This article was downloaded by:[University of Santiago de Compostela] On: 21 July 2008 Access Details: [subscription number 789041077] Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Food Additives & Contaminants Part A - Chemistry, Analysis, Control, Exposure & Risk Assessment Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713599661 Optimization of microwave-assisted extraction with saponification (MAES) for the determination of polybrominated flame retardants in aquaculture samples N. M. Fajar a ; A. M. Carro a ; R. A. Lorenzo a ; F. Fernandez a ; R. Cela a a Qu mica Anal tica, Nutrici n y Bromatolog a, University of Santiago de Compostela, Santiago de Compostela, Spain First Published: August 2008 To cite this Article: Fajar, N. M., Carro, A. M., Lorenzo, R. A., Fernandez, F. and Cela, R. (2008) 'Optimization of microwave-assisted extraction with saponification (MAES) for the determination of polybrominated flame retardants in aquaculture samples', Food Additives & Contaminants, 25:8, 1015 — 1023 To link to this article: DOI: 10.1080/02652030801905435 URL: http://dx.doi.org/10.1080/02652030801905435 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
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This article was downloaded by:[University of Santiago de Compostela]On: 21 July 2008Access Details: [subscription number 789041077]Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Food Additives & ContaminantsPart A - Chemistry, Analysis, Control, Exposure& Risk AssessmentPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713599661
Optimization of microwave-assisted extraction withsaponification (MAES) for the determination ofpolybrominated flame retardants in aquaculture samplesN. M. Fajar a; A. M. Carro a; R. A. Lorenzo a; F. Fernandez a; R. Cela a
a Qu mica Anal tica, Nutrici n y Bromatolog a, University of Santiago deCompostela, Santiago de Compostela, Spain
First Published: August 2008
To cite this Article: Fajar, N. M., Carro, A. M., Lorenzo, R. A., Fernandez, F. and Cela, R. (2008) 'Optimization ofmicrowave-assisted extraction with saponification (MAES) for the determination of polybrominated flame retardants inaquaculture samples', Food Additives & Contaminants, 25:8, 1015 — 1023
To link to this article: DOI: 10.1080/02652030801905435URL: http://dx.doi.org/10.1080/02652030801905435
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf
This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction,re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expresslyforbidden.
The publisher does not give any warranty express or implied or make any representation that the contents will becomplete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should beindependently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with orarising out of the use of this material.
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Food Additives and ContaminantsVol. 25, No. 8, August 2008, 1015–1023
Optimization of microwave-assisted extraction with saponification (MAES) for thedetermination of polybrominated flame retardants in aquaculture samples
N.M. Fajar, A.M. Carro*, R.A. Lorenzo, F. Fernandez and R. Cela
Quımica Analıtica, Nutricion y Bromatologıa, University of Santiago de Compostela, Santiago de Compostela, Spain
(Received 10 October 2007; final version received 9 January 2008)
The efficiency of microwave-assisted extraction with saponification (MAES) for the determination of sevenpolybrominated flame retardants (polybrominated biphenyls, PBBs; and polybrominated diphenyl ethers,PBDEs) in aquaculture samples is described and compared with microwave-assisted extraction (MAE).Chemometric techniques based on experimental designs and desirability functions were used for simultaneousoptimization of the operational parameters used in both MAES and MAE processes. Application of MAES tothis group of contaminants in aquaculture samples, which had not been previously applied to this type ofanalytes, was shown to be superior to MAE in terms of extraction efficiency, extraction time and lipid contentextracted from complex matrices (0.7% as against 18.0% for MAE extracts). PBBs and PBDEs were determinedby gas chromatography with micro-electron capture detection (GC-mECD). The quantification limits for theanalytes were 40–750 pg g�1 (except for BB-15, which was 1.43 ng g�1). Precision for MAES-GC-mECD(%RSD511%) was significantly better than for MAE-GC-mECD (%RSD520%). The accuracy of bothoptimized methods was satisfactorily demonstrated by analysis of appropriate certified reference material(CRM), WMF-01.
microwave-assisted decomposition (MAD) combinedwith MAS or MAE to eliminate organochloridepesticides from sediments and mussels; and simulta-neous MAE of PAHs from fish samples and hydrolysisof lipids with KOH (Xiong et al. 2000; Zhang et al.2000; Hernandez-Borges et al. 2006; Pena et al. 2006).An MAE procedure that includes gentle saponificationenabled shortening the analysis time and avoiding thealkaline decomposition of organochlorine compoundsin oyster samples (Carro et al. 2002).
