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Essential-Oil Analysis of Irradiated Spices by Using Comprehensive Two-Dimensional Gas Chromatography [ChemPlusChem](ChemPlusChem Via Acquire Media NewsEdge) Four different spices were packaged either under 100 % nitrogen or using ambient air, and then gamma-irradiated at different doses. The essential oils of the original spices and of those irradiated were obtained by hydrodistillation. Changes to the essential-oil compositions due to irradiation and effects of packaging were detected and compared by using comprehensive two-dimensional gas chromatography (GC × GC). The use of GC × GC provided better separation and more reliable results. It enabled ready detection of some peaks that coeluted with other peaks in the first-dimension column (1 D). Differences in detected peaks were observed after irradiation of samples packaged under ambient air, with additional peak(s) noted. Two peaks appeared after irradiation of aerobically packaged rosemary, black pepper and thyme, and one peak increased in relative abundance in the samples. No changes in these three peaks were noted after irradiation of samples packaged under 100 % N2. These three essential-oil components were identified by using GC × GC quadrupole time-of-flight mass spectrometry (Q-TOFMS). The changes after irradiation were mainly due to packaging type, and no effect of the irradiation dose was observed in the study. Keywords : essential oils · gas chromatography · irradiation · mass spectrometry · natural products Introduction Irradiation is a novel method of food sterilisation. It is found to be effective in inactivating the microbial load and in increasing the shelf life of many herbs and spices.[1] However, it has some undesired effects on food. Gamma irradiation has been found to increase oxidation in several spices.[2] Oxidation of ground black pepper increased as the gamma-irradiation dose increased.[3] It has been postulated that gamma irradiation increased radical formation in several spices including black pepper and rosemary.[4] The combination of modified atmosphere packaging (MAP) with irradiation is promising for controlling the extent of oxidation. MAP inhibits oxidation in many foods. The applications of irradiation combined with MAP have been reviewed by Cumming and Blank.[5] Packaging of irradiated seasoned ground beef in reduced O2 resulted in lower oxidation than that in air packaging.[6] Undesired effects of irradiation, such as lipid oxidation, can be avoided by eliminating O2 from the package of food products.[7] Gamma irradiation is a common technique for spice sterilisation, and its effects on essential-oil components should be monitored and evaluated carefully. Essential oils are volatile hydrocarbon and oxygenated hydrocarbon constituents of plants, and they include terpenoid and non-terpenoid compounds such as alcohols, ketones, aldehydes, acids, esters and so forth.[8] Gas chromatography (GC) is widely used for the analysis of essential oils. Comprehensive two-dimensional gas chromatography (GC ^ GC) is an emerging technique in qualitative and quantitative analysis of essential oils. It normally involves direct coupling of a first-dimension column (1D) with a shorter second-dimension column (2D) of different polarity, and incorporates a modulator device.[9] Various advantages of the method include improved separation, better signal/noise (S/N) ratio, and structured chromatograms. Dimandja et al.[10] reported a two- to threefold increase in separation ability of GC ^ GC relative to GC when mint essential oil was used as an analyte. Hyphenated techniques are used to combine the separation ability of GC with the identification ability of other techniques, most importantly mass spectrometry (MS). The specific coupling of GC ^GC with MS methods has been reviewed recently.[11] There have been several studies reporting the analysis of essential oils using GC ^ GC;[12] however, no studies evaluated the use of GC ^ GC for the analysis of irradiated foodstuffs. The aim of this study was to evaluate changes in the essential-oil composition of thyme, rosemary, black pepper and cumin arising from the use of irradiation and O2 in the package headspace, assessed by using GC ^ GC. Results and Discussion The 1D chromatograms of samples irradiated at different doses were generally similar, which is to be expected since 1DGC does not have the clarity to reveal the full chemical composi- tion of volatile components, owing to the likely overlap of compounds. However, when GC ^GC analysis was employed specific