J. Agric. Food Chem. 2004, 52, 2123−2132
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Polycyclic Aromatic Hydrocarbons and Olive Pomace Oil MARIÄA D. GUILLEÄ N,* PATRICIA SOPELANA,
AND
GEMMA PALENCIA
Tecnologı´a de Alimentos, Facultad de Farmacia, Universidad del Paı´s Vasco, Paseo de la Universidad No. 7, 01006 Vitoria, Spain
The occurrence of polycyclic aromatic hydrocarbons (PAHs) in five samples of olive pomace oil has been studied to determine the contamination degree of this type of oil and to evaluate if specific purification steps must be introduced during its manufacture. The PAHs present have been determined by gas chromatography-mass spectrometry. A high number of PAHs, with a wide range of molecular weights and in very high concentrations, have been found in four of the samples studied. A very high number of alkyl derivatives and, in many cases, in higher concentrations than their respective parent PAHs, have also been identified. One of the samples, however, presents a more reduced number of PAHs and in significantly lower concentrations than the others. These findings reveal that it is necessary to introduce adequate cleanup steps in the manufacturing process of olive pomace oil, which can give rise to oils with a relatively low content of PAHs. Some carcinogenic PAHs have also been identified, both unalkylated and alkylated. KEYWORDS: Polycyclic aromatic hydrocarbons; olive pomace oil; gas chromatography; mass spectrometry
INTRODUCTION
The presence of polycyclic aromatic hydrocarbons (PAHs) in a wide variety of vegetable oils has been revealed by the studies of many authors (1-9). The occurrence of PAHs in edible oils is attributed mainly to environmental contamination of the vegetable raw material and to contamination coming from some operations carried out during their processing, such as seed drying or solvent extraction, although some authors indicate that the endogenous origin of some PAHs cannot be totally discarded (5, 10). Among all of the vegetable oils, olive oil is highly appreciated not only for its excellent organoleptic and nutritious characteristics but also for its known healthy properties (11). For these reasons, its consumption is increasing continuously. Different types of oils can be obtained from the fruit of the olive tree: virgin olive oil, obtained solely by mechanical or other physical means under conditions that do not lead to alterations in the oil and which has not undergone any treatment other than washing, decantation, centrifugation and filtration; refined olive oil, obtained from virgin olive oils by refining methods that do not lead to alterations in the initial glyceridic structure; olive oil, which is a blend of refined and virgin olive oil fit for consumption as it is; and olive pomace oil, which is obtained by treating the solid residue remaining after the extraction of olive oil (olive pomace) with solvents, excluding oils obtained from re-esterification processes and from any mixture of oils with other properties. * Corresponding author (telephone 34-45-013081; fax 34-45-013014; e-mail
[email protected]).
Olive pomace oil contains proportions of the different acyl groups similar to those in olive and in virgin olive oils, slightly lower in oleic and higher in linoleic and saturated acyl groups (12); however, even though olive pomace is a rich source of antioxidants (13), its oil is slightly poorer than virgin olive oil in natural antioxidant components, probably due to their elimination during refining (14). It must also be noted that the manufacturing processes of each type of oil derived from the olives are different, to such an extent that it can determine the PAH contamination level of the final product. The influence of the manufacturing process on PAH contamination is clear in the case of some types of vegetable oils, such as coconut oil, where very high concentrations of this type of contaminant have been found in the crude product (without refining), which can also appear in the refined oil if no specific cleanup stage is carried out during its processing (5, 15). A wide range of PAH concentrations have been found in olive oils and in virgin olive oils (1, 3, 4, 6, 8, 9, 16, 17); however, little attention has been paid to olive pomace oil, which is usually employed in the frying or cooking of foods and also in canned food. To the best of our knowledge, only two papers have been published on the occurrence of PAHs in this type of oil (9, 18); moreover, they refer to only a reduced number of these toxic contaminants. It must be pointed out that until now the European Union has not established legal limits for PAHs in edible oils. However, in Spain, a legal disposition was put into effect in July 2001 (19) limiting the concentrations of eight PAHs in olive pomace oils: benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[e]pyrene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene, and benzo[ghi]perylene; a similar
10.1021/jf035259q CCC: $27.50 © 2004 American Chemical Society Published on Web 03/12/2004
