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Supercritical fluid extraction and high-performance liquid chromatography–fluorescence detection method for polycyclic aromatic hydrocarbons investigation in vegetable oil
Food Control 16 (2005) 59–64 www.elsevier.com/locate/foodcont
Supercritical fluid extraction and high-performance liquid chromatography–fluorescence detection method for polycyclic aromatic hydrocarbons investigation in vegetable oil ~a M.A. Lage Yusty *, J.L. Cortizo Davin Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Pharmacy, University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain Received 2 March 2003; received in revised form 18 November 2003; accepted 24 November 2003
Abstract In spite of the fact that food processes, that involve drying and smoking, may cause polycyclic aromatic hydrocarbon contamination, an extraction clean/up procedure carried out by SFE was developed in order to isolate polycyclic aromatic hydrocarbons from oil vegetable samples for subsequent HPLC–FL determination. The detection and quantification limits obtained were <1.55 lg kg1 oil and <2.55 lg kg1 oil, respectively, allowed to check the presence of seven of the eight PAHs with legal limit in olive–pomace oil: benzo[a]anthracene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[ah]anthracene and benzo[ghi]perilene. In brief, the method permits the evaluation of edible oil safety and, therefore, consumers protection. 2003 Elsevier Ltd. All rights reserved. Keywords: Polycyclic aromatic hydrocarbons; Oil; SFE; HPLC–FL
1. Introduction Polycyclic aromatic hydrocarbon (PAH) are a group of well-known carcinogenic compounds yielded from incomplete combustion of organic compounds and geochemical processes. They have been the subject of much concern in recent years due their toxic potential. They are known as highly stable contaminants present in many foods (Speer, Steeg, Horstmann, K€ uhn, & Montang, 1990), and they are included in the list that contains the top 20 of hazardous substances, the 2001 CERCLA priority list of hazardous substances. Moreover are between the substances most frequently found in completed exposure pathways (ATSDR, 2001). In the vegetables oils, it has been suggested that the main sources of contamination are: contamination of plant material, mainly through the air,
*
Corresponding author. Tel.: +34-81-594626; fax: +34-81-594912. E-mail address: [email protected] (M.A. Lage Yusty).
0956-7135/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2003.11.008
drying the plant material with smoke before extraction, and contamination through the extraction solvent (Pupin & Figueiredo Toledo, 1996). The refining process can drastically reduce the amount of these contaminants. The deodorization process seems to have little effect on high molecular PAHs and removes mainly ‘‘light’’ PAHs (up to four aromatic rings), while higher condensed ‘‘heavy’’ PAHs are mainly removed by charcoal treatment (Balenovic, Petrovic, & Perkovac, 1995; Dennis, Massey, Cripps, Venn, Howarth, & Lee, 1991; Moret & Conte, 2000). In consequence of the Benzo[a]pyrene detection in olive–pomace oil (Council Regulation, 2001), formed by heating during the elaboration process, Spain has adopted a legal limit for PAHs content in these oils (Orden 25 de Julio 2001) with a maximum tolerable limit of 2 lg kg1 oil for each of the following PAHs: benzo[a]anthracene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[ah]anthracene, benzo[ghi]perilene and indeno[1,2, 3-cd]pyrene, and a maximum tolerable limit of 5 lg kg1 oil for the sum of the eight analytes.
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M.A. Lage Yusty, J.L. Cortizo Davi~na / Food Control 16 (2005) 59–64
Major problems associated with the determination of PAHs in complex matrices, such a vegetable oils, are the low analyte level (lg kg1 ) and the diversity of potential interferences present. The widely used procedures for the extraction of PAHs from the rest of lipids are saponification of lipids by alcoholic KOH, followed by liquid–liquid partition and solid phase purification (Balenovic et al., 1995; Win, Luther, Jacob, Vaessen, & Boenke, 1998), liquid–liquid partition followed by clean up (Menichini, Bocca, Merli, Ianni, & Monfredini, 1991a; Menichini, Domenico, Bonanni, Corradetti, Mazzanti, & Zucchetti, 1991b; Pupin & Figueiredo Toledo, 1995, 1996; Vazquez Troche, Garcıa Falc on, Gonz alez Amigo, Lage Yusty, & Simal Lozano, 2000) and supercritical fluid extraction (SFE) (Dankers, Groenenboom, Scholtis, & Van der Heiden, 1993; Janda, Bartle, & Clifford, 1993; Langenfeld, Hathorne, Miller, & Pawliszyn, 1995; Moret & Conte, 2000; Turner, Sparr Eskilsson, & Bj€ orklund, 2002). The analytical determination has been carried out by HPLC with fluorescence detector (Balenovic et al., 1995; Burdaspal, Legarda, & Sanchez, 2001; Chen, Wang, & Chiu, 1996; Codina, Vaquero, Comellas, & Broto-Puig, 1994; Garcıa Falc on, G onzalez Amigo, Lage Yusty, L opez de Alda Villaiz an, & Simal Lozano, 1996; Lodovici, Dolara, Casalini, Ciapellano, & Testolin, 1995; M€ akel€ a & Pyy, 1995; Moret, Bortolomeazzi, & Conte, 1996a; Nieva Cano, Rubio Barroso, & Santos Delgado, 2001; Pupin & Figueiredo Toledo, 1995, 1996; Stijn Van, Kerhoff, & Vandeginste, 1996; Vaessen, Wagstaffe, & Lindsey, 1990; V azquez Troche et al., 2000), UV detector (Bazylac & Maslowska, 1990; Vaessen et al., 1990) or both, UV and FL (Joe, Salemme, & Fazio, 1984; Vaessen et al., 1990) or with GC coupled with FID or MS (Aamot, Krane, & Steinnes, 1987; Balenovic et al., 1995; Kolarovic & Traitler, 1982; Menichini et al., 1991a, 1991b; Saeed, Al-Bloushi, & AlMatrouk, 1995; Turner et al., 2002; Vaessen et al., 1990). The aim of this paper was the determination of eight PAHs limited in oils by HPLC with fluorescence detector, follow to supercritical fluid extraction.
