Food Additives and Contaminants, May 2005; 22(5): 482–489
Application of accelerated solvent extraction followed by gel performance chromatography and high-performance liquid chromatography for the determination of polycyclic aromatic hydrocarbons in mussel tissue ` 1, O. PARDO1, P. MARTI´1, & A. PASTOR2 V. YUSA 1
Public Health Laboratory of Valencia. Generalitat Valenciana, C/Micer Masco´ 31, 46010-Valencia, Spain, and Analytical Chemistry Department, University of Valencia, Spain
2
(Received 29 July 2004 ; revised 1 February 2005; accepted 18 February 2005)
Abstract Accelerated solvent extraction (ASE) has been evaluated as a fast alternative to methanolic saponification for the extraction of 12 polycyclic aromatic hydrocarbons (PAHs) from mussel tissue. Several solvent systems and different operating conditions were investigated. The mixture dichloromethane-acetone (1:1, v/v) gave the best recoveries at 125 C and 1500 psi, in a total time of 10 min. No yield difference was found between freeze-drying (Fd) or drying the wet mussel with diatomaceous earth (Ded) prior to extraction. The ASE method was validated using the standard reference material SRM 2977, a freeze-dried mussel tissue with naturally present organic contaminants. The performance characteristics of the ASE method (trueness: 70–110%; precision: 4–14% and limit of quantification (LOQ): 0.1–0.25 mg/kg) meet the criteria established by the European Union for quantitative methods of analysis for official control of organic residues and contaminants. ASE provides a 24 times faster extraction than MSE and reduces 12 times the volume of solvent required.
Keywords: Accelerated solvent extraction, ASE, polycyclic aromatic hydrocarbons, mussel, gel performance chromatography
Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. PAHs are formed by the incomplete combustion of organic matter and are generated whenever fossil fuels or vegetation are burned (Moffat et al. 1999). PAHs containing four fused rings, such as benzo[a]anthracene and chrysene, are weakly carcinogenic. Many of the larger PAHs such as dibenzo[a,h]anthracene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene are known to have the potential to produce potent carcinogens (Nisbet and La Goy 1992; Law 2002; European Commission 2002a). The contamination of the atmosphere with PAHs results in the compounds becoming deposited in the aquatic environment. Sources of contamination can also include oil spills and run-off from land of industrial effluent. PAHs become concentrated in marine sediments which constitute a potential pollution reservoir for PAHs release under specific conditions. Bottom-feeding fish and filter-feeding
invertebrates are the most susceptible species to contamination. Most organisms have a high bio-transformation potential for PAHs having as a result no significant bio-magnification in the aquatic food chain. However, filter-feeding bivalves (e.g., mussels and oysters) filter large volumes of water and have a low metabolic capacity for PAH, so the compounds tend to persist more in these invertebrates (Bolger et al. 1996). In general, PAH analysis of fish and shellfish involves alcoholic saponification, several liquid/ liquid extractions and column clean up procedures. In some cases direct extraction by Soxlhet or sonication is used. The most frequently used techniques for further clean-up are adsorption chromatography (on silica gel, alumina or florisil) and gel permeation chromatography. Some methods also include partitioning of PAHs between polar aprotic solvent (e.g., dimethyl formamide (DMF), dimethyl sulphoxide (DMSO) and nonpolar extraction solvents. A general view of analytical procedures used for PAHs determination
Correspondence: V. Yusa`. Email:
[email protected] ISSN 0265–203X print/ISSN 1464–5122 online ß 2005 Taylor & Francis Group Ltd DOI: 10.1080/02652030500077452
