High performance solid-phase extraction cleanup method coupled with gas chromatography-triple quadrupole mass spectrometry for analysis of polychlorinated naphthalenes and dioxin-like polychlorinated biphenyls in complex samples

Journal of Chromatography A, 1448 (2016) 1–8

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High performance solid-phase extraction cleanup method coupled with gas chromatography-triple quadrupole mass spectrometry for analysis of polychlorinated naphthalenes and dioxin-like polychlorinated biphenyls in complex samples Fang Li a,b,1 , Jing Jin a,1 , Dongqin Tan a,b , Jiazhi Xu a,b , Dhanjai a , Yuwen Ni a , Haijun Zhang a , Jiping Chen a,∗ a Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, No. 457 Zhongshan Road, Dalian 116023, China b University of Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e

i n f o

Article history: Received 14 January 2016 Received in revised form 14 April 2016 Accepted 14 April 2016 Available online 16 April 2016 Keywords: Cleanup Solid-phase extraction Magnesium oxide Polychlorinated naphthalenes Dioxin-like polychlorinated biphenyls

a b s t r a c t A solid-phase extraction (SPE) cleanup method was developed to purify the sample extracts for the analysis of polychlorinated naphthalenes (PCNs) and dioxin-like polychlorinated biphenyls (dl-PCBs). Monodisperse magnesium oxide (MgO) microspheres and basic alumina were used as SPE adsorbents. Important parameters of the SPE procedure were optimized, including the amount of basic alumina and the type and volume of the washing and elution solvents. The optimized SPE cleanup method exhibited excellent purification performance for the removal of organochlorinated compounds, lipid compounds, sulfur, and pigments. Additionally, it was found that the retention activities of congeners differed with the number and position of the chlorine substituents in PCNs. In this study, an analytical method based on a combination of accelerated solvent extraction (ASE) coupled with SPE cleanup and gas chromatographytriple quadrupole mass spectrometry (GC–MS/MS) is proposed for the analysis of PCNs and dl-PCBs in complex samples (sediment, pine needle, and scallop samples). The analytical method demonstrates good linearity, acceptable recovery (63–148%) and precision (relative standard deviations less than 26%). The limits of detection (LODs) of PCN and dl-PCB congeners were in the range of 0.6–19.1 pg g−1 and 0.4–8.6 pg g−1 , respectively. The PCNs and dl-PCBs levels in these samples ranged from 0.16 to 3.07 ng g−1 dry weight (dw) and from undetectable to 0.07 ng g−1 dw, respectively. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Polychlorinated naphthalenes (PCNs) and polychlorinated biphenyls (PCBs) are two typical kinds of persistent organic pollutants (POPs) that have significant adverse effects on ecosystems and human health [1,2]. These POPs have been listed in Annexes A and C of the Stockholm Convention on POPs [3]. Commercial PCN and PCB mixtures were widely used as dielectrics, capacitor fluids, lubricant oil additives, and wood and paper preservatives [4]. Although their productions have been banned, PCNs and PCBs can still be generated in industrial thermal processes and leaked from products and landfill and are subsequently released into the environment and

∗ Corresponding author. E-mail address: [email protected] (J. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.chroma.2016.04.037 0021-9673/© 2016 Elsevier B.V. All rights reserved.

accumulated in organisms [5,6]. Therefore, the analysis of PCNs and dioxin-like PCBs (dl-PCBs) in environmental and biological samples is essential for evaluating the pollution caused by them. PCNs and dl-PCBs have been detected in numerous environmental and biological samples [7,8]. As reported, highresolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) is the most common analytical technique for PCNs and dl-PCBs. Until recently, gas chromatography-tandem mass spectrometry with high selectivity and sensitivity has been applied to the analysis of PCNs and dl-PCBs [9,10]. Owing to the poor tolerance of this instrumental technique to non-volatile matrices, it is necessary to select a sample cleanup strategy with high purification performance. Column chromatography, combined with the use of silica gel, alumina, and Florisil is often used to remove nonpolar and polar co-extracted interfering substances from extracts [7,11,12]. Although satisfactory efficiency can be achieved, column chromatography usually needs a large amount of solvent,