2. Experimental 2.1. Reagents Benzo[a]anthracene (BaA) and benzo[a]pyrene (BaP) standards were purchased from Aldrich; benzo[b]fluoranthene (BbF), benzo[ghi]perilene (BghiP), benzo[k]fluoranthene (BkF) and indene[1,2,3-cd]pyrene (Ind) standards from Sugelabor; benzo[e]pyrene (BeP) and dibenzo[ah]anthracene (DBaA) standards from Dr. Ehrenstorfer Gmbh. All chemicals were analytical grade. Acetonitrile, methanol and silicagel 60 were supplied from Merck. Ultra pure water was obtained with a Milli
Q filter system (Millipore, Bedford, MA EEUU) and Helio (SEO N-50), CO2 SFC tube plongeur and CO2 siphon were from the Sociedad Espa~ nola de Oxıgeno. 2.2. Preparation of standards Stocks solutions containing 100 mg l1 of benzo[a]anthracene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[ah]anthracene and indeno[1,2,3-cd]pyrene and 1000 mg l1 of benzo[ghi]perilene were prepared in acetonitrile and were stored at 4 C in volumetric flasks (with glass stoppers) wrapped with aluminium foil to avoid possible light degradation. A global standard solution of PAHs was prepared with an aliquot of each individual standard solution and subsequent dilution with acetonitrile. 2.3. Apparatus A Hewlett Packard 7680 A Supercritical fluid extractor, controlled by a PC running SFE software was used to perform extraction and clean-up. A SpectraPhysics liquid chromatograph equipped with a P100 isocratic pump, a Tecknokroma reversed-phase Tracer Tr–C-160 C18 precolumn and a Hypersil Green PAH column (5 lm particle size; 10 cm · 4.6 mm I.D.), a FL2000 fluorescence detector, and a Datajet integrator connected via Labnet to a PC running Winner on Windows (WOW) data processing software was used for HPLC. Water circulation through a thermostatted water-bath was used to keep the column temperature at 31 ± 0.1 C. 2.4. Procedure 2.4.1. Extraction and clean-up Fluids used were supercritical carbon dioxide with cosolvent methanol. The modifier (200 ll) was added directly onto the sample in the extraction thimble. Silicagel partially deactived (15%) was used for adsorption chromatography, follow purification in Soxhlet with acetonitrile for 6 h. The sample (1 g) homogenized with 2 g silicagel was placed into a 7 ml extraction thimble, and 1 g silicagel was added. Before the extraction, two layers of Whatman filter with the diameter of the thimble were placed just above the cap at the bottom of the thimble. An initial 10 min static equilibration period at 110 C and 283 bar, was followed by a 50 min in the dynamic or continuous flow mode at a rate of 1.5 ml/min of supercritical CO2 and a density of 0.6 g/ml. The variable diameter nozzle was heated at 60 C; analyte collection was performed with a solid stainless-steels (SS) trap at 75 C and the PAHs were eluted with seven 1.5 ml portions of acetonitrile; during extraction the trap and
M.A. Lage Yusty, J.L. Cortizo Davi~na / Food Control 16 (2005) 59–64
nozzle were kept at 70 and 60 C, respectively. The acetonitrile of the seventh vial was discarded. The acetonitrile extract collected (9 ml) was concentrated to a volume of 1 ml and filtrated through a 0.5 lm pore-size MFS-25 PTFE filter [Micro Filtration Systems (MFS)]. 2.4.2. Chromatographic method An aliquot (50 ll) of the acetonitrile solution was injected into the HPLC system and eluted with acetonitrile–water (78–22, v/v) at a constant flow-rate of 0.5 ml/min. For detection and quantification, two excitation (Ex) and emission (Em) wavelengths programmes had been used: programme 1 for BaA (Ex 270 nm, Em 390 nm), BbF (Ex 300 nm, Em 440 nm), BkF (Ex 296 nm, Em 426 nm), BaP (Ex 296 nm, Em 406 nm), DBaA (Ex 300 nm, Em 400 nm), BghiP (Ex 296nm, Em 406 nm) and Ind (Ex 300 nm, Em 464 nm); programme 2 for BeP (Ex 290 nm, Em 398 nm). Quantitative analysis of the eight PAHs was performed using external standard calibration with a reference standard solution diluted appropriately.