Polycyclic aromatic hydrocarbons in mussel tissue in fish and shellfish is summarized in Table I. These routine methods are time and solvent consuming. Accelerated solvent extraction utilizes organic solvents under elevated temperature (50–200 C) and pressures (1000–2000 psi) to extract organic pollutants from environmental matrices (Fitzpatrick et al. 2000; Dean 1998). This technique increases the speed of the extraction process with low solvent consumption. The main parameters for method optimization are solvent, temperature and time. Pressure, between the operating values (1000–2000 psi), is not considered a critical experimental parameter (Dionex 2000). Various authors have studied the effects of these variables on the performance of ASE and the recoveries of PAHs from environmental matrices (Popp et al. 1997; Saim et al. 1998; Heemken et al. 1997; Richter 1996; Schantz et al. 1997b). However, less attention has being paid to the ASE extraction of PAHs from biological matrices (fish and mussel tissue) and foodstuffs. This paper describes applications of the ASE for the determination of PAHs in fish and shellfish. A number of investigations were performed to optimize the procedure. Various solvents or solvent mixtures were investigated. The dependence of the extraction yield from different parameters such as the static extraction time, static cycles, the oven temperature, was studied. The influence of freezedrying the sample prior to extraction was also investigated. Under these optimized conditions, ASE was evaluated for the extraction of PAHs from a certified reference material SRM 2977 (organics in freeze-dried mussel tissue). The results were compared with those of a methanolic saponification extraction (MSE). Experimental Sample preparation The mussels (Mytilus edulis) used in this study was purchased fresh from a retail fish outlet in the metropolitan area of Valencia. The tissue of 4 kg of mussels was removed from the shell, combined and homogenized (Ultra-Turrax TR-50, Germany). The material was divided into containers and stored at 18 C until analysed. Five containers were randomly selected and tested for the presence of PAHs in the mussel tissue. Fortification of thawed mussel tissue was performed as follows: An analytical aliquot of approximately 4 g was weighed; 0.2 ml of a 100 ng/ml of PAHs mixture in acetonitrile was added directly to the mussel tissue and mixed with a spatula. This resulted in a contamination level of approximately
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5 ng/g. Then, the sample was kept overnight at 4 C before its extraction. In order to investigate the effects of sample drying, mussel tissue was freeze-dried with a Telstar freeze dryer, model: Cryodos-80 (Terrassa, Spain). The moisture content was calculated with a HR73 halogen moisture analyzer of Mettler-Toledo. Standards and solvents Solutions of 10 ng/ml in acetonitrile of anthracene (Ant), fluoranthene (Fl), pyrene (Py), benzo[a]anthracene (Bz[a]ant), chrysene (Chr), benzo[e]pyrene (Bz[e]py), benzo[b]fluoranthene (Bz[b]fl), benzo[k]fluoranthene (Bz[k]fl), benzo[a]pyrene (Bz[a]py), dibenzo[a,h]anthracene (Dbz[a,h]ant), benzo[ghi]perylene (Bz[ghi]per) and indene[1,2,3c,d]pyrene (I[1,2,3-c,d]py), were supplied by Dr Ehrenstorfer GmbH (Augbsburg, Germany). SRM 2977 (organic contaminants and trace elements in freeze-dried mussel tissue) was obtained from Standard Reference Material Program, NIST (Gaithersburg, MD 20899). All solvents used were HPLC or analytical grade. The acetone, dichloromethane (DCM), hexane, methanol were purchased from Scharlau (Barcelona, Spain). The Na2SO4 anhydrous was from J. T. Baker (Deventer, Holland), and the diatomaceous earth was from Aldrich. Methanolic saponification extraction (MSE) The extraction procedure was realized according to Lawrence et al. (1984). Approximately 30 g of sample was saponified with 11.2 g of KOH and 100 ml of H2O/methanol (1:9, v/v) for 3.5–4 h under reflux. The mixture was cooled, diluted with 100 ml of H2O/methanol (2:8, v/v), and extracted twice with 100 ml of cyclohexane. The aqueous/ methanol layer was then discarded. The cyclohexane extracts were combined, washed with 100 ml of H2O/ methanol (1:1, v/v) and 2 100 ml of H2O, filtered through anhydrous sodium sulfate, and concentrated on a rotary evaporator at 40 C. Prior to GPC cleanup, the extracts were dissolved to 5 ml with dichloromethane. Accelerated solvent extraction procedure Extractions were carried out using Dionex ASE 200 Accelerated Solvent Extractor. Mussel tissue (4 g) was ground and dried with diatomaceous earth (DE) with a pestle and mortar and placed into 11 ml or 22 ml stainless-steel cells containing a cellulose filter in the cell outlet. The sample cells were then closed, to finger tightness, and situated in the carousel of the ASE 200 system.
