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Comparative investigation of irradiated meat by the methods of electron paramagnetic resonance and gas chromatography
Spectrochimica Acta Part A 54 (1998) 2421 – 2426
Comparative investigation of irradiated meat by the methods of electron paramagnetic resonance and gas chromatography N.D. Yordanov a,*, V. Gancheva a, R. Tarandjiiska b, R. Velikova b, L. Kulieva b, B. Damyanova b, S. Popov b b
a Institute of Catalysis, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Abstract A comparative study of irradiated pork meat containing bone was made by the methods of electron paramagnetic resonance (EPR) and gas chromatography (GC). In this investigation EPR has the advantage to be a very fast and unambiguous method even in the cases of thermal treatment of bones. On the other hand, GC analysis is a time consuming procedure however, it becomes very valuable for meat samples that contain no bones. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Irradiation; Meat; Electron paramagnetic resonance; Gas chromatography
1. Introduction The g-irradiation treatment of meat is recommended as one of the most simple, clean, convenient and useful methods of sterilization and therefore of prolongation of the storage period. On the other hand, according to the requirements of the sanitary control this manipulation must be unambiguously identified. Approaches based on electron paramagnetic resonance (EPR) spectroscopy and gas-chromatography (GC) and/or GC-mass spectrometry (GC-MS) are internation-
* Corresponding author. Tel.: +359-2-724917; fax: +3592-756116; e-mail: [email protected].
ally recognized and recommended for this purpose [1–4]. Investigations of irradiated meat have shown that EPR is a very promising method if the sample contains bone. Radiation induces defects in the bone which are extremely stable with time and very easily detected [5,6]. Irradiated samples show an asymmetric signal due to hydroxyapatite free radicals generated in the bone and the appearance of this signal is unambiguous indication of irradiation treatment. The GC method is based on the changes in the triacylglycerol molecules upon irradiation [7]. Radiation causes cleavage of the triacylglycerol molecules resulting in the formation of Cn − 1 and Cn − 2:1 hydrocarbons (HC) from the respective Cn
1386-1425/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 1 4 2 5 ( 9 8 ) 0 0 2 2 2 - 4
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N.D. Yordano6 et al. / Spectrochimica Acta Part A 54 (1998) 2421–2426
acyl moieties. Because HC with odd number of carbon atoms, as well as unsaturated HC, are not normal constituents of the meat, their presence in a sample is unambiguous indication for radiation treatment [8,9]. Based on this finding an approach has been developed for detection of irradiation treatment of fat containing food [3,4,10,11]. Another transformation of the triacylglycerol fatty acids due to irradiation is the cleavage of the acyl–oxygen bond in the triacylglycerols. The reaction results in the formation of 2-alkylcyclobutanones that contain the same number of carbon atoms as the parent fatty acid and an alkyl group located in ring position 2. Such compounds have not been found as natural components in meat, so their presence in a sample is also an indication for a previous irradiation.
2. Experimental
2.1. Samples and their treatment The pork meat with bones was purchased from the local market. The irradiation was carried out in a INRNE g-irradiator with a dose rate of 0.36 kGy h − 1. The overall average absorbed dose was 8 kGy. The bones were separated and the meat was minced. All samples (nonirradiated and irradiated) were kept in refrigerator at − 20°C.