3. Results and discussion SFE drastically reduce analysis time, volume of solvents consumed and sample manipulation, and are also suitable for routine analyses. This technique eliminate the clean-up step. In a previous work, we have obtained the highest recoveries of PAHs with alumina or silicagel adsorbents (Laffon Lage, Garcıa Falc on, G onzalez Amigo, Lage Yusty, & Simal Lozano, 1997). The silicagel, previous extraction by soxhlet with acetonitrile, did not show interferences in the retention times of PAHs studied and therefore it was used in this paper. The use of silicagel as adsorbent eliminates the clean-up step procedure because it is integrated in the extraction process (G onzalez Amigo, Garcıa Falc on, Lage Yusty, & Simal Lozano, 2000). Different CO2 density were assayed between 0.55 and 0.70 g ml1 . High density produce oil dragging together with the analytes, while low density does not produce analyte adequate extraction, therefore a 0.60 g ml1 density was selected. The solid-phase trap was of SS for these matrix, with better results than Octadecylsilane (ODS), used for us in previous papers (G onzalez Amigo et al., 2000). Small volumes of solvent (9 ml) and their concentration to 1 ml previous HPLC–FL analysis allow to achieve the aim. The excitation and emission wavelength programmes used for the detection and the quantification of eight PAHs (Table 1) correspond to the optimum excitation– emission wavelength have been determined by spectrofluorimetry.
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Table 1 Wavelength of excitation (kexc ) and emission (kem ) of eight PAHs PAH
kexc (nm)
kem (nm)
BaA BeP BbF BkF BaP DBaA BghiP Ind
270 290 300 296 296 300 296 300
390 398 440 426 406 400 406 464
3.1. Quantification and the linearity of the instrumental response PAHs peaks were identified by comparison of sample chromatograms with the chromatogram of the PAHs standard. Quantification was carried out by the external standard method. The calibration line was constructed by regressing mean (n ¼ 3) peak height on standard concentration, except Ind (LQ very high). The range used for each PAH is included in Table 2. Calibration curves were highly linear with determination coefficients between 0.9847 and 0.9998. 3.2. Detection and quantification limits Detection and quantification limits for PAHs in acetonitrile were calculated following the ACS guidelines (ACS, 1980) and are included in Table 2. 3.3. Precision and recovery Method precision and recovery were determined applying the full procedure to three replicate samples of ‘‘olive oil’’ spiked with the eight PAHs in the range of concentrations used. First, it was confirmed that the PAHs levels in this sample were below the detection limits. The results were showed in Table 3, Figs. 1 and 2. HPLC–FL is accepted as the more sensitivity technique for the PAHs determination. Table 2 Detection limits (LD) and quantification limits (LQ) and PAHs concentrations range using of HPLC–FL PAH
LD (lg l1 )
LQ (lg l1 )
Range (lg l1 )
BaA BeP BbF BkF BaP DBaA BghiP Ind
0.389 1.55 0.468 0.0750 0.276 0.467 0.670 10.1
0.706 2.55 0.836 0.201 0.434 0.797 1.60 21.3
1, 2.5, 4, 5 and 6 2.5, 4, 5, 6 and 7 1, 2.5, 3, 4 and 5 0.25, 0.5, 1, 2.5 and 4 1, 2, 3, 4 and 5 1, 2.5, 4, 5 and 6 1.6, 2.5, 4, 5 and 6 a
a: LQ very high. lg l1 of acetonitrile ¼ lg kg1 of oil.
