- Email: [email protected]
Control of contamination of olive oil by sunflower seed oil in bottling plants by GC-MS of fatty acid methyl esters
Food Control 14 (2003) 463–467 www.elsevier.com/locate/foodcont
Control of contamination of olive oil by sunflower seed oil in bottling plants by GC-MS of fatty acid methyl esters J. Gamazo-V azquez, M.S. Garcıa-Falc on, J. Simal-G andara
*
Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology, Ourense Campus, University of Vigo, E-32004 Ourense, Spain Received 23 January 2002; received in revised form 19 August 2002; accepted 20 August 2002
Abstract This paper describes a quality control method for the detection of contamination of olive oil by seedoil, such as may occur as the result of carryover in bottling lines, with the aim of diagnosing if cross-contamination has occurred and by how much. The method is based on gas chromatography of the methyl esters of the oil components, with detection by mass spectrometry (GC-MSD). In the example presented (contamination by sunflower seed oil), it was identified the optimal discriminatory parameter, which was the ratio between the peak areas of the oleic and linoleic derivatives in chromatograms obtained using full-scan MS between 35 and 350 amu; this ratio would allow diagnosis of contamination of olive oil by sunflower seed oil at least at the 1% level with >95% certainty in bottling plants. It is important that each bottling plant establish its own reference sample collection and identify its optimal discriminant parameters following the proposed procedures. Ó 2002 Elsevier Ltd. All rights reserved. Keywords: Olive oil; Bottling plant; Contamination by carryover; Fatty acid methyl esters; GC-MSD
1. Introduction Consumer protection agencies must watch for the purity of olive oil, and bottling companies that are respectful of the law must watch for accidental contamination of olive oil by less expensive oils such as seed oils and take care to prevent it. Accidental contamination may, for example, occur due to carryover when olive oil is bottled in a bottling line that has previously been used to bottle seedoil. Much work has been done on the authenticity of olive oil monitoring its adulteration by seed oils (Firestone, Carson, & Reina, 1988; EEC 2568, 1991; Wesley, Barnes, & McGill, 1995; Wesley, Pacheco, & McGill, 1996; Baeten, Meurens, Morales, & Aparicio, 1996; Favier, Bicanic, Cozijnsen, Van Veldhuizen, & Helander, 1998; Marigheto, Kemsley, Defernez, & Wilson, 1998; Storelli & Gambacorta, 1998; Andrikopoulos, Giannakis, & Tzamtzis, 2001). Taking into account that composition of oils varies largely due to species, vari-
eties, growing place, weather conditions. . . these methods are not able to detect adulteration levels lower than 5%. In this paper we are looking for lower levels of cross-contamination in the bottling plant since it is possible to collect oil samples from the line supply tanks and use them as reference oils. Here we describe the chemical and instrumental procedures involved in a sensitive and simple method proposed by us (Sarria-Vidal, de la Monta~ na-Miguelez, & Simal-Gandara, 1997), based on gas chromatography of the methyl esters of the oil components with detection by mass spectrometry, for the detection and quantitation of contamination of olive oil by seedoil with the aim of diagnosing if cross-contamination has occurred in bottling lines, once identified the optimal discriminant parameters.
2. Methods 2.1. Samples and chemicals
*
Corresponding author. Tel.: +34-988-387000; fax: +34-988387001. E-mail address: [email protected] (J. Simal-Gandara). 0956-7135/02/$ - see front matter Ó 2002 Elsevier Ltd. All rights reserved. doi:10.1016/S0956-7135(02)00102-0
Samples of olive oil and sunflower seed oil were collected from the supply tanks of the bottling company ‘‘Aceites Abril’’ (Ourense, Spain). Mixtures containing
464
J. Gamazo-Vazquez et al. / Food Control 14 (2003) 463–467
1%, 5%, 10%, 25% and 50% of sunflower seed oil were made up and homogenized for 5 min in an ultrasonic bath. Pending use, pure samples and homogenized mixtures were kept at 0–4 °C in amber glass bottles with screw-on lids. Boron trifluoride in methanol (12% w/w) and triheptadecanoin (glyceryl trimargarate) were purchased from Sigma-Aldrich (St.Louis, MO, USA). Analytical grade n-heptane, dry methanol (max. water content 0.01%), anhydrous sodium sulphate and potassium hydroxide (85 wt% pure pellets) were supplied by Panreac (Barcelona, Spain). Analytical grade C-50 helium was supplied by Carburos Met alicos (Vigo, Spain).