The aim of the present study was to develop a newmethod to extract PBBs and PBDEs from feedintended for farmed fish, and from scallops, clamsand mussels using microwave energy combined withsaponification (MAES) and to compare its efficiencywith MAE. The results showed that MAES does notneed additional clean-up steps because the lipidcontent of the extracts is lowered by a factor of 26 ascompared with MAE. Brominated compounds weredetermined by GC-�ECD due to high sensitivity ofthis detector to molecules that contains electronegativeatoms. To demonstrate the accuracy of the method, acertified reference material (WMF-01) was analysedapplying MAES and MAE and the values of PBDEsconcentrations were compared (Stapleton et al. 2007).
Materials and methods
Chemicals
Sulphuric acid (99%), silica gel (60 A pore size,0.040–0.063mm, 230–400 mesh), high-performanceliquid chromatography (HPLC)-grade methanol andethanol (99.9%) were from Merck (Darmstadt,Germany). Anhydrous sodium sulphate (99%) wasfrom BDH (Poole, UK). A mixture of PBDEs(10 mgml�1) in cyclohexane: BDE-47 (2,20,4,40-tetra-bromodiphenyl ether, 42.5%), BDE-99 (2,20,4,40,5-pentabromodiphenyl ether, 39.3%), BDE-100(2,20,4,40,6-pentabromodiphenyl ether, 10.9%), BDE-153 (2,20,4,40,5,50-hexabromodiphenyl ether, 1.9%),BDE-154 (2,20,4,40,5,60-hexabromodiphenyl ether,2.7%) was supplied by Dr. Ehrenstorfer (Augsburg,Germany). BB-15 (4,40-dibromobiphenyl, 99.8%) andBB-49 (2,20,4,50-tetrabromobiphenyl, 97%) were pur-chased as solids from Supelco (Bellefonte, PA, USA).A stock PBBs standard solution (400 ngml�1) was usedto prepare the working solutions by dilution, except forthe PBDEs (1400 ngml�1), which were used at thestarting concentration.
Samples
Feed for turbot and trout and samples of scallop, clamand mussel were obtained from cultivation areas on theGalician coast, Spain. The samples were triturated andhomogenized in a grinder before processing.
Optimization experiments were carried out on a
pooled (composite) sample of a 50 g homogenate ofsmall trout feed spiked with 100ml of n-hexanecontaining 1200 ml of the mixture of PBDEs at10 mgml�1, 105 ml of BB-15 solution at 22.0 mgml�1,and 150 ml of BB-49 solution at 14.2 mgml�1. The
n-hexane was removed by evaporation to air-dryness.In preliminary experiments, samples of 0.5, 1.0 and1.5 g were taken to evaluate the homogeneity. Relativestandard deviations lower than 10% for all the studiedcompounds were obtained when the amount of sampleused for analysis was above 1 g, and, therefore, a
minimum sample size of 1 g was used for subsequentexperiments. Then samples were stored at roomtemperature in glass bottles out of light exposureuntil the analysis.
A certified reference material (CRM) WMF-01,which contains organic contaminants in fish tissue, wasobtained from Wellington Laboratories (Guelph, ON,Canada).
MAES and MAE conditions: experimental design
Microwave-assisted extraction was carried out in aMillestone Ethos oven (Bergamo, Italy). The natureand volume of solvent (hexane 15ml mixed with 1mlwater) and the power (400W) were fixed according theresults of a previous study about the determination of15 representative organochlorine pesticides, polychlori-
nated biphenyls, PBBs and PBDEs (Carro et al. 2007).The other experimental conditions were established onthe basis of the results of the optimization studies.
The use of chemometric tools such as experimentaldesign improves yields at all stages of the analyticalprocess, and good-quality data can be obtained withminimal experimental effort (Hernandez-Borges et al.
2006; Pena et al. 2006; Serodio et al. 2007). We used acentral composite design of a spherical domain with�¼ 1.682 for MAES optimization. The design con-sisted of three factors with five levels: extractiontemperature (50, 65, 85, 105 and 120�C), extractiontime (1, 3, 6, 9 and 11min) and volume of solution of
1M KOH in methanol (5, 9, 15, 21 and 25ml). Theproposed experimental design (14 experiments plusthree central points) is shown in Table 1. For MAEoptimization, five levels for extraction temperature(25, 40, 65, 90 and 105�C) and four levels for extractiontime (3, 6, 7 and 9min) were selected using a
pentagonal design with three central points. In bothcases, the mathematical models were constructedconsidering the percentage recovery of each compoundstudied as the response. The experimental plan and theresponses obtained are shown in Table 1.