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Samples
Extraction
Purification
Partition
Determination
References
Fish Canned mussels, oysters, fish, smoked meat Smoked fish, meat, cheese Smoked fish Fish, shellfish Mussel, fish
Methanolic KOH/cyclohexane Methanolic KOH/cyclohexane
Silica gell/Sephadex LH 20 Florisil/Toluene
DMF-water DMSO, hexane
GC-FID HPLC-FL/GC-MS
Grimmer et al. 1975 Lawrence et al. 1984
Sonication/hexane
Silica/hexane; C18/hexane
DMSO
HPLC-FL
Garcı´a et al. 1999
Methanolic KOH/cyclohexane Ethanolic KOH/2,2,4 -trimethyl pentane Soxhlet-extracted with hexane, acetone, diethyl ether. Methanolic KOH/cyclohexane Soxlhet extraction with metanol and dichloromethane; hexane Ethanolic KOH/pentane
Silica gel/cyclohexane Florisil/Tolueno Cyclohexane
Caffeine, formic acid HPLC-FL DMSO, 2,2,4-trimethylpentane HPLC-FL DMSO GC-MS
Karl et al. 1996 Humason et al. 1982 Rainio et al. 1986
Silica-alumina
-
HPLC-FL
Murray et al. 1991
Alumina(HPLC)/dichloromethane, pentane; Silica(HPLC/dichloromethane, pentane Silica gel/dichloromethane Bio-Beads S-X3/chloroform (GPC)
-
GC-MS
Baumard et al. 1997
-
GC-FI GC-MS HPLC-FL
White et al. 2002 Eickhoff et al. 2003 Cejpek et al. 1995
Silica gel/cyclohexane; Sephadex LH-20/ isopropane Florisil/hexane Silica gel/ethyl ether-n-hexane BioBeads S-X3/cyclohexane-ethyl acetate (GPC)
DMF, water
HPLC-FL; GC-MS Lawrence et al. 1986
DMF, water
GC-MS HPLC-FL GC-MS
Pointet et al. 2000 Takatsuki et al. 1985 Speer et al. 1990
BioBeads SX-3/dichlorometane-hexane. Florisil/hexane Silica gel/dichlorometane SPE, ODS/acetonitrile SPE(aminopropylsilane)/dichloromethane-hexane Aluminium oxide-silica gel/toluene Silica/isooctane Silica gel/cyclohexane Bio-Beads SX-3/dichloromethane-cyclohexane
-
GC-MS
Birkholz et al. 1998
DMF-water -
GC-MS HPLC-FL HPLC-FL GC-FID HPLC-FL HPLC-UV HPLC-FL
Easton et al. 2002 Dafflon et al. 1995 Schantz et al. 1997 Mostafa et al. 2002 Dennis et al. 1983 Kannappan et al. 1999 Musial et al. 1986
Mussel Mussel Fish Crabs Protein/or fat-rich foods Fish
Methanolic KOH/hexane Methanolic KOH/pentane Sonication/chloroform Methanolic KOH/cyclohehane
Fish Fish, shellfish Smoked meat, bacon, fish Fish
Soxhlet-extracted with dichloromethane Ethanolic KOH/n-hexane Methanolic KOH/cyclohexane
Fish Fish, meat Mussel Seafood Fish, meat Fish, shellfish Shellfish
Methanolic KOH/pentane Methanolic KOH/cyclohexane Soxhlet extracted using hexane/acetone Methanolic KOH/n-hexane Methanolic KOH/isooctane Extraction with dichloromethane Ethanolic KOH/2,2,4-trimethylpentane
Soxhlet extracted with dichlorometane
V. Yusa` et al.
Table I. A survey of analytical procedures used for PAH determination in fish and shellfish.
Polycyclic aromatic hydrocarbons in mussel tissue The optimized conditions were as follows: Oven temperature of 125 C with 5 min heat-up time under a pressure of 1500 psi and one static cycle with a static time of 5 min. The flush volume amounted to 60% of the extraction cell volume. The extracted analytes were purged from the sample cell using pressurized nitrogen (125–150 psi) for 1 min. The sample extracts were dried by sodium sulfate which was added to the extraction bottle and concentrated to 0.5 ml at 40 C under nitrogen. Prior to the GPC clean-up, the extracts were dissolved to 5 ml with dichloromethane.