350 amu (scan time 0.9 s, interscan time 0.1 s); singleion monitoring at m=z 55, 67 and 74 (SIM1; these signals are characteristic of mono-unsaturated, diunsaturated and saturated FAMEs, respectively; see Fig. 1); and single-ion monitoring at m=z 67, 81 and 95 (SIM2; these are three of the main signals of diunsaturated FAMEs such as the methyl ester of linoleic acid; see Fig. 1). The GC-MS apparatus was linked to a PC running software for data acquisition and processing. Under the GC-MS conditions used, FAMEs eluted in order of increasing molecular weight and, for a given molecular weight, in order of decreasing saturation.
2.2. Saponification and derivatization conditions
3. Results and discussion
Samples were saponified and derivatized in one pot, as follows. A solution of about 0.125 g of oil in 50 ml of heptane was made up, and 10 ml of this solution was transferred to a 250 ml round-bottomed flask. After addition of 5 ml of the internal standard solution (3 mg/ ml triheptadecanoin in n-heptane) the mixture was treated with 10 ml of a 0.2 M solution of potassium hydroxide in methanol. A condenser was connected and the mixture was refluxed for 10 min. Methanolic boron trifluoride solution (5 ml) was added through the condenser, and refluxing was continued for a further 5 min. The reaction mixture was allowed to cool to room temperature, saturated with anhydrous sodium sulphate, shaken well, and left until the phases separated. The upper (n-heptane) layer was drawn off, transferred to a small stoppered tube containing solid anhydrous sodium sulphate, and stored in a refrigerator until subjected to GC-MS. These FAME solutions were stable for more than two weeks.
3.1. Selection of FAMEs markers for the detection of olive oil contamination
2.3. GC-MS instrument and analytical conditions Gas chromatography of FAMEs was performed on a 0:20 mm ði:d:Þ 60 m fused silica column lined with a 0.20 lm film of polyethyleneglycol (TR-Wax, from Teknokroma, Barcelona, Spain). Samples (2 lL) were injected in split mode (split/column flow ratio 10:1). The columnhead pressure of the carrier gas (helium) was 200 kPa at the initial oven temperature, and its flow rate 0.8 ml/min. The injection temperature was 225 °C; the oven temperature was 100 °C for 1 min, rose to 250 °C over 15 min and was held at this temperature for 14 min (total run time 30 min). The output from the GC column entered the ionization chamber of the mass spectrometer via an interface tube maintained at 250 °C. MS (EI, 70 eV, ion source temperature 200 °C, solvent delay 13 min) was performed in three modes: full scan between 35 and
Eight samples of each pure oil and each mixture were separately saponified, derivatized and analyzed as above. Every sample was processed only once since any kind of errors during saponification, derivatization and analysis are corrected by doing ratios between FAMEs. To screen for parameters capable of discriminating between pure olive oil and olive oil contaminated by 1% of sunflower seed oil, the eight samples of the pure and 1%contaminated oils were subjected to all three GC-MS modes (full scan, SIM1 and SIM2), and in each case the chromatogram peak areas corresponding to the main FAMEs were measured. Two kinds of potential discriminatory parameters were considered: the ratios between the peak areas of the main oil FAMEs (those of palmitic, stearic, oleic and linoleic acids) and that of the internal standard (heptadecanoic acid), and the ratios between the peak areas of one oil FAME and another (using area ratios rather than the areas themselves corrects for inefficiency and variability in the saponification–derivatization procedure and for chromatographic error). Each potential discriminatory parameter was thus defined by the area ratios and MS mode it involved. For each oil (pure olive oil and the 1% mixture), the distributions of the potential discriminatory parameters were found not to differ significantly from normal (Kolmogorov–Smirnov test, 7 df, p 6 0:05). Parameters were identified as valid discriminatory markers if there was no value of the parameter that was compatible both with the hypothesis that it was the mean of the distribution for the pure oil (as evaluated at the 5% significance level by a two-tailed StudentÕs t test using the data for the pure oil) and with the hypothesis that it was the mean of the distribution for the mixture (as evaluated analogously using the data for the mixture); to this end, symmetric 95% confi-
J. Gamazo-Vazquez et al. / Food Control 14 (2003) 463–467
465
Fig. 1. Full scan mass spectra of (a) a typical monounsaturated FAME (methyl oleate), (b) a typical di-unsaturated FAME (methyl linoleate), and (c) a typical saturated FAME (methyl palmitate).