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time, and labor. Additionally, multi-step operation always results in low recoveries for low chlorinated congeners. In other words, it is important to select a suitable cleanup method for achieving high purification performance and low cost. Solid-phase extraction (SPE) is one of the most promising cleanup techniques with the benefits of simple operation, low consumption of time and solvent, and low cost. Commercial SPE cartridges containing C18 , NH2 , HLB, and WAX have been frequently used for the extraction and cleanup of POPs in human blood [13,14] and human milk [15,16]. Similarly, SPE adsorbents such as silica gel and alumina have also attracted the interest of scientists [17–19]. SPE-based automated cleanup systems pre-packed with multilayer silica, basic alumina, and PX-21 carbon adsorbents have been developed and used to analyze POPs in complex samples, and have demonstrated superior reproducibility, high sample throughput, and low contamination [20,21]. However, these commercial SPE adsorbents always have low selectivity and poor purification performance. In recent decades, many types of highly selective materials have been used as SPE adsorbents for POPs analysis, such as carbon nanotubes [22], titanium dioxide nanotubes [23], molecularly imprinted polymers [24,25], graphene [26], and metalorganic frameworks [27]. However, the large-scale production and broader application of most of these new adsorbents is hindered by complex synthetic procedures, requirement of large volumes of toxic solvent, or high cost. We previously reported that monodisperse magnesium oxide (MgO) microspheres synthesized via a simple, controllable, and non-toxic seed-induced precipitation approach can be used as SPE adsorbents for the selective separation of polycyclic aromatic hydrocarbons [28,29]. As reported, there were special retention interactions between monodisperse MgO microspheres and aromatic rings [28], as well as alumina and coplanar molecules [30]. This study attempts to develop a cleanup method based on MgO microspheres and basic alumina as SPE adsorbents to purify sample extracts for the analysis of PCNs and dl-PCBs (congener substitution listed in Tables S1 and S2 in the Supporting information). Certain parameters of the SPE procedure were optimized, including the amount of basic alumina, and the type and volume of the washing and elution solvents. Additionally, the retention activities of target compounds on the developed SPE column were also investigated. An analytical method combining accelerated solvent extraction (ASE), SPE cleanup, and gas chromatography-triple quadrupole mass spectrometry (GC–MS/MS) analysis is proposed for the analysis of PCNs and dl-PCBs in complex samples (sediment, pine needle, and scallop samples).

pentachlorobenzene (penta-CBs) were obtained from SigmaAldrich (St Lous, Mo, USA). Tetrachlorobenzene (1,2,4,5-tetra-CB) and hexachlorobenzene (HCB) were purchased from Shanghai Jingchun Chemical Reagents Co., Ltd. (Shanghai, China) and Shanghai Chemical Reagents Co., Ltd. (Shanghai, China), respectively. Standard solution of short-chain chlorinated paraffins (SCCPs, 100.0 ␮g mL−1 ) with a chlorine content of 55.5% was obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Di-isobutyl phthalate (DIBP) and sulfur powder were purchased from Acros Organics (Geel, Belgium) and Tianjin Damao Chemical Reagent Factory (Tianjin, China), respectively. Bean oil was obtained from a supermarket (Dalian, China). Standard stock solutions were prepared in nonane and further diluted with n-hexane to obtain the working solutions. All standard solutions were stored at −20 ◦ C. Monodisperse MgO microspheres were prepared using a facile seed-induced precipitation method [28]. Basic alumina (Activity Super I, 63−200 ␮m, pH = 10) was purchased from MP Biomedicals (Eschwege, Germany). SPE cartridges (3 mL, polypropylene) were obtained from Dalian Sipore Co., Ltd. (Dalian, China). 2.2. Optimization of SPE procedure The empty cartridge was packed with different amounts (0.1–1.0 g) of basic alumina and 0.3 g of monodisperse MgO microspheres from the bottom up. Monodisperse MgO microspheres acted as the cleanup phase to remove various impurities rich in electrons. Basic alumina sorbent was used to further remove impurities and to retain target compounds. The SPE column was conditioned with 5.0 mL of n-hexane before loading the standard mixture of PCNs in n-hexane (1.0 mL, 10 ng mL−1 ). The column was then washed with 5.0 mL of n-hexane, followed by elution with a mixture of n-hexane/DCM (95:5, v/v). The collected elution fraction was reduced to 200 ␮L for GC–MS/MS analysis. Based on the optimal amount of basic alumina, the mixture of PCNs and dlPCBs in n-hexane (1.0 mL, 20 ng mL−1 ) was also loaded into the SPE column to determine the optimal volume of washing and eluting solvent. Next, the SPE column was washed with 3 × 5.0 mL of n-hexane, followed by elution with 3 × 5.0 mL of n-hexane/DCM (95:5, v/v). All fractions were concentrated and solvent-exchanged to 500 ␮L of n-hexane containing 5.0 ng 13 C12 -labeled PCBs (EC4979). Finally, the working solutions of sulfur and lipid compounds (0.8 mg mL−1 sulfur, 2.9 mg mL−1 DIBP, and 3.0 mg mL−1 bean oil), SCCPs (1.0 mL, 40 ␮g mL−1 ), PCDD/Fs, and chlorobenzenes (CBs, 1.0 mL, 10 ng mL−1 ) were performed to evaluate the purification efficiency of the optimized SPE cleanup method. 2.3. Sample collection and preparation