differences were observed (Figure 1). (Full GC ^ GC data of the non-irradiated oils are shown in Figures S1-S4 in the Supporting Information). It is clearly shown in Figure 1 that the same profile of most of the components is obtained and they do not change as a result of irradiation ; this illustrates the consistency of the sampling and injection processes, and the peak-location reproducibility in 2D space, and also highlights that the changes that are observed are real changes in the identified chemical species. Two of the peaks (peaks 1 and 3) emerged after irradiation of air-packaged (AP) samples at all doses (7, 12 and 17 kGy). One of the peaks (peak 2) increased in relative abundance after irradiation of the AP samples. Peak 2 was initially identi- fied as cumin aldehyde by using a flame-ionisation detector (FID) through comparison with an external standard. Peak 1 was tentatively identified as 4-(1-methylethyl)-1,3-cyclohexa- diene-1-methanol (CAS number 1413-55-4), and peak 3 as 2- caren-10-al by using GC ^ GC accurate-mass time-of-flight mass spectrometry (TOFMS). Peak 2 was subsequently confirmed to be cumin aldehyde by GC ^GC-TOFMS analysis, which supports the result obtained by FID. It has also been observed in a previ- ous study that cumin aldehyde was not detected in essential oils of modified-atmosphere-packaged and irradiated thyme, rosemary and black pepper.[13] National Institute of Standards and Technology (NIST) library matches were 750, 840 and 760 for peaks 1-3, respectively. Since the newly detected compounds were oxygenated monoterpenes, it seems reasonable to assume that the genera- tion of these new compounds is linked to the O2 content in the atmospheric environment. Moreover, the increase of these components was negligible in MAP samples, thus the 100 % N2 environment suppresses their formation. Components 1 and 3 in rosemary, black pepper and thyme essential oils were not detectable in samples packaged under 100 % N2 even after irradiation at all doses. The amount of component 2 in these samples after irradiation was no differ- ent to levels in the control (P> 0.05). The peaks (4-(1-methyl- ethyl)-1,3-cyclohexadiene-1-methanol, cumin aldehyde and 2- caren-10-al) that were observed only in essential oils of air- packaged and irradiated rosemary, black pepper and thyme did not change in cumin essential oil (or the changes were not significant), as they existed in all samples and cumin aldehyde is the principal component of cumin essential oil. There is the potential for the production of these essential-oil compounds to be used as an indicator of the irradiation of spices, and fur- ther study will address this possibility. Essential-oil analysis of irradiated spices by using GC ^ GC provided significantly better separation than with 1D GC. The comparison of the 1D and 2D profiles of thyme essential oil is presented in Figure 2. It is clearly shown in Figure 3 that 4-(1- methylethyl)-1,3-cyclohexadiene-1-methanol, cumin aldehyde and 2-caren-10-al have 1D retention times that overlap with various other compounds in the sample. Compound 1 elutes on the 1D column with the same retention time as other peaks in essential oils of air-packaged, irradiated spices used in this study. Compound 2 coelutes with another compound in black pepper, and peak 3 overlaps with other compounds in 1D. Compound 3 has the same 1D retention time as carvacrol, which was reported as one of major compounds of thyme es- sential oil.[13] Positive identification of minor oxidation prod- ucts, or those that exhibit small changes arising from the treat- ment process, is considerably compromised if a single-column GC method is used, as a result of these overlapping interfer- ences. Mitrevski et al.[14] reviewed the use of GC ^ GC for the analy- sis of illicit drugs, and concluded that GC ^ GC provides better separation and more peaks than 1D GC. The general merits of multidimensional and comprehensive 2D GC technologies, ap- proaches to implementation and applications have been out- lined recently, in particular with a focus on system design for increased separation.[15] There have been studies reporting the effects of gamma irra- diation on the oxidation of foods.[16] It was observed elsewhere that MAP reduces the undesired effects of irradiation on the quality of several spices.[13] It was reported that thiobarbituric acid reactive substances (TBARS) of methanol extracts of ore- gano increased with the irradiation dose ; however, the influ- ence of O2 inside the packaging was not evaluated in that study.