dence intervals (CIs) were calculated using StudentÕs t distribution with 7 df, and parameters for which the CIs for the pure olive oil and the 1% mixture did not overlap were identified as valid discriminatory markers.
3.2. Selection of FAMEs markers for the quantitation of olive oil contamination The parameters identified as valid discriminatory markers were the ratio of linoleic FAME peak area to
466
J. Gamazo-Vazquez et al. / Food Control 14 (2003) 463–467
Table 1 Symmetric 95% CIs for the parameters defined by MS mode and the ratio of the peak areas of natural oil and triheptadecanoin FAMEs, for pure olive oil and olive oil with 1% of sunflower seed oil Fatty acid
95% CIs Full scan
Palmitic Stearic Oleic Linoleic
SIM1
SIM2
Pure olive oil
Olive:sunflower seed 99:1
Pure olive oil
Olive:sunflower seed 99:1
Pure olive oil
Olive:sunflower seed 99:1
0.119–0.158 0.030–0.040 0.857–1.12 0.045–0.060
0.138–0.147 0–0.180 1.07–1.14 0.064–0.072
0.107–0.147 0.020–0.030 0.477–0.636 0.025–0.035
0.121–0.139 0.022–0.027 0.594–0.652 0.034–0.039
0.112–0.155 0.026–0.033 3.55–4.65 0.515–0.701
0.132–0.156 0.022–0.033 4.38–4.88 0.704–0.819
In bold, CIs of parameters deemed valid for discrimination between the two oils.
Table 2 Symmetric 95% CIs for the parameters defined by MS mode and the ratio of the peak areas of natural oil FAMEs, for pure olive oil and olive oil with 1% of sunflower seed oil Fatty acid ratio
95% CIs Full scan
Palmitic/stearic Palmitic/linoleic Oleic/palmitic Oleic/stearic Oleic/linoleic Linoleic/stearic
SIM1
SIM2
Pure olive oil
Olive:sunflower seed 99:1
Pure olive oil
Olive:sunflower seed 99:1
Pure olive oil
Olive:sunflower seed 99:1
3.82–4.07 2.38–2.91 6.77–7.64 27.1–29.6 17.8–19.9 1.39–1.64
2.50–4.21 1.95–2.26 7.43–8.17 19.4–32.6 15.7–16.9 1.18–1.97
4.37–6.08 3.83–4.59 4.05–4.81 20.3–25.7 17.2–20.1 1.07–1.41
4.39–6.51 3.17–4.04 4.42–5.27 23.1–28.4 16.3–18.0 1.36–1.64
3.50–5.36 0.199–0.240 28.3–34.5 118–153 6.20–7.43 16.8–23.5
4.40–6.90 0.165–0.220 29.0–36.3 139–226 5.83–6.38 23.1–36.5
In bold, CIs of parameters deemed valid for discrimination between the two oils.