These experimental designs led to response surfacesthat served for a first graphical approach to optimiza-tion. Moreover, the use of multi-criteria optimization
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based on the construction of a desirability function foreach individual response enabled the identification ofthe best operational conditions to simultaneous extrac-tion of PBBs and PBDEs using MAES or MAE (Lewiset al. 1999). Each individual desirability function waschosen from a family of linear or exponentialcontinuous functions, and varied from zero (undesir-able response) to 100 (optimal response). The overalldesirability function was estimated as the geometricaverage of the individual desirability functions.NEMROD�W software was used for the generationand the evaluation of the experimental designs(Mathieu et al. 2000). This software incorporates analgorithm in the optimization process to find themaximum of the overall desirability function corre-sponding to the optimal experimental conditions.
Clean-up of extracts
The extracts produced by MAES or MAE weresubjected to clean-up to avoid interferences and toprotect the chromatographic columns from damage.Among the several clean-up procedures developed fororganohalogenated pollutants extracts, those based onsolid-phase extraction (SPE) are the most convenient
in practice (Gomara et al. 2006; Karlsson et al. 2006;Carro et al. 2007). In this case, 3 g of acid silica gelwere used to guarantee lipid removal. Acidic silicagel was prepared adding 4 g of H2SO4 to 6 g of silica gel(Rodil et al. 2007). With the optimized parameters, thelipid content of the extracts after SPE was less than0.06% and 0.04% of the original lipid content asmeasured gravimetrically when MAE (18%) or MAES(0.7%) were respectively applied. The extract wasconcentrated to 0.5ml in a TurboVap II Station(Zymark, Hopkinton, MA, USA) and finally driedin a nitrogen blow down concentrator. Thecomplete schemes for MAES and MAE are shown inFigure 1.
Chromatographic conditions
The clean extracts were analysed by GC-mECD on anAgilent Technologies 6980N (Avondale, PA, USA) gaschromatograph equipped with a microelectron-capturedetector. Gas chromatography was carried out on a30m� 0.32mm i.d. HP-5 (5% phenyl-methylpolysi-loxane) (Agilent Technologies) fused silica column(0.25 mm film thickness). Helium (purity 99.999%)(Carburos Metalicos, A Coruna, Spain) was used as
Table 1. Experimental plans and responses obtained (percentage recovery) in each experiment.
the carrier gas at a constant pressure of 13 psi. The
initial temperature was 60�C, held for 2min, then
ramped at 30�Cmin�1 up to 170�C, and held for 2min,
followed by a second ramp of 12�Cmin�1 up to 280�Cand held for 10min. The injector, operated in splitless
mode for 1min, and detector temperatures were fixed
at 250 and 285�C, respectively.
Results and discussion
Optimization of extraction procedures
MAES
The results of the central composite design (Table 1)
indicated that low values of the three factors have a
negative effect on extraction yield. The only factor with
a direct statistically significant effect for all regression
models, except those for BDE-153 and BDE-154, was
the volume of KOH solution. Significant interactions
between the main factors were also observed. Thus, amulti-criteria methodology that incorporates certaincompromise experimental conditions to fulfil theexpectations of the analyst was required (Yusa et al.2006a, b; Carro et al. 2007).
Individual desirability functions were defined foreach analyte and these individual functions werecombined to an overall desirability function thatreflects the success in extracting the seven compoundsfrom samples. In this case, non-linear partial desir-ability functions were chosen for the responses to bemaximized. Values below 50% were considered notacceptable (zero desirability), whereas values above70% were considered optimal, although the achieve-ment of greater recoveries by using these functions canbe expected. The maximum of the overall desirabilityfunction corresponded to 65�C and 3min of extractiontemperature and time, and 9ml of 1M KOH inmethanol, and the predicted percentage recoveries at
25 min of cooling
Elution with 15 mL of hexane
MAES
1g feed sample + 15 mL hexane + 9 mL methanolic 2M KOH, 3 min,
65°C, stirring 200 W
MAE
1g feed sample + 15 mL hexane + 1 mL water, 9 min, 75°C, stirring
200 W
Centrifuge 5 min, 3000 rpm
Clean-up on SPE column, (3g acidic silica)
Concentrate organic phase 0.5 mL in TurboVap10 psi
Concentrate to dryness in MiniVap
Reconstruct with 200 μL of hexane
Inject 1 μL in GC/mECD
Figure 1. Scheme of sample preparation for MAES and MAE procedures.