2. Materials and methods 2.1. Reagents and materials Pesticide residue grade n-hexane, dichloromethane (DCM), and nonane were obtained from J. T. Baker (Phillipsburg, NJ, USA). Standard solutions of PCNs (5.0 ␮g mL−1 PCN-MXA and PCN-MXC) were purchased from the Wellington Laboratories Inc. (Guelph, Canada). Standard solutions of dl-PCBs (EPA method 1668 calibration solution), isotopically labeled 13 C10 tetra-octa PCN mixture (1.0 ␮g mL−1 , ECN-5102), d7 2-chloronaphthalene (100.0 ␮g mL−1 ), 13 C −labeled PCBs (1.0 ␮g mL−1 , EC-4937 and 5.0 ␮g mL−1 , 12 EC-4979), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs, EDF-5493) were purchased from the Cambridge Isotope Laboratories Inc. (Andover, MA, USA). Monochlorobenzene (monoCB), dichlorobenzenes (1,2- and 1,3-di-CBs), and trichlorobenzene (1,2,3-tri-CB) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Trichlorobenzenes (1,2,4- and 1,3,5tri-CBs), tetrachlorobenzenes (1,2,3,4- and 1,2,3,5-tetra-CBs), and

Sediment samples (SD 1, 2) were taken with a stainless steel grab sampler from the Ningbo area in September 2013. Pine needle samples (PN 1, 2) were collected from a chemical industry in November 2013, handpicked at heights between 1.5 and 2.0 m above the ground from a 1-year-old branch with a pair of acetonecleaned stainless steel scissors. Scallop samples (SL 1, 2) were purchased from a supermarket in Dalian in 2013. These samples were freeze-dried with Labconco Freeze Dry System/Freezone® 4.5, and were then ground into powders. Sediment samples were sieved (<250 ␮m), and stored at −20 ◦ C in brown glass bottles. Biological samples were also stored at −20 ◦ C in pre-cleaned self-sealing aluminum/polyethylene bags with zippers until analysis. Prior to extraction, one nanogram of the surrogate internal standard, including the 13 C10 -labeled PCN mixture, d7 -labeled CN 2, and 13 C12 -labeled PCB mixture (EC-4937) was spiked into samples, followed by equilibration for 12 h. Sediment samples (5.0 g), pine needle samples (2.0 g), and scallop samples (2.0 g) were extracted by ASE (Dionex 350, Dionex Corporation). Previously describe ASE

F. Li et al. / J. Chromatogr. A 1448 (2016) 1–8

conditions [31] were used with some modification. DCM/n-hexane (1:1, v/v) was used as the extraction solvent under the following conditions: pre-heating 5 min to 100 ◦ C, followed by application of a pressure of 15 000 kPa for 10 min and over four cycles. The extracts were subsequently concentrated, and solvent-exchanged with approximately 1.0 mL n-hexane. Further, the SPE cleanup procedure was performed according to the optimized conditions in Section 2.2. Elution fractions were concentrated, and solventexchanged with 100 ␮L n-hexane containing 1.0 ng 13 C12 -labeled PCBs mixture (EC-4979) for GC–MS/MS analysis. 2.4. Instrumental analysis 2.4.1. GC–MS/MS PCNs, dl-PCBs, PCDD/Fs, and CBs were analyzed using a gas chromatograph-triple quadrupole mass spectrometer (GC–MS/MS, ThermoFisher Scientific). The capillary column was a non-polar DB5 MS column (60 m × 0.25 mm × 0.25 ␮m, Restek Inc., USA). The GC oven temperature was programmed as follows: from 90 ◦ C (2 min) to 200 ◦ C (10 ◦ C min−1 ) and up to 280 ◦ C (2 ◦ C min−1 , 20 min) for PCN analysis; and from 120 ◦ C (1 min) to 180 ◦ C (10 ◦ C min−1 ) and then up to 280 ◦ C (5 ◦ C min−1 , 20 min) for dl-PCB analysis. For tetra- to octa-CDD/Fs, the column temperature was programmed as follows: from 140 ◦ C (1 min) to 200 ◦ C (20 ◦ C min−1 , 1 min), up to 220 ◦ C (5 ◦ C min−1 , 16 min), up to 235 ◦ C (5 ◦ C min−1 , 7 min) and onwards to 310 ◦ C (5 ◦ C min−1 , 10 min); and from 50 ◦ C (1 min) to 220 ◦ C (10 ◦ C min−1 , 20 min) for CB analysis. All samples were injected in a splitless mode (1 min), and the injection volume was 1.0 ␮L. The injector and transfer line temperatures were 270 ◦ C and 280 ◦ C, respectively. The mass spectrometer was operated in the electron impact source (EI, 70 eV), and the source temperature was set at 250 ◦ C. Quantitative analysis of PCNs and dl-PCBs was performed in the selected reaction monitoring (SRM) mode. The emission current was fixed at 50 ␮A. Argon (1.5 mTorr) was used as the collision gas. The MS/MS operating condition for PCNs was according to a previously reported method [13] with some modifications. MS/MS parameters of PCNs and dl-PCBs are summarized in Tables S3 and S4, and the corresponding chromatograms of PCNs and dl-PCBs are shown in Figs. S1 and S2 of the Supporting information. The PCDD/Fs and CBs were determined using the selected ion monitoring (SIM) mode, and ion with m/z 322.0/306.0, 356.0/340.0, 390.0/374.0, 424.0/408.0, and 458.0/442.0 were monitored for PCDD/Fs analysis, while ions with m/z 112.0, 146.0, 180.0, 213.0, 250.0, and 284.0 were monitored for chlorobenzenes analysis. 2.4.2. Gel permeation chromatography A P230 high-pressure with constant-flow pump and a column (ID/length 25 mm × 450 mm) packed with the cross-linked divinylbenzene-styrene copolymer (SX-3 Bio-Beads) was applied with DCM for elution at a flow rate of 5.0 mL min−1 . Data obtained by Biotronik UV detector BT3030 were collected at a wavelength of 254 nm. The sample extracts were concentrated, and solvent-exchanged with approximately 1.0 mL DCM. The collecting time was set to 26.0–40.0 min. The collected fraction was concentrated, and solvent-exchanged with nonane for instrumental analysis. 2.5. Data analysis An isotope dilution method was employed for the analysis of PCNs and dl-PCBs in samples. It was assumed that the response of individual congeners of each homologue was equal to that of the 13 C-labled congeners with the same degree of chlorination, except for mono- through tri-CN congeners. Congener d7 -CN 2 was used as the internal standard for mono-CN and di-CN congeners, and 13 C10 -