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(ex 250 nm, em 390 nm), fluoranthene (ex 280 nm, em 450 nm), pyrene (ex 238 nm, em 398 nm), benzo[a]anthracene and chrysene (ex 280 nm, em 389 nm), benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene (ex 268 nm, em 398 nm), dibenzo[a,h]anthracene, benzo[ghi]perylene and indene[1,2,3-c,d]pyrene (ex 300 nm, em 466 nm). Quantitation was performed by external standard calibration with a seven points calibration curves ranging from 0.1–100 ng/ml.
Results and discussion Gel permeation chromatography clean-up
Effect of extraction solvent
The clean-up of mussel tissue extracts was performed using gel permeation chromatography (GPC). For the GPC the Waters Gel Permeation Chromatography Clean Up System (Waters 515 high-pressure liquid chromatography pump, Waters 717 sample input module, tandem columns Envirogel GPC Clean Up 19 mm 150 mm and 19 mm 300 mm, and a Waters 2487 UV detector, 254 nm) together with a Gilson FC 204 Fraction Collector was used. The conditions were as follows: mobile phase: methylene chloride; flow rate: 5 ml/min; 2 ml injection out of the 5 ml sample loop volume; the cycle programme was: 15 min 30 s waste time, 5 min collect cycle, 5 min wash cycle; column temperature: ambient. Before each multiple-sample procedure, the GPC was calibrated by establishing an elution profile with a calibration solution which consisted of corn oil, bis(2-ethylhexyl)phthalate, Methoxychlor, Perylene (EPA 1994; Dionex 2000). The calibration chromatograms were examined to ensure that the relative retention times (RRTs) and peak shapes were as expected. The collected PAH fraction was evaporated to dryness at 40 C under nitrogen and the residues were dissolved in 1 ml of acetonitrile.
The polarity of the extraction solvent should closely match that of the target compounds, but, in some cases, solvent mixtures of polar and non-polar solvents give higher recoveries. Two individual solvents (hexane and methylene chloride) and two solvent mixtures (dichloromethaneacetone and hexane-acetone), both 1:1, v/v, were tested as extraction solvents under the standard operating conditions described above. These solvents or solvents mixtures were chosen from those used in conventional MSE methods and in ASE methods for environmental matrices. Table II illustrates the effect of the solvent on the yield of PAHs. Statistical treatment of the data was given on the 12 PAHs investigated. As the DCM-acetone (1:1,v/v) mixture has been reported as a suitable solvent mixture for the extraction of PAHs from soils and other environmental matrices (Saim et al. 1998; Richter et al. 1996), the statistical significance extraction data of other solvents were compared with those of the dichloromethaneacetone solvent mixture, using a two-sample t-test approach (Gardiner 1997) (see Table III). The PAHs relative recoveries were not statistically significant for dichloromethane, except for benzo[a]pyrene. However, statistical significance was noted for both hexane and acetone-hexane. All the statistically significant results shown in Table III gave lower relative recoveries than these of the dichloromethane-acetone extraction. The low relative recoveries obtained from hexane are attributable to its lower polarity (Saim et al. 1998). It is difficult to explain the low yield obtained with the hexane-acetone (1:1, v/v) solvent mixture that has also been reported for the extraction of PAHs from different solid matrices (Heemken et al. 1997; Popp et al. 1997). Consequently, the mixture dichloromethane-acetone was chosen for all further investigations of PAHs in mussel tissue.