2.2. Sampling and measurements 2.2.1. Electron paramagnetic resonance (EPR) Small pieces (5× 2.5 ×2.5 mm) of meat bone without marrow were cut from both non-irradiated (control) and irradiated samples. Control and irradiated samples were separated into three groups. The first was of fresh cut bone, the second was dried at 60°C for 3 h and the third was boiled in water for 1 h and than dried at 60°C for 3 h. The EPR measurements were performed on a ADANI PS100.X portable spectrometer at 315 K. Details about this spectrometer are available in the literature [12]. In all cases the operating conditions were as follows: center field, 339 mT; sweep width, 8 mT; microwave power, 2 mW; modula-
tion amplitude, 0.4 mT; sweep time, 160 s; time constant, 200 ms. The first EPR measurements were done 24 h after irradiation
2.2.2. Gas-chromatography 2.2.2.1. Hydrocarbon analysis. Irradiated and control samples of the same batch (20 g each) of the mincemeat were refluxed for 60 min with 100 ml n-hexane to extract the total lipids. One milliliter of 20:0 fatty acid (50 mg ml − 1) was added to the extracts and the HC were isolated by column chromatography on deactivated Florisil (20 g for each sample) with n-hexane as eluent, according to [1]. The final volume was adjusted to 1 ml (rotary evaporator). HC were determined on Hewlett–Packard 5890A gas chromatograph equipped with a 25 m×0.2 mm (i.d.) HP-5 capillary column (film thickness 0.53 m) and a Shimadzu R3A electronic integrator under the following chromatographic conditions: detector temperature: 300°C injector temperature: 300°C oven temperature programme: from 40 to 200°C with a rate of 4°C min − 1 injection volume: 2 ml injection mode: splitless carrier gas: nitrogen at linear velocity 40 cm s−1 GC/MS identification was performed on Hewlett–Packard 5890 gas chromatograph with a Hewlett–Packard 5972 series mass selective detector equipped with a HP-5 column (20 m ×0.25 mm i.d., 0.3 mm film thickness). Temperature programme: injector: 200°C transfer line: 270°C oven temperature programme: 55°C for 2 min then to 155°C with 12 °C min − 1 and finally to 230°C with 5°C min − 1 injection volume: 1 ml injection mode: splitless carrier gas: He MSD mode: full scan from 50 to 300 emu EI ionisation 70 eV
N.D. Yordano6 et al. / Spectrochimica Acta Part A 54 (1998) 2421–2426
2.2.2.2. 2 -Alkylcyclobutanone analysis. The 2alkylcyclobutanones were isolated from 20 g mince meat by gentle reflux for 6 h with 100 ml n-hexane in Soxlet extractor exactly as described [2]. An aliquot (200 mg of the lipid in 5 ml) was applied on a small column of deactivated Florisil and eluted with 150 ml hexane in 5 ml portions with 2 to 5 ml min − 1. 2-Alkylcyclobutanones were then eluted from the column with 150 ml 1% diethylether in hexane. The volume was reduced to 5 ml in rotary evaporator, evaporated to dryness under nitrogen and the residue was resuspended in 200 ml cyclohexanone. 2.2.2.3. Analysis of fatty acid methyl esters. Another sample of the same batch was extracted as above to isolate the total lipids. An aliquot of the hexane extract (10 mg of lipid) was transferred to a test tube, the solvent was evaporated to dryness under nitrogen. The residue was redissolved in 1 ml of hexane and transmethylated with 1% sulphuric acid in methanol [13]. The 2-alkylcyclobutanones composition was determined by GC/MS under the following conditions. Instrument: Hewlett – Packard 5890 gas chromatograph with a Hewlett – Packard 5972 series mass selective detector equipped with HP-5 column (20 m ×0.25 mm i.d., 0.3 mm film thickness) Temperature programme: injector: 250°C transfer line: 280°C oven temperature programme: 55°C for 1 min, 15°C min − 1, to 300°C for 5 min injection volume: 1 ml injection mode: splitless solvent delay: 6 min multiplier voltage: autotune value carrier gas: He – MSD mode: SIM of ions m/z 98 and m/z 112 EI ionisation: 70 eV
2423
ated bone with removed marrow present an asymmetric signal due to the free radicals, generated by irradiation, in the main constituent of bone-hydroxyapatite. Some angular-dependence of the positioning of the sample in respect to the direction of the magnetic field was found as this reported for tooth enamel [14] but it is not important for the purposes of the studies reported here. Thus it was neglected. It was found that the EPR signal is not influenced by heating or boiling the sample (Fig. 1) and also on keeping up to 6 months. Its presence may be considered as unambiguous evidence for radiation treatment of the meat.
3.2. Gas-chromatography 3.2.1. Hydrocarbons The HC fractions has been examined by gas chromatography in the presence of eicosane (20:0) as an internal standard. Preliminary identification was made by retention times (RT) after comparison with authentical standards.
3. Results and discussions
3.1. Electron paramagnetic resonance There is no observable EPR signal in all nonirradiated samples. The freshly cut pieces of irradi-
Fig. 1. EPR spectra of irradiated (D= 8 kGy) bone (a) without any treatment, (b) boiled and dried and (c) dried.