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approach was put into practice in Italy (20). Previously, DGF had proposed 25 µg/kg as a limit for total PAHs and 5 µg/kg for the sum of heavy PAHs (10) and, recently, the Canadian Food Inspection Agency has suggested correcting results on the basis of toxic equivalency factors (TEFs) (21). Due to the few studies on the presence of PAHs in olive pomace oil, in this paper some samples of this oil have been studied. The study has been carried out following a classical scheme for PAH isolation and subsequent separation, identification, and quantification by gas chromatography-mass spectrometry (GC-MS) operating in scan and in selective ion monitoring (SIM) modes. The aim of this work is to study those PAHs present in the samples selected and capable of determination by GC-MS, to determine the degree of PAH contamination of olive pomace oil, and to assess the need to introduce adequate cleanup steps for PAHs during the manufacture of this oil. MATERIALS AND METHODS Samples. The samples are five different commercial olive pomace oils, designated OP1, OP2, OP3, OP4, and OP5. Samples OP1-OP4 were acquired prior to the year 2001, before the Spanish order of July 25th (19), and OP5 was acquired during the year 2002, after the previous order. These olive pomace oils are blends of refined olive pomace oil and virgin olive oil; the latter is added in a small proportion to improve the organoleptic properties of refined olive pomace oil. Each sample was studied in duplicate. Reagents and Materials. The solvents employed were n-hexane for analyses, dimethyl sulfoxide (DMSO) for spectroscopy, cyclohexane and methanol, both of HPLC grade (99.9+%), and dichloromethane (99.8%). Other reagents and materials used were potassium hydroxide, sodium chloride, anhydrous sodium sulfate, and Supelclean LC-Si solid phase extraction (SPE) tubes, 3 mL (500 mg). All solvents, reagents, and materials mentioned are commercially available from Riedel-de Hae¨n (Seelze, Germany), Merck (Darmstadt, Germany), Aldrich (Steinheim, Germany), Panreac (Barcelona, Spain), Symta (Madrid, Spain), and Supelco (Bellefonte, PA). Four groups of PAH standards were used for the identification and quantification of the PAHs present in the samples: (1) A commercial mix of PAHs dissolved in a mixture of dichloromethane/benzene (75:25), contained naphthalene, acenaphthene, acenaphthylene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[c]phenanthrene, benz[a]anthracene, chrysene, 7,12dimethylbenz[a]anthracene, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, 3-methylcholanthrene, indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene, benzo[ghi]perylene, dibenzo[a,l]pyrene, dibenzo[a,i]pyrene, and dibenzo[a,h]pyrene, in concentrations of ∼500 µg/mL. This mix was obtained from Supelco (Bellefonte, PA). (2) Commercial individual cyclohexane solutions of 1,7-dimethylnaphthalene, 1,4-dimethylnaphthalene, 1,5-dimethylnaphthalene, 1-methylphenanthrene, 3,6-dimethylphenanthrene, 2,3-dimethylanthracene, 9,10-dimethylphenanthrene, 2-methylfluoranthene, 1-methylfluoranthene, 11H-benzo[c]fluorene, 1-methylpyrene, 6-methylbenz[a]anthracene, 7-methylbenz[a]anthracene, 3-methylchrysene, 2-methylchrysene, 5-methylchrysene, 4-methylchrysene, 6-methylchrysene, 1-methylchrysene, dibenz[a,j]anthracene, benzo[b]chrysene, picene, anthanthrene, coronene, and dibenzo[a,e]pyrene were in concentrations of ∼10 µg/ mL. All of these solutions were purchased from Symta (Madrid, Spain). (3) Pure PAHs included 1,6-dimethylnaphthalene, 2,6-dimethylnaphthalene, o-terphenyl, 2-methylanthracene, 9-methylanthracene, m-terphenyl, 11H-benzo[a]fluorene, 11H-benzo[b]fluorene, benzo[e]pyrene, and perylene, all of which were obtained from Sigma-Aldrich (Steinheim, Germany); 2,3-dimethylnaphthalene, from Symta (Madrid, Spain); and p-terphenyl, from Merck (Darmstadt, Germany). All of these standards were used to prepare different solutions in dichloromethane or cyclohexane. (4) Naphthalene-d8, acenaphthene-d10, phenanthrene-d10, pyrene-d10, p-terphenyl-d14, chrysene-d12, and perylene-d12 were used as internal
Guille´n et al. standards. These were acquired as pure compounds from Supelco (Bellefonte, PA) except for pyrene-d10, which was purchased from Symta (Madrid, Spain), and different solutions containing a mixture of them were prepared in dichloromethane or cyclohexane. It must be pointed out that some of the PAH standards abovementioned are cancer suspect agents, so precautions must be taken in the handling of these compounds. The purity of all pure PAH standards ranged from 97 to 99.5%. Methodology. Before the treatment of the samples for the determination of PAHs is begun, two aspects related to the procedure must be noted. First, it must be guaranteed that all of the glassware is free of PAHs. For this purpose, it is recommended that all the glass be cleaned with dichloromethane, several times, in an ultrasonic bath, concentrating the washing solvent and analyzing the concentrate by GC-MS in SIM mode to check for the absence of residual contamination. Second, the purity of the solvents employed should be carefully monitored in order to avoid the incorporation