heptadecanoic FAME peak area under both full scan and SIM2 MS modes (Table 1), and the ratios of palmitic FAME peak area and oleic FAME peak area to linoleic FAME peak area under full scan mode (Table 2). For the parameters selected, symmetric 95% CIs for all the other mixtures were calculated following GC-MS with the appropriate MS mode (the normality of the corresponding distributions was checked as before, and for each parameter the variances of the various distributions were shown not to be significantly inhomoge-
neous). Parameters for which there was no overlap among the CIs of the various oils were considered suitable for quantitation of contamination. The two linoleic/heptadecanoic ratios and the oleic/linoleic ratio were identified as suitable for quantitation of contamination (Tables 3 and 4). Of these, the oleic/linoleic ratio may be regarded as the best marker because its determination requires no internal standard and is therefore not subject to errors in the weighing of standard and sample.
Table 3 Symmetric 95% CIs for the parameters defined by the ratio of the peak areas of linoleic and triheptadecanoin FAMES with full scan or SIM2 MS mode, for pure olive oil, pure sunflower seed oil, and each mixture of the two
Table 4 Symmetric 95% CIs for the parameters defined by the ratio of the peak areas of palmitic and oleic FAMEs to linoleic FAME under full scan MS mode, for pure olive oil, pure sunflower seed oil, and each mixture of the two
Oil (Olive:sunflower seed) Olive oil 99:1 95:5 90:10 75:25 50:50 Sunflower seed oil
95% CIs
Oil (olive:sunflower seed)
Full scan
SIM2
0.045–0.060 0.064–0.072 0.083–0.095 0.116–0.123 0.142–0.268 0.270–0.336 0.627–0.886
0.515–0.701 0.704–0.819 0.993–1.11 1.38–1.47 1.73–3.16 3.75–4.03 9.19–10.8
In bold, CIs of parameters deemed valid for quantification of contamination.
Olive oil 99:1 95:5 90:10 75:25 50:50 Sunflower seed oil
95% CIs Palmitic/linoleic
Oleic/linoleic
2.91–2.38 2.26–1.95 1.86–1.43 1.75–1.16 1.05–0.042 0.398–0.381 0.164–0.085
19.9–17.8 16.9–15.7 12.4–11.7 8.95–8.26 6.17–3.37 3.01–1.70 0.522–0.412
In bold, CIs of parameters deemed valid for quantification of contamination.
J. Gamazo-Vazquez et al. / Food Control 14 (2003) 463–467
4. Conclusion The above results show that the proposed method, based on saponification, derivatization and GC-MS of FAMEs, can be used in bottling plants to discriminate with good efficiency between pure olive oil and olive oil contaminated by a seed oil at the 1% level.
Acknowledgement This work was supported by ‘‘Aceites Abril’’ (Ourense, Spain).
References Andrikopoulos, N. K., Giannakis, I. G., & Tzamtzis, V. (2001). Analysis of olive oil and seed oil triglycerides by capillary gas chromatography as a tool for the detection of the adulteration of olive oil. Journal of Chromatographic Science, 39(4), 137–145. Baeten, V., Meurens, M., Morales, M. T., & Aparicio, R. (1996). Detection of virgin olive oil adulteration by Fourier transform Raman spectroscopy. Journal of Agricultural and Food Chemistry, 44, 2225–2230.