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this point ranged from 72 to 84%. Under theseexperimental conditions the constraints imposed onthe individual desirability functions are totally fulfilledand an overall desirability rate of 80% was reached.The global desirability was very sensitive to smallchanges in the analysed factors, as can be seen fromcontour plots of isodesirability curves (Figure 2a–c).Grey regions represent null desirability and, thus,unsuitable experimental conditions.
Although the saponification processes reported inthe literature generally use a solution of 1M KOH(methanolic or ethanolic) (Llompart et al. 2001; Ohtaet al. 2002; Wittmann et al. 2003), we studied the effectof KOH concentration in the MAES of the sevenanalytes of interest. Thus, under the optimal conditions
we found for MAES, experiments (n¼ 4) were carried
out with 1M, 2M and a saturated solution of KOH.
The best recoveries were obtained with 2M KOH and
with the saturated solution. Since the lowest relative
standard deviations (RSDs) were observed for
2M KOH, this solution was chosen for further
experiments.
MAE
The results of the pentagonal design (Table 1) showed
that high values of both factors have a positive effect in
the extraction yields, but only the extraction time had a
statistically significant effect for BDE-153. According
to the information reported by the design (Table 1), the
Figure 2. Contour plot of the overall desirability function in the space of the MAES/MAE factors: extraction time andtemperature for MAES (a), KOH volume and extraction temperature for MAES (b), KOH volume and extraction time forMAES (c), and extraction time and temperature for MAE (d). Grey zones correspond to zero desirability and contour lines 0.20,0.40, 0.60, 0.80 and 1.00 to 20, 40, 60, 80 and 100% of the overall desirability function.
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experiment run 1 (75�C and 9min) provides the best
extraction yields for PBBs and PBDEs. Non-linear
partial desirability functions were easily built support-
ing these results. Figure 2d shows the two-dimensional
plot of this overall desirability function in the
experimental domain studied; grey zones correspond-
ing to zero desirability. The maximum in the desir-
ability surface appears at 75�C and 9min of extraction.
Since this optimum is at the upper limit of the design,
some additional experiments with the extraction time
extended to 12 and 15min were made. No better
recoveries were achieved, proving that 9min is indeed a
global maximum.
Performance evaluation of the analytical method
To the best of our knowledge, there are no reports on
the application of MAES combined with GC-mECD to
the analysis of PBBs and PBDEs in aquaculture
samples. The performance of the method was tested
using spiked trout feed samples. The retention times,
linear ranges, coefficients of determination (R2) of
calibration lines, and limits of detection and quantifi-
cation are listed in Table 2. Good correlations were
obtained within the intervals studied, with coefficients
of determination equal to or above 0.997 (with the
exceptions of BDE-153 and BDE-154). The limits of
detection (LODs), defined as the minimum amount of
analyte that produces a peak with a signal-to-noise
ratio of 3, were below 220 pg g�1 except for BB-15
(430 pg g�1). The limits of quantification (LOQs)
were below 750 pg g�1 except for BB-15 (1.43 ng g�1)
(Table 2). These detection limits were lower than
most of previously reported for biological samples
applying other analytical procedures (Bayen et al.
2004; Karlsson et al. 2006; Naert and Van
Peteghem 2007).Analytical quality parameters of the proposed
MAES were assessed with spiked trout feed.
Unspiked trout feed samples were previously analysed
in triplicate for the compounds of interest and none of
the investigated analytes were detected. Typical chro-
matograms obtained for small trout feed with and
without spiking, CRM WMF-01 and a standard
solution (200 ngml�1 of BB-15 and BB-49, respec-
tively, and 700 ngml�1 of the total penta-BDE
standard) are shown in Figure 3.
Comparison between MAES and MAE techniques
To evaluate the applicability of the proposed MAES
method for determining PBBs and PBDEs in
aquaculture samples, the accuracy (percentage recov-
ery) and the precision (percentage RSD) of the
results were compared with those obtained when
MAE was used. The comparative study was made
using spiked small trout feed samples. Six indepen-
dent extractions were performed to evaluate the
reproducibility of the experimentally optimized
extraction methods. The right panel of Table 2
shows the average recoveries and precision obtained
for each analyte after applying MAES or MAE. The
recovery and the repeatability were improved by the
use of MAES owing to its higher efficiency to
destroy lipids (Table 2).CRM WMF-01, which consists of fish tissue
containing organic contaminants, was also used to
compare the results obtained by MAES and MAE in
terms of accuracy and precision. Figure 4 shows the
certified most probable values and 95% confidence
intervals for the concentrations of analytes in CRM
WMF-01, together with corresponding data obtained
by MAES-GC-�ECD and MAE-GC-�ECD (six
replicates). The average concentrations obtained
using MAES was within the certified 95% confidence
intervals for all analytes. By contrast, when MAE was
used, the concentrations of BDE-99, BDE-153 and
BDE-154 were below the lower limit of the interval.