of impurities and even of additional PAHs to the samples of study. The scheme of the methodology employed for the study of the oils is shown in Figure 1. The extraction of PAHs from the oil samples was carried out by taking as the starting point the liquid-liquid partition scheme with DMSO described by Natusch and Tomkins (22); it must be pointed out that the ratio of 2 volumes of water to 1 volume of DMSO employed for these authors was changed to a ratio of water/DMSO of 2.4/1, because a better separation of phases in the subsequent extraction of PAHs with cyclohexane was observed with the latter proportion. This same ratio has also been used by Garcı´a Falco´n et al. (23) in the determination of benzo[a]pyrene in lipid soluble liquid smoke. The cleanup procedure by SPE silica tubes was performed according to that in a previous paper (24). Samples OP3 and OP4 were subjected to an alkaline treatment after the whole procedure, because their analyses by GC-MS in scan mode showed that they contained fatty acids. For this purpose, ∼11.2 g of potassium hydroxide dissolved in 100 mL of a mixture of methanol and distilled water (9:1 v/v) and boiling chips were added to the previous samples, and the whole mixture was refluxed for 4 h. The resulting mixture was diluted with 100 mL of methanol/water (8:2, v/v), and PAHs were again extracted with cyclohexane. This extract was concentrated to a smaller volume in a rotary evaporator at 40 °C, dried over anhydrous sodium sulfate, and concentrated to 1 mL in a rotary evaporator first and then under a nitrogen stream. This second extract was injected directly into the gas chromatograph. When the duplicates of the samples needing alkaline treatment were performed, this step was included after the extraction of PAHs with cyclohexane. The identification and quantification of PAHs was carried out with a Hewlett-Packard gas chromatograph model HP 6890 series, equipped with a mass selective detector 5973 and a Hewlett-Packard Vectra XM series 4 computer. The column used was a fused-silica capillary column (60 m long × 0.25 mm i.d. × 0.25 µm film thickness), coated with a nonpolar stationary phase (HP-5MS, 5% phenyl methyl siloxane). The operation conditions were the following: the oven temperature was set initially at 50 °C (0.50 min hold), increased to 130 °C at 8 °C/min and again increased to 290 °C at a rate of 5 °C/min (50 min hold); the temperatures of the ion source and the quadrupole mass analyzer were kept at 230 and 150 °C, respectively; helium with a purity of 99.999% was used as carrier gas at a constant flow of 1.0 mL/min; injector and transference line temperatures were held at 290 and 300 °C, respectively; pulsed splitless mode was used for injection with a pressure pulse of 30 psi, and 1 µL of each sample was introduced into the gas chromatograph. The data acquisition modes employed were scan and SIM. Scan mode was used to determine the type of compounds present in the samples, whereas SIM was used to identify and quantify the PAHs present. Identification of the compounds was based on their retention times and on the relative abundances of the specific ions selected for each PAH. Quantification in SIM mode is based on the measurement of the peak area corresponding to the most abundant ion of each compound and was carried out by the internal standard quantification method. For this purpose, a calibrant solution was prepared with the different PAH standards previously mentioned and was spiked with the same
PAHs and Olive Pomace Oil
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Figure 1. Scheme of the methodology employed for the study of the olive pomace oil samples. internal standard solution as the one used to spike the samples. Naphthalene-d8 was used for quantification of naphthalene and its methyl derivatives; acenaphthene-d10 for acenaphthylene and acenaphthene; phenanthrene-d10 for phenanthrene, anthracene, and their methyl derivatives; pyrene-d10 for fluoranthene and pyrene; p-terphenyl-d14 for m-terphenyl, p-terphenyl, benzofluorenes, and methylfluoranthenes/ pyrenes; chrysene-d12 for benz[a]anthracene, chrysene, and their methyl derivatives; and, finally, perylene-d12 for PAHs with higher molecular weights. The response factors of each compound, relative to the internal standard chosen for its quantification, were calculated for each sample. The response of the detector in SIM mode to different concentrations of analytes is linear within 4 magnitude orders (∼0.01-100 ng), with a correlation coefficient >0.99. RESULTS AND DISCUSSION
In relation to the methodology, it is worthwhile mentioning that every procedure for the determination of any contaminant in low concentrations, as is usually the case of PAHs, should aim to obtain extracts as free as possible from compounds which can hinder the identification and quantification of the analytes of interest. Therefore, given that obtaining of clean extracts is often a difficult task in the study of complex matrices such as foodstuffs, the incorporation of additional interfering compounds at any stage of the process should be avoided or minimized.
