume injection, derivatized with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), and analyzed by gas chromatography–mass spectrometry (GC–MS), was developed for the determination of six 1,2,3-benzotriazoles and six 1,3-benzothiazoles in aquatic matrices. It was demonstrated that MTBSTFA had a better overall performance compared with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The method detection limits in tap water, river water and effluent samples were 0.050–1.3 ng L−1 , 0.057–1.8 ng L−1 and 0.10–4.0 ng L−1 , respectively. Mean recoveries of the target analytes at different aquatic matrices, ranged from 43% to 131% with relative standard deviations (RSDs) below 17%. The method was successfully employed to river water and effluent sewage samples collected from a sewage treatment plant in Germany. Seven target compounds were detected with the maximum concentration up to 2.9 g L−1 for 4-Me-BTR in the effluent sample. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Benzothiazoles (BTH, substances containing the 1,3benzothiazole skeleton) and benzotriazoles (BTR, substances containing the 1,2,3-benzotriazole skeleton) are two kinds of highproduction-volume chemicals and are widely used in a variety of industrial and consumer products [1–3]. For example, the annual production of BTRs in the U.S. was over 9000 tons in 1999 and a much greater production worldwide is expected [4]. As organic corrosion inhibitors, they play a significant role in the protection of materials from deteriorating. BTR derivatives can “slow-down” the corrosion of metal surfaces by forming metal-BTR complexes [5]. Further, they are often used in rubber production as vulcanization accelerator and as corrosion inhibitors in dishwashing agents [6,7]. BTH are most commonly used as vulcanization accelerators in rubber production and as biocides in paper manufacturing [8].
∗ Corresponding author. Tel.: +86 20 89102511; fax: +86 20 84451672. E-mail addresses: [email protected] (W. Xu), [email protected] (T. Licha).
Owing to their wide application and resistance to biodegradation, both classes of compounds have been detected in raw and treated wastewater at g L−1 concentration levels and in surface and ground water at ng L−1 concentration levels [9–14]. They have been classified as emerging pollutants due to their potential adverse effects on aquatic organisms, microbial communities and mammals even at low concentrations [15–17]. Several BTH and BTR, such as benzothiazole (BTH) and 5-methyl-1H-benzotriazole (5-Me-BTR) have been proven to be toxic to luminescent bacteria, plants and aquatic animals [18–20]. 1H-BTR was found to be mutagenic in bacteria cell systems (Salmonella, Escherichia coli) and is classified as a suspected human carcinogen [18]. A number of studies indicated that BTH and BTR could possess estrogenic effects in laboratory animals [21,22]. Liquid chromatography combined with mass spectrometry (MS) or tandem mass spectrometry (MS–MS) is often used for their determination due to the fact that most BTH and BTR are rather polar compounds [8,23,24]. Recently, gas chromatography–mass (GC–MS) or tandem mass spectrometry has increasingly been applied in the determination of BTH and BTR due to some of their advantages, such as high selectivity, good isomer separation, and reduced matrix effects [11,25–27]. However, both techniques
http://dx.doi.org/10.1016/j.chroma.2015.10.017 0021-9673/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: W. Xu, et al., Simultaneous determination of trace benzotriazoles and benzothiazoles in water by largevolume injection/gas chromatography–mass spectrometry, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.10.017
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Fig. 1. Chemical structures and CAS numbers of BTR and BTH compounds analyzed.
often experience matrix effects when analyzing complex environmental matrices [28–30]. In addition, the limits of quantifications (LOQs) of most methods (generally higher than several or 10 ng L−1 ) were not sensitive enough to determine these compounds in trace levels in surface water, groundwater or seawater, etc. In this study, we developed a method for the determination of six BTR and six BTH (Fig. 1) as tert-butyldimethylsilyl derivatives using GC–MS based on large-volume injection (LVI). Thus, the main objectives of the present study were to compare and select most effective silylation reagent, and to optimize operating conditions for LVI technique, silylation and SPE extraction. The developed method was applied to screen these compounds in river water and effluent samples from a sewage treatment plant (STP) in Germany. 2. Experimental 2.1. Chemicals and materials Analytical standards (≥97%) of six BTH including benzothiazole (BTH), 2-aminobenzothiazole (2-Amin-BTH), 2-hydroxybenzothiazole (2-OH-BTH), 2-methylthiobenzothiazole (MTBT), 2-methylbenzothiazole (2-Me-BTH), 2-(morpholinothio)benzothiazole (2-Mo-BTH) and six BTRs including 1H-benzotriazole (1H-BTR), 1H-hydroxybenzotriazole (OH-BTR), 4-methyl1H-benzotriazole (4-Me-BTR) and 5-methyl-1H-benzotriazole (5-Me-BTR), 5,6-dimethyl-1H-benzotriazole (XTR), and 5chloro-1H-benzotriazole (5-Cl-BTR) were purchased from Sigma–Aldrich (Germany). Silylation reagents, N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA, 97%) and two mixtures N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, 98%) with 1% trimethylchlorosilane (TMCS) and N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide (MTBSTFA, 99%) with 1% tert-butyldimethylchlorosilane (TBDMCS) were purchased from Restek Corporation (Germany). 1H-BTR-d4 and BTH-d4 used as surrogate and internal standard respectively were obtained from Sigma–Aldrich (Germany). GC grade n-hexane, cyclohexane, methanol (MeOH), acetonitrile (ACN), dichloromethane and acetone were obtained from Merck (Darmstadt, Germany). Ultra-pure water was prepared with a Milli-Q water purification system (Schwalbach, Germany). Oasis HLB cartridges (500 mg, 6 mL) were purchased from Waters. Unless noted otherwise, chemicals used in the analysis were purer than the analytical grade.