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The final identification was performed by GC/ MS and comparison with the library spectra. Saturated HC with even number of carbon atoms (12:0,14:0,16:0) were detected in the blank (control) sample. Two characteristic groups of at least four compounds with RT in the region of 18–20 min (ahead of 12:0 HC) and 24 – 27 min (ahead of 14:0 HC) were detected in both blank and irradiated samples and assigned to impurities generated during the isolation step. The n-hexane that was used as a solvent at all stages of the protocol was free of impurities as confirmed by a separated GC run under the same experimental conditions. In agreement with suggested mechanism, the irradiated samples contained the expected types of saturated and unsaturated HC with either even number, or odd number of carbon atoms, that were not found in the control sample, namely: 114:1, 15:0, 1 – 16:1, 1,7 – 16:2, 17:0, 8 – 17:1, 6,9– 17:2 (Fig. 2a and b). According to [1] a 1,7,10– 16:3 HC, derived from the linoleic acid (9,12–18:) should appear in very low amounts. A peak with the adequate retention time was detected in the irradiated mincemeat as seen from the Fig. 2b. The mass spectrum of that peak unambiguously confirmed that the compound is a 16:3 HC, but due to some overlapping of the peaks in GC and the presence of additional interfering fragments in the mass spectrum, it was not possible to unambiguously determine the exact double bond positions. According to the MS spectrum the irradiated sample contains also 1 – 15:1. The possible source is 16:1 acyl moiety (the amount of 16:1 fatty acid in the sample was found to be 1.5%). However, the corresponding Cn − 2:1 HC, 14:2, was not registered in the MS spectrum. The reason could be that the respective small peak should have a RT about 27 min which means that it is most probably overlapped by some compound of the group that emerges just ahead the tetradecane HC (Fig. 2a and b). There are other small peaks in the GC/MS traces of the irradiated samples, but the low amounts of the corresponding HC prevent their identification. In order to determine their structures, the HC fraction should be additionally subfractionated into saturated and unsaturated
Table 1 Main fatty acid in minced pork and their radiation-induced hydrocarbons(HC) as determined by GLC and results are the mean of three separated analysisa FA
16:0 18:0 18:1 18:2 a
Content in the total
Radiation induced HC
fat (%)
Cn−l:X b
Cn−2:X+1
%
32.4 14.6 43.0 10.2
15:0 17:0 17:1 17:2
14:1 16:1 16:2 16:3
32.0 14.4 44.1 9.9
The standard deviation of the analysis does not exceed 5%
rel. b
X denotes the number of double bonds.
HC. Silver ion chromatography which is widely applied in the analysis of lipids [15] seems very promising, as has been recently shown [16]. In order to verify the analysis of the unambigously identified radiation-induced HC, a comparison was made between their content in the sample and the content of the parent fatty acids. The results are presented in Table 1. The close agreement between the two sets of result reveals that the radiation-induced HC has been correctly identified and quantified by GC and GC-MS. The analysis discussed above was first performed 8 days after the irradiation. Isolated HC (hexane solutions) and irradiated meat samples were than stored at −20°C. After 3 months of storage the analytical procedure was repeated. The freshly isolated HC and the stored HC hexane solutions showed no differences in the composition.
3.2.2. 2 -Alkylcyclobutanones The 2-alkylcyclobutanones, formed by the irradiation, contain the same number of carbon atoms as the parent fatty acid and the alkyl group is always located in ring position 2 [17]. The 2-alkylcyclobutanones which were analysed in the interlaboratory studies were 2-dodecylcyclobutanone (DCB) and 2-tetradecylcyclobutanone (TCB), which are formed from palmitic and stearic acid, respectively, during irradiation. These compounds have never been found in nonirradiated foods.
N.D. Yordano6 et al. / Spectrochimica Acta Part A 54 (1998) 2421–2426
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Fig. 2. GC-MS chromatogram of irradiated (D =8 kGy) pork meat: (a) peaks eluting in the interval of 14 – 21 min; (b) peaks eluting in the interval 22–32 min.
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The hexane fraction of nonirradiated and irradiated mincemeat that was expected to contain 2-alkylcyclobutanones was examined by GC/ EIMS operated in the selected ion-monitoring mode for ions m/z 98 and m/z 112. The respective mass spectra for the nonirradiated and irradiated samples differ significantly. DCB and TCB were found to have a characteristic m/z 98/m/z 112 ratio: 4.0–4.5:1 for DCB and 3.8 – 4.2:1 for TCB. In the GC/MS of nonirradiated sample peaks with such a ratios were not found. In the irradiated samples the ratio m/z 98 to m/z 112 was found to be 4.1:1. The signal to noise ratio was 4:1. It has been assumed that this way the presence of at least one of the expected 2-alkylcyclobutanones was proved, indicating radiation treatment. Since 2-alkylcyclobutanone standards were not available it was not possible to distinguish between DCB and TCB species.