As has already been mentioned, it is necessary to emphasize the importance of the purity of the solvents employed for the extraction of PAHs because, given the large volumes often employed in these methodologies and the concentration degree in the final extracts, they can represent a significant source of interference. Nor must it be forgotten that many organic solvents are derived from coal tar or petroleum, materials very rich in PAHs, so the potential presence of PAHs should also be considered (25). However, as far as we know, there are very few studies dealing with the determination of PAHs in commercial solvents (26), and very little work has been done in the past decades. To verify if the solvents used for the determination of PAHs in oils contained PAHs, it was decided to study two commercial brands of hexane (H1 and H2) and two of cyclohexane (CH1 and CH2). Moreover, in the case of cyclohexane, samples from two bottles of the same brand were also taken (designated A and B), to determine if there were differences from one to another. The results obtained, expressed as micrograms per liter, are presented in Table 1. It can be observed that there are great differences in the PAH contents of the solvents studied, not only between solvents but also between brands of the same solvent, even though these differences are more noticeable in the case of hexane. Thus,
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Table 1. PAHs Identified in Different Commercial Brands of n-Hexane (H1 and H2) and Cyclohexane (CH1 and CH2), and in Different Bottles of the
Same Brand of Cyclohexane (A and B), and Their Concentrations, in Micrograms per Liter, Expressed as Mean Value ± Standard Deviation PAH
H1
H2
CH1-A
CH1-B
CH2-A
CH2-B
naphthalene 2-methylnaphthalene 1-methylnaphthalene 2,6-dimethylnaphthalene 1,7-dimethylnaphthalene 1,6-dimethylnaphthalene 1,5-dimethylnaphthalene fluorene phenanthrene 3-methylphenanthrene 2-methylphenanthrene 9-methylphenanthrene 1-methylphenanthrene dimethylphenanthrene or isomer 1 dimethylphenanthrene or isomer 2 dimethylphenanthrene or isomer 3 dimethylphenanthrene or isomer 5 dimethylphenanthrene or isomer 6 dimethylphenanthrene or isomer 7 dimethylphenanthrene or isomer 8 dimethylphenanthrene or isomer 9 dimethylphenanthrene or isomer 10 fluoranthene pyrene 1-MFtc + 11H-benzo[a]fluorene methylfluoranthene or isomer 4 methylfluoranthene or isomer 5 1-methylpyrene o-terphenyl m-terphenyl p-terphenyl methylchrysene or isomer 3 2-methylchrysene
0.27 ± 0.02 −a 0.05 ± 0.01 − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −
3.33 ± 0.06 5.09 ± 0.05 4.17 ± 0.07 11.16 ± 0.09 13.44 ± 0.82 11.49 ± 0.14 2.85 ± 0.02 1.34 ± 0.01 2.92 ± 0.04 3.17 ± 0.04 4.26 ± 0.04 4.14 ± 0.02 2.30 ± 0.00 1.38 ± 0.06 1.58 ± 0.00 1.20 ± 0.06 6.13 ± 0.04 2.77 ± 0.00 1.61 ± 0.04 0.84 ± 0.02 1.10 ± 0.02 0.33 ± 0.01 − 0.20 ± 0.02 0.19 ± 0.00 0.14 ± 0.00 0.27 ± 0.01 0.14 ± 0.00 − − − 0.19 ± 0.01 0.08 ± 0.00
0.09 ± 0.01 0.01 ± 0.00 0.01 ± 0.00
