Chemosphere 74 (2009) Contents lists available at ScienceDirect. Chemosphere. journal homepage:

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1 Chemosphere 74 (2009) Contents lists available at ScienceDirect Chemosphere journal homepage: Validation of high-throughput measurement system with microwave-assisted extraction, fully automated sample preparation device, and gas chromatography-electron capture detector for determination of polychlorinated biphenyls in whale blubber Hiroyuki Fujita a, *, Katsuhisa Honda a, Noriaki Hamada b, Genta Yasunaga c, Yoshihiro Fujise c a Department of Environmental Science for Industry, Ehime University, Tarumi Matsuyama, Ehime , Japan b Miura Institute of Environmental Science, Miura Co. Ltd., Tsuji Hojyo Matsuyama, Ehime , Japan c The Institute of Cetacean Research, 4-5, Toyomi-cho, Chuo-ku, Tokyo , Japan article info abstract Article history: Received 14 July 2008 Received in revised form 28 October 2008 Accepted 28 October 2008 Available online 17 December 2008 Keywords: Polychlorinated biphenyls Microwave-assisted extraction Sample preparation device Gas chromatography-electron capture detector Whale blubber Validation of a high-throughput measurement system with microwave-assisted extraction (MAE), fully automated sample preparation device (SPD), and gas chromatography-electron capture detector (GC-ECD) for the determination of polychlorinated biphenyls (PCBs) in minke whale blubber was performed. PCB congeners accounting for >95% of the total PCBs burden in blubber were efficiently extracted with a small volume (20 ml) of n-hexane using MAE due to simultaneous saponification and extraction. Further, the crude extract obtained by MAE was rapidly purified and automatically substituted to a small volume (1 ml) of toluene using SPD without using concentrators. Furthermore, the concentration of PCBs in the purified and concentrated solution was accurately determined by GC-ECD. Moreover, the result of accuracy test using a certified material (SRM 1588b; Cod liver oil) showed good agreement with the NIST certified concentration values. In addition, the method quantification limit of total-pcb in whale blubbers was 41 ng g 1. This new measurement system for PCBs takes only four hours. Consequently, it indicated this method is the most suitable for the monitoring and screening of PCBs in the conservation of the marine ecosystem and safe distribution of foods. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Polychlorinated biphenyls (PCBs) have been widely used in electrical equipments such as transformers and capacitors during (UNEP, 2001). In spite of the prohibition of their production and usage, PCBs are still present in the environment due to their lipophilic and highly persistent character. Particularly, top marine predators such as whales and seals tend to accumulate PCBs in their fatty tissues (Connell, 1988; Woodley et al.,1991; Gauthier et al., 1997). PCB residual levels in these animals have often exceeded the provisional limit of 0.5 lgg 1 (fresh weight) provided by the Japanese Ministry of Health in 1973 for seafood. Therefore, the continuous monitoring of PCBs in marine organisms and investigative whaling (JARPN) is essential for the conservation of the marine ecosystem and safe distribution of seafood. However, the conventional method of PCB analysis involves several complicated processes, takes a long time, and consumes a large volume of organic solvents. As a step toward the improvement of analytical techniques, the microwave-assisted extraction (MAE) * Corresponding author. Tel.: ; fax: address: fujita_hiroyuki@miuraz.co.jp (H. Fujita). method has been widely noted as a new extraction technique for PCBs in environmental solid samples and biological tissues (Vetter et al., 1998; Natalia et al., 2007). This extraction technique is used for reducing time and as a work-saving practice; the recovery of PCBs is highly efficient as compared to that obtained by conventional extraction methods, e.g., by using Soxhlet or liquid liquid separation. In the EPA method 3546, this extraction technique has been approved for the extraction of organochlorine compounds in environmental solid samples such as soil and sediments. In respect of the purification technique for environmental analytical chemistry, certain instruments of automation systems have been investigating and reported for the purification procedure of dioxin-like PCBs, polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans (dioxins) and polybrominated diphenylethers (Focant and De Pauw, 2002; Focant et al., 2004). These automation systems are utilized by a few certification-testing laboratories for dioxins routine analysis. However, there are not utilized purification system with respect to PCBs routine monitoring and screening analysis and the handling is not still simple, and classical column chromatography have been using as the main purification method for PCB analysis. Therefore, further methods are essential for speedy PCB measurements in biological samples /$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi: /j.chemosphere