Analysis of extracts by HPLC-Fl For HPLC analysis a Waters 2695 separation module equipped with a fluorescence detector Waters 2475 was used. The PAHs were separated inside a Waters PAHs C18 column (250 4.6 mm I.D.) at a column temperature of 30 C. Acetonitrile and water were used as the mobile phase. The flow rate was 1.2 ml/min. The composition gradient of the mobile phase started with 60% acetonitrile and was increased up to 100% in 20 min. This percentage was kept constant for 19 min until the end of the analysis. For detection, the following excitation (ex) and emission (em) wavelength programme was used: anthracene
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Table II. Extraction of PAHs from spiked mussel tissue using different solvents. Recovery (%) [R.S.D. %]. Compound Ant Fl Py Bz[a]ant Chr Bz[e]py Bz[b]fl Bz[k]fl Bz[a]py Dbz[a,h]ant Bz[ghi]per I[1,2,3-c,d]py
CH2Cl2-acetone Hexane Acetone-hexane CH2Cl2 76 85 83 83 90 104 92 101 91 90 93 93
[14] [5] [10] [5] [8] [8] [8] [4] [9] [10] [6] [4]
73 82 85 68 74 73 92 97 63 84 70 68
[3] [7] [4] [4] [5] [4] [2] [4] [3] [9] [9] [4]
66 71 80 64 68 73 95 91 61 87 72 76
[6] [7] [6) [6] [7] [6] [9] [7] [8] [8] [8] [5]
74 82 83 83 88 100 103 106 78 85 96 94
[8] [7] [5] [12] [12] [10] [12] [7] [9] [5] [11] [6]
(n ¼ 5). ASE operating conditions: T ¼ 125 C; p ¼ 1500 psi; heat time ¼ 5 min; static time ¼ 5 min; cycles ¼ 1.
Table III. Statistically significant solvent extraction data for individual PAHs compared with dichloromethane-acetone extraction ( p-values reported). Compound Ant Fl Py Bz[a]ant Chr Bz[e]py Bz[b]fl Bz[k]fl Bz[a]py Dbz[a,h]ant Bz[ghi]per I[1,2,3-c,d]py
Table IV. Effect of static times and cycles on the recovery of PAHs from mussel tissue. Recovery % [R.S.D%]
Hexane
Acetone-hexane
CH2Cl2
0.002 0.001 0.004 0.000
0.000 0.001 0.001
0.006
0.021 0.000
0.000 0.000
0.001 0.000
Static time 2 cycles Compound Ant Fl Py Bz[a]ant Chr Bz[e]py Bz[b]fl Bz[k]fl Bz[a]py Dbz[a,h]ant Bz[ghi]per I[1,2,3-c,d]py
1 min 77 90 84 85 93 96 102 97 79 85 92 93
[7] [9] [9] [11] [10] [13] [10] [10] [8] [11] [7] [9]
5 min 72 91 84 89 94 103 106 98 91 88 101 96
[9] [5] [5] [8] [7] [4] [7] [5] [5] [9] [6] [13]
1 cycle 10 min 80 91 83 88 98 103 97 97 91 88 98 98
[14] [10] [7] [12] [11] [13] [10] [11] [15] [15] [11] [13]
5 min 76 85 83 83 90 104 92 101 91 90 93 93
[14] [5] [10] [5] [8] [8] [8] [4] [7] [10] [6] [4]
(n ¼ 5). ASE operating conditions: T ¼ 125 C; p ¼ 1500 psi; heat up time ¼ 5 min; solvent: DCM-acetone (1:1, v/v).
were used by Richer (1996) for environmental matrices. In accordance with the results, one 5-min static cycle was considered able to obtain quantitative recoveries. Freeze-drying effect
0.029
Statistical significance was determined using a 2-sample t-test, at 95% confidence level, p < 0.05.