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M.A. Lage Yusty, J.L. Cortizo Davi~na / Food Control 16 (2005) 59–64
Table 3 Methods recovery and precision PAH
%R
RSD (%)
BaA BeP BbF BkF BaP DBaA BghiP
93.3 48.0 57.3 57.5 46.4 27.5 18.4
0.73 8.6 5.8 6.2 2.1 5.3 6.6
The limits obtained for others authors were often higher. Pupin and Figueiredo Toledo (1995), have analysed benzo[a]pyrene in oils by liquid–liquid partition, clean-up and HPLC–FL determination The limit of detection obtained was 0.5 lg/kg oil, with a recoveries >76.7% and relative standard deviations <15.0%. Burdaspal et al., 2001 similarly, have found a limit of detection of 0.57 lg/kg oil and different recoveries and RSD depending of range of concentration (89% and 21.9%, and 104% and 4.3% for 0.67 and 2.25 lg/kg, respectively). Menichini et al. (1991a), have analysed PAHs in oils by liquid–liquid extraction, clean-up, and GC–FID have obtained. The limit of detection obtained was 3 lg/kg oil, with a recoveries between 50% and 85% and RSD 4–
18%. The limit of detection obtained by Cejpek, Hajslova, Kocourek, Tomaniova, and Cmolik, 1998, was 2 lg/kg oil by liquid–liquid partition, clean-up and espectrofluorimetry with a recoveries between 75% and 105%. G onzalez Amigo et al. (2000), analyse PAHs in bird tissue by supercritical fluid extraction and HPLC–FL determination have obtained recoveries >90% and relative standard deviations 6 7.7%. Detection and quantitation limits were <0.65 and <1.27 l/l except for Ind. This method is valid for detection and quantification of the PAHs evaluated (except for Ind) by their high detection limit, because the quantification limits obtained (expressed in lg kg1 ) are lower than the legal limits permitted. It can decide if the oil is apt or not for human consumption.
4. Conclusions An extraction/clean-up procedure by SFE was developed for PAHs separation from vegetable oil samples and a HPLC–FL method was optimised for determination of the levels of seven of the eight PAHs with legal limit in these samples: BaA, BeP, BbF, BkF,
Fig. 1. HPLC–FL chromatogram of an extract of sample and spiked oil (programme 1).
Fig. 2. HPLC–FL chromatogram of an extract of sample and spiked oil (programme 2).
M.A. Lage Yusty, J.L. Cortizo Davi~na / Food Control 16 (2005) 59–64
BaP, DBaA and BghiP and to evaluate the edible oil safety.
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carbons in smoked foods. Journal of Association of Official Analytical Chemists, 67(6), 1076–1082. Kolarovic, L., & Traitler, H. (1982). Determination of polycyclic aromatic hydrocarbons in vegetable oils by caffeine complexation and glass capillary gas chromatography. Journal of Chromatography, 237, 263–272. Laffon Lage, B., Garcıa Falc on, M. S., G onzalez Amigo, S., Lage Yusty, M. A., & Simal Lozano, J. (1997). Comparison of supercritical fluid extraction and conventional liquid–solid extraction for the determination of benzo(a)pyrene in water-soluble smoke. Food Additives and Contaminants, 14(5), 469– 474. Langenfeld, J. J., Hathorne, S. B., Miller, D. J., & Pawliszyn, J. (1995). Kinetic study of supercritical fluid extraction of organic contaminants from heterogeneous environmental samples with carbon dioxide and elevated temperatures. Analytical Chemistry, 67, 1727– 1736. Lodovici, M., Dolara, P., Casalini, C., Ciapellano, S., & Testolin, G. (1995). Polycyclic aromatic hydrocarbon contamination in the Italian diet. Food Additives and Contaminants, 12(5), 703–713. M€akel€a, M., & Pyy, L. (1995). Effect of temperature on retention time reproducibility and on the use of programmable fluorescence detection of fifteen polycyclic aromatic hydrocarbons. Journal of Chromatography A, 699, 49–57. Menichini, E., Bocca, A., Merli, F., Ianni, D., & Monfredini, F. (1991a). Polycyclic aromatic hydrocarbons in olive oils on the Italian market. Food Additives and Contaminants, 8(3), 363–369. Menichini, E., Domenico, A., Bonanni, L., Corradetti, E., Mazzanti, L., & Zucchetti, G. (1991b). Reliability assessment of a gas chromatographic method for polycyclic aromatic hydrocarbons in olive oil. Journal of Chromatography, 555, 211–220. Moret, S., Bortolomeazzi, S. R., & Conte, L. S. (1996a). Determinazione HPLC degli idrocarburi policiclic aromatici negli oli di oliva: comparazione di alcuni metodi di estracione e purificazione. Rivista Italiana Sostanze grasse, 73, 141–146. Moret, S., & Conte, L. S. (2000). Polycyclic aromatic hydrocarbons in edible fat and oils: occurrence and analytical methods. Journal of Chromatography A, 882, 