2 1070 H. Fujita et al. / Chemosphere 74 (2009) In this report, we investigated the validation of a high-throughput measurement system for PCBs in whale blubber, examining followed by, (1) Detail of profile patterns of PCB congeners in whale blubber by high resolution gas chromatography high resolution mass spectrometry (HRGC-HRMS), (2) The extraction efficiency and precision of PCBs using MAE. Furthermore, (3) purification and recovery rates of PCBs using the fully automated sample preparation device (SPD), (4) the identification of PCB congeners on gas chromatography-electron capture detector (GC- ECD) chromatograms and (5) the validation of the new PCB measurement system combined with MAE, SPD, and GC-ECD using standard reference materials. 2. Experimental 2.1. Chemicals and reagents A mixture of 62 native congeners of PCBs (2.0 lgml 1 of each congener, BP-MS; Wellington Laboratories Inc., Canada) was used as the PCB standard solution for calibration by GC-ECD and HRGC-HRMS. 13 C 12 -labelled PCB standard solution (#3, #15, #28, #52, #118, #153, #180, #194, #208, and #209; EC-4189, 1.0 lgml 1 for individual PCB congeners; Cambridge Isotope Laboratories, Inc., USA) was used as the surrogate standard solution for quantification by HRGC-HRMS. The determinations of 209 PCB congeners were performed by an extracted/purified fly ash solution. In order to examine the interference in PCB analyses by GC- ECD, a 10 ± 1 pg ll 1 standard solution of the organochlorine pesticides (OCPs) was prepared by mixing the compounds of HCB, a- HCH, b-hch, d-hch, cis-chlordane, trans-chlordane, p,p 0 -DDT, p,p 0 -DDD (Wako Pure Chemical Industries Ltd., Japan), c-hch, o,p 0 -DDD (GL Sciences Inc., Japan), o,p 0 -DDT, o,p 0 -DDE, p,p 0 -DDE (Nisio Industries Ltd., Japan), oxychlordane, cis-nonachlor, and trans-nonachlor (AccuStandard Inc., USA). For PCBs and pesticides, analytical grade solvents of ethanol, n-hexane, dichloromethane, and toluene were purchased from Wako Pure Chemical Industries Ltd., Japan, and the reagent for KOH was obtained from Kanto Chemical Co. Inc., Japan Samples Thirty-five samples of minke whale (Balaenoptera acutrostrata) blubber were obtained from the Western North Pacific in 2003 and 2004 under the Japanese Whale Research Program (JARPN II). These samples were provided by the Japan Institute of Cetacean Research and stored in polyethylene bags at 20 C until analysis. The standard reference material (SRM 1588b; Organics in Cod Liver Oil) for the accuracy test was obtained from USA National Institute of Standards and Technology (NIST). The sub-reference material (whale blubber oil) was prepared from minke whale blubber for the validation of the new PCB measurement system in this study Liquid liquid extraction (LLE) and purification of the general method For liquid liquid extraction of PCBs, the epidermis was eliminated from the blubber and homogenized; then, approximately 10 g of the blubber was accurately weighed into a funnel. Then, 40 ml of ethanolic 1 M KOH and surrogate standard were added to the funnel and mixed for 1 h. The mixture was allowed to stand for 30 min, and then 40 ml of water and n-hexane each were added into the funnel and mixed. Thereafter, the saponified layer was extracted with 40 ml of n-hexane. This procedure was repeated two times. All of the n-hexane layers were combined and then washed with 100 ml of water. The extracts were filtered through 20 g of anhydrous Na 2 SO 4 and finally concentrated to 10 ml. Two milliliters of each of the extracts was purified using multilayer silica gel column chromatography composed of two components 4 g of silica gel impregnated with sulfuric acid (44% mass fraction: 44% H 2 SO 4 Si) and 3 g of silica gel impregnated with silver nitrate (10% mass fraction: 10% AgNO 3 Si). All of the PCB fractions were eluted with 90 ml of 2% dichloromethane in n-hexane. The eluate was concentrated and finally substituted with ll of decane MAE system MAE of PCBs in whale blubber was performed using a Model 7295 microwave sample preparation system (OI Analytical, USA). The poly-tetrafluoroethylene-co-perfluoropropylvinylether (PFA)- lined sample vessel is airtight, pressure-resistant (upto 200 psi), and with a volume of 60 ml; the inner pressure is monitored by an exclusive pressure sensor, and the microwave power is automatically controlled; the magnetron power output, being 950 W, is available to be set up to 100% of the power (2450 MHz). The operating conditions of this preparation system can be optimized by adjusting the three parameters of the magnetron power rate, the irradiating time, and the pressure value MAE method For performing MAE of PCBs in whale blubber, 2 5 g of the samples was accurately weighed into the sample vessels. Approximately 10 ml of the saponifying solution (ethanolic 1 M KOH: water = 1:1) and 20 ml of n-hexane were added as an extractant. MAE was carried out up to 20 psi with increments of 5 psi at 5 40% of magnetron power output for min. After cooling the vessels until room temperature, 5 10 ml of the n-hexane layer was pipetted and then cleaned up by multilayer silica gel column chromatography or SPD. The concentration of PCBs in one of the purified solutions was determined by GC-ECD or HRGC-HRMS after the addition of the surrogate standard solution to the extracts Principle and application of SPD for PCB analysis The purification for PCB analysis was performed using SPD (SPD-600GC; Miura Co. Ltd., Japan), which was originally developed for the purification method of dioxins analysis as high accuracy and rapidly. Fujita et al. investigated the purification effects by heating chemical impregnated silica gel columns, multilayer silica gel column. On this heating purification method, they reported that removal effects of various contaminants in crude extracts from environmental samples were highly efficient. For example, on 10% AgNO 3 Si at 60 C, effectiveness of chemical reaction with silver ions and sulfur compounds progressed, and while on 44% H 2 SO 4 Si at 60 C, their removal effects of mono- and poly-aromatic hydrocarbon compounds was excellent, and were so far better than those at room temperature. In the case of the combination of 10% AgNO 3 Si and 44% H 2 SO 4 Si, chemical impregnated multilayer silica gel column, the purification effects by heating at 60 C could be highly synergistic effect as compared to those at room temperature. It indicate that these results are useful for purification of sediment and biological samples enriched in sulfur compounds, and also for reduction of analytical time, amounts of solvents and contaminations in the capillary column and detector spot. (Kishino et al., 2004; Fujita et al., 2004; Miyawaki et al., 2007).