3

CN 42 was used as that for tri-CN congeners. Quantification of PCN and dl-PCB congeners was performed by using a relative response factor (RRF, labeled to native) of the congener at the same level of chlorination and the similar retention time. The concentration of each homologue equals to the sum of the concentration of all congeners. The value below LOQ was replaced by half of the LOQ.

3. Results and discussion 3.1. Effect of the amount of sorbents To obtain the maximum purification efficiencies of SPE column, the amount of adsorbents was optimized by 0.3 g of MgO and varying amounts of basic alumina from 0.1 to 1.0 g. It was found that 0.1 g of basic alumina was not enough for retaining targets. PCN congeners (penta- through octa-CN) could be detected in the washing fraction (4–39%). Five milliliters of n-hexane/DCM (95:5, v/v) eluted 41–88%, 36–62%, 4–37%, and 2.8–32% of PCN congeners from the SPE columns packed with 0.3, 0.5, 0.8 g, and 1.0 g of basic alumina, respectively. These results demonstrate that 0.8 g of basic alumina or more have stronger retention for PCN congeners. In other words, 0.3 g of MgO and 0.3–0.5 g of basic alumina was suitable for retaining PCNs. Furthermore, fewer background signals and interferences were obtained for sediment sample purified by an SPE column packed with 0.5 g of basic alumina (Fig. S3). Therefore, the optimal composition for packing SPE column from the bottom up was 0.5 g of basic alumina and 0.3 g of MgO.

3.2. Retention activities of individual PCN congeners To assess the retention activities of individual PCN congeners, PCN standard mixtures in n-hexane (1.0 mL, 20 ng mL−1 ) were loaded into the SPE column. Then, the mixture was successively eluted with 10.0 mL of different volume ratios of n-hexane/DCM. As listed in Table 1, the recoveries of PCN congeners showed an increasing trend with decreasing volume ratios of n-hexane/DCM. With the exception of the congener CN 48, PCN congeners were well-eluted by n-hexane/DCM mixtures. It was found that n-hexane could not elute any PCN congeners from the developed SPE column. When the volume ratios of n-hexane/DCM were less than 98:2, acceptable recoveries (46–77.6%) of these congeners were obtained. In other words, all PCN congeners can be eluted under the same condition. The n-hexane/DCM (99:1, v/v) eluted 66.7% of CN 6, while it did not elute congeners CN 2 and CN 13. Decreasing the volume ratio of n-hexane/DCM to 98.75:1.25 led to the recoveries of 26.2% and 68.2% for CN 2 and CN 13, respectively. It was indicated that the retention order of low chlorinated congeners is as follows: CN 2 > CN 13 > CN 6. It was also observed that n-hexane/DCM (99:1, v/v) recovered 47.1% of CN 36, 20.1% of CN 28, and 30.7% of CN 27. At n-hexane/DCM volume ratios less than 98.25:1.75, four tetraCN congeners were completely eluted. However, CN 48 showed the strongest retention compared to other PCN congeners. The results showed the following order of retention of tetra-CN congeners: CN 48 > CN 46 > CN 28 > CN 27 > CN 36. Similarly, penta-CN and hexa-CN congeners also showed different retention activities on the developed SPE column. These results indicate that the retention activities of individual PCN congeners on the developed SPE column were different from the number and position of chlorine substituents. The possible reason is that the interaction between target compounds and adsorbents was activated or deactivated, depending on how the relative electron density of the naphthalene ring impacted the electron-withdrawing effect of chlorine substituent in PCNs [30].

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Table 1 PCNs recoveries from the developed SPE column with MgO microspheres and basic alumina as SPE adsorbents as a function of the volume ratios of n-hexane/DCM (n = 3).