4. Conclusions The present study shows that in respect to identification of radiation treatment of meat containing bone EPR has the advantage to be very fast (sampling and analysis takes ca. 45 min) and unambiguous method. Moreover, bones may be studied as freshly cut of, dried or even after boiling. On the other hand, the GC approach gives also unambiguous results but it is time-consuming procedure. However, it becomes very valuable when no bones are available in the studied samples.
Acknowledgements The financial support of the EU (Project CIPA CT 94-0134) and National Scientific Foundation (Project X-419) is gratefully acknowledged.
.
References [1] European Standard prEN 1784, Foodstuffs — detection of irradiated food containing fat — gas chromatogrphic analysis of hydrocarbons, European Committee for Standardization, Brussels, 1996. [2] European Standard prEN 1785, Foodstuffs — detection of irradiated food containing fat — gas chromatigraphic/ Mass spectrometric analysis of 2-alkylcyclobutanones, European Committee for Standardization, Brussels, 1996. [3] G.A. Schreiber, G. Schulzki, A. Spiegelberg, N. Helle, K.W. Bogl, J. Assoc. Off. Anal. Chem. 77 (1994) 1202. [4] M.H. Stevenson, W. Meier, D.J. Kilpatrick, BCR report, Commission of the European Communities, Luxemburg, 1994, (Report EUR/159/69/en). [5] M.F. Desrosiers, M.G. Simic, J. Agric. Food Chem. 36 (1988) 601. [6] J. Raffi, M.H. Stevenson, M. Kent, J.M. Thiery, J.-J. Belliardo, Int. J. Food Sci. Technol. 27 (1992) 111. [7] M.F. Dubravcic, W.W. Nawar, J. Am. Oil Chem. Soc. 45 (1968) 656. [8] W.W. Nawar, Z.R. Zhu, Y.J. Yoo, in: D.E. Johnston, M.H. Stevenson (Eds.), Food Irradiation and the Chemist, The Royal Society of Chemistry, London, 1990, pp. 13 – 24. [9] W.W. Nawar, Z.R. Zhu, H.P.L.C. Wan, E. DeGroote, Y. Chen, T. Aciukewicz, in: C.H. McMurray, E.M. Stewart, R. Gray, J. Pearce (Eds.), Detection Methods for Irradiated Foods. Current Status, The Royal Society of Chemistry, Cambridge, 1994, pp. 241 – 248. [10] K.M. Morehouse, Y. Ku, Radiat. Phys. Chem. 42 (1993) 359. [11] A. Spiegelberg, G. Schulzki, N. Helle, K.W. Bogl, G.A. Schreiber, Radiat. Phys. Chem. 43 (1994) 433. [12] V.N. Lyniov, in: N.D. Yordanov (Ed.), Electron Magnetic Resonance of Disordered Systems, World Scientific, Singapore, 1991, p. 53. [13] W.W. Christie, Gas Chromatography and Lipids, A Pactical Guide, The Oily Press, Ayr, 1989, p. 68. [14] P. Cevc, M. Schara, C. Ravnik, Radiat. Res. 51 (1972) 581. [15] B. Nikolova-Damyanova, in: W.W. Christie (Ed.), Advances in Lipid Methodology — One, The Oily Press, Ayr, 1992, pp. 181 – 237. [16] M. Hartmann, J. Ammon, H.P.L.C. Berg, Lebensm. Unters. Forsch. A 204 (1997) 231. [17] A.V. Crone, M.V. Hand, J.T.G. Hamilton, N.D. Sharma, D.R. Boyd, M.H. Stivenson, J. Sci. Food Agric. 62 (1993) 361.