3 H. Fujita et al. / Chemosphere 74 (2009) Column heater Sample collection vial Step 1; n-hexane 65mL Purification column Column joint Concentration column Step 2; Toluene 1.0mL Fig. 1. Schematic diagram of the column flow channel of SPD-600GC. We applied the above-mentioned techniques and developed the device for PCBs analysis. Fig. 1 shows the schematic diagram of the column flow channel, which is as follows: The upper column is a multilayer silica gel column (Purification column; 200 mm 12.5 mm I.D.), and the lower one is a c-alumina column (Concentration column; 0.5 g, 30 mm 6 mm I.D.). These columns were obtained from the Miura Institute of Environmental Science, Miura Co. Ltd., Japan. The crude extract obtained by MAE or liquid liquid extraction was directly applied on top of the purification column, followed by setting the column parts and starting the program sequence. After heating the purification column to 60 C, the PCBs in the purification column were eluted with 65 ml of n-hexane maintained at 60 C, and then, the PCBs trapped in the concentration column were reversibly eluted with 1.0 ml of toluene maintained at approximately 60 C. A special feature of this device has nothing the valve, equipping a function of switching flow line by air pressure. When n-hexane on Step 1 flows, a solenoide air valve seals up a space of sample collection vial. While, at Step 2 flowing toluene for recovering PCBs, a solenoide air valve open a space of sample collection vial and shut the upper flow line of n-hexane at the same time. Therefore, there is little possibility of occurring carryover of PCBs; because of this device have nothing the valve contaminated by PCBs. To observe the recovery rates of PCBs by SPD, the recovery tests were examined in several crude extracts of the blubber obtained by liquid liquid extraction. The individual extracts were purified using SPD and the conventional clean-up technique (multilayer silica gel column chromatography), and the PCB concentrations in these purified solutions were measured by HRGC-HRMS. On the basis of the collected data, the recovery rates of PCBs by SPD were calculated. And, in order to confirm effects of purification in SPD, Full scan chromatograms of eluate from multilayer silica gel column and SPD were compared. The interferences in the eluate purified by SPD were also checked as follows: The OCPs standard solution (1.0 ml) was applied to the purification column and followed by starting the program sequence similar to the operation mentioned above; the OCP chromatograms in the purified solution were obtained by GC-ECD measurement. On the basis of the comparisons between the OCP chromatograms before/after the pretreatment by SPD, the influence of interferences for the PCB determination were evaluated Instrumental analysis The determination of PCBs were analyzed using GC-ECD (Agilent 6890N GC Micro-ECD; Agilent Technologies, DE, USA), equipped with a DB-5 ms capillary column (60 m, 0.25 mm I.D., 0.25 lm; J&W Scientific, USA) and the detector of the radioactive source 63 Ni at a constant flow rate of 0.7 ml N 2 min 1 as a carrier gas. The initial column temperature was maintained at 100 C for 1 min, and then increased to 190 C at the rate of 20 Cmin 1. Then, the temperature was increased to 250 C at the rate of 2 C min 1, raised to 280 C at the rate of 6 C min 1, and finally maintained at 280 C for 9.5 min. The injection port was operated in the splitless mode at 270 C, and the detector was maintained at 300 C. The quantification of PCBs was performed using external standard calibration, and the average response factor was generated from four point calibrations. Integration and calculations were accomplished using Agilent Chemstation software (Rev. B SR1 [260]). The linearity and detection limit (DL) were validated by Agilent Technologies: The linearity for quantification was in the range of ng g 1 ; the DLs of PCB congeners by GC-ECD were approximately 0.05 ng g 1 (Chanel and Chang, 2000). The DLs and quantification limits (QL) in this procedure were also calculated from the standard deviations of the repeated analyses using the sub-reference material, and the DL and QL in this case appeared as SD 3 and SD 10, respectively. The quantification and congener pattern for total PCBs of minke whales by HRGC-HRMS was performed using Agilent 6890N GC and JMS-700S (JEOL, Tokyo, Japan). The gas chromatographic separations of PCB congeners were performed on HT8-PCB fused silica capillary column (60 m, 0.25 mm I.D., 0.2 lm film thickness; SGE, USA). Ultra-high-purity helium was used at a constant flow rate of 1.0 ml min 1 as a carrier gas. The oven temperature program for the measurements of tetra- to hepta-pcbs was maintained at 120 C for 1 min and then increased to 180 C at the rate of 20 C min 