The multifactor analysis of variance (ANOVA) was
performed to compare the results obtained with the
two extraction techniques. No significant effect of
the extraction procedure (p¼ 0.4415) was found for the
Table 2. Optimization of the chromatographic analysis of spiked small trout feed: comparative results of MAES and MAE foraccuracy and precision studies.
Performance of MAES-GC-mECD method MAES-GC-mECD (n¼ 6) MAE-GC-mECD (n¼ 6)
recovery data in the CRM sample. Significant effects
of the factor analyte (different PBDEs) (p¼ 0.0000)
and of the interaction extraction technique� analyte
(p¼ 0.0004) were detected. The conclusion is that the
global behaviour of MAES and MAE procedures has
been similar and CRM WMF-01 has been successfully
used to validate MAES/MAE-GC-mECD. However,
the results obtained for each analysed pollutant has
been statistically different depending of the extractiontechnique selected.
Application of the method to real-world samples
Apart of CRM WMF-01, the reliability of the MAESproposed method was checked by analysing in quad-ruplicate, real aquaculture samples including (feed for
min12 13 14 15 18 19
Area response
170
0
1000020000300004000050000
010000200003000040000
50000
01000020000300004000050000
10000200003000040000
50000
BB-15
BDE-154 BDE-153BDE-100
BDE-99BDE-47BB-49
(A)
Area response
min13 14 15 16 17 18 19
BB-49
12
BB-15
BDE-99
BDE-153BDE-154BDE-100
(B)
(C)
(D)
16
BDE-47
Area response
12 13 14 15 16 17 18 19 min
Area response
BDE-47
BDE-100 BDE-99 BDE-154
BDE-153
min12 13 14 15 16 17 18 19
Figure 3. Chromatograms of a standard solution (200 ngml�1 of BB-15 and BB-49, respectively, and 700 ngml�1 of the totalpentaBDE standard) (A), MAES extract from spiked small trout feed (B), non-spiked small trout feed extract obtained byMAES procedures (C), and MAES extract from the certified reference material WMF-01 (D).
0
20
40
60
80
100
120
140
160
BDE-47 BDE-100 BDE-99 BDE-154 BDE-153
Con
cent
ratio
n (n
g g−1
)
MAEMAESWMF-01
Figure 4. Comparative study of the accuracy of extraction of certified reference material WMF-01 by MAES and MAE (n¼ 6)and quantification by GC-mECD.
Food Additives and Contaminants 1021
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small and big trout and turbot, scallop, clam andmussels) without spiking (Table 3). In order to confirmthe results obtained by GC-mECD, a selective gaschromatography in combination with tandem massspectrometry (GC-MS/MS) method was used (Carroet al. 2007). BB-49 in scallop and turbot feed samplesand BDE-47 in turbot feed samples were quantifiedby GC-mECD, presenting amounts of 1.48� 0.1,1.23� 0.01 and 1.57� 0.07 ng g�1, respectively, whichwere quantified by GC-MS/MS (0.5 ng g�1 for LOQ ofBB-49 and 0.8 and 2.5 ng g�1 for LOD and LOQ ofBDE-47, respectively).
Conclusions
Both optimizedMAES andMAE processes can be usedfor the determination of PBBs and PBDEs in aqua-culture samples including fish feed. The results showedthat MAES does not need additional clean-up stepsbecause the lipid content of the obtained extracts islowered by a factor of 26 as compared with MAE. Bothprocedures have comparable reproducibility, but theMAES procedure provided more accurate results foraquaculture feed and certified reference material. Theimprovement is particularly significant for the heavierPBDEs compounds. The simple, quick and efficientMAES-GC-mECD method that was optimized in thisstudy would be appropriate for the quantification ofPBBs and PBDEs far below the currently concentrationlevels in contaminated aquaculture samples.
Acknowledgements
The authors wish to thank the Galician Government,Xunta de Galicia, for financial support (ProjectNo. PGIDIT06PXIB237039PR).
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