467
EEC 2568 (1991). Commission Regulation (2568/91/EEC) of 11th July (1991), on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis. Favier, J. P., Bicanic, D., Cozijnsen, J., Van Veldhuizen, B., & Helander, P. (1998). CO2 laser infrared optothermal spectroscopy for quantitative adulteration studies in binary mixtures of extra virgin olive oil. Journal of the American Oil ChemistsÕ Society, 75, 359–362. Firestone, D., Carson, K. L., & Reina, R. J. (1988). Update on control of olive oil adulteration and misbranding in the United States. Journal of the American Oil ChemistsÕ Society, 65, 788–792. Marigheto, N. A., Kemsley, E. K., Defernez, M., & Wilson, R. H. (1998). A comparison of mid-infrared and Raman spectroscopies for the authentication of edible oils. Journal of the American Oil ChemistsÕ Society, 75, 987–992. Sarria-Vidal, M. A., de la Monta~ na-Miguelez, J., & Simal-Gandara, J. (1997). Toward a test of overall migration from the coated face of a recycled paperboard food contact material into fatty food simulants. Journal of Agricultural and Food Chemistry, 45, 2701–2707. Storelli, M., & Gambacorta, G. (1998). Analisi di Identita di Oli di Oliva: Approccio Metodologico. La Rivista Italiana delle Sostanze Gras, LXXV, 383–389. Wesley, I. J., Barnes, R. J., & McGill, A. E. J. (1995). Measurement of adulteration of olive oils by near-infrared spectroscopy. Journal of the American Oil ChemistsÕ Society, 72, 289–292. Wesley, I. J., Pacheco, F., & McGill, A. E. J. (1996). Identification of adulterants in olive oils. Journal of the American Oil ChemistsÕ Society, 73, 515–518.
Control of contamination of olive oil by sunflower seed oil in bottling plants by GC-MS of fatty acid methyl esters J. Gamazo-V azquez, M.S. Garcıa-Falc on, J. Simal-G andara
*
Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology, Ourense Campus, University of Vigo, E-32004 Ourense, Spain Received 23 January 2002; received in revised form 19 August 2002; accepted 20 August 2002
Abstract This paper describes a quality control method for the detection of contamination of olive oil by seedoil, such as may occur as the result of carryover in bottling lines, with the aim of diagnosing if cross-contamination has occurred and by how much. The method is based on gas chromatography of the methyl esters of the oil components, with detection by mass spectrometry (GC-MSD). In the example presented (contamination by sunflower seed oil), it was identified the optimal discriminatory parameter, which was the ratio between the peak areas of the oleic and linoleic derivatives in chromatograms obtained using full-scan MS between 35 and 350 amu; this ratio would allow diagnosis of contamination of olive oil by sunflower seed oil at least at the 1% level with >95% certainty in bottling plants. It is important that each bottling plant establish its own reference sample collection and identify its optimal discriminant parameters following the proposed procedures. Ó 2002 Elsevier Ltd. All rights reserved. Keywords: Olive oil; Bottling plant; Contamination by carryover; Fatty acid methyl esters; GC-MSD
1. Introduction Consumer protection agencies must watch for the purity of olive oil, and bottling companies that are respectful of the law must watch for accidental contamination of olive oil by less expensive oils such as seed oils and take care to prevent it. Accidental contamination may, for example, occur due to carryover when olive oil is bottled in a bottling line that has previously been used to bottle seedoil. Much work has been done on the authenticity of olive oil monitoring its adulteration by seed oils (Firestone, Carson, & Reina, 1988; EEC 2568, 1991; Wesley, Barnes, & McGill, 1995; Wesley, Pacheco, & McGill, 1996; Baeten, Meurens, Morales, & Aparicio, 1996; Favier, Bicanic, Cozijnsen, Van Veldhuizen, & Helander, 1998; Marigheto, Kemsley, Defernez, & Wilson, 1998; Storelli & Gambacorta, 1998; Andrikopoulos, Giannakis, & Tzamtzis, 2001). Taking into account that composition of oils varies largely due to species, vari-
eties, growing place, weather conditions. . . these methods are not able to detect adulteration levels lower than 5%. In this paper we are looking for lower levels of cross-contamination in the bottling plant since it is possible to collect oil samples from the line supply tanks and use them as reference oils. Here we describe the chemical and instrumental procedures involved in a sensitive and simple method proposed by us (Sarria-Vidal, de la Monta~ na-Miguelez, & Simal-Gandara, 1997), based on gas chromatography of the methyl esters of the oil components with detection by mass spectrometry, for the detection and quantitation of contamination of olive oil by seedoil with the aim of diagnosing if cross-contamination has occurred in bottling lines, once identified the optimal discriminant parameters.