CN 2 CN 6 CN 13 CN 36 CN 28 CN 27 CN 48 CN 46 CN 52 CN 50 CN 53 CN 67 CN 69 CN 72 CN 73 CN 75 a b

100:0

99:1

98.75:1.25

98.5:1.5

98.25:1.75

98:2

97:3

96:4

95:5

90:10

70:30

nda nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd 66.7 (5)b nd 47.1 (16) 20.1 (16) 30.7 (18) nd nd 51.9 (14) 19.4 (14) 2.5 (19) 6.4 (11) nd 34.6 (12) nd 33.0 (20)

26.2 (11) nd 68.2 (12) 15.1 (18) 47.2 (11) 45.8 (5) nd 53.1 (18) 7.1 (22) 45.0 (3) 53.9 (17) 48.2 (10) 46.2 (20) 20.8 (16) nd 28.0 (13)

36.1 (16) nd 7.2 (23) 2.8(6) 5.7 (17) 1.1 (17) nd 17.9 (18) nd nd 17.2 (15) 10.4 (12) 20.5 (14) nd 28.6 (17) nd

3.5 (21) nd nd nd 1.8 (20) nd nd nd nd nd nd nd nd nd 17.4 (15) nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd 71.2 (10) nd nd nd nd nd nd nd nd nd

Not detected. Mean (relative standard deviation, RSD, %).

3.3. Effect of washing and elution solvents The type and volume of the washing and elution solvent are crucial factors for assuring the elution efficiency of target analytes without interferences during SPE. As reported, it was difficult to elute PCNs and dl-PCBs with n-hexane, while n-hexane/DCM (95:5, v/v) was able to elute them from an alumina column [12]. Therefore, the experiments were performed with different elution volumes (5.0–15.0 mL) of n-hexane and n-hexane/DCM (95:5, v/v) to assess the elution efficiency of PCNs and dl-PCBs from the developed SPE columns. As listed in Tables 2 and 3, none of the PCN and dl-PCB congeners were eluted by n-hexane, but PCN and dl-PCB congeners were well-recovered using n-hexane/DCM (95:5, v/v). In fraction 4, the recoveries of PCN congeners were more than 75% (relative standard deviation, RSD <20%), except for CN 75 (66.9%). However, CN 2 and CN 73 were also detected in fraction 5, indicating that 5.0 mL of n-hexane/DCM (95:5, v/v) was not enough for the elution of CN 2 and CN 73. Surprisingly, it was found that CN 48 was not recovered by n-hexane and n-hexane/DCM (95:5, v/v). When the volume ratio of n-hexane/DCM was decreased to 70:30, the recovery of CN 48 was up to 75.8% (fraction 7). As reported, the proportion

of CN 48 in combustion processes was significantly less than other congeners [32] and it also holds lower relative potency factor [33]. Further, more interfering substances can be eluted as the polarity of the eluent was increased. Therefore, CN 48 was not analyzed in this paper. In contrast, dl-PCB congeners were well-eluted (>84%) with 5.0 mL of n-hexane/DCM (95:5, v/v), except for PCB 81. The recovery of PCB 81 reached a maximum (85.3%) with 10.0 mL of eluent (fractions 4 and 5). Clearly, the elution of PCN and dl-PCB congeners is dependent on the nature of individual congeners and the polarity of eluent. The optimized cleanup conditions are described as follows: one milliliter of sample extracts in n-hexane are loaded, and then washed with 5.0 mL of n-hexane, followed by eluting PCNs and dl-PCBs with 10.0 mL of n-hexane/DCM (95:5, v/v).

3.4. Purification performance Many interfering substances, including lipid compounds, sulfur, pigments, and other analogs are generally present in sample extracts. They can decrease the sensitivity of instruments by increasing the background signal, and lead to unsatisfactory chromatograms and false positive/negative results. Therefore, it is

Table 2 Elution efficiencies (recovery, %) of individual congeners of PCNs in different fractions (n = 3). Compounds

F−1a

F−2b

F−3b

F−4c

F−5c

F−6c

F−7d

F−8d

CN 2 CN 6 CN 13 CN 36 CN 28 CN 27 CN 48 CN 46 CN 52 CN 50 CN 53 CN 66 CN 69 CN 72 CN 73 CN 75

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nde nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

76.8 (7)f 96.6 (7) 93.4 (10) 91.9 (10) 89.8 (8) 85.5 (3) nd 86.5 (10) 83.0 (9) 84.6 (11) 82.8 (11) 82.3 (10) 78.5 (12) 86.7 (10) 42.3 (16) 66.9 (14)

14.1 (18) nd nd nd nd nd nd nd nd nd nd nd nd nd 35.9 (19) nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd 75.8 (8) nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

a b c d e f

F-1: loaded with samples and first eluted by 5.0 mL of n-hexane. F-2 and F-3: eluted by 5.0 mL of n-hexane, respectively. F-4, F-5 and F-6: eluted by 5.0 mL of n-hexane/DCM (95:5, v/v), respectively. F-7 and F-8: eluted by 5.0 mL of n-hexane/DCM (70:30, v/v), respectively. Not detected. Mean (relative standard deviation, RSD, %).