Comparative investigation of irradiated meat by the methods of electron paramagnetic resonance and gas chromatography N.D. Yordanov a,*, V. Gancheva a, R. Tarandjiiska b, R. Velikova b, L. Kulieva b, B. Damyanova b, S. Popov b b
a Institute of Catalysis, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Abstract A comparative study of irradiated pork meat containing bone was made by the methods of electron paramagnetic resonance (EPR) and gas chromatography (GC). In this investigation EPR has the advantage to be a very fast and unambiguous method even in the cases of thermal treatment of bones. On the other hand, GC analysis is a time consuming procedure however, it becomes very valuable for meat samples that contain no bones. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Irradiation; Meat; Electron paramagnetic resonance; Gas chromatography
1. Introduction The g-irradiation treatment of meat is recommended as one of the most simple, clean, convenient and useful methods of sterilization and therefore of prolongation of the storage period. On the other hand, according to the requirements of the sanitary control this manipulation must be unambiguously identified. Approaches based on electron paramagnetic resonance (EPR) spectroscopy and gas-chromatography (GC) and/or GC-mass spectrometry (GC-MS) are internation-
* Corresponding author. Tel.: +359-2-724917; fax: +3592-756116; e-mail: [email protected].
ally recognized and recommended for this purpose [1–4]. Investigations of irradiated meat have shown that EPR is a very promising method if the sample contains bone. Radiation induces defects in the bone which are extremely stable with time and very easily detected [5,6]. Irradiated samples show an asymmetric signal due to hydroxyapatite free radicals generated in the bone and the appearance of this signal is unambiguous indication of irradiation treatment. The GC method is based on the changes in the triacylglycerol molecules upon irradiation [7]. Radiation causes cleavage of the triacylglycerol molecules resulting in the formation of Cn − 1 and Cn − 2:1 hydrocarbons (HC) from the respective Cn
1386-1425/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 1 4 2 5 ( 9 8 ) 0 0 2 2 2 - 4
2422
N.D. Yordano6 et al. / Spectrochimica Acta Part A 54 (1998) 2421–2426
acyl moieties. Because HC with odd number of carbon atoms, as well as unsaturated HC, are not normal constituents of the meat, their presence in a sample is unambiguous indication for radiation treatment [8,9]. Based on this finding an approach has been developed for detection of irradiation treatment of fat containing food [3,4,10,11]. Another transformation of the triacylglycerol fatty acids due to irradiation is the cleavage of the acyl–oxygen bond in the triacylglycerols. The reaction results in the formation of 2-alkylcyclobutanones that contain the same number of carbon atoms as the parent fatty acid and an alkyl group located in ring position 2. Such compounds have not been found as natural components in meat, so their presence in a sample is also an indication for a previous irradiation.
2. Experimental
2.1. Samples and their treatment The pork meat with bones was purchased from the local market. The irradiation was carried out in a INRNE g-irradiator with a dose rate of 0.36 kGy h − 1. The overall average absorbed dose was 8 kGy. The bones were separated and the meat was minced. All samples (nonirradiated and irradiated) were kept in refrigerator at − 20°C.
2.2. Sampling and measurements 2.2.1. Electron paramagnetic resonance (EPR) Small pieces (5× 2.5 ×2.5 mm) of meat bone without marrow were cut from both non-irradiated (control) and irradiated samples. Control and irradiated samples were separated into three groups. The first was of fresh cut bone, the second was dried at 60°C for 3 h and the third was boiled in water for 1 h and than dried at 60°C for 3 h. The EPR measurements were performed on a ADANI PS100.X portable spectrometer at 315 K. Details about this spectrometer are available in the literature [12]. In all cases the operating conditions were as follows: center field, 339 mT; sweep width, 8 mT; microwave power, 2 mW; modula-
tion amplitude, 0.4 mT; sweep time, 160 s; time constant, 200 ms. The first EPR measurements were done 24 h after irradiation