Standard stock solutions (10 mg mL−1 ) of all analytes were prepared in MeOH. Mixed standards solution (1 g mL−1 ) was prepared in MeOH/H2 O (1:1, v/v) for recovery tests by spiking various concentration levels into aquatic matrices. The calibration mixed standards were prepared from stock solutions through serial dilutions with n-hexane. All mixed standards solution was stored at 4 ◦ C in a refrigerator for up to 3 months. 2.2. Sample collection and preparation Tap water from the laboratory was used for the development and optimization of the method. One grab sample of effluent was collected from a municipal STP, which was located in a city with a population of around 120,000 inhabitants. The STP consisted of a mechanical treatment for the separation of solids followed by activated sludge treatment. One river water grab sample was collected from the Göttingen section of the River Leine (Germany). The river sample was taken directly from the river bank. All water samples were stored in pre-cleaned 1 L brown glass bottles (Fisher scientific, Schwerte, Germany) and kept at 5 ◦ C in a refrigerator. The samples were analyzed within one week. After complete precipitation, the supernatant was used for analysis instead of a filtered aliquot. 2.3. Sample extraction and derivatization The final optimized SPE method was described as follows. The water samples (1 L for tap and river water and 100 mL for sewage water) were acidified to pH 3.0 using 3 M HCl. All blanks and samples were spiked with a known amount of surrogate before extraction. The samples were then passed through Oasis HLB cartridges at a flow rate of 5 mL min−1 . The cartridges had previously been conditioned using 10 mL of MeOH and 10 mL of acidified Milli-Q water (pH 3.0). Thereafter, the cartridges were washed with 10 mL of acidified Milli-Q water/MeOH (95:5, v/v) (pH 3.0) and 10 mL of Milli-Q water, respectively, and then dried under vacuum for 30 min. The analytes were eluted by 4 × 2 mL MeOH/ACN (1:1, v/v). The solvent was evaporated to dryness under a gentle stream of nitrogen, and reconstituted in 960 L of acetone, 20 L of internal standard (1.0 g mL−1 ) and 20 L of silylation reagent. After being completely mixed, the mixtures were then transferred to a 2 mL amber glass vial. The sealed vials were incubated at 38 ◦ C for 1 h before instrument analysis. For the determination of recoveries
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and matrix effects, 1 L of each, tap water, river water, and sewage water was fortified separately with appropriate amount of target analytes and surrogate. The solutions were treated by the same procedure as described earlier for the field samples. 2.4. LVI-GC–MS analysis Determination of the target compounds was performed by 7890A gas chromatograph coupled to a 5975C Mass Selective Detector (Agilent Technologies, USA). Separation of the different compounds was achieved in a DB-5MS fused-silica capillary column (30 m × 0.25 mm id × 0.25 m film thickness) from Agilent. Helium was used as the carrier gas with the flow rate of 1.0 mL min−1 . The GC oven temperature was programmed from 35 ◦ C (hold 0.5 min) to 132 ◦ C (hold for 3 min) at a rate of 12 ◦ C min−1 and then from 132 ◦ C to 320 ◦ C (hold for 6 min) at 15 ◦ C min−1 . The transfer line temperature was set at 280 ◦ C. The analytes were detected in selected ion monitoring (SIM) that operated in electron impact (EI) mode at 70 eV, with the ion source temperature at 250 ◦ C. Prior to the quantitation process, mass spectra and GC retention times of each compound from m/z 100 to 400 were obtained in full scan mode. The strongest m/z of each compound was selected for quantification. LVI was accomplished by using a cooled injection system equipped with SkyTM inlet liners from Restek. The solvent was removed in solvent vent mode at a vent flow of 100 mL min−1 . The split vent was closed 0.1 min after the injection was complete. At the same time, the CIS temperature was increased from 35 ◦ C (hold for 2 min) to 325 ◦ C at a rate of 600 ◦ C min−1 and hold for 5 min. During method development, the injection volume, injection speed, and injection temperature as well as vent flow and vent time were optimized. Other LVI parameters were directly obtained by using the Solvent Elimination Calculation System (SECS) according to different solvents. In the final method, 100 L of the extract was injected with an injection speed of 0.9 L s−1 and at an injection temperature of 35 ◦ C into an empty single-baffled glass liner. 2.5. Quality assurance and quality control The identification of each compound was ensured by the retention times and maximal characteristic m/z ratio of each compound, and two or more qualifier ions were used for confirmation if applicable (Table S1). Quantification of the target compounds was obtained using the internal standard method. A procedural blank and a matrix spike duplicate were analyzed in sequence to check for contamination and for peak identification and quantification. Laboratory blanks were analyzed along with the samples to assess potential sample contamination. The recovery data for each target compound in these matrices were corrected by the corresponding non-spike samples. There were no target compounds found in procedural blanks and in tap water. In addition, surrogate was added to all samples to monitor each step of the sample preparation. 3. Results and discussion 3.1. LVI-GC–MS optimization Many factors and parameters of the LVI system can remarkably affect the sensitivity and selectivity of measurement due to its complex injection process [31,32]. In this work, the injection volume was systematically increased (50, 80, 100, 120 and 150 L) and was finally set at 100 L. It was found that when the injection volume was over 100 L the chromatogram background increased and more importantly the signal enhancement slowed down with the corresponding increase of injection volume (Fig. S1). Vent flow
3
in the range of 80–100 mL min−1 was found appropriate for the target analytes in this study. Sensitivity evidently declined when the vent flow was higher than 120 mL min−1 or lower than 60 mL min−1 (Fig. S2). Vent time and injection speed are two interrelated factors and can significantly affect the system sensitivity [33]. The sensitivity showed small differences when the injection speed ranged from 0.6 L s−1 to 1.0 L s−1 and was finally fixed at 0.9 L s−1 based on a vent flow of 100 mL min−1 . The vent time should be little longer than the needle spends inside the inlet. Therefore, the vent time is usually set at 0.05 min longer than the injection time. However, it was found that if vent time was too long (Fig. S3), the sensitivity of all analytes declined, especially for the non-silylated analytes (BTH, MTBT, 2-Me-BTH and 2-Mo-BTH). The changes of initial inlet temperature (35 ◦ C, 40 ◦ C and 50 ◦ C) were investigated and showed small differences in peak areas. It was finally set at 35 ◦ C. Instrumental operating conditions, such as oven temperature programming were optimized to obtain better separation and peak shapes. The extracted ion chromatograms (EIC) total ion chromatograms (TIC) of each target compound are shown in Fig. 2 and Fig. S4, respectively. All target compounds showed good peak shapes and sensitivities, especially for the silylation derivatives. The reproducibility experiments were performed by spiking different levels of target analytes into matrix samples on three different days. Good reproducibility of all target analytes was obtained with standard deviations (RSDs) below 14%.
