AUTOMATED CLEAN UP METHOD FOR THE ...

AUTOMATED CLEAN UP METHOD FOR THE DETERMINATION OF PCBs AND PBDEs IN FISH. G. Mariani1 , Manni A.2, H. Skejo1 and G. Umlauf1 1.

European Commission – DG Joint Research Centre, Institute for Environment and Sustainability. Via E. Fermi 2749, 21027 Ispra (VA), Italy

2

Chemical, Materials and Environmental Engineering Dept., University of Rome “La Sapienza”, Via Eudossiana, 18, 00184 Rome, Italy

Introduction: Organohalogen compounds such as certain organochlorinated Pesticides (OCPs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and brominated diphenylethers (PBDEs) are toxic and persistent and have been detected in various environmental compartments. All these compounds fall under or are candidate to the Stockholm Convention and they are subject to United Nations Environment Programme (UNEP) monitoring programmes1. In the past, different complex automated clean-up systems have been developed and dedicated to the analysis of dioxins in environmental samples. Later these methodologies have been adapted to include other conventional or emerging persistent organic compounds such as PCBs and PBDEs2, 3, 4. These methodologies involve different types of adsorption media such as acidic silica/silica, alumina and active carbon and are useful if multiresidual analysis with PCDD/Fs analysis in a final step is applied. Often the monitoring of these chemicals do not include all these compounds, but is limited to only a few classes excluding the PCDD/Fs, so that a reduced number of clean up steps are needed and time and cost expenses reduced.. In this study we tested the flexibility of an automated clean-up system used for the multiresidual analysis5 developing a shorter and simpler automated method for the simultaneous analysis of PCBs and PBDEs in fish. For this purpose we reduced the clean up steps to GPC followed by acidic silica/silica columns. The automated clean up system (J2 Scientific, Missouri, USA) consists of a gel permeation chromatography (GPC) with autosampler (AccuPrep MPSTM), an evaporator system (AccuVapTM) and three solid phase module able to manage the acid silica/neutral silica, basic alumina and active carbon columns (Dioxin/SPE module) in-line. The system processes the samples in sequence (see Figure).

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The system was tested with natural fish certified reference material EDF-2525 (CIL, Andover MA).

Material and Methods Analytical determinations: 10g of reference fish sample EDF-2525 were lyophilized and submitted to the extraction. The material was processed in quadruplicate. Analytical blank were analysed in parallel. The extraction was carried out by Soxhlet for 24h with a mixture of acetone/n-hexane 1/1 after spiking with 13 C-labelled internal standards (12 Dioxin Like and 7 indicators PCB and in accordance with IUPAC nomenclature: BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183; BDE-197, BDE-207 and BDE-209 ) The extract was dried under nitrogen flow and the lipid content was diluted to 5ml with a mixture of cyclohexane/ethyl acetate 1/1 and injected into a 5ml loop of automated GPC system. The GPC column was 2.5cm x 32 cm filled with BioBeads SX-3 resin working at a flow rate of 5ml/min using cyclohexane/ethyl acetate 1/1. The eluate was collected between 23:30 and 45 min (107 ml). The sample collected was concentrated under nitrogen flow to 0.5ml and then diluted with n-hexane to 5ml. These 5ml were loaded automatically on the acid silica/neutral silica column and eluted with 75ml of nhexane at a flow rate of 6ml/min. This fraction was collected, concentrated to 100μl, spiked with 13Clabelled recovery standards (PCB-111, PCB-170, BDE-126 and BDE-206) and submitted to instrumental analysis. Instrumental analysis: The instrumental analysis of PBDEs and PCBs were based on isotope dilution using HRGC-HRMS (high resolution gas chromatography – high resolution mass spectrometry) for quantification on the basis of EPA16146 and EPA 16687. PBDEs were analyzed on double HRGC (Thermo Trace GC Ultra, Thermo Electron, Bremen, Germany), were coupled with a DFS high resolution mass spectrometer HRMS (Thermo Electron, Bremen, Germany) operating in the EI-mode at 45 eV with a resolution of >10000. For tri- to octa-brominated congeners two ions of the isotopic molecular cluster were recorded, for nonaand deca-brominated congeners two isotopic ions of the cluster M+-2Br were recorded for both native and labeled congeners. The quantified isomers were identified through comparison of retention times of the corresponding standard and the isotopic ratio of the two ions recorded. PBDEs were analyzed on a Sol-Gel-1ms capillary column, 15 m long with 0.25 mm i.d. and 0.1 µm film (SGE, Victoria, Australia). The following gas-chromatographic conditions were applied: PTV injector with temperature program from 110 to 300 °C at 14.5 °C sec-1, constant flow at 1.0 ml min-1 of He, GC-MS interface at 300 °C and a GC program rate: 110 °C with a 1 min. hold, then 20 °C min-1 to 300 °C and a final hold at 300 °C for 6 min. Mono-ortho PCBs and Indicator-PCBs were analyzed on a GC (HP-6890, Hewlett Packard, Waldbronn, Germany) coupled with a VG Autospec Ultima high resolution mass spectrometer (Micromass, Manchester, UK) operating in EI-mode at 36 eV with a resolution of >10000. PCBs were separated on HT-8 capillary column, 60 m long with 0.25 mm i.d.(inner diameter) and 0.25 µm film (SGE, Victoria, Australia). Gas chromatographic conditions for PCBs were: Split/splitless injector at 280 °C, constant flow at 1.5 ml min-1 of He, GC-MS interface at 280 °C and a GC program rate: Starting from 120 °C with 20 °C min-1 to 180 °C, 2 °C min-1 to 260 °C, and 5 °C min-1 to 300 °C isotherm for 4 min.