F. Li et al. / J. Chromatogr. A 1448 (2016) 1–8

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Table 3 Elution efficiencies of individual congeners of dl-PCBs in different fractions (n = 3). Compounds PCB 77 PCB 81 PCB 123 PCB 118 PCB 114 PCB 105 PCB 126 PCB 167 PCB 156 PCB 157 PCB 169 PCB 189 a b c d e

F−1a d

nd nd nd nd nd nd nd nd nd nd nd nd

F−2b

F−3b

nd nd nd nd nd nd nd nd nd nd nd nd

F−4c e

nd nd nd nd nd nd nd nd nd nd nd nd

87.1 (6) 48.0 (4) 92.5 (5) 92.4 (9) 91.5 (9) 88.2 (7) 84.9 (4) 91.3 (4) 86.4 (7) 89.0 (9) 89.8 (7) 89.9 (7)

F−5c

F−6c

nd 37.3 (8) nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd

F-1: loaded with samples and first eluted by 5.0 mL of n-hexane. F-2 and F-3: eluted by 5.0 mL of n-hexane, respectively. F-4, F-5 and F-6: eluted by 5.0 mL of n-hexane/DCM (95:5, v/v), respectively. not detected. Mean (relative standard deviation, RSD,%).

necessary to remove these interferences during the cleanup procedures.

except for mono-CB (about 40%). These results demonstrate a rough elution order from the developed SPE columns: CBs > PCNs and dlPCBs > PCDD/Fs and SCCPs. The finding suggests that the developed SPE cleanup method could effectively separate PCNs and dl-PCBs from potential co-extracts such as SCCPs, PCDD/Fs, and CBs.

3.4.1. Organochlorinated compounds The selective separation of PCNs and dl-PCBs from several potential organochlorinated compounds (SCCPs, PCDD/Fs, and CBs) was evaluated. SCCPs in the washing and elution fractions were determined according to we previously reported instrumental method [34]. SCCPs were not detected in two fractions, indicating that they were still retained on the SPE column. Similarly, PCDD/F congeners were not eluted by n-hexane and n-hexane/DCM (95:5, v/v) from the developed SPE column. More than 75% of individual chlorobenzene congeners were washed with 5.0 mL of n-hexane,

3.4.2. Lipid, sulfur, and pigments The elimination of lipid compounds and sulfur from sample extracts is very important for POP analysis. In this work, the working solution (1.0 mL, in DCM) was directly loaded on a GPC. The equal working solution was purified by the optimized SPE cleanup method, after which the elution fraction was analyzed by GPC. No lipid compounds and sulfur were detected in the elution frac-

Table 4 Method validation data. Accuracy (recovery, %) and precision (relative standard deviation, RSD, %) obtained by GC–MS/MS analysis for PCNs and dl-PCBs in sediment, pine needle and scallop samples (n = 4). Sediment sample

Pine needle sample

Scallop sample

−1

0.2 ng g

2.0 ng g

0.5 ng g

5.0 ng g

0.5 ng g−1

5.0 ng g−1

PCN congeners CN 2 CN 6 CN 13 CN 36 CN 28 CN 27 CN 46 CN 52 CN 50 CN 53 CN 66 CN 69 CN 72 CN 73 CN 75

115 (16)a 113 (16) 111 (18) 176 (17) 121 (19) 106 (13) 154 (16) 141 (14) 117 (15) 140 (19) 127 (15) 108 (15) 127 (15) 140 (19) 86 (26)

66 (4) 76 (4) 84 (2) 91 (3) 84 (6) 84 (7) 86 (6) 77 (14) 85 (5) 74 (5) 95 (6) 91 (5) 88 (8) 93 (2) 45 (20)

91 (17) 109 (8) 129 (6) 177 (12) 147 (18) 123 (14) 181 (12) 148 (10) 139 (8) 121 (8) 112 (9) 124 (14) 125 (10) 175 (20) 50 (19)

60 (3) 77 (5) 86 (7) 93 (3) 87 (4) 89 (4) 82 (4) 82 (7) 84 (5) 76 (7) 87 (2) 84 (2) 84 (4) 86 (5) 77 (15)

102 (7) 104 (9) 109 (17) 191 (13) 128 (9) 121 (13) 137 (5) 135 (17) 101 (11) 115 (8) 123 (19) 107 (16) 115 (11) 130 (12) 104 (24)

60 (6) 73 (9) 75 (8) 84 (12) 80 (10) 83 (10) 77 (10) 76 (10) 79 (7) 70 (7) 83 (9) 80 (8) 82 (10) 81 (13) 63 (9)

dl-PCB congeners PCB 77 PCB 81 PCB 123 PCB 118 PCB 114 PCB 105 PCB 126 PCB 167 PCB 156 PCB 157 PCB 169 PCB 189