3.2. Optimization of the silylation reaction Silylation has been widely used in analytical chemistry for the derivatization of a variety of polar compounds, which could result in reduced polarity, enhanced volatility and increased thermal stability, and enables the GC–MS analysis of many compounds [34]. Further, silylation often enables better GC separation and the application of special detection techniques. However, previous studies generally determined benzotriazoles or benzothiazoles directly by GC–MS, without any derivatization [25,27,35]. BTH are proven to be thermally label compounds [36,37]. Derivatization could greatly enhance their thermal stability and, thus, leads to a better method performance and enhanced reproducibility [37]. Compared with the un-derivatized analytes from previous studies [25], the derivatized benzotriazoles and benzothiazoles showed good peak shapes and improved the sensitivities greatly, particularly for the compounds containing the relatively active hydrogens ( OH and NH2 ). BSTFA, MSTFA and MTBSTFA are three preferred silylation reagents in a number of previous studies [38]. MTBSTFA-derivates produce characteristic fragmentation patterns presenting mainly the fragments of [M]+ , [M−15]+ (cleavage of a methyl from the molecular ion) and [M−57]+ (cleavage of the t-butyl moiety), of which [M−57]+ is generally dominant in the mass spectrogram [39]. BSTFA and MSTFA-derivates, both yielding trimethylsilyl (TMS) derivatives, mainly show the fragments [M]+ , [M−15]+ (cleavage of a methyl from the molecular ion) and [M−31]+ (cleavage of the trimethylsilyl ether moiety followed by cyclization involving the silyl group). Due to the protection of TBDMS group from moisture, the tert-butyldimethylsilyl (t-BDMS) derivatives produced from MTBSTFA are more stable to hydrolysis than the corresponding TMS derivatives. As demonstrated in previous studies, TBDMS derivatives are formed more easily and have, thus, higher sensitivities (10–100 times) as well as repeatabilities than the corresponding TMS derivatives [39]. TBDMS derivatives of BTH and BTR are also dominated by intensive [M−57]+ ions, regardless of whether one or two active hydrogen were silylated. The strongest BSTFA and MSTFA-derivates of both BTH and BTR are [M−31]+ in this study. Similar to previous studies, the sensitivity of t-BDMS
Please cite this article in press as: W. Xu, et al., Simultaneous determination of trace benzotriazoles and benzothiazoles in water by largevolume injection/gas chromatography–mass spectrometry, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.10.017
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Fig. 2. Extracted ion chromatograms (EIC) of the quantitative ions for BTR and BTH standards at a concentration of 40 ng mL−1 each.
derivatives of both BTH and BTR with MTSTFA is remarkably higher than that of TMS derivatives, especially for XTR, 1H-BTR and OHBTR (more than 10 times higher) [38,40]. In comparison to the non-silylated analytes, the sensitivity of other compounds greatly improved after silylation with MTSTFA. It had been documented that the reactivity of amines especially secondary amines toward silylation is harder than for alcohol and phenol functional groups [39]. Most compounds in this study are secondary amines with the reactive hydrogens on this group. Therefore, a strong silylation reagent is needed and the silylation conditions need to be optimized in order to achieve high sensitivities. Compared to nonpolar organic solvents such as n-hexane, cyclohexane, and octane, acetone was proved to be able to facilitate the reaction and have the
best overall performance in the analysis of both BTH and BTR. MTBSTFA (20 L) was added to acetone for achieving a high degree of derivate. Although, the excess use of the agent is of utmost importance more than 20 L of it would be a waste of reagent. Additionally, the large volume injection will remarkably increase the response of the several large contaminating peaks of the silylation reagent if more MTBSTFA were added. Hence, it is understandable that high purity silylation reagent (>99%) will decrease the contaminating peaks. The derivatization temperature and time required for the derivatization process were optimized (Fig. S5). The derivatization reaction is complete within 1 h at both 38 ◦ C and 48 ◦ C. A small decrease in response for several targets was found after 1 h at 48 ◦ C most probably due to thermal degradation (hydrolysis). Therefore,
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DCM
MeOH
ACN
5
MeOH/DCM (1:1)
MeOH/ACN (1:1)
Recovery (%)
120 100 80 60 40 20 0 BTH
MTBT
2-AminBTH
2-OHBTH
2-MeBTH
2-MoBTH
1H-BTR
4-MeBTR
5-MeBTR
XTR
OH-BTR 5-Cl-BTR
Fig. 3. Effect of the extraction solvents on analyte recoveries (n = 3, spiking level: 30 ng L−1 ). Table 1 Recoveries (%) of target compounds in tap water, river water and effluent samples for each target compound. Compounds
Tap water −1
BTH MTBT 2-Amin-BTH 2-OH-BTH 2-Me-BTH 2-Mo-BTH 1H-BTR 4-Me-BTR 5-Me-BTR XTR OH-BTR 5-Cl-BTR
River water −1
−1
−1
Sewage water
20 ng L
50 ng L
100 ng L
20 ng L
50 ng L
100 ng L
20 ng L−1
50 ng L−1
100 ng L−1
48 ± 7 73 ± 4 101 ± 1 102 ± 4 57 ± 4 93 ± 5 91 ± 5 95 ± 2 103 ± 6 89 ± 4 107 ± 16 94 ± 5
50 ± 5 67 ± 4 112 ± 3 111 ± 2 52 ± 5 94 ± 4 90 ± 4 111 ± 2 96 ± 7 108 ± 4 96 ± 9 95 ± 4
52 ± 5 68 ± 2 99 ± 2 114 ± 8 59 ± 4 90 ± 6 104 ± 7 101 ± 5 116 ± 2 89 ± 11 102 ± 2 90 ± 7
43 ± 2 72 ± 1 109 ± 1 110 ± 3 56 ± 3 98 ± 2 68 ± 6 96 ± 2 94 ± 3 82 ± 8 117 ± 6 83 ± 2
52 ± 3 62 ± 6 103 ± 5 115 ± 15 66 ± 2 96 ± 8 85 ± 4 93 ± 2 98 ± 12 94 ± 5 103 ± 9 82 ± 4
54 ± 6 67 ± 3 104 ± 5 109 ± 7 66 ± 3 86 ± 8 92 ± 6 77 ± 9 94 ± 7 83 ± 4 89 ± 5 83 ± 11
43 ± 6 66 ± 4 114 ± 13 75 ± 10 55 ± 8 93 ± 8 99 ± 6 82 ± 3 88 ± 2 83 ± 6 101 ± 17 88 ± 8
41 ± 9 71 ± 3 131 ± 4 83 ± 4 52 ± 6 97 ± 4 83 ± 4 81 ± 3 90 ± 5 98 ± 16 97 ± 3 88 ± 9
51 ± 6 65 ± 3 129 ± 7 89 ± 5 67 ± 8 95 ± 11 94 ± 9 82 ± 4 93 ± 9 76 ± 6 99 ± 6 104 ± 6