Static time and extraction cycles study In order to optimize the extraction time four different alternatives were tested: Two 1-min static cycles, 5-min static cycles, 10-min static cycles and one 5-min static cycle. The results are shown in Table IV. One-way ANOVA (Gardiner 1997) test was applied to each PAH for the four extraction modes in order to evaluate any statistically significant differences. Only benzo[a]pyrene displayed significant differences (I-value < 0.5). For this PAHs a two 1-min static cycles yielded statistically lower relative recoveries than one 5-min static cycle (two samples t-test, p < 0.5). The use of static cycles was developed to introduce fresh solvent during the extraction process in order to mimic Soxlhet extraction. Static cycles have been proved to be useful for sample types with a very high concentration of analyte, or samples with difficult-to-penetrate matrices (Dionex 2000). The EPA method 3545 (1996) suggest only one 5 minute static extraction cycle. Similar conditions
Sample drying prior to extraction enhances the extraction yield of samples that contain water. Wet mussel tissue samples (85% moisture) are normally DE-dried (DEd) as pointed out in 2.4. The recoveries of PAHs from DE-dried tissue was compared with freeze-dried (Fd) mussel using the following ASE conditions: 125 C, 1500 psi, 5 min heat up time, 5 min static time, 1 cycle, dichloromethane:acetone (1:1, v/v) as the extraction solvent. As shown in Figure 1, the two drying methods yield similar relative recoveries (no statistical significance). However freeze-drying procedure adds time to sample preparation (24–48 h) and increases the potential for analyte losses. However if the samples are to be kept frozen for a long time the freeze-drying makes them more stable and homogenous. Temperature effects The use of solvents at elevated temperatures should give enhanced performance compared to extraction at near room temperatures. The use of higher temperatures increases the capacity of solvents to solubilize analytes and increases the diffusion rate. In addition, increased temperatures also decrease the viscosity of liquid solvents (better penetration of matrix particles) and can disrupt the strong solute-matrix interactions caused by van der Waals
Polycyclic aromatic hydrocarbons in mussel tissue Ded
120
487
Fd
% Recovery
100 80 60 40 20
h] an t Bz [g hi ]p I[1 er ,2 ,3 -c ,d ]p y
py a]
bz [a , D
Bz [
]fl
]fl [k Bz
Bz [b
[e
]p y
hr Bz
C
t a] an
Py
Bz [
Fl
An t
0
Figure 1. Effect of sample preparation on the recovery of PAHs from spiked mussel tissue. ASE conditions: 125 C; 1500 psi; static time 5 min; DCM-acetone (1:1, v/v); 1 cycle; DEd: diatomaceous earth drying; Fd: freeze-drying; (n ¼ 5). Error bar ¼ standard deviation.
60˚C
120
110˚C
125˚C
150˚C
100
% Recovery
80
60
40
20
nt Bz [g hi ]p er I[1 ,2 ,3 -c ,d ]p y
h] a a, bz [
D
Bz [a ]p y
Bz [k ]fl
Bz [b ]fl
Bz [e ]p y
C hr
Bz [a ]a nt
Py
Fl
An t
0
Figure 2. Effect of temperature on the recovery of PAHs from spiked mussel tissue. ASE conditions: 1500 psi; static time 5 min; DCM-acetone (1:1, v/v); 1 cycle; (n ¼ 5). Error bar ¼ standard deviation.
forces, hydrogen bonding, and dipole attractions of the solute molecules and active sites of the matrix (Richter 1996). We studied the effect of temperature on the recovery of PAHs from spiked mussel tissue. The oven temperature was varied from 60–150 C using the following ASE conditions: 1500 psi, 5 min static time, 1 cycle, dichloromethane:acetone (1:1, v/v) as the extraction solvent. The results are given in Figure 2. An increase in the temperature gave improved recoveries but the concentration of analytes extracted levelled off at 125 C. Similar results of dependence of PAHs yield from the temperature
in environmental matrices have been found by Saim et al. (1998), Richter et al. (1996) and Schantz et al. (1997b). Extraction of SRM 2977 with ASE and MSE It is well known that compounds spiked on a matrix will extract much quicker than compounds that are naturally incurred in the material. For that reason it is necessary to validate the ASE optimized method with an incurred mussel tissue. The ASE optimized method (solvent: dichloromethane-acetone (1:1, v/v); temperature: 125 C; pressure: 1500 psi; heat-up time: 5 min; static
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V. Yusa` et al.
time: 5 min; cicles: 1) was compared with MSE on the extraction of PAHs from the SRM 2977, freeze-dried mussel tissue. The results are showed in Table V. As the SRM 2977 was extracted five times, using each extraction technique, it is possible to evaluate for statistical significance for each PAHs using a two sample t-test approach, at 95% confidence level. All the PAH determinations were not significant ( p > 0.05). For all PAHs, trueness of the ASE method calculated from repeated analyses of the certified reference material (see Table V), was between the guideline ranges for the deviation of the experimentally determined mean mass fraction from the certified values adopted by the European Community, that are as follows: 30% to þ10% for >1 mg/Kg to 10 mg/Kg; and 20% to þ10% for 10 mg/Kg (European Community 2002b). The precision did not exceed the level calculated by the Horwitz equation (Horwitz 1982). In the same way, ASE method meets the proposed EU method criterion (European Community 2004) that quotes an acceptable recovery range of 50–120% for analysis of benzo(a)pyrene in foodstuffs. The calculated limit of quantification (LOQ) expressed as the analyte concentration corresponding to the sample blank value plus 10 standard deviations above the average blank signal (Eurachem 1998), is lower than the tested levels for all PAHs. Ten independent sample blanks were run under repeatability conditions, and the noise levels at appropriate points in the chromatograms were measured. The LOQ in g/Kg were calculated taking into account the dilution factor, the final extract volume (ml) and the weight of the test portion (g).