106 (19) 109 (17) 116 (19) 95 (16) 120 (14) 94 (19) 99 (16) 108 (14) 95 (15) 103 (18) 101 (13) 102 (12)

75 (7) 76 (10) 77 (2) 73 (5) 73 (5) 71 (5) 67 (9) 69 (10) 80 (4) 78 (3) 78 (5) 77 (3)

121 (15) 123 (11) 108 (11) 101 (13) 119 (13) 123 (15) 118 (22) 118 (11) 117 (13) 127 (18) 135 (8) 118 (11)

78 (4) 80 (6) 83 (8) 81 (3) 83 (5) 82 (5) 76 (6) 80 (5) 79 (4) 80 (3) 75 (1) 81 (12)

140 (10) 122 (10) 118 (8) 100 (13) 99 (18) 98 (15) 99 (8) 109 (14) 93 (11) 109 (11) 89 (6) 125 (14)

87 (4) 87 (2) 88 (3) 85 (1) 88 (3) 87 (2) 81 (1) 79 (6) 83 (4) 85 (4) 81 (4) 88 (3)

a

Mean (RSD,%).

−1

−1

−1

6

F. Li et al. / J. Chromatogr. A 1448 (2016) 1–8

Table 5 Concentrations (ng g−1 dw) and STEQs (pg TEQ g−1 ) of PCNs and dl-PCBs congeners in sediment, pine needle and scallop samples (n = 3). SD 1

SD 2

PN 1

PN 2

SL 1

SL 2

CN congeners CN 2 CN 6 CN 13 CN 36 CN 28 CN 27 CN 46 CN 52 CN 50 CN 53 CN 66 CN 69 CN 72 CN 73 CN 75 74 PCNse TEQPCNs

0.74 (6)a nac 0.04 (6) nd nd 0.03 (16) nd 0.02 (8) nd nd 0.03 (7) nd nd nd nd 3.07 0.01

0.11 (16) na 0.29 (11) nd nd nd nd 0.01 (11) nd nd 0.05 (10) nd nd 0.41 (14) nd 2.99 0.14

ndb na 0.03 (19) nd nd nd nd nd nd nd nd nd nd 0.24 (15) nd 2.43 0.03

nd na nd nd nd nd nd nd nd nd nd nd nd nd nd 0.53 0.04

nd na 0.01 (21) nd nd nd nd nd nd nd nd nd nd nd nd 0.25 0.03

nd na
dl-PCB congeners PCB 77 PCB 81 PCB 123 PCB 118 PCB 114 PCB 105 PCB 126 PCB 167 PCB 156 PCB 157 PCB 169 PCB 189 dl-PCBs TEQPCBs

nd 0.02 (15) nd 0.03 (1) nd 0.01 (15) nd 0.009 (17) 0.004 (20) nd nd
nd 0.004 (20) nd 0.008 (17) nd nd nd
nd nd nd nd nd nd nd
nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd nd nd nd nd nd nd
nd nd nd nd nd nd nd
a b c d e

Mean (relative standard deviation, RSD, %). nd: not detected. na: not available. Data of congener CN 6 was not available as the co-eluting of congener CN 5, CN 7, CN 8, CN 11, and CN 12.
tion. This evaluation procedure was repeated using an extract from a pine needle sample. As shown in Fig. 1, lipid compounds and pigments in the extract are well-removed by the developed SPE cleanup method. These results demonstrate that the developed SPE cleanup method can be used to remove lipid compounds, sulfur, and pigments from sample extracts.

3.4.3. Comparison with other cleanup methods Purification performance of the developed SPE cleanup method was compared with that of two conventional cleanup methods. A sediment extract was cleaned up by GPC, the developed SPE cleanup method, and two-stage open column chromatography [12]. The sample was then measured using GC–MS/MS in the fullscan mode (m/z 50–500), and the gas chromatographic conditions were identical to those of PCNs (the retention time ranged from 13 to 52 min). As shown in Fig. 2, the total ion chromatograms of sample extracts purified by GPC, SPE and column chromatography have background signals within the range of 12–55 min,

Fig. 1. GPC chromatograms of pine needle sample extracts before and after cleaned up using the developed SPE column.

Fig. 2. Comparison of total ion chromatograms of sediment sample purified using three different cleanup methods.

F. Li et al. / J. Chromatogr. A 1448 (2016) 1–8

7

Table 6 Comparison of analytical parameters of the proposed analytical method and other two analytical methods used for the determination of PCNs and dl-PCBs in complex samples. Proposed analytical method

EPA method 1668A

Conventional analytical methoda

Brief description

ASE SPE cleanup GC–MS/MS analysis

Soxhlet extraction Multilayer silica gel column and alumina column cleanup HRGC/HRMS analysis

Operational step Sorbent consumption Solvent consumption Analytical time Recovery range Extracts color Cost

Simple Less <150 mL <2.5 h 40–157% No or light brown Low

Soxhlet extraction/Dean-stark GPC or silica gel or Florisil or Carbopak or anthropogenic isolation column cleanup HRGC/HRMS analysis Complex Large >600 mL >30 h 21–197% No High

a

Complex Large >600 mL >30 h 40–120% No High

Zhao et al. [12].