245–253. Nieva Cano, M. J., Rubio Barroso, S., & Santos Delgado, M. J. (2001). Determination of PAHs in food samples by HPLC with fluorometric detection following sonication extraction without sample clean-up. Analyst, 126, 1326–1331. ORDEN de 25 de julio de 2001 por la que se establecen lımites de determinados hidrocarburos aromaticos policıclicos en aceite de orujo de oliva. BOE no. 178 de 26 de julio: 27397–27398. Pupin, A. M., & Figueiredo Toledo, M. C. (1995). Benzo(a)pyrene in olive oils on the Brazilian market. Food Chemistry, 55(2), 185–188. Pupin, A. M., & Figueiredo Toledo, M. C. (1996). Benzo(a)pyrene in Brazilian vegetable oils. Food Additives and Contaminants, 13(6), 639–646. Saeed, T., Al-Bloushi, A., & Al-Matrouk, K. (1995). Assessment of levels of polycyclic aromatic hydrocarbons in the oil from Kuwait Oil Lakes. Archives of Environmental Contaminants and Toxicology, 29, 45–51. Speer, K., Steeg, E., Horstmann, P., K€ uhn, T. H., & Montang, A. (1990). Determination and distribution of polycyclic aromatic hydrocarbons in native vegetable oils, smoked fish products, mussels and oysters, and bream from the river Elbe. Journal of High Resolution Chromatography, 13, 104–111. Stijn Van, F., Kerhoff, M. A. T., & Vandeginste, B. G. M. (1996). Determination of polycyclic aromatic in edible oils and fats by online donor acceptor complex chromatography and high-performance liquid chromatography with fluorescence detection. Journal of Chromatography A, 750, 263–273. Turner, C., Sparr Eskilsson, C., & Bj€ orklund, E. (2002). Collection in analytical fluid extraction. Journal of Chromatography A, 947, 1–22.
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Vaessen, H. A. M. G., Wagstaffe, P. J., & Lindsey, A. S. (1990). Reference materials for PAHs in foodstuffs: II Results of a further intercomparison of methods in experienced laboratories. Fresenius Journal of Analytical Chemistry, 336, 503–510. V azquez Troche, S., Garcıa Falc on, M. S., Gonzalez Amigo, S., Lage Yusty, M. A., & Simal Lozano, J. (2000). Enrichment of
benzo(a)pyrene in vegetable oils and determination by HPLC– FL. Talanta, 51, 1069–1076. Win, T., Luther, W., Jacob, J., Vaessen, H. A., & Boenke, A. (1998). Reference materials for PAHs in foodstuffs: results of the certification exercise of two coconut oil reference materials. Fresenius Journal of Analytical Chemistry, 360, 640–644.
Supercritical fluid extraction and high-performance liquid chromatography–fluorescence detection method for polycyclic aromatic hydrocarbons investigation in vegetable oil ~a M.A. Lage Yusty *, J.L. Cortizo Davin Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Pharmacy, University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain Received 2 March 2003; received in revised form 18 November 2003; accepted 24 November 2003
Abstract In spite of the fact that food processes, that involve drying and smoking, may cause polycyclic aromatic hydrocarbon contamination, an extraction clean/up procedure carried out by SFE was developed in order to isolate polycyclic aromatic hydrocarbons from oil vegetable samples for subsequent HPLC–FL determination. The detection and quantification limits obtained were <1.55 lg kg1 oil and <2.55 lg kg1 oil, respectively, allowed to check the presence of seven of the eight PAHs with legal limit in olive–pomace oil: benzo[a]anthracene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[ah]anthracene and benzo[ghi]perilene. In brief, the method permits the evaluation of edible oil safety and, therefore, consumers protection. 2003 Elsevier Ltd. All rights reserved. Keywords: Polycyclic aromatic hydrocarbons; Oil; SFE; HPLC–FL
1. Introduction Polycyclic aromatic hydrocarbon (PAH) are a group of well-known carcinogenic compounds yielded from incomplete combustion of organic compounds and geochemical processes. They have been the subject of much concern in recent years due their toxic potential. They are known as highly stable contaminants present in many foods (Speer, Steeg, Horstmann, K€ uhn, & Montang, 1990), and they are included in the list that contains the top 20 of hazardous substances, the 2001 CERCLA priority list of hazardous substances. Moreover are between the substances most frequently found in completed exposure pathways (ATSDR, 2001). In the vegetables oils, it has been suggested that the main sources of contamination are: contamination of plant material, mainly through the air,
*
Corresponding author. Tel.: +34-81-594626; fax: +34-81-594912. E-mail address: [email protected] (M.A. Lage Yusty).