38 ◦ C and 1 h were selected as derivatization temperature and time, respectively. 3.3. Solid phase extraction The HLB cartridge has been evaluated as the most effective cartridge for the extraction of various types of compounds including pharmaceuticals, endocrine-disrupting chemicals and as well as BTR and BTH from aquatic environmental samples. The HLB cartridge mainly benefits from its stronger ability to present both hydrophobic and hydrophilic intermolecular forces to targets than C18 or charcoal sorbent in the other cartridges [11,41]. Therefore, this study did not test the differences in extracting the targets using various types of cartridges. The SPE method was optimized through changing water pH (3.0, 5.0, 7.0) and elution solvents at a spiking concentration of 40 ng L−1 each in tap water. The recoveries were generally much higher at pH 3.0 than at pH 5.0 and 7.0. In comparison to BTR, BTH were significantly influenced by pH in their recoveries as all are below 50% at pH 5.0 and 7.0. The elution solvent was evaluated to be one of the key factors influencing the recoveries due to the polarity difference. Only elution with MeOH/ACN (1:1, v/v) gave relatively good recoveries for all targets (Fig. 3). For BTH, 2-Me-BTH and MTBT, only ACN and MeOH/ACN gave recoveries greater than 40%, other solvents showed recoveries lower than 30% or even 20%. Fig. 3 demonstrates that the best recoveries of the three compounds were merely a little higher than 50% even when using MeOH/ACN as elution solvent. Although recoveries for the three compounds were lower than 50% in river and sewage water due to matrix effects (Table 1), they are in the range
−1
−1
or even better compared to other studies (<20%) [23,42]. In Asimakopoulos’s study (2013), BTH and 2-Me-BTH recoveries were improved through increasing ionic strength by spiking the sample with -glucuronidase (GUS) [41]. It was suggested that glutathione could protect the thiol group of MTBT from oxidation in the aquatic environment [23]. However, both -glucuronidase and glutathione containe active hydrogen and could consume the silylation reagent. Hence, they were not used in this study. 3.4. Method performance The targets were quantified by an internal standard calibration curve prepared at concentrations ranging from 1.0 to 100 ng mL−1 (except for 2-Amin-BTH and 2-OH-BTH, see Table S2). BTH-d4 was spiked at a concentration of 20 ng in all standard solutions. A good inter-day repeatability was observed with RSD of 8.8% (n = 3). The instrumental limits of detection (LOD) were calculated from the signal-to-noise (S/N) ratio of 3 for the standards, and the limit of quantification (LOQ) of the whole method was defined as a ratio of 10 by spiking samples prior to the extraction and analysis. The LOQs in the tap water, river water and effluent samples were 0.050–1.3 ng L−1 , 0.057–1.8 ng L−1 and 0.1–4.0 ng L−1 , respectively (Table 2). Due to the high background levels of contamination in effluent samples, LOQs slightly increased in such samples. These values are similar to or better than those achieved in most literatures [11,25,28,43]. OH-BTR owned the highest LOQ which corresponded with the previous study [9]. Recoveries of target analytes were performed by spiking 20 ng, 50 ng and 100 ng in samples of tap water, river water and effluent,
Please cite this article in press as: W. Xu, et al., Simultaneous determination of trace benzotriazoles and benzothiazoles in water by largevolume injection/gas chromatography–mass spectrometry, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.10.017
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Table 2 Limits of detection (LOD) and limit of quantification (LOQ) of all target analytes in tap water, river water and sewage water. Compounds
BTH MTBT 2-Amin-BTH 2-OH-BTH 2-Me-BTH 2-Mo-BTH 1H-BTR 4-Me-BTR 5-Me-BTR XTR OH-BTR 5-Cl-BTR
Tap water (ng L−1 )
River water (ng L−1 )
Sewage water (ng L−1 )
LOD
LOQ
LOD
LOQ
LOD
LOQ
0.13 0.20 0.023 0.015 0.25 0.33 0.11 0.030 0.056 0.35 0.40 0.030
0.43 0.67 0.080 0.050 0.83 1.1 0.38 0.10 0.19 1.2 1.3 0.10
0.15 0.25 0.030 0.017 0.30 0.35 0.12 0.032 0.061 0.37 0.55 0.032
0.50 0.83 0.10 0.057 1.0 1.2 0.40 0.11 0.20 1.2 1.8 0.11
0.18 0.38 0.042 0.030 0.34 0.38 0.21 0.035 0.072 0.65 1.2 0.045
0.60 1.27 0.14 0.10 1.1 1.3 0.70 0.12 0.24 2.2 4.0 0.15
4. Conclusions A novel sensitive analytical methodology was developed for the determination of six benzotriazoles and six benzothiazoles in aqueous samples. MTBSTFA forms sensitive, reproducible and stable derivatives, BSTFA and MSTFA do not. Rather low LOQs were obtained based on the LVI technique, allowing the determination of trace level concentrations in aquatic samples. Satisfactory recoveries for the target analytes were obtained for different matrixes. The method was also successfully applied to determine the analytes in river water and sewage samples effluent. Seven analytes were detected in the river water and sewage effluent with high level of 4-Me-BTR and 4-Me-BTR (up to 2900 ng L−1 ). The result confirms the ubiquitous presence of these compounds in the aquatic environments. Acknowledgments
Table 3 Concentrations of the target compounds detected in the river water and effluent samples. Compounds
River water (ng L−1 )
Sewage water (ng L−1 )
BTH MTBT 2-OH-BTH 1H-BTR 4-Me-BTR 5-Me-BTR 5-Cl-BTR
18 ± 4.3a 76 ± 8.6
116 ± 8.5 738 ± 34 121 ± 17 1717 ± 85 2900 ± 126 2271 ± 142 442 ± 58
a b
Mean ± standard deviation (n = 3).
respectively using the optimized methods. Good recoveries ranging from 48 to 116% were achieved for the target analytes in tap water (Table 1), with a RSD of 1.0–16% (n = 3). The figures in river water and effluents were 43–117% (RSD of 1.0–15%) and 41–131% (RSD of 3.0–17%), respectively. The high RSD was mainly due to the relatively high base line with large-volume injection when analyzing real samples and, thus, the signal to noise ratio does not improve linearly with larger volumes. If analyte concentrations in river water or effluent samples exceeded 100 ng L−1 , then the extracts were diluted to be within the calibration range of the instrument and reanalyzed. In the recovery experiments all results were calibrated by the isotope-labeled internal standard to compensate adverse matrix effects.
3.5. Application to environmental samples The optimized methodology was successfully applied to the analysis of the target compounds in river water and sewage effluent. Generally, three BTH (BTH, MTBT and 2-OH-BTH) and four BTR (1H-BTR, 4-Me-BTR, 5-Me-BTR and 5-Cl-BTR) were detected in the river water and sewage effluent sampled (Table 3). The detected compounds in the river water ranged from 11 to 76 ng L−1 which were much lower than those found in the rivers Main, Hengstbach, and Hegbach [10] and in the rivers Elbe and Weser [31]. High levels of the BTRs were found in the sewage effluent with concentrations up to 2900 ng L−1 of 4-Me-BTR. As it can be observed, both 4-MeBTR and 5-Me-BTR, were detected in high level. This is probably a consequence of their high production and wide use. Their concentrations obtained are similar to those reported in wastewaters from Australia and Germany [11,44].