2. Methods 2.1. Samples and chemicals
*
Corresponding author. Tel.: +34-988-387000; fax: +34-988387001. E-mail address: [email protected] (J. Simal-Gandara). 0956-7135/02/$ - see front matter Ó 2002 Elsevier Ltd. All rights reserved. doi:10.1016/S0956-7135(02)00102-0
Samples of olive oil and sunflower seed oil were collected from the supply tanks of the bottling company ‘‘Aceites Abril’’ (Ourense, Spain). Mixtures containing
464
J. Gamazo-Vazquez et al. / Food Control 14 (2003) 463–467
1%, 5%, 10%, 25% and 50% of sunflower seed oil were made up and homogenized for 5 min in an ultrasonic bath. Pending use, pure samples and homogenized mixtures were kept at 0–4 °C in amber glass bottles with screw-on lids. Boron trifluoride in methanol (12% w/w) and triheptadecanoin (glyceryl trimargarate) were purchased from Sigma-Aldrich (St.Louis, MO, USA). Analytical grade n-heptane, dry methanol (max. water content 0.01%), anhydrous sodium sulphate and potassium hydroxide (85 wt% pure pellets) were supplied by Panreac (Barcelona, Spain). Analytical grade C-50 helium was supplied by Carburos Met alicos (Vigo, Spain).
350 amu (scan time 0.9 s, interscan time 0.1 s); singleion monitoring at m=z 55, 67 and 74 (SIM1; these signals are characteristic of mono-unsaturated, diunsaturated and saturated FAMEs, respectively; see Fig. 1); and single-ion monitoring at m=z 67, 81 and 95 (SIM2; these are three of the main signals of diunsaturated FAMEs such as the methyl ester of linoleic acid; see Fig. 1). The GC-MS apparatus was linked to a PC running software for data acquisition and processing. Under the GC-MS conditions used, FAMEs eluted in order of increasing molecular weight and, for a given molecular weight, in order of decreasing saturation.
2.2. Saponification and derivatization conditions
3. Results and discussion
Samples were saponified and derivatized in one pot, as follows. A solution of about 0.125 g of oil in 50 ml of heptane was made up, and 10 ml of this solution was transferred to a 250 ml round-bottomed flask. After addition of 5 ml of the internal standard solution (3 mg/ ml triheptadecanoin in n-heptane) the mixture was treated with 10 ml of a 0.2 M solution of potassium hydroxide in methanol. A condenser was connected and the mixture was refluxed for 10 min. Methanolic boron trifluoride solution (5 ml) was added through the condenser, and refluxing was continued for a further 5 min. The reaction mixture was allowed to cool to room temperature, saturated with anhydrous sodium sulphate, shaken well, and left until the phases separated. The upper (n-heptane) layer was drawn off, transferred to a small stoppered tube containing solid anhydrous sodium sulphate, and stored in a refrigerator until subjected to GC-MS. These FAME solutions were stable for more than two weeks.