0956-7135/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2003.11.008
drying the plant material with smoke before extraction, and contamination through the extraction solvent (Pupin & Figueiredo Toledo, 1996). The refining process can drastically reduce the amount of these contaminants. The deodorization process seems to have little effect on high molecular PAHs and removes mainly ‘‘light’’ PAHs (up to four aromatic rings), while higher condensed ‘‘heavy’’ PAHs are mainly removed by charcoal treatment (Balenovic, Petrovic, & Perkovac, 1995; Dennis, Massey, Cripps, Venn, Howarth, & Lee, 1991; Moret & Conte, 2000). In consequence of the Benzo[a]pyrene detection in olive–pomace oil (Council Regulation, 2001), formed by heating during the elaboration process, Spain has adopted a legal limit for PAHs content in these oils (Orden 25 de Julio 2001) with a maximum tolerable limit of 2 lg kg1 oil for each of the following PAHs: benzo[a]anthracene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[ah]anthracene, benzo[ghi]perilene and indeno[1,2, 3-cd]pyrene, and a maximum tolerable limit of 5 lg kg1 oil for the sum of the eight analytes.
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M.A. Lage Yusty, J.L. Cortizo Davi~na / Food Control 16 (2005) 59–64
Major problems associated with the determination of PAHs in complex matrices, such a vegetable oils, are the low analyte level (lg kg1 ) and the diversity of potential interferences present. The widely used procedures for the extraction of PAHs from the rest of lipids are saponification of lipids by alcoholic KOH, followed by liquid–liquid partition and solid phase purification (Balenovic et al., 1995; Win, Luther, Jacob, Vaessen, & Boenke, 1998), liquid–liquid partition followed by clean up (Menichini, Bocca, Merli, Ianni, & Monfredini, 1991a; Menichini, Domenico, Bonanni, Corradetti, Mazzanti, & Zucchetti, 1991b; Pupin & Figueiredo Toledo, 1995, 1996; Vazquez Troche, Garcıa Falc on, Gonz alez Amigo, Lage Yusty, & Simal Lozano, 2000) and supercritical fluid extraction (SFE) (Dankers, Groenenboom, Scholtis, & Van der Heiden, 1993; Janda, Bartle, & Clifford, 1993; Langenfeld, Hathorne, Miller, & Pawliszyn, 1995; Moret & Conte, 2000; Turner, Sparr Eskilsson, & Bj€ orklund, 2002). The analytical determination has been carried out by HPLC with fluorescence detector (Balenovic et al., 1995; Burdaspal, Legarda, & Sanchez, 2001; Chen, Wang, & Chiu, 1996; Codina, Vaquero, Comellas, & Broto-Puig, 1994; Garcıa Falc on, G onzalez Amigo, Lage Yusty, L opez de Alda Villaiz an, & Simal Lozano, 1996; Lodovici, Dolara, Casalini, Ciapellano, & Testolin, 1995; M€ akel€ a & Pyy, 1995; Moret, Bortolomeazzi, & Conte, 1996a; Nieva Cano, Rubio Barroso, & Santos Delgado, 2001; Pupin & Figueiredo Toledo, 1995, 1996; Stijn Van, Kerhoff, & Vandeginste, 1996; Vaessen, Wagstaffe, & Lindsey, 1990; V azquez Troche et al., 2000), UV detector (Bazylac & Maslowska, 1990; Vaessen et al., 1990) or both, UV and FL (Joe, Salemme, & Fazio, 1984; Vaessen et al., 1990) or with GC coupled with FID or MS (Aamot, Krane, & Steinnes, 1987; Balenovic et al., 1995; Kolarovic & Traitler, 1982; Menichini et al., 1991a, 1991b; Saeed, Al-Bloushi, & AlMatrouk, 1995; Turner et al., 2002; Vaessen et al., 1990). The aim of this paper was the determination of eight PAHs limited in oils by HPLC with fluorescence detector, follow to supercritical fluid extraction.