Results and Discussion: The levels of the analytical blanks obtained during the clean-up process were from 10 to 100 times lower of the reported concentrations for all compounds studied. The blank level was not subtracted. Table 1 displays the PCBs average results obtained for 4 parallel samples and the reference values of the certified sample EDF-2525. The ratio between the average concentration detected and the reference value ranged from a minimum of 0.8 to a maximum of 1.2 and the RSD were all under 8%. The recoveries were all in the interval of the 5060% with the exceptions of congeners PCB-28 and PCB-52 that were 30 and 33 % respectively, probably lost during the concentration steps due at their higher volatility. Table 2 shows the average concentrations of 21 PBDEs congeners obtained for 4 parallel samples and the reference values of certified sample EDF-2525.

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For 10 of the PBDEs congeners certified values were available. The ratio between the average concentration detected and the reference value ranged from a minimum of 0.8 to a maximum of 1.09 with the exception of BDE-209 with a ratio of 0.43, where a large interval of confidence was certified (545 ± 1999 pg/g). However, the average concentration of BDE-209 we measured (233 pg/g) was within the range of the CRM. The RSD for the congeners quantified with the respective labeled internal standards was below 11% for the others ranged from a minimum of 1% for BDE-49 to the maximum of 28% for BDE203. The recoveries were all in the interval of the 50-60% with the exceptions of congeners BDE-209 that was 40%.

Conclusions -

The system assembled with GPC and 3 three solid phase module used for the adsorption chromatography with acid silica/silica allows to process up to 27 samples in one sequence. The method has demonstrated good accuracy and repeatability both PCBs and PBDEs. The software and hardware of the automated clean up system allowed easily to modify or to create a new method suitable at our purpose without any modification of the instrument.

References: 1 - http://www.chem.unep.ch/pops/ 2 -Abad E., Saulo J., Caixach J. and Rivera J. (2000) J. Chromatography A 893: 383-391 3 - Pirard, C., De Pauw, E., Focant, J.F. (2003). J. Chromatogr. A 998: 169-181. 4 - Thomsen, C., Nicolaysen, T., Broadwell, S.L., Huag, L.S., Becher, G. (2004) Organohalogen Compd. 66: 145-152. 5 - Mariani G., Amalfitano L., Manni A, Mueller A., Skejo H., Umlauf G. (2009) Organohalogen Compd. 71: 2796-2801. 6 - U.S. EPA., 2003. Method 1614: 7 - U.S. EPA., 1999. Method 1668, revision A:

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Table 1: Concentrations of PCBs in Fish Reference Material EDF-2525 Standard Deviation (n=4)

Reference Value ± 2 St. Dev.

Average Result JRC / Reference Value Ratio

Analyte

Average Results (n=4)

Non Ortho - PCBs

pg/g

PeCB-105 PeCB-114 PeCB-118 PeCB-123 HxCB-156 HxCB-157 HxCB-167 HpCB-189

46006 3137 107199 2742 11847 2628 6642 1010

1606 112 2548 195 503 156 276 52

3 4 2 7 4 6 4 5

50100 ± 15700 3410 ± 1550 122000 ± 38000 3280 ± 2020 13100 ± 2620 3380 ± 1010 7060 ± 3020 1440 ± 498

0.9 0.9 0.9 0.8 0.9 0.8 0.9 0.7

8428 29338 83410 164873 259015 110066

274 949 4037 5124 14882 6336

3 3 5 3 6 6

7100 ± 1260 27100 ± 12100 82700 ± 21400 178000 ± 27800 226000 ± 71200 108000 ± 23600

1.2 1.1 1.0 0.9 1.1 1.0

RSD %

pg/g

Indicator-PCBs TriCB-28 TeCB-52 PeCB-101 HxCB-138 HxCB-153 HpCB-180

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Table 2: Concentrations of PBDEs in Fish Reference Material EDF-2525

Analyte

Average Results (n=4)

Standard Deviation (n=4)

RSD %

pg/g BDE-17 BDE-28 BDE-47 BDE-49 BDE-66 BDE-71 BDE-77 BDE-85 BDE-99 BDE-100 BDE-119 BDE-153 BDE-154 BDE-183 BDE-184 BDE-196 BDE-197 BDE-203 BDE-206 BDE-207 BDE-209

Organohalogen Compounds

17.0 339 7305 577 311 96.4 20.4 9.97 2311 1825 418 2114 2528 133 14.9 4.44 11.6 54.8 32.7 25.6 233

Reference Value ± 2 St. Dev.

Average Result JRC/Reference Value Ratio

pg/g 2.50 24.6 249 8.50 16.5 9.97 2.32 2.45 122 24.6 4.81 93.8 64.3 8.53 2.29 0.65 1.58 15.2 7.05 2.08 26.4

15 7 3 1 5 10 11 25 5 1 1 4 3 6 15 15 14 28 22 8 11

312 ± 202 9080 ± 2620 524 ± 274 262 ± 81

1.09 0.80 1.10 1.19

2280 ± 472 1720 ± 566

1.01 1.06

2030 ± 506 2550 ± 1000 137 ± 47.8

1.04 0.99 0.97

545 ± 1999

0.43

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