The limits of quantification for the ASE method ranged from 0.10 mg/Kg Bz[a]ant to 0.25 mg/Kg I[1,2,3-c,d]py (for the MSE method the LOQs ranged from 0.01 mg/Kg Bz[a]ant to 0.03 mg/Kg I[1,2,3-c,d]py). The ASE method meets the proposed EU maximum limit of quantification (0.9 mg/ Kg) for methods of analysis for benzo(a)pyrene in foodstuffs (European Community 2004). Conclusions The studies carried out have evidenced that ASE is an alternative to MSE for extraction of PAHs from mussel tissue. The ASE method developed through this study (10 minutes, 25 ml) provides a 24 times faster extraction than MSE and reduce 12 times the volume of solvent required. Among the examined solvents, dichloromethaneacetone (1:1, v/v) provides the highest extraction yields with operating conditions as follows: Temperature, 125 C; pressure, 1500 psi; heat-up time, 5 min; static time, 5 min; cycles, 1. No influence was found between freeze-drying or drying with diatomaceous earth the wet mussel prior to extraction. Automation of PAHs analysis by application of ASE followed by GPC provides a high productivity, and the performance characteristics (trueness, repeatability and limit of quantification) of the method are according to the requirements for quantitative methods of the European Community.
Acknowledgments The authors acknowledge the financial support of the Escuela Valenciana de Estudios de Salud (EVES) de la Generalitat Valenciana.
Table V. Extraction of PAHs from SRM 2977: comparison of ASE and MSE.
Compound Ant* Fl Py Bz[a]ant Chr* Bz[e]py Bz[b]fl* Bz[k]fl Bz[a]py Bz[ghi]per I[1,2,3-c,d]py
ASE
MSE
References
a
a
Baumard P, Budzinski H, Garrigues P. 1997. Analytical procedure for the analysis of PAHs in biological tissues by gas chromatography coupled to mass spectrometry: Application to mussels. Fresenius Journal of Analytical Chemistry 359:502–509. Birkholz DA, Coutts RT, Hudey SE. 1998. Determination of polycyclic aromatic compounds in fish tissue. Journal of Chromatography 449:251–260. Bolger M, Henry SH, Carrington CD. 1996. American Fisheries Society Symposium 18:837–843. Cejpek K, Hajslova Z, Merhaut J. 1995. Simplified extraction and clean-up procedure for the determination of PAHs in fatty and protein-rich matrices. International Journal of Environmental Analytical Chemistry 61:65–80. Dafflon O, Gobet H, Kock H, Bosset JO. 1995. Le dosage des hydrocarbures aromatiques polycycliques dans le poisson, les produits carne´s et le fromage par chromatographie liquide a` haute performance. Travaux Pratiques de Chimie Analytique 86:534–555.
Certified Mean Recovery Mean Recovery concentrationa,b range range (RSD %) (RSD %) 84 35.1 3.8 78.9 3.5 20.34 0.78 49 2 13.1 1.1 11.01 0.28 41 8.35 0.72 9.53 0.43 4.84 0.81
7.6 33.3 76.4 19.5 47 13 10.8 3.9 1.4 9.1 4.6
70–85 73–90 82–93 79–93 82–110 72–100 88–110 93–108 78–106 78–109 85–97
7.5 35 72 20 46 11 10 3.8 1.4 8 4.4
58–84 62–87 68–84 74–98 51–82 73–105 73–98 85–105 78–113 68–100 72–97
(n ¼ 5). ASE operating conditions: 125 C, 1500 psi, static time: 5 min., 1cycle, solvent: DCM-acetone (1:1,v/v); aConcentrations in mg/Kg dry weight; bThe dispersion is expressed as expanded uncertainty; *Reference concentrations.
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