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Simultaneous determination of trace benzotriazoles and benzothiazoles in water by large-volume injection/gas chromatography–mass spectrometry Weihai Xu a,b,∗ , Wen Yan a , Tobias Licha b a b
CAS Key Laboratory of Marginal Sea Geology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China Department Applied Geology, Geoscience Centre of the University of Göttingen, Göttingen 37077, Germany
a r t i c l e
i n f o
Article history: Received 15 July 2015 Received in revised form 8 October 2015 Accepted 8 October 2015 Available online xxx Keywords: Benzotriazole Benzothiazole Large-volume injection Silylation GC–MS
a b s t r a c t Benzotriazole (BTR) and benzothiazole (BTH) derivatives have been acknowledged as emerging pollutants due to their widespread contamination in the environment and their adverse effects on aquatic organisms. A rapid and reliable analytical method, based on solid phase extraction (SPE) and large-volume injection, derivatized with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), and analyzed by gas chromatography–mass spectrometry (GC–MS), was developed for the determination of six 1,2,3-benzotriazoles and six 1,3-benzothiazoles in aquatic matrices. It was demonstrated that MTBSTFA had a better overall performance compared with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The method detection limits in tap water, river water and effluent samples were 0.050–1.3 ng L−1 , 0.057–1.8 ng L−1 and 0.10–4.0 ng L−1 , respectively. Mean recoveries of the target analytes at different aquatic matrices, ranged from 43% to 131% with relative standard deviations (RSDs) below 17%. The method was successfully employed to river water and effluent sewage samples collected from a sewage treatment plant in Germany. Seven target compounds were detected with the maximum concentration up to 2.9 g L−1 for 4-Me-BTR in the effluent sample. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Benzothiazoles (BTH, substances containing the 1,3benzothiazole skeleton) and benzotriazoles (BTR, substances containing the 1,2,3-benzotriazole skeleton) are two kinds of highproduction-volume chemicals and are widely used in a variety of industrial and consumer products [1–3]. For example, the annual production of BTRs in the U.S. was over 9000 tons in 1999 and a much greater production worldwide is expected [4]. As organic corrosion inhibitors, they play a significant role in the protection of materials from deteriorating. BTR derivatives can “slow-down” the corrosion of metal surfaces by forming metal-BTR complexes [5]. Further, they are often used in rubber production as vulcanization accelerator and as corrosion inhibitors in dishwashing agents [6,7]. BTH are most commonly used as vulcanization accelerators in rubber production and as biocides in paper manufacturing [8].
∗ Corresponding author. Tel.: +86 20 89102511; fax: +86 20 84451672. E-mail addresses: [email protected] (W. Xu), [email protected] (T. Licha).
Owing to their wide application and resistance to biodegradation, both classes of compounds have been detected in raw and treated wastewater at g L−1 concentration levels and in surface and ground water at ng L−1 concentration levels [9–14]. They have been classified as emerging pollutants due to their potential adverse effects on aquatic organisms, microbial communities and mammals even at low concentrations [15–17]. Several BTH and BTR, such as benzothiazole (BTH) and 5-methyl-1H-benzotriazole (5-Me-BTR) have been proven to be toxic to luminescent bacteria, plants and aquatic animals [18–20]. 1H-BTR was found to be mutagenic in bacteria cell systems (Salmonella, Escherichia coli) and is classified as a suspected human carcinogen [18]. A number of studies indicated that BTH and BTR could possess estrogenic effects in laboratory animals [21,22]. Liquid chromatography combined with mass spectrometry (MS) or tandem mass spectrometry (MS–MS) is often used for their determination due to the fact that most BTH and BTR are rather polar compounds [8,23,24]. Recently, gas chromatography–mass (GC–MS) or tandem mass spectrometry has increasingly been applied in the determination of BTH and BTR due to some of their advantages, such as high selectivity, good isomer separation, and reduced matrix effects [11,25–27]. However, both techniques
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Fig. 1. Chemical structures and CAS numbers of BTR and BTH compounds analyzed.
often experience matrix effects when analyzing complex environmental matrices [28–30]. In addition, the limits of quantifications (LOQs) of most methods (generally higher than several or 10 ng L−1 ) were not sensitive enough to determine these compounds in trace levels in surface water, groundwater or seawater, etc. In this study, we developed a method for the determination of six BTR and six BTH (Fig. 1) as tert-butyldimethylsilyl derivatives using GC–MS based on large-volume injection (LVI). Thus, the main objectives of the present study were to compare and select most effective silylation reagent, and to optimize operating conditions for LVI technique, silylation and SPE extraction. The developed method was applied to screen these compounds in river water and effluent samples from a sewage treatment plant (STP) in Germany. 2. Experimental 2.1. Chemicals and materials Analytical standards (≥97%) of six BTH including benzothiazole (BTH), 2-aminobenzothiazole (2-Amin-BTH), 2-hydroxybenzothiazole (2-OH-BTH), 2-methylthiobenzothiazole (MTBT), 2-methylbenzothiazole (2-Me-BTH), 2-(morpholinothio)benzothiazole (2-Mo-BTH) and six BTRs including 1H-benzotriazole (1H-BTR), 1H-hydroxybenzotriazole (OH-BTR), 4-methyl1H-benzotriazole (4-Me-BTR) and 5-methyl-1H-benzotriazole (5-Me-BTR), 5,6-dimethyl-1H-benzotriazole (XTR), and 5chloro-1H-benzotriazole (5-Cl-BTR) were purchased from Sigma–Aldrich (Germany). Silylation reagents, N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA, 97%) and two mixtures N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, 98%) with 1% trimethylchlorosilane (TMCS) and N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide (MTBSTFA, 99%) with 1% tert-butyldimethylchlorosilane (TBDMCS) were purchased from Restek Corporation (Germany). 1H-BTR-d4 and BTH-d4 used as surrogate and internal standard respectively were obtained from Sigma–Aldrich (Germany). GC grade n-hexane, cyclohexane, methanol (MeOH), acetonitrile (ACN), dichloromethane and acetone were obtained from Merck (Darmstadt, Germany). Ultra-pure water was prepared with a Milli-Q water purification system (Schwalbach, Germany). Oasis HLB cartridges (500 mg, 6 mL) were purchased from Waters. Unless noted otherwise, chemicals used in the analysis were purer than the analytical grade.