1. The temperature was raised to 280 C at the rate of 2 C min 1 and finally maintained at 280 C for 3 min. On the other hand, for mono-, di-, tri-, octa-, nona-, and deca-pcb, the temperature was initially set as 120 C, and after maintaining it for 1 min, the temperature was increased to 180 C at the rate of 20 Cmin 1. Finally, the temperature was raised to 280 C at the rate of 4 C min 1 and maintained for 9.5 min. For all of the PCB measurements, the injector temperature was maintained at 280 C, and 2 ll of sample was injected in the splitless injection mode. The HRMS was operated in the electron ionization mode, using selected ion monitoring at an electron energy of 38 ev and an ionizing current of 500 la. The resolution of the instrument was routinely more than 10,000 (10% valleys). Two isotope ions, M + and (M+2) + or (M+2) + and (M+4) +, which are known as a relative abundance, were monitored in each of the PCB congeners and 13 C-labelled surrogate standards. Concentrations of the identified PCB congeners were calculated using the mean relative response factors determined from standard calibration runs. The procedure QLs were in the range of 0.1 to 1.0 pg g 1 for mono- to deca-chlorinated congeners. The initial test of purification procedure was undertaken using gas chromatography low resolution mass spectrometry (GC- LRMS). GC-LRMS is Agilent 6890 N GC equipped with a JMS-K9 (JEOL, Tokyo, Japan). A capillary column and GC temperature programme were same GC-ECD procedure. PCBs fraction from silica gel column clean-up procedure and SPD was concentrated 50 ll

4 1072 H. Fujita et al. / Chemosphere 74 (2009) Table 1 The concentrations of 173 PCB congeners in 35 minke whale blubber detected by HRGC-HRMS. Below all data were obtained by the conventional method. Congeners pg/g fresh weight % to total PCBs (b) (a) Average Maximum Minimum 1Cl # # # Cl # #8, # # # #13, # Cl # #18 * # # # # # # # # # # #28 * #20, # # # # Cl # # #53 * # # # #52, #69 * # #49 * #48, #47 * #44 * # # # #71 * # # # # #74 * # #66 * # # # # # # Cl # # # # # #102, #93, * #98, #95 # #91 * # #92 * #84 * # # #101 * # #99 * #112, # Table 1 (continued) Congeners pg/g fresh weight % to total PCBs (b) (a) Average Maximum Minimum # # #86, * #117, #97 #125, #116 #87, * #115 #85 * #120, * #110 # # #109, #107 # #118 * # #105 * # Cl # # # # #136 * # # #151 * #135 * #144, #147 * #149, #139 * # # # # # # #146 * #132 * #153 * # #137 * #130 * #164, #163 * #138 * #158 * # # #128 * #167 * #156 * # # Cl # # #179 * # #178 * # #182, #187 * #183 * # #174 * # #177 * #171 * # #180 * # # #170 * # #

5 H. Fujita et al. / Chemosphere 74 (2009) Table 1 (continued) Congeners pg/g fresh weight % to total PCBs (b) toluene and analyzed by GC-LRMS full scan mode (mass range: ), respectively. 3. Results and discussion (a) Average Maximum Minimum 8Cl #202 * # # # # #198, #199 * # #203 * # #194 * # Cl # # # Cl # Total-PCBs (a) The congeners marked with an asterisk (*) in this table demonstrated a QL value > 200 pg/g in GC-ECD measurement. (b) Relative ratio (%) of each congeners to Total PCBs concentration Profile of PCB congeners in whale blubber In order to clarify the detailed profiles of PCB congeners in minke whale blubber, the concentration of total PCBs in 35 samples of whale blubber were prepared by LLE, multilayer silica gel column, and determined by HRGC-HRMS. The results presented in Table 1 show that the sum burden of 173 congeners among all the PCB congeners (209 congeners) could be actually represented as the total PCBs because the other PCB congeners (36 congeners) were either not detected or were less than the QL obtained by the HRGC-HRMS method. In the case of the PCB measurement by GC-ECD, considering the DL (0.05 ng g 1 ) and QL (0.2 ng g 1 ) of GC-ECD, PCBs with a higher concentration than the QL value included 65 congeners of tri- to octa-chlorinated PCBs, which are marked by asterisks in Table 1; their sum burden accounted for approximately 95% of the total PCB burden. On the other hand, the concentrations of the other congeners, i.e., mono-, di-, nona-, and deca-chlorinated congeners, was very low; moreover, their sum burden accounted for less than 1% of the total PCB burden. It is known that the relatively low residue levels of mono-, di-, nona-, and deca-chlorinated congeners in whale blubber are similar to those in environmental and biological samples, as mentioned in several reports (Ross et al., 2000; Andersen et al., 2001; Hobbs et al., 2001, 2003; Wurl and Obbard, 2005). Consequently, these results indicate that in the case of GC-ECD determination, the measurement of tri- to octa-chlorinated PCB congeners, i.e., 65 congeners, is essential for learning the residue levels of total PCBs in whale blubber Extraction efficiency with MAE In general, the method of LLE has been