2.2.2. Gas-chromatography 2.2.2.1. Hydrocarbon analysis. Irradiated and control samples of the same batch (20 g each) of the mincemeat were refluxed for 60 min with 100 ml n-hexane to extract the total lipids. One milliliter of 20:0 fatty acid (50 mg ml − 1) was added to the extracts and the HC were isolated by column chromatography on deactivated Florisil (20 g for each sample) with n-hexane as eluent, according to [1]. The final volume was adjusted to 1 ml (rotary evaporator). HC were determined on Hewlett–Packard 5890A gas chromatograph equipped with a 25 m×0.2 mm (i.d.) HP-5 capillary column (film thickness 0.53 m) and a Shimadzu R3A electronic integrator under the following chromatographic conditions: detector temperature: 300°C injector temperature: 300°C oven temperature programme: from 40 to 200°C with a rate of 4°C min − 1 injection volume: 2 ml injection mode: splitless carrier gas: nitrogen at linear velocity 40 cm s−1 GC/MS identification was performed on Hewlett–Packard 5890 gas chromatograph with a Hewlett–Packard 5972 series mass selective detector equipped with a HP-5 column (20 m ×0.25 mm i.d., 0.3 mm film thickness). Temperature programme: injector: 200°C transfer line: 270°C oven temperature programme: 55°C for 2 min then to 155°C with 12 °C min − 1 and finally to 230°C with 5°C min − 1 injection volume: 1 ml injection mode: splitless carrier gas: He MSD mode: full scan from 50 to 300 emu EI ionisation 70 eV
N.D. Yordano6 et al. / Spectrochimica Acta Part A 54 (1998) 2421–2426
2.2.2.2. 2 -Alkylcyclobutanone analysis. The 2alkylcyclobutanones were isolated from 20 g mince meat by gentle reflux for 6 h with 100 ml n-hexane in Soxlet extractor exactly as described [2]. An aliquot (200 mg of the lipid in 5 ml) was applied on a small column of deactivated Florisil and eluted with 150 ml hexane in 5 ml portions with 2 to 5 ml min − 1. 2-Alkylcyclobutanones were then eluted from the column with 150 ml 1% diethylether in hexane. The volume was reduced to 5 ml in rotary evaporator, evaporated to dryness under nitrogen and the residue was resuspended in 200 ml cyclohexanone. 2.2.2.3. Analysis of fatty acid methyl esters. Another sample of the same batch was extracted as above to isolate the total lipids. An aliquot of the hexane extract (10 mg of lipid) was transferred to a test tube, the solvent was evaporated to dryness under nitrogen. The residue was redissolved in 1 ml of hexane and transmethylated with 1% sulphuric acid in methanol [13]. The 2-alkylcyclobutanones composition was determined by GC/MS under the following conditions. Instrument: Hewlett – Packard 5890 gas chromatograph with a Hewlett – Packard 5972 series mass selective detector equipped with HP-5 column (20 m ×0.25 mm i.d., 0.3 mm film thickness) Temperature programme: injector: 250°C transfer line: 280°C oven temperature programme: 55°C for 1 min, 15°C min − 1, to 300°C for 5 min injection volume: 1 ml injection mode: splitless solvent delay: 6 min multiplier voltage: autotune value carrier gas: He – MSD mode: SIM of ions m/z 98 and m/z 112 EI ionisation: 70 eV
2423
ated bone with removed marrow present an asymmetric signal due to the free radicals, generated by irradiation, in the main constituent of bone-hydroxyapatite. Some angular-dependence of the positioning of the sample in respect to the direction of the magnetic field was found as this reported for tooth enamel [14] but it is not important for the purposes of the studies reported here. Thus it was neglected. It was found that the EPR signal is not influenced by heating or boiling the sample (Fig. 1) and also on keeping up to 6 months. Its presence may be considered as unambiguous evidence for radiation treatment of the meat.
3.2. Gas-chromatography 3.2.1. Hydrocarbons The HC fractions has been examined by gas chromatography in the presence of eicosane (20:0) as an internal standard. Preliminary identification was made by retention times (RT) after comparison with authentical standards.
3. Results and discussions
3.1. Electron paramagnetic resonance There is no observable EPR signal in all nonirradiated samples. The freshly cut pieces of irradi-
Fig. 1. EPR spectra of irradiated (D= 8 kGy) bone (a) without any treatment, (b) boiled and dried and (c) dried.