2. Experimental 2.1. Reagents Benzo[a]anthracene (BaA) and benzo[a]pyrene (BaP) standards were purchased from Aldrich; benzo[b]fluoranthene (BbF), benzo[ghi]perilene (BghiP), benzo[k]fluoranthene (BkF) and indene[1,2,3-cd]pyrene (Ind) standards from Sugelabor; benzo[e]pyrene (BeP) and dibenzo[ah]anthracene (DBaA) standards from Dr. Ehrenstorfer Gmbh. All chemicals were analytical grade. Acetonitrile, methanol and silicagel 60 were supplied from Merck. Ultra pure water was obtained with a Milli
Q filter system (Millipore, Bedford, MA EEUU) and Helio (SEO N-50), CO2 SFC tube plongeur and CO2 siphon were from the Sociedad Espa~ nola de Oxıgeno. 2.2. Preparation of standards Stocks solutions containing 100 mg l1 of benzo[a]anthracene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[ah]anthracene and indeno[1,2,3-cd]pyrene and 1000 mg l1 of benzo[ghi]perilene were prepared in acetonitrile and were stored at 4 C in volumetric flasks (with glass stoppers) wrapped with aluminium foil to avoid possible light degradation. A global standard solution of PAHs was prepared with an aliquot of each individual standard solution and subsequent dilution with acetonitrile. 2.3. Apparatus A Hewlett Packard 7680 A Supercritical fluid extractor, controlled by a PC running SFE software was used to perform extraction and clean-up. A SpectraPhysics liquid chromatograph equipped with a P100 isocratic pump, a Tecknokroma reversed-phase Tracer Tr–C-160 C18 precolumn and a Hypersil Green PAH column (5 lm particle size; 10 cm · 4.6 mm I.D.), a FL2000 fluorescence detector, and a Datajet integrator connected via Labnet to a PC running Winner on Windows (WOW) data processing software was used for HPLC. Water circulation through a thermostatted water-bath was used to keep the column temperature at 31 ± 0.1 C. 2.4. Procedure 2.4.1. Extraction and clean-up Fluids used were supercritical carbon dioxide with cosolvent methanol. The modifier (200 ll) was added directly onto the sample in the extraction thimble. Silicagel partially deactived (15%) was used for adsorption chromatography, follow purification in Soxhlet with acetonitrile for 6 h. The sample (1 g) homogenized with 2 g silicagel was placed into a 7 ml extraction thimble, and 1 g silicagel was added. Before the extraction, two layers of Whatman filter with the diameter of the thimble were placed just above the cap at the bottom of the thimble. An initial 10 min static equilibration period at 110 C and 283 bar, was followed by a 50 min in the dynamic or continuous flow mode at a rate of 1.5 ml/min of supercritical CO2 and a density of 0.6 g/ml. The variable diameter nozzle was heated at 60 C; analyte collection was performed with a solid stainless-steels (SS) trap at 75 C and the PAHs were eluted with seven 1.5 ml portions of acetonitrile; during extraction the trap and
M.A. Lage Yusty, J.L. Cortizo Davi~na / Food Control 16 (2005) 59–64
nozzle were kept at 70 and 60 C, respectively. The acetonitrile of the seventh vial was discarded. The acetonitrile extract collected (9 ml) was concentrated to a volume of 1 ml and filtrated through a 0.5 lm pore-size MFS-25 PTFE filter [Micro Filtration Systems (MFS)]. 2.4.2. Chromatographic method An aliquot (50 ll) of the acetonitrile solution was injected into the HPLC system and eluted with acetonitrile–water (78–22, v/v) at a constant flow-rate of 0.5 ml/min. For detection and quantification, two excitation (Ex) and emission (Em) wavelengths programmes had been used: programme 1 for BaA (Ex 270 nm, Em 390 nm), BbF (Ex 300 nm, Em 440 nm), BkF (Ex 296 nm, Em 426 nm), BaP (Ex 296 nm, Em 406 nm), DBaA (Ex 300 nm, Em 400 nm), BghiP (Ex 296nm, Em 406 nm) and Ind (Ex 300 nm, Em 464 nm); programme 2 for BeP (Ex 290 nm, Em 398 nm). Quantitative analysis of the eight PAHs was performed using external standard calibration with a reference standard solution diluted appropriately.
3. Results and discussion SFE drastically reduce analysis time, volume of solvents consumed and sample manipulation, and are also suitable for routine analyses. This technique eliminate the clean-up step. In a previous work, we have obtained the highest recoveries of PAHs with alumina or silicagel adsorbents (Laffon Lage, Garcıa Falc on, G onzalez Amigo, Lage Yusty, & Simal Lozano, 1997). The silicagel, previous extraction by soxhlet with acetonitrile, did not show interferences in the retention times of PAHs studied and therefore it was used in this paper. The use of silicagel as adsorbent eliminates the clean-up step procedure because it is integrated in the extraction process (G onzalez Amigo, Garcıa Falc on, Lage Yusty, & Simal Lozano, 2000). Different CO2 density were assayed between 0.55 and 0.70 g ml1 . High density produce oil dragging together with the analytes, while low density does not produce analyte adequate extraction, therefore a 0.60 g ml1 density was selected. The solid-phase trap was of SS for these matrix, with better results than Octadecylsilane (ODS), used for us in previous papers (G onzalez Amigo et al., 2000). Small volumes of solvent (9 ml) and their concentration to 1 ml previous HPLC–FL analysis allow to achieve the aim. The excitation and emission wavelength programmes used for the detection and the quantification of eight PAHs (Table 1) correspond to the optimum excitation– emission wavelength have been determined by spectrofluorimetry.