widely used for the extraction of PCBs in adipose tissues. Table 2 shows the extraction efficiency of PCBs using MAE as a percentage against the PCB concentrations obtained by LLE. Both cleanup were performed by Table 2 Extraction efficiencies from MAE and recoveries from SPD. Congeners Extraction efficiency (%) (a) Recoveries from SPD (%) (b) Average RSD Average RSD 3Cl # # Cl # #52, # # #48, # # # # # Cl #102, #93, #98, # # #92, # # # #86, #117, # #87, # # #120, # # # Cl # # # #144, # #149, # # # # # # #164, # # # # # # Cl # # # #182, # # # # # # # # Cl # #198, # # # # (a) 2 g, 3 g, 4 g and 5 g of blubbers were treated by MAE and LLE, respectively. And both purification were performed by silica gel column, determined by HRGC-HRMS. Relative efficiencies of MAE to LLE procedure were shown. (b) Individual five blubbers were analyzed by MAE and SPD, Simultaneously, those samples were analyzed by LLE and Silica gel column, determined by HRGC-HRMS. Relative recoveries of MAE/SPD procedure to conventional procedure were calculated. multilayer silica gel column. The averages of PCB extraction efficiencies obtained in individual samples, i.e. for 2 g, 3 g, 4 g, and 5 g, ranged from 78% to 103%, and their relative standard deviations (RSD, 1 13%) were relatively low. Even if the extracts were pipetted from no-accurately 20 ml n-hexane layer added the non-saponificated lipids of blubber, this result indicates that this MAE procedure can keep up the precision of efficiency and quantification, up to 5 g fresh

6 1074 H. Fujita et al. / Chemosphere 74 (2009) weight. The sample weight for routine analysis are normally about 2 g, and sufficiently fulfill the quantification limit, below one-tenth of the provisional limit 0.5 lgg 1 (fresh weight). Therefore, after MAE, the operating is only pipetting without concentration processes, this extraction procedure is suitable into the routine monitoring and screening method for PCBs from whale blubbers. It is known that the degradation of highly chlorinated congeners may not occur during the process of saponification at room temperature (Booij and Berg, 1994; Greizerstein et al., 1997). In contrast, several researchers investigated the effects of saponification conditions on the determination of PCBs and reported that the decomposition of highly chlorinated congeners at high-temperature saponification was reduced by adding water to ethanolic KOH (Smedes and de Boer, 1997; Numata et al., 2005). In this study, the relatively high extraction efficiency of highly chlorinated congeners would also be considered due to using ethanolic KOH with the added water. Some previous studies have also shown that MAE is faster and a less time-consuming extraction method for the quantitative determination of PCBs in environmental samples. Vetter et al. examined the method of MAE for PCB analysis in seal blubber and cod livers (Vetter et al., 1998). They reported that good results were obtained using the microwave transformer Weflon due to the indirect rise of the temperature of the nonpolar solvent (n-hexane). Xiong et al. investigated the optimized conditions for MAE during the saponification process of biological tissues and the decomposition of OCPs with methanolic 1 M KOH (Xiong et al., 2000). However, further evaluation of the complex processes involved, such as the concentration of extracts, solvent substitution, and filtration after MAE, are required. In this study, the MAE method could be simultaneously carried out for both the saponification of adipose tissues and the extraction of PCBs Purification procedure of PCBs with SPD In order to evaluate the application of SPD in total PCB analysis, the PCB extracts obtained from five blubber samples using MAE were purified by SPD equipped with the purification and concentration columns mentioned above (Chapter 2.6), determined by HRGC-HRMS. The results were compared with those obtained by the multilayer silica gel column and HRGC-HRMS. Table 2 shows the PCB recoveries using SPD as a percentage against those obtained by the conventional method. In general, relatively high and stable recoveries of PCBs were observed in almost all the PCB congeners, indicating that SPD-600GC is an instrument delivering high performance. But no shown in Table 2, certain highly chlorinated PCB congeners (#188, #184, #202, #200, #197, #199, #208, #207, and #209) and lower chlorinated congeners showed relatively low recoveries, although their burdens were negligible with respect to the total PCB burden. It seems reasonable to con- Fig. 2. Chromatograms of individual purification procedures. (a) and (b) shows full scan total ion chromatograms by GC-LRMS treated a whale blubber by multilayer silica gel column and SPD, respectively. (c) Shows GC-ECD chromatogram prepared a whale blubber by SPD.