Standard stock solutions (10 mg mL−1 ) of all analytes were prepared in MeOH. Mixed standards solution (1 g mL−1 ) was prepared in MeOH/H2 O (1:1, v/v) for recovery tests by spiking various concentration levels into aquatic matrices. The calibration mixed standards were prepared from stock solutions through serial dilutions with n-hexane. All mixed standards solution was stored at 4 ◦ C in a refrigerator for up to 3 months. 2.2. Sample collection and preparation Tap water from the laboratory was used for the development and optimization of the method. One grab sample of effluent was collected from a municipal STP, which was located in a city with a population of around 120,000 inhabitants. The STP consisted of a mechanical treatment for the separation of solids followed by activated sludge treatment. One river water grab sample was collected from the Göttingen section of the River Leine (Germany). The river sample was taken directly from the river bank. All water samples were stored in pre-cleaned 1 L brown glass bottles (Fisher scientific, Schwerte, Germany) and kept at 5 ◦ C in a refrigerator. The samples were analyzed within one week. After complete precipitation, the supernatant was used for analysis instead of a filtered aliquot. 2.3. Sample extraction and derivatization The final optimized SPE method was described as follows. The water samples (1 L for tap and river water and 100 mL for sewage water) were acidified to pH 3.0 using 3 M HCl. All blanks and samples were spiked with a known amount of surrogate before extraction. The samples were then passed through Oasis HLB cartridges at a flow rate of 5 mL min−1 . The cartridges had previously been conditioned using 10 mL of MeOH and 10 mL of acidified Milli-Q water (pH 3.0). Thereafter, the cartridges were washed with 10 mL of acidified Milli-Q water/MeOH (95:5, v/v) (pH 3.0) and 10 mL of Milli-Q water, respectively, and then dried under vacuum for 30 min. The analytes were eluted by 4 × 2 mL MeOH/ACN (1:1, v/v). The solvent was evaporated to dryness under a gentle stream of nitrogen, and reconstituted in 960 L of acetone, 20 L of internal standard (1.0 g mL−1 ) and 20 L of silylation reagent. After being completely mixed, the mixtures were then transferred to a 2 mL amber glass vial. The sealed vials were incubated at 38 ◦ C for 1 h before instrument analysis. For the determination of recoveries
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and matrix effects, 1 L of each, tap water, river water, and sewage water was fortified separately with appropriate amount of target analytes and surrogate. The solutions were treated by the same procedure as described earlier for the field samples. 2.4. LVI-GC–MS analysis Determination of the target compounds was performed by 7890A gas chromatograph coupled to a 5975C Mass Selective Detector (Agilent Technologies, USA). Separation of the different compounds was achieved in a DB-5MS fused-silica capillary column (30 m × 0.25 mm id × 0.25 m film thickness) from Agilent. Helium was used as the carrier gas with the flow rate of 1.0 mL min−1 . The GC oven temperature was programmed from 35 ◦ C (hold 0.5 min) to 132 ◦ C (hold for 3 min) at a rate of 12 ◦ C min−1 and then from 132 ◦ C to 320 ◦ C (hold for 6 min) at 15 ◦ C min−1 . The transfer line temperature was set at 280 ◦ C. The analytes were detected in selected ion monitoring (SIM) that operated in electron impact (EI) mode at 70 eV, with the ion source temperature at 250 ◦ C. Prior to the quantitation process, mass spectra and GC retention times of each compound from m/z 100 to 400 were obtained in full scan mode. The strongest m/z of each compound was selected for quantification. LVI was accomplished by using a cooled injection system equipped with SkyTM inlet liners from Restek. The solvent was removed in solvent vent mode at a vent flow of 100 mL min−1 . The split vent was closed 0.1 min after the injection was complete. At the same time, the CIS temperature was increased from 35 ◦ C (hold for 2 min) to 325 ◦ C at a rate of 600 ◦ C min−1 and hold for 5 min. During method development, the injection volume, injection speed, and injection temperature as well as vent flow and vent time were optimized. Other LVI parameters were directly obtained by using the Solvent Elimination Calculation System (SECS) according to different solvents. In the final method, 100 L of the extract was injected with an injection speed of 0.9 L s−1 and at an injection temperature of 35 ◦ C into an empty single-baffled glass liner. 2.5. Quality assurance and quality control The identification of each compound was ensured by the retention times and maximal characteristic m/z ratio of each compound, and two or more qualifier ions were used for confirmation if applicable (Table S1). Quantification of the target compounds was obtained using the internal standard method. A procedural blank and a matrix spike duplicate were analyzed in sequence to check for contamination and for peak identification and quantification. Laboratory blanks were analyzed along with the samples to assess potential sample contamination. The recovery data for each target compound in these matrices were corrected by the corresponding non-spike samples. There were no target compounds found in procedural blanks and in tap water. In addition, surrogate was added to all samples to monitor each step of the sample preparation. 3. Results and discussion 3.1. LVI-GC–MS optimization Many factors and parameters of the LVI system can remarkably affect the sensitivity and selectivity of measurement due to its complex injection process [31,32]. In this work, the injection volume was systematically increased (50, 80, 100, 120 and 150 L) and was finally set at 100 L. It was found that when the injection volume was over 100 L the chromatogram background increased and more importantly the signal enhancement slowed down with the corresponding increase of injection volume (Fig. S1). Vent flow
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in the range of 80–100 mL min−1 was found appropriate for the target analytes in this study. Sensitivity evidently declined when the vent flow was higher than 120 mL min−1 or lower than 60 mL min−1 (Fig. S2). Vent time and injection speed are two interrelated factors and can significantly affect the system sensitivity [33]. The sensitivity showed small differences when the injection speed ranged from 0.6 L s−1 to 1.0 L s−1 and was finally fixed at 0.9 L s−1 based on a vent flow of 100 mL min−1 . The vent time should be little longer than the needle spends inside the inlet. Therefore, the vent time is usually set at 0.05 min longer than the injection time. However, it was found that if vent time was too long (Fig. S3), the sensitivity of all analytes declined, especially for the non-silylated analytes (BTH, MTBT, 2-Me-BTH and 2-Mo-BTH). The changes of initial inlet temperature (35 ◦ C, 40 ◦ C and 50 ◦ C) were investigated and showed small differences in peak areas. It was finally set at 35 ◦ C. Instrumental operating conditions, such as oven temperature programming were optimized to obtain better separation and peak shapes. The extracted ion chromatograms (EIC) total ion chromatograms (TIC) of each target compound are shown in Fig. 2 and Fig. S4, respectively. All target compounds showed good peak shapes and sensitivities, especially for the silylation derivatives. The reproducibility experiments were performed by spiking different levels of target analytes into matrix samples on three different days. Good reproducibility of all target analytes was obtained with standard deviations (RSDs) below 14%.