12–30 min, and 35–55 min, respectively. However, fewer background signals and interferences were obtained for the sediment sample purified by the developed SPE cleanup method and by twostage open column chromatography. In contrast, the developed SPE cleanup method is superior to two-stage open column chromatography, owing to less organic solvent consumption and simple operation. 3.5. Method validation In this work, an analytical method combining ASE, SPE cleanup, and GC–MS/MS analysis is proposed to analyze PCNs and dl-PCBs in complex samples. Performance parameters of the proposed analytical method were validated in terms of the linear dynamic ranges (LDRs), limits of detection (LODs), limits of quantification (LOQs), the accuracy, and precision. LDRs of PCN and dl-PCB congeners were established using sediment, pine needle, and scallop samples spiked with PCNs and dl-PCBs at five levels. According to EPA method 1668A, the relative response vs. concentration in spiked samples was computed over the LDR; and RSD of RRF should be less than 20% in LDR. As listed in Tables S5 and S6, good linearity was obtained for PCN and dl-PCB congeners with the RSDs of RRF less than 23% within two orders of magnitude in a dynamic concentration range. The LODs and LOQs were determined with blank samples spiked at 2.0 ng mL−1 , and calculated as the analyte concentration, providing peaks for which the signal-to-noise ratio was 3 and 10, respectively. The LODs of PCN and dl-PCB congeners in sediment, pine needle, and scallop samples were in the range of 0.6–19.1 pg g−1 and 0.4–8.6 pg g−1 , respectively. The corresponding LOQs in these samples ranged from 1.9 to 63.8 pg g−1 and 1.2–28.7 pg g−1 , respectively. The accuracy and precision of the analytical method were assessed by spiking two concentration levels (1.0 ng and 10.0 ng) of the PCN and dl-PCB mixtures into blank sample matrices. As listed in Table 4, the recoveries of PCN and dl-PCB congeners generally ranged from 63% to 148% with RSDs less than 26%. These results demonstrate that the proposed analytical method provides good sensitivity, satisfactory accuracy and precision for the analysis of PCNs and dl-PCBs in complex samples.

licate analysis of PCNs and dl-PCBs in these samples. Congeners CN 36, CN 28, CN 50, CN 53, CN 69, CN 72, and CN 75 were not detected in any samples. The total concentrations of PCNs were in the range of 0.16–3.07 ng g−1 dry weight (dw) with low chlorinated naphthalenes as the dominant homologues. dl-PCBs concentrations in these samples ranged from undetectable to 0.07 ng g−1 dw. Congeners PCB 77, PCB 123, PCB 114, PCB 126, and PCB 169 were also not detected in these samples. According to relative potency factors of PCNs [35] and WHO-toxic equivalency factors of dl-PCBs [36], the corresponding total toxic equivalents of PCNs and dl-PCBs in these samples were 0.02–0.14 pg TEQ g−1 . The performance parameters of the proposed analytical method were compared with those of EPA Method 1668A [37] and a conventional analytical method [12]. As listed in Table 6, the proposed analytical method exhibits excellent merits in terms of equivalent purification efficiency, simple operation, less analytical time, and low cost. Furthermore, it is environmentally friendly as it greatly reduces the consumption of adsorbents and organic solvents.

4. Conclusions A high-performance SPE cleanup method based on the combination of monodisperse MgO microspheres and basic alumina was developed to purify sample extracts for the analysis of PCNs and dlPCB. The cleanup method showed excellent purification efficiency, simple operation, and less solvent consumption. The number and position of chlorine substituents was observed to impact the retention activities of individual PCN congeners. Combined with ASE and GC–MS/MS analysis, the developed SPE cleanup method was successfully applied to the analysis of PCNs and dl-PCBs in sediment, pine needle, and scallop samples. This analytical method with the isotope dilution technique obtained satisfactory recoveries, good accuracy and precision, and wide LDR with low LODs. With the increasing attention to environmental pollution caused by dioxinlike compounds, this rapid, simple, and environmentally friendly method is a promising method for the analysis of PCNs and dl-PCBs in various environmental and biological samples.

3.6. Application to the analysis of real samples Acknowledgments To evaluate the applicability of the proposed analytical method, it was used to analyze PCNs and dl-PCBs in sediment, pine needle, and scallop samples. The recoveries of the surrogate internal standards of PCNs and dl-PCBs in these samples were in the range of 40–137% and 39–143%, respectively. Chromatographic peaks representing all congeners of PCNs were discovered up to a value of 54 with some co-elution [9]. Table 5 shows the results of the trip-

This work was supported by the National Natural Science Foundation of China (Grant No. 21205117), the Special Environmental Protection Foundation for Public Welfare Project (201309030), the Special Fund for Agro-Scientific Research in the Public Interest (201503108), and the National Basic Research Program of China (973 Program, Grant No. 2015CB453100).

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