7 H. Fujita et al. / Chemosphere 74 (2009) clude that these highly chlorinated congeners were elute from the concentration column packed with c-alumina. However, it have been known previously that an activated c-alumina column have been eliminated interferences from large amount of saturated cyclic and aliphatic hydrocarbons (Smedes and de Boer, 1997; Aries et al., 2004; Fujita et al., 2005). Therefore, we examined the comparison of two purification procedure, silica gel column and SPD. Fig. 2 shows three chromatograms, Fig. 2a and b are total ions chromatograms by GC-LRMS (these vertical axis maximum scale are same.) of PCBs fractions from silica gel column and SPD, respectively. Both the vertical axis of chromatograms shows same scale. Fig. 2c is GC-ECD chromatograms of PCBs fraction treated whale blubber extracts by SPD. Although co-elution of PCBs with saturated hydrocarbons goes on using n-hexane from silica gel column, it indicate no strong retention occurs on SPD with an activated c- alumina for non-polarity of saturated hydrocarbons. Consequently, purification procedure for PCBs analysis of whale blubbers requires the additional clean up step to free interferences. This SPD equipped highest activated c-alumina effectively eliminated the interferences as saturated and aliphatic hydrocarbons etc., furthermore, is expected by reducing overload of the detector of analytical instrument Interferences as OCPs The concentrations of PCBs in the blubber samples pretreated using MAE and SPD were determined by GC-ECD, and their chromatograms are shown in Fig. 3a. Each peak on the GC-ECD chromatograms was identified from the retention times of the PCB standard as results reported later (Rogers et al., 2004). The chromatograms presented in Fig. 3a demonstrate a good peak separation for almost all the PCB congeners, indicating that the Hz HCH HCB -HCH -HCH -HCH Oxy-Chlordane trans-chlordane o,p -DDE cis-chlordane trans-nonachlor p,p -DDE o,p -DDD cis-nonachlor, p,p -DDD o,p -DDT p,p -DDT min Hz 450 HCB p,p -DDE o,p -DDE trans-chlordane cis-chlordane trans-nonachlor min Hz #90,101,113 #99 #119,150 #116,148,85,p,p -DDE #92, min Fig. 3. GC-ECD chromatograms: (a) OCPs standard solution, (b) OCPs standard solution prepared by SPD, and (c) the extract of the blubber prepared by MAE and SPD.