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The final identification was performed by GC/ MS and comparison with the library spectra. Saturated HC with even number of carbon atoms (12:0,14:0,16:0) were detected in the blank (control) sample. Two characteristic groups of at least four compounds with RT in the region of 18–20 min (ahead of 12:0 HC) and 24 – 27 min (ahead of 14:0 HC) were detected in both blank and irradiated samples and assigned to impurities generated during the isolation step. The n-hexane that was used as a solvent at all stages of the protocol was free of impurities as confirmed by a separated GC run under the same experimental conditions. In agreement with suggested mechanism, the irradiated samples contained the expected types of saturated and unsaturated HC with either even number, or odd number of carbon atoms, that were not found in the control sample, namely: 114:1, 15:0, 1 – 16:1, 1,7 – 16:2, 17:0, 8 – 17:1, 6,9– 17:2 (Fig. 2a and b). According to [1] a 1,7,10– 16:3 HC, derived from the linoleic acid (9,12–18:) should appear in very low amounts. A peak with the adequate retention time was detected in the irradiated mincemeat as seen from the Fig. 2b. The mass spectrum of that peak unambiguously confirmed that the compound is a 16:3 HC, but due to some overlapping of the peaks in GC and the presence of additional interfering fragments in the mass spectrum, it was not possible to unambiguously determine the exact double bond positions. According to the MS spectrum the irradiated sample contains also 1 – 15:1. The possible source is 16:1 acyl moiety (the amount of 16:1 fatty acid in the sample was found to be 1.5%). However, the corresponding Cn − 2:1 HC, 14:2, was not registered in the MS spectrum. The reason could be that the respective small peak should have a RT about 27 min which means that it is most probably overlapped by some compound of the group that emerges just ahead the tetradecane HC (Fig. 2a and b). There are other small peaks in the GC/MS traces of the irradiated samples, but the low amounts of the corresponding HC prevent their identification. In order to determine their structures, the HC fraction should be additionally subfractionated into saturated and unsaturated
Table 1 Main fatty acid in minced pork and their radiation-induced hydrocarbons(HC) as determined by GLC and results are the mean of three separated analysisa FA
16:0 18:0 18:1 18:2 a
Content in the total
Radiation induced HC
fat (%)
Cn−l:X b
Cn−2:X+1
%
32.4 14.6 43.0 10.2
15:0 17:0 17:1 17:2
14:1 16:1 16:2 16:3
32.0 14.4 44.1 9.9
The standard deviation of the analysis does not exceed 5%
rel. b
X denotes the number of double bonds.
HC. Silver ion chromatography which is widely applied in the analysis of lipids [15] seems very promising, as has been recently shown [16]. In order to verify the analysis of the unambigously identified radiation-induced HC, a comparison was made between their content in the sample and the content of the parent fatty acids. The results are presented in Table 1. The close agreement between the two sets of result reveals that the radiation-induced HC has been correctly identified and quantified by GC and GC-MS. The analysis discussed above was first performed 8 days after the irradiation. Isolated HC (hexane solutions) and irradiated meat samples were than stored at −20°C. After 3 months of storage the analytical procedure was repeated. The freshly isolated HC and the stored HC hexane solutions showed no differences in the composition.
3.2.2. 2 -Alkylcyclobutanones The 2-alkylcyclobutanones, formed by the irradiation, contain the same number of carbon atoms as the parent fatty acid and the alkyl group is always located in ring position 2 [17]. The 2-alkylcyclobutanones which were analysed in the interlaboratory studies were 2-dodecylcyclobutanone (DCB) and 2-tetradecylcyclobutanone (TCB), which are formed from palmitic and stearic acid, respectively, during irradiation. These compounds have never been found in nonirradiated foods.
N.D. Yordano6 et al. / Spectrochimica Acta Part A 54 (1998) 2421–2426
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Fig. 2. GC-MS chromatogram of irradiated (D =8 kGy) pork meat: (a) peaks eluting in the interval of 14 – 21 min; (b) peaks eluting in the interval 22–32 min.
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N.D. Yordano6 et al. / Spectrochimica Acta Part A 54 (1998) 2421–2426
The hexane fraction of nonirradiated and irradiated mincemeat that was expected to contain 2-alkylcyclobutanones was examined by GC/ EIMS operated in the selected ion-monitoring mode for ions m/z 98 and m/z 112. The respective mass spectra for the nonirradiated and irradiated samples differ significantly. DCB and TCB were found to have a characteristic m/z 98/m/z 112 ratio: 4.0–4.5:1 for DCB and 3.8 – 4.2:1 for TCB. In the GC/MS of nonirradiated sample peaks with such a ratios were not found. In the irradiated samples the ratio m/z 98 to m/z 112 was found to be 4.1:1. The signal to noise ratio was 4:1. It has been assumed that this way the presence of at least one of the expected 2-alkylcyclobutanones was proved, indicating radiation treatment. Since 2-alkylcyclobutanone standards were not available it was not possible to distinguish between DCB and TCB species.
4. Conclusions The present study shows that in respect to identification of radiation treatment of meat containing bone EPR has the advantage to be very fast (sampling and analysis takes ca. 45 min) and unambiguous method. Moreover, bones may be studied as freshly cut of, dried or even after boiling. On the other hand, the GC approach gives also unambiguous results but it is time-consuming procedure. However, it becomes very valuable when no bones are available in the studied samples.
Acknowledgements The financial support of the EU (Project CIPA CT 94-0134) and National Scientific Foundation (Project X-419) is gratefully acknowledged.
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