3.2. Optimization of the silylation reaction Silylation has been widely used in analytical chemistry for the derivatization of a variety of polar compounds, which could result in reduced polarity, enhanced volatility and increased thermal stability, and enables the GC–MS analysis of many compounds [34]. Further, silylation often enables better GC separation and the application of special detection techniques. However, previous studies generally determined benzotriazoles or benzothiazoles directly by GC–MS, without any derivatization [25,27,35]. BTH are proven to be thermally label compounds [36,37]. Derivatization could greatly enhance their thermal stability and, thus, leads to a better method performance and enhanced reproducibility [37]. Compared with the un-derivatized analytes from previous studies [25], the derivatized benzotriazoles and benzothiazoles showed good peak shapes and improved the sensitivities greatly, particularly for the compounds containing the relatively active hydrogens ( OH and NH2 ). BSTFA, MSTFA and MTBSTFA are three preferred silylation reagents in a number of previous studies [38]. MTBSTFA-derivates produce characteristic fragmentation patterns presenting mainly the fragments of [M]+ , [M−15]+ (cleavage of a methyl from the molecular ion) and [M−57]+ (cleavage of the t-butyl moiety), of which [M−57]+ is generally dominant in the mass spectrogram [39]. BSTFA and MSTFA-derivates, both yielding trimethylsilyl (TMS) derivatives, mainly show the fragments [M]+ , [M−15]+ (cleavage of a methyl from the molecular ion) and [M−31]+ (cleavage of the trimethylsilyl ether moiety followed by cyclization involving the silyl group). Due to the protection of TBDMS group from moisture, the tert-butyldimethylsilyl (t-BDMS) derivatives produced from MTBSTFA are more stable to hydrolysis than the corresponding TMS derivatives. As demonstrated in previous studies, TBDMS derivatives are formed more easily and have, thus, higher sensitivities (10–100 times) as well as repeatabilities than the corresponding TMS derivatives [39]. TBDMS derivatives of BTH and BTR are also dominated by intensive [M−57]+ ions, regardless of whether one or two active hydrogen were silylated. The strongest BSTFA and MSTFA-derivates of both BTH and BTR are [M−31]+ in this study. Similar to previous studies, the sensitivity of t-BDMS
Please cite this article in press as: W. Xu, et al., Simultaneous determination of trace benzotriazoles and benzothiazoles in water by largevolume injection/gas chromatography–mass spectrometry, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.10.017
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Fig. 2. Extracted ion chromatograms (EIC) of the quantitative ions for BTR and BTH standards at a concentration of 40 ng mL−1 each.
derivatives of both BTH and BTR with MTSTFA is remarkably higher than that of TMS derivatives, especially for XTR, 1H-BTR and OHBTR (more than 10 times higher) [38,40]. In comparison to the non-silylated analytes, the sensitivity of other compounds greatly improved after silylation with MTSTFA. It had been documented that the reactivity of amines especially secondary amines toward silylation is harder than for alcohol and phenol functional groups [39]. Most compounds in this study are secondary amines with the reactive hydrogens on this group. Therefore, a strong silylation reagent is needed and the silylation conditions need to be optimized in order to achieve high sensitivities. Compared to nonpolar organic solvents such as n-hexane, cyclohexane, and octane, acetone was proved to be able to facilitate the reaction and have the
best overall performance in the analysis of both BTH and BTR. MTBSTFA (20 L) was added to acetone for achieving a high degree of derivate. Although, the excess use of the agent is of utmost importance more than 20 L of it would be a waste of reagent. Additionally, the large volume injection will remarkably increase the response of the several large contaminating peaks of the silylation reagent if more MTBSTFA were added. Hence, it is understandable that high purity silylation reagent (>99%) will decrease the contaminating peaks. The derivatization temperature and time required for the derivatization process were optimized (Fig. S5). The derivatization reaction is complete within 1 h at both 38 ◦ C and 48 ◦ C. A small decrease in response for several targets was found after 1 h at 48 ◦ C most probably due to thermal degradation (hydrolysis). Therefore,
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140
DCM
MeOH
ACN
5
MeOH/DCM (1:1)
MeOH/ACN (1:1)
Recovery (%)
120 100 80 60 40 20 0 BTH
MTBT
2-AminBTH
2-OHBTH
2-MeBTH
2-MoBTH
1H-BTR
4-MeBTR
5-MeBTR
XTR
OH-BTR 5-Cl-BTR
Fig. 3. Effect of the extraction solvents on analyte recoveries (n = 3, spiking level: 30 ng L−1 ). Table 1 Recoveries (%) of target compounds in tap water, river water and effluent samples for each target compound. Compounds
Tap water −1
BTH MTBT 2-Amin-BTH 2-OH-BTH 2-Me-BTH 2-Mo-BTH 1H-BTR 4-Me-BTR 5-Me-BTR XTR OH-BTR 5-Cl-BTR
River water −1
−1
−1
Sewage water
20 ng L
50 ng L
100 ng L
20 ng L
50 ng L
100 ng L
20 ng L−1
50 ng L−1
100 ng L−1
48 ± 7 73 ± 4 101 ± 1 102 ± 4 57 ± 4 93 ± 5 91 ± 5 95 ± 2 103 ± 6 89 ± 4 107 ± 16 94 ± 5
50 ± 5 67 ± 4 112 ± 3 111 ± 2 52 ± 5 94 ± 4 90 ± 4 111 ± 2 96 ± 7 108 ± 4 96 ± 9 95 ± 4
52 ± 5 68 ± 2 99 ± 2 114 ± 8 59 ± 4 90 ± 6 104 ± 7 101 ± 5 116 ± 2 89 ± 11 102 ± 2 90 ± 7
43 ± 2 72 ± 1 109 ± 1 110 ± 3 56 ± 3 98 ± 2 68 ± 6 96 ± 2 94 ± 3 82 ± 8 117 ± 6 83 ± 2
52 ± 3 62 ± 6 103 ± 5 115 ± 15 66 ± 2 96 ± 8 85 ± 4 93 ± 2 98 ± 12 94 ± 5 103 ± 9 82 ± 4
54 ± 6 67 ± 3 104 ± 5 109 ± 7 66 ± 3 86 ± 8 92 ± 6 77 ± 9 94 ± 7 83 ± 4 89 ± 5 83 ± 11
43 ± 6 66 ± 4 114 ± 13 75 ± 10 55 ± 8 93 ± 8 99 ± 6 82 ± 3 88 ± 2 83 ± 6 101 ± 17 88 ± 8
41 ± 9 71 ± 3 131 ± 4 83 ± 4 52 ± 6 97 ± 4 83 ± 4 81 ± 3 90 ± 5 98 ± 16 97 ± 3 88 ± 9
51 ± 6 65 ± 3 129 ± 7 89 ± 5 67 ± 8 95 ± 11 94 ± 9 82 ± 4 93 ± 9 76 ± 6 99 ± 6 104 ± 6