8 1076 H. Fujita et al. / Chemosphere 74 (2009) GC-ECD [ng/g] y = 1.10x R = HRGC-HRMS [ng/g] Fig. 4. Correlation between the total-pcb concentrations measured by GC-ECD and GC-HRMS. Table 3 Comparison in concentrations of PCBs in the SRM 1588b. GC-ECD determination of PCBs is certainly possible. However, peaks derived from coelutions with other PCB congeners or interferences were observed for several congeners (e.g., #92/84, #119/ 150 and 116/148/85). Generally, almost all the environmental samples were widely contaminated by the OCPs, which generally coexisted with PCBs in the extract solutions. As for the determination of PCBs by GC- ECD using DB-5 ms, some peaks of PCBs are known to overlap with OCP peaks (Vetter et al., 1994). Fig. 3 shows three chromatograms on same retention time: (a) the OCP standard solution consisting of the 16 congeners mentioned above; (b) the OCP standard solution treated by SPD; and (c) the blubber sample extract pretreated by MAE and SPD. The results imply that seven compounds, i.e., HCHs, trans-chlordane, o,p 0 -DDD, cis-nonachlor, p,p 0 -DDD, o,p 0 -DDT, and p,p 0 -DDT were completely removed from the OCP standard solution. In contrast, 10% of trans-chlordane, 6.5% of cis-chlordane, and 27% of trans-nonachlor remained in solution, and the compounds of o,p 0 -DDE and p,p 0 -DDE could not be removed almost at all. Nevertheless, the sum burden of the PCB congeners overlapping with these OCPs was only 1.2% of the total PCBs burden, indicat- Congeners (a) Certified or reference concentration (ng/g) MAE/SPD/GC-ECD (n = 5) Ave. (ng/g) SD (ng/g) RSD (%) PCB #18 (2,2 0,5-Trichlorobiphenyl) 7.96 ± 0.45(b) #12 (3,4-Dichlorobiphenyl) #13 (3,4 0 -Dichlorobiphenyl) PCB #28 (2,4,4 0 -Trichlorobiphenyl) 27.8 ± 1.4(b) #31 (2,4 0,5-Trichlorobiphenyl) 8.52 ± 0.50(b) PCB #52 (2,2 0,5,5 0 -Tetrachlorobiphenyl) 82.4 ± 1.7(b) #73 (2,3 0,5 0,6-Tetrachlorobiphenyl) PCB #49 (2,2 0,4,5 0 -Tetrachlorobiphenyl) 29.8 ± 0.6(b) PCB #44 (2,2 0,3,5 0 -Tetrachlorobiphenyl) 32.4 ± 2.7(b) i PCB #74 (2,4,4 0,5-Tetrachlorobiphenyl) 40 ± 11(c) PCB #66 (2,3 0,4,4 0 -Tetrachlorobiphenyl) 51.7 ± 7.5(b) i #95 (2,2 0,3,5 0,6-Pentachlorobiphenyl) 37.7 ± 8.2(b) PCB #101 (2,2 0,4,5,5 0 -Pentachlorobiphenyl) 127 ± 9(b) #90 (2,2 0,3,4 0,5-Pentachlorobiphenyl) PCB #99 (2,2 0,4,4 0,5-Pentachlorobiphenyl) 101 ± 12(b) PCB #87 (2,2 0,3,4,5 0 -Pentachlorobiphenyl) 54.6 ± 2.1(b) PCB #110 (2,3,3 0,4 0,6-Pentachlorobiphenyl) 69.3 ± 4.8(b) PCB #151 (2,2 0,3,5,5 0,6-Hexachlorobiphenyl) 48.1 ± 5.4(b) #82 (2,2 0,3,3 0,4-Pentachlorobiphenyl) 8.2 ± 1.6(c) PCB #149 (2,2 0,3,4 0,5 0,6-Hexachlorobiphenyl) 90 ± 11(b) #123 (2 0,3,4,4 0,5-Pentachlorobiphenyl) PCB #118 (2,3 0,4,4 0,5-Pentachlorobiphenyl) 172 ± 7(b) #140 (2,2 0,3,4,4 0,6 0 -Hexachlorobiphenyl) #139 (2,2 0,3,4,4 0,6-Hexachlorobiphenyl) PCB #153 (2,2 0,4,4 0,5,5 0 -Hexachlorobiphenyl) 275 ± 4(b) PCB #105 (2,3,3 0,4,4 0 -Pentachlorobiphenyl) 59.2 ± 1.2(b) PCB #138 (2,2 0,3,4,4 0,5 0 -Hexachlorobiphenyl) 212 ± 29(b) J # 163 (2,3,3 0,4 0,5,6-Hexachlorobiphenyl) 42.2 ± 3.2(c) #164 (2,3,3 0,4 0,5 0,6-Hexachlorobiphenyl) PCB #178 (2,2 0,3,3 0,5,5 0,6-Heptachlorobiphenyl) 28 ± 1(c) #129 (2,2 0,3,3 0,4,5-Hexachlorobiphenyl) PCB #187 (2,2 0,3,4 0,5,5 0,6-Heptachlorobiphenyl) 43.6 ± 7.2(b) #182 (2,2 0,3,4,4 0,5,6 0 -Heptachlorobiphenyl) PCB #183 (2,2 0,3,4,4 0,5 0,6-Heptachlorobiphenyl) 28.9 ± 1.8(b) #162 (2,3,3 0,4 0,5,5 0 -Hexachlorobiphenyl) PCB #128 (2,2 0,3,3 0,4,4 0 -Hexachlorobiphenyl) 39.9 ± 6.0(b) PCB #167 (2,3 0,4,4 0,5,5 0 -Hexachlorobiphenyl) 12.2 ± 0.8(c) PCB #177 (2,2 0,3,3 0,4 0,5,6-Heptachlorobiphenyl) 5.18 ± PCB #156 (2,3,3 0,4,4 0,5-Hexachlorobiphenyl) 18.0 ± 2.1(b) PCB #180 (2,2 0,3,4,4 0,5,5 0 -Heptachlorobiphenyl) 98.5 ± 6.3(b) PCB #170 (2,2 0,3,3 0,4,4 0,5-Heptachlorobiphenyl) 41.9 ± 3.8(b) J # 190 (2,3,3 0,4,4 0,5,6-Heptachlorobipheny l) PCB #194 (2,2 0,3,3 0,4,4 0,5,5 0 -Octachlorobiphenyl) 13.5 ± 1.2(b) PCB #206 (2,2 0,3,3 0,4,4 0,5,5 0,6-Nonachlorobiphenyl) 3.75 ± 0.07(b) (a) When two or more congeners are known to coelute using GC-ECD on DB-5 ms column, the PCB congener listed first is the major component, and the additional congeners may be present as minor components detected by GC-HRMS. (b) NIST-certified concentration values. (c) NIST-reference concentration values.