3.1. Selection of FAMEs markers for the detection of olive oil contamination
2.3. GC-MS instrument and analytical conditions Gas chromatography of FAMEs was performed on a 0:20 mm ði:d:Þ 60 m fused silica column lined with a 0.20 lm film of polyethyleneglycol (TR-Wax, from Teknokroma, Barcelona, Spain). Samples (2 lL) were injected in split mode (split/column flow ratio 10:1). The columnhead pressure of the carrier gas (helium) was 200 kPa at the initial oven temperature, and its flow rate 0.8 ml/min. The injection temperature was 225 °C; the oven temperature was 100 °C for 1 min, rose to 250 °C over 15 min and was held at this temperature for 14 min (total run time 30 min). The output from the GC column entered the ionization chamber of the mass spectrometer via an interface tube maintained at 250 °C. MS (EI, 70 eV, ion source temperature 200 °C, solvent delay 13 min) was performed in three modes: full scan between 35 and
Eight samples of each pure oil and each mixture were separately saponified, derivatized and analyzed as above. Every sample was processed only once since any kind of errors during saponification, derivatization and analysis are corrected by doing ratios between FAMEs. To screen for parameters capable of discriminating between pure olive oil and olive oil contaminated by 1% of sunflower seed oil, the eight samples of the pure and 1%contaminated oils were subjected to all three GC-MS modes (full scan, SIM1 and SIM2), and in each case the chromatogram peak areas corresponding to the main FAMEs were measured. Two kinds of potential discriminatory parameters were considered: the ratios between the peak areas of the main oil FAMEs (those of palmitic, stearic, oleic and linoleic acids) and that of the internal standard (heptadecanoic acid), and the ratios between the peak areas of one oil FAME and another (using area ratios rather than the areas themselves corrects for inefficiency and variability in the saponification–derivatization procedure and for chromatographic error). Each potential discriminatory parameter was thus defined by the area ratios and MS mode it involved. For each oil (pure olive oil and the 1% mixture), the distributions of the potential discriminatory parameters were found not to differ significantly from normal (Kolmogorov–Smirnov test, 7 df, p 6 0:05). Parameters were identified as valid discriminatory markers if there was no value of the parameter that was compatible both with the hypothesis that it was the mean of the distribution for the pure oil (as evaluated at the 5% significance level by a two-tailed StudentÕs t test using the data for the pure oil) and with the hypothesis that it was the mean of the distribution for the mixture (as evaluated analogously using the data for the mixture); to this end, symmetric 95% confi-
J. Gamazo-Vazquez et al. / Food Control 14 (2003) 463–467
465
Fig. 1. Full scan mass spectra of (a) a typical monounsaturated FAME (methyl oleate), (b) a typical di-unsaturated FAME (methyl linoleate), and (c) a typical saturated FAME (methyl palmitate).
dence intervals (CIs) were calculated using StudentÕs t distribution with 7 df, and parameters for which the CIs for the pure olive oil and the 1% mixture did not overlap were identified as valid discriminatory markers.
3.2. Selection of FAMEs markers for the quantitation of olive oil contamination The parameters identified as valid discriminatory markers were the ratio of linoleic FAME peak area to
466
J. Gamazo-Vazquez et al. / Food Control 14 (2003) 463–467
Table 1 Symmetric 95% CIs for the parameters defined by MS mode and the ratio of the peak areas of natural oil and triheptadecanoin FAMEs, for pure olive oil and olive oil with 1% of sunflower seed oil Fatty acid
95% CIs Full scan
Palmitic Stearic Oleic Linoleic
SIM1
SIM2
Pure olive oil
Olive:sunflower seed 99:1
Pure olive oil
Olive:sunflower seed 99:1
Pure olive oil
Olive:sunflower seed 99:1
0.119–0.158 0.030–0.040 0.857–1.12 0.045–0.060
0.138–0.147 0–0.180 1.07–1.14 0.064–0.072
0.107–0.147 0.020–0.030 0.477–0.636 0.025–0.035
0.121–0.139 0.022–0.027 0.594–0.652 0.034–0.039
0.112–0.155 0.026–0.033 3.55–4.65 0.515–0.701
0.132–0.156 0.022–0.033 4.38–4.88 0.704–0.819
In bold, CIs of parameters deemed valid for discrimination between the two oils.