61
Table 1 Wavelength of excitation (kexc ) and emission (kem ) of eight PAHs PAH
kexc (nm)
kem (nm)
BaA BeP BbF BkF BaP DBaA BghiP Ind
270 290 300 296 296 300 296 300
390 398 440 426 406 400 406 464
3.1. Quantification and the linearity of the instrumental response PAHs peaks were identified by comparison of sample chromatograms with the chromatogram of the PAHs standard. Quantification was carried out by the external standard method. The calibration line was constructed by regressing mean (n ¼ 3) peak height on standard concentration, except Ind (LQ very high). The range used for each PAH is included in Table 2. Calibration curves were highly linear with determination coefficients between 0.9847 and 0.9998. 3.2. Detection and quantification limits Detection and quantification limits for PAHs in acetonitrile were calculated following the ACS guidelines (ACS, 1980) and are included in Table 2. 3.3. Precision and recovery Method precision and recovery were determined applying the full procedure to three replicate samples of ‘‘olive oil’’ spiked with the eight PAHs in the range of concentrations used. First, it was confirmed that the PAHs levels in this sample were below the detection limits. The results were showed in Table 3, Figs. 1 and 2. HPLC–FL is accepted as the more sensitivity technique for the PAHs determination. Table 2 Detection limits (LD) and quantification limits (LQ) and PAHs concentrations range using of HPLC–FL PAH
LD (lg l1 )
LQ (lg l1 )
Range (lg l1 )
BaA BeP BbF BkF BaP DBaA BghiP Ind
0.389 1.55 0.468 0.0750 0.276 0.467 0.670 10.1
0.706 2.55 0.836 0.201 0.434 0.797 1.60 21.3
1, 2.5, 4, 5 and 6 2.5, 4, 5, 6 and 7 1, 2.5, 3, 4 and 5 0.25, 0.5, 1, 2.5 and 4 1, 2, 3, 4 and 5 1, 2.5, 4, 5 and 6 1.6, 2.5, 4, 5 and 6 a
a: LQ very high. lg l1 of acetonitrile ¼ lg kg1 of oil.
62
M.A. Lage Yusty, J.L. Cortizo Davi~na / Food Control 16 (2005) 59–64
Table 3 Methods recovery and precision PAH
%R
RSD (%)
BaA BeP BbF BkF BaP DBaA BghiP
93.3 48.0 57.3 57.5 46.4 27.5 18.4
0.73 8.6 5.8 6.2 2.1 5.3 6.6
The limits obtained for others authors were often higher. Pupin and Figueiredo Toledo (1995), have analysed benzo[a]pyrene in oils by liquid–liquid partition, clean-up and HPLC–FL determination The limit of detection obtained was 0.5 lg/kg oil, with a recoveries >76.7% and relative standard deviations <15.0%. Burdaspal et al., 2001 similarly, have found a limit of detection of 0.57 lg/kg oil and different recoveries and RSD depending of range of concentration (89% and 21.9%, and 104% and 4.3% for 0.67 and 2.25 lg/kg, respectively). Menichini et al. (1991a), have analysed PAHs in oils by liquid–liquid extraction, clean-up, and GC–FID have obtained. The limit of detection obtained was 3 lg/kg oil, with a recoveries between 50% and 85% and RSD 4–
18%. The limit of detection obtained by Cejpek, Hajslova, Kocourek, Tomaniova, and Cmolik, 1998, was 2 lg/kg oil by liquid–liquid partition, clean-up and espectrofluorimetry with a recoveries between 75% and 105%. G onzalez Amigo et al. (2000), analyse PAHs in bird tissue by supercritical fluid extraction and HPLC–FL determination have obtained recoveries >90% and relative standard deviations 6 7.7%. Detection and quantitation limits were <0.65 and <1.27 l/l except for Ind. This method is valid for detection and quantification of the PAHs evaluated (except for Ind) by their high detection limit, because the quantification limits obtained (expressed in lg kg1 ) are lower than the legal limits permitted. It can decide if the oil is apt or not for human consumption.
4. Conclusions An extraction/clean-up procedure by SFE was developed for PAHs separation from vegetable oil samples and a HPLC–FL method was optimised for determination of the levels of seven of the eight PAHs with legal limit in these samples: BaA, BeP, BbF, BkF,
Fig. 1. HPLC–FL chromatogram of an extract of sample and spiked oil (programme 1).
Fig. 2. HPLC–FL chromatogram of an extract of sample and spiked oil (programme 2).
M.A. Lage Yusty, J.L. Cortizo Davi~na / Food Control 16 (2005) 59–64
BaP, DBaA and BghiP and to evaluate the edible oil safety.
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