9 H. Fujita et al. / Chemosphere 74 (2009) ing that these PCB congeners were possibly negligible in the total PCB quantifications. Xiong et al. investigated the microwave-assisted decomposition of OCPs, a-hch, b-hch, d-hch, c-hch, p,p 0 -DDE, p,p 0 -DDD, o,p 0 - DDT, p,p 0 -DDT, aldrin, and dieldrin in mussel samples (Xiong et al., 2000). According to their results, a-hch, b-hch, d-hch, c- HCH, p,p 0 -DDD, o,p 0 -DDT, and p,p 0 -DDT could be transformed, but decomposition for 30 45% of p,p 0 -DDE, aldrin, and dieldrin did not occur. In addition, their results demonstrated that dieldrin and its decomposing products were perfectly eliminated from the crude extracts by rinsing with concentrated H 2 SO 4 (Carro et al., 2002). The purification technique using SPD combined with MAE can be used for the removal of OCPs from crude extracts. However, since several PCB congeners could not be determined the detailed constitution of PCB congeners needs to be clarified further Comparison of GC-ECD and HRGC-HRMS In order to confirm the specificity of MAE/SPD/GC-ECD determination, nine samples of whale blubber were selected, and their PCB concentrations were analyzed according to the following procedure. The solutions prepared by MAE and SPD were equally divided into two vials, and then their PCB concentrations were quantified by GC-ECD and HRGC-HRMS, respectively. The correlation between the concentrations of total PCBs obtained by the two detectors is shown in Fig. 4. The concentrations of the total PCBs by GC-ECD was approximately 1.1 times higher than those obtained by HRGC-HRMS; further, they were significantly correlated (correlation coefficient = 0.998, P < 0.05). The slightly high values obtained by GC-ECD compared with those obtained with HRGC-HRMS might be due to the overlap of OCPs such as persistent organic pollutants, polychlorinated naphtalenes, PCDD/DFs, etc., as mentioned in previous reports (Ong and Hites, 1994; Hong et al., 2004). However, the total PCBs values using GC-ECD increases nearly linearly with an increase in PCB concentrations of blubbers using HRGC-HRMS Accuracy and precision In order to evaluate the accuracy and precision of the new method that combines MAE, SPD, and GC-ECD, a repeated analysis (n = 5) of PCBs was examined in SRM 1588(b). Table 3 shows the concentrations of PCB congeners obtained by the repeated analysis with certified concentration values. Almost all the congeners examined showed a good agreement with the certified values, and their RSDs except for those of three congeners, i.e., #18, #183, and #206 ranged from 1% to 7%. However, compared with the certified values, relatively low values were found in #183/162 and #206; this might be due to their relatively low recoveries on the concentration column of SPD. Coelutions of PCB congeners were observed in two pairs, #28/#31 and #66/#95. However the sum of #28 and #31was good agreement with the sum of their certified values, and the sum of #65 and #95, too. Furthermore, the value of congener #87 obtained in this study showed a good agreement with the certified value, which is most likely a result of the removal of dieldrin which generally coelutes with the #87 congener as reported later (Lefkovitz et al., 2001). Next, the repeated analysis (n = 5) of PCBs using MAE, SPD, and GC-ECD was also performed for the sub-reference material. The standard deviations (SD) were calculated as the repeatability and the DL/QL of this method. Almost all the RSDs for the PCB congeners showed lower values as compared with the maximum allowable RSD (15%) that was expected by a previous report (Shah et al., 1992). The QL of the total PCB calculated from that of the individual PCB congeners was 41 ng g 1, and this value was less than one-tenth of the provisional limit, i.e., 500 ng g Conclusions The validation of an analytical method for PCBs using MAE, SPD, and GC-ECD was performed for blubber samples of minke whales. The results indicated that PCBs in whale blubber could be efficiently extracted with a small volume (20 ml) of n-hexane using MAE because of the simultaneous effects of saponification and extraction; further, its crude extract could be rapidly purified and automatically substituted to a small volume (1 ml) of toluene using SPD without using concentrators. 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