Table 2 Symmetric 95% CIs for the parameters defined by MS mode and the ratio of the peak areas of natural oil FAMEs, for pure olive oil and olive oil with 1% of sunflower seed oil Fatty acid ratio
95% CIs Full scan
Palmitic/stearic Palmitic/linoleic Oleic/palmitic Oleic/stearic Oleic/linoleic Linoleic/stearic
SIM1
SIM2
Pure olive oil
Olive:sunflower seed 99:1
Pure olive oil
Olive:sunflower seed 99:1
Pure olive oil
Olive:sunflower seed 99:1
3.82–4.07 2.38–2.91 6.77–7.64 27.1–29.6 17.8–19.9 1.39–1.64
2.50–4.21 1.95–2.26 7.43–8.17 19.4–32.6 15.7–16.9 1.18–1.97
4.37–6.08 3.83–4.59 4.05–4.81 20.3–25.7 17.2–20.1 1.07–1.41
4.39–6.51 3.17–4.04 4.42–5.27 23.1–28.4 16.3–18.0 1.36–1.64
3.50–5.36 0.199–0.240 28.3–34.5 118–153 6.20–7.43 16.8–23.5
4.40–6.90 0.165–0.220 29.0–36.3 139–226 5.83–6.38 23.1–36.5
In bold, CIs of parameters deemed valid for discrimination between the two oils.
heptadecanoic FAME peak area under both full scan and SIM2 MS modes (Table 1), and the ratios of palmitic FAME peak area and oleic FAME peak area to linoleic FAME peak area under full scan mode (Table 2). For the parameters selected, symmetric 95% CIs for all the other mixtures were calculated following GC-MS with the appropriate MS mode (the normality of the corresponding distributions was checked as before, and for each parameter the variances of the various distributions were shown not to be significantly inhomoge-
neous). Parameters for which there was no overlap among the CIs of the various oils were considered suitable for quantitation of contamination. The two linoleic/heptadecanoic ratios and the oleic/linoleic ratio were identified as suitable for quantitation of contamination (Tables 3 and 4). Of these, the oleic/linoleic ratio may be regarded as the best marker because its determination requires no internal standard and is therefore not subject to errors in the weighing of standard and sample.
Table 3 Symmetric 95% CIs for the parameters defined by the ratio of the peak areas of linoleic and triheptadecanoin FAMES with full scan or SIM2 MS mode, for pure olive oil, pure sunflower seed oil, and each mixture of the two
Table 4 Symmetric 95% CIs for the parameters defined by the ratio of the peak areas of palmitic and oleic FAMEs to linoleic FAME under full scan MS mode, for pure olive oil, pure sunflower seed oil, and each mixture of the two
Oil (Olive:sunflower seed) Olive oil 99:1 95:5 90:10 75:25 50:50 Sunflower seed oil
95% CIs
Oil (olive:sunflower seed)
Full scan
SIM2
0.045–0.060 0.064–0.072 0.083–0.095 0.116–0.123 0.142–0.268 0.270–0.336 0.627–0.886
0.515–0.701 0.704–0.819 0.993–1.11 1.38–1.47 1.73–3.16 3.75–4.03 9.19–10.8
In bold, CIs of parameters deemed valid for quantification of contamination.
Olive oil 99:1 95:5 90:10 75:25 50:50 Sunflower seed oil
95% CIs Palmitic/linoleic
Oleic/linoleic
2.91–2.38 2.26–1.95 1.86–1.43 1.75–1.16 1.05–0.042 0.398–0.381 0.164–0.085
19.9–17.8 16.9–15.7 12.4–11.7 8.95–8.26 6.17–3.37 3.01–1.70 0.522–0.412
In bold, CIs of parameters deemed valid for quantification of contamination.
J. Gamazo-Vazquez et al. / Food Control 14 (2003) 463–467
4. Conclusion The above results show that the proposed method, based on saponification, derivatization and GC-MS of FAMEs, can be used in bottling plants to discriminate with good efficiency between pure olive oil and olive oil contaminated by a seed oil at the 1% level.
Acknowledgement This work was supported by ‘‘Aceites Abril’’ (Ourense, Spain).
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