38 ◦ C and 1 h were selected as derivatization temperature and time, respectively. 3.3. Solid phase extraction The HLB cartridge has been evaluated as the most effective cartridge for the extraction of various types of compounds including pharmaceuticals, endocrine-disrupting chemicals and as well as BTR and BTH from aquatic environmental samples. The HLB cartridge mainly benefits from its stronger ability to present both hydrophobic and hydrophilic intermolecular forces to targets than C18 or charcoal sorbent in the other cartridges [11,41]. Therefore, this study did not test the differences in extracting the targets using various types of cartridges. The SPE method was optimized through changing water pH (3.0, 5.0, 7.0) and elution solvents at a spiking concentration of 40 ng L−1 each in tap water. The recoveries were generally much higher at pH 3.0 than at pH 5.0 and 7.0. In comparison to BTR, BTH were significantly influenced by pH in their recoveries as all are below 50% at pH 5.0 and 7.0. The elution solvent was evaluated to be one of the key factors influencing the recoveries due to the polarity difference. Only elution with MeOH/ACN (1:1, v/v) gave relatively good recoveries for all targets (Fig. 3). For BTH, 2-Me-BTH and MTBT, only ACN and MeOH/ACN gave recoveries greater than 40%, other solvents showed recoveries lower than 30% or even 20%. Fig. 3 demonstrates that the best recoveries of the three compounds were merely a little higher than 50% even when using MeOH/ACN as elution solvent. Although recoveries for the three compounds were lower than 50% in river and sewage water due to matrix effects (Table 1), they are in the range
−1
−1
or even better compared to other studies (<20%) [23,42]. In Asimakopoulos’s study (2013), BTH and 2-Me-BTH recoveries were improved through increasing ionic strength by spiking the sample with -glucuronidase (GUS) [41]. It was suggested that glutathione could protect the thiol group of MTBT from oxidation in the aquatic environment [23]. However, both -glucuronidase and glutathione containe active hydrogen and could consume the silylation reagent. Hence, they were not used in this study. 3.4. Method performance The targets were quantified by an internal standard calibration curve prepared at concentrations ranging from 1.0 to 100 ng mL−1 (except for 2-Amin-BTH and 2-OH-BTH, see Table S2). BTH-d4 was spiked at a concentration of 20 ng in all standard solutions. A good inter-day repeatability was observed with RSD of 8.8% (n = 3). The instrumental limits of detection (LOD) were calculated from the signal-to-noise (S/N) ratio of 3 for the standards, and the limit of quantification (LOQ) of the whole method was defined as a ratio of 10 by spiking samples prior to the extraction and analysis. The LOQs in the tap water, river water and effluent samples were 0.050–1.3 ng L−1 , 0.057–1.8 ng L−1 and 0.1–4.0 ng L−1 , respectively (Table 2). Due to the high background levels of contamination in effluent samples, LOQs slightly increased in such samples. These values are similar to or better than those achieved in most literatures [11,25,28,43]. OH-BTR owned the highest LOQ which corresponded with the previous study [9]. Recoveries of target analytes were performed by spiking 20 ng, 50 ng and 100 ng in samples of tap water, river water and effluent,
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Table 2 Limits of detection (LOD) and limit of quantification (LOQ) of all target analytes in tap water, river water and sewage water. Compounds
BTH MTBT 2-Amin-BTH 2-OH-BTH 2-Me-BTH 2-Mo-BTH 1H-BTR 4-Me-BTR 5-Me-BTR XTR OH-BTR 5-Cl-BTR
Tap water (ng L−1 )
River water (ng L−1 )
Sewage water (ng L−1 )
LOD
LOQ
LOD
LOQ
LOD
LOQ
0.13 0.20 0.023 0.015 0.25 0.33 0.11 0.030 0.056 0.35 0.40 0.030
0.43 0.67 0.080 0.050 0.83 1.1 0.38 0.10 0.19 1.2 1.3 0.10
0.15 0.25 0.030 0.017 0.30 0.35 0.12 0.032 0.061 0.37 0.55 0.032
0.50 0.83 0.10 0.057 1.0 1.2 0.40 0.11 0.20 1.2 1.8 0.11
0.18 0.38 0.042 0.030 0.34 0.38 0.21 0.035 0.072 0.65 1.2 0.045
0.60 1.27 0.14 0.10 1.1 1.3 0.70 0.12 0.24 2.2 4.0 0.15
4. Conclusions A novel sensitive analytical methodology was developed for the determination of six benzotriazoles and six benzothiazoles in aqueous samples. MTBSTFA forms sensitive, reproducible and stable derivatives, BSTFA and MSTFA do not. Rather low LOQs were obtained based on the LVI technique, allowing the determination of trace level concentrations in aquatic samples. Satisfactory recoveries for the target analytes were obtained for different matrixes. The method was also successfully applied to determine the analytes in river water and sewage samples effluent. Seven analytes were detected in the river water and sewage effluent with high level of 4-Me-BTR and 4-Me-BTR (up to 2900 ng L−1 ). The result confirms the ubiquitous presence of these compounds in the aquatic environments. Acknowledgments
Table 3 Concentrations of the target compounds detected in the river water and effluent samples. Compounds
River water (ng L−1 )
Sewage water (ng L−1 )
BTH MTBT 2-OH-BTH 1H-BTR 4-Me-BTR 5-Me-BTR 5-Cl-BTR
18 ± 4.3a 76 ± 8.6
116 ± 8.5 738 ± 34 121 ± 17 1717 ± 85 2900 ± 126 2271 ± 142 442 ± 58
a b
Mean ± standard deviation (n = 3).
respectively using the optimized methods. Good recoveries ranging from 48 to 116% were achieved for the target analytes in tap water (Table 1), with a RSD of 1.0–16% (n = 3). The figures in river water and effluents were 43–117% (RSD of 1.0–15%) and 41–131% (RSD of 3.0–17%), respectively. The high RSD was mainly due to the relatively high base line with large-volume injection when analyzing real samples and, thus, the signal to noise ratio does not improve linearly with larger volumes. If analyte concentrations in river water or effluent samples exceeded 100 ng L−1 , then the extracts were diluted to be within the calibration range of the instrument and reanalyzed. In the recovery experiments all results were calibrated by the isotope-labeled internal standard to compensate adverse matrix effects.
3.5. Application to environmental samples The optimized methodology was successfully applied to the analysis of the target compounds in river water and sewage effluent. Generally, three BTH (BTH, MTBT and 2-OH-BTH) and four BTR (1H-BTR, 4-Me-BTR, 5-Me-BTR and 5-Cl-BTR) were detected in the river water and sewage effluent sampled (Table 3). The detected compounds in the river water ranged from 11 to 76 ng L−1 which were much lower than those found in the rivers Main, Hengstbach, and Hegbach [10] and in the rivers Elbe and Weser [31]. High levels of the BTRs were found in the sewage effluent with concentrations up to 2900 ng L−1 of 4-Me-BTR. As it can be observed, both 4-MeBTR and 5-Me-BTR, were detected in high level. This is probably a consequence of their high production and wide use. Their concentrations obtained are similar to those reported in wastewaters from Australia and Germany [11,44].
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Please cite this article in press as: W. Xu, et al., Simultaneous determination of trace benzotriazoles and benzothiazoles in water by largevolume injection/gas chromatography–mass spectrometry, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.10.017