[17] The advantage of MAP combined with irradiation was reported in other studies. The higher the O2 level, the higher the oxidation in seasoned ground beef.[6b] It has been suggest- ed that irradiation alters hydrocarbons by oxidation or hydrox- ylation and initiates new components by accordingly changing the chemical structure.[18] We did not find any studies evaluat- ing the effects of irradiation on foodstuffs or the combination irradiation with MAP by using comprehensive two-dimensional gas chromatography. The retention-time, area-ratio (area of the component/area of the internal standard) and peak-width data of component 1 are given in Table 1; the respective data for component 2 are given in Table 2 ; and Table 3 lists the respective data for com- ponent 3 for each of rosemary, black pepper, thyme and cumin essential oils. The 4-(1-methylethyl)-1,3-cyclohexadiene-1-methanol and 2- caren-10-al peaks emerged after irradiation of aerobically pack- aged rosemary, black pepper and thyme, but their relative abundances did not depend on the applied dose (P > 0.05). Peak 2 (cumin aldehyde) was significantly higher in AP than in MAP in irradiated rosemary, black pepper and cumin (P < 0.05), but no significant change was observed on increasing the dose (P>0.05). Peak 2 in the essential oil of irradiated cumin was significantly higher in AP than in MAP (P < 0.01). Peaks 1 and 3 did not change as a result of the irradiation dose or O2 content in the package. Conclusion The changes in volatile oils extracted from a variety of spices were evaluated by using GC^GC. This approach provided good separation and ready visualisation in 2D contour plots of new compounds produced upon irradiation. Whereas these changes were almost impossible to be recognised in 1DGC, GC^GC offers more detailed analysis and more reliable com- parison through their 2D profiles. The results of this study indi- cate that comprehensive two-dimensional gas chromatography is a useful technology for the comparison of samples with minor differences and where potential volatile molecular changes in samples might not be known in advance. In this specific case, it will be valuable in future studies to detect the irradiation treatment of spices, or by extension, comparison of other samples through their volatile profiles. As the com- pounds that were the focus of this study were only detectable as resolved peaks in GC ^ GC, it can be used in the detection and separation of these components in other studies with es- sential oils. It was observed that irradiation in the presence of oxygen may produce a number of new compounds, or alters the amount of existing compounds in the essential oils of spices. Some of the changes are initiated by oxidation under the effect of irradiation. The extent of changes in compounds at- tributable to oxidation were significantly lower or in some in- stances not detected in samples packaged under 100 % N2 prior to the irradiation process. Experimental Section Materials The unsterilised rosemary (Rosmarinus officinalis L.), black pepper (Piper nigrum L.), thyme (Thymus vidgaris L.) and cumin (Cuminum cyminum L.) were obtained in ground form from a local manufac- turer (Bagdat Baharat, Ankara, Turkey). Essential-oil standard com- pounds were provided by Australian Botanical Products (Hallam, VIC, Australia), and 2,4-dimethylphenol was purchased from Merck (Darmstadt, Germany). Sample preparation The spices were packaged under either 100% N2 (0% O2; modified atmosphere packaging (MAP) or ambient air (aerobic packaging ; AP). Polyethylene terephthalate/polyethylene-ethylene vinyl alco- hol copolymer-polyethylene (PET/PE-EVOH)) or low-density poly- ethylene (LDPE) pouches were used as packaging material, respec- tively. The gas mixture was obtained by using a gas mixer (PBI Dansensor Map Mix 9000, Ringsted, Denmark) and was fed to a packaging machine (Multivac C200, Multivac Sepp Haggenm^ller GmbH & Co. KG, Wolfertschwenden, Germany). Samples were gamma-irradiated at 7, 12 or 17 kGy at room temperature in a com- mercial irradiation facility (Gamma-Pak Sterilizasyon San. ve Tic. A.S. , Tekirdag, Turkey) with a 60Co source. The average applied dose rate was 2.0 kGy h^1 and was measured using Amber 3042 dosimeters (Harwell Dosimeters Ltd, Oxfordshire, UK). Each treat- ment was replicated twice. Essential oils from irradiated samples were obtained by hydrodistil- lation. A sample of spice (100 g) was mixed with distilled water (1600 mL), and they were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. Each sample was extracted in duplicate runs. The oils were dried using anhydrous sodium sulfate and stored in amber vials sealed under nitrogen. A dilution (100 mLL^1)inn-hexane was prepared just before analysis. 2,4-Dimethylphenol at 100 mL L^1 concentration was prepared in n- hexane and used as an internal standard (IS). Equal volumes of di- luted oil and IS were mixed before GC injection. GC ^GC-FID analysis An Agilent 7890A gas chromatograph (Agilent Technologies, Mul- grave, VIC, Australia) with an FID detector and a longitudinally modulated cryogenic system (LMCS, Model 2.02, Chromatography Concepts, Doncaster, VIC, Australia) was used for GC^GC analysis of the essential oils. A nonpolar 30 m^0.25 mm (inside diameter; ID)^0.25mmfilmthickness(df) DB-5MS UIphase (AgilentTechnolo- gies) column was used as the 1D column and a moderately polar 2 m^0.15 mm^0.15 mm df VF-200ms phase (Agilent Technologies) was used as the 2D column. An aliquot (1 mL) of the sample was in- jected by an autosampler (7683, Agilent Technologies) at a 10 :1 split ratio. The oven temperature program was started at 50 8C (hold 0.5 min), increased at a rate of 108C min^1 to 220 8C, and then increased at 208Cmin^1 to 2608C (hold 3 min). The tempera- tures of the injector and detector were both 280 8C. The detector acquisition rate was 100 Hz. The modulation period (PM) was set to 6.0 s, and the cryogenic trap was held at approximately ^20 8C. Hy- drogen was used as the carrier gas at 1.5 mL min^1. All 1D GC ex- periments were conducted under the conditions described above, with the modulation process turned off. GC ^GC-Q-TOFMS analysis The identification of unknown peaks was performed by using an Agilent 7890A gas chromatograph connected to a 7200 accurate- mass Q-TOFMS GC/MS instrument (Agilent Technologies). A HP- 5mscolumn(30m^0.25mm ID^0.25mmdf; AgilentTechnologies) was used as the 1D column, and a BPX50 column (1.2 m ^ 0.1 mm ID ^ 0.1 mm df; SGE, Ringwood, Australia) was used as the 2D column. The columns were connected with a deactivated pressfit (Restek, Bellefonte, USA), and a LMCS was used as the modulator. The end of the 2D column was connected to a Deans switch (DS) device, and then to the Q-TOFMS by a 0.8 m (0.1 mm ID) deactivat- ed fused-silica (dfs) column. The other DS outlet was closed, and the device was employed to back-flush the column set, and for column change without venting the Q-TOFMS. An aliquot (1 mL) of sample (50 mL L^1 essential oil in hexane) was injected at a 10:1 split ratio. The oven temperature program was started at 50 8C (hold 0.5 min), increased at 108Cmin^1 to 220 8C, and then in- creased at 20 8C min^1 to 260 8C (hold 3 min). The temperature of the injector was 280 8C; the PM was set to 6.0 s, and the cryogenic trap was kept at approximately ^20 8C. Helium was used as the carrier gas at 1 mL min^1. Electron ionisation (EI) was performed at 70 eV energy, and the TOFMS ion-source temper- ature was 230 8C. The monitored mass range was from m/z 40 to m/z 500 at the maximum acquisition rate attainable with the TOFMS instrument : 50 spectra per second acquisition rate. Data analysis Analysis of the variance was applied to data to detect statistical differences by using IBM SPSS Statistics 21 (IBM Corp. , NY, USA) software. Tukey's multiple comparison test was applied to compare means when a significant difference was observed. MassHunter version B.06.00 (Agilent Technologies) was used for TOFMS data acquisition and processing, and the NIST algorithm was used for spectral searching through the MS library (NIST11). Data from GC ^ GC-FID analyses were processed by using Chemsta- tion software (Agilent Technologies). Data visualisation was per- formed by raw-data export in csv file format, converted to a 2D matrix by using in-house software (2D GC converter), and finally presented by using the Transform software (version 3.3, Fortner Re- search LLC, USA). Acknowledgements This study was partly supported by the Research Fund of Istanbul Technical University. The Development Foundation of Istanbul Technical University and Tincel Cultural Foundation supported C.K. for research collaboration at Monash University. The spice samples used in this study were donated by Bagdat Baharatlari Gida San. ve Tic. Ltd. Sti. (Ankara, Turkey) and irradiated by Gamma-Pak Sterilizasyon San. ve Tic. A.S. (Tekirdag, Turkey). Korozo Ambalaj San. ve Tic. A.S. (Istanbul, Turkey) is acknowl- edged for providing packaging material. We acknowledge fund- ing from the ARC under Discovery program grant DP01095335. P.J.M. acknowledges the Australian Research Council (ARC) for a DORA fellowship. [1] a) W. Y. Hsu, A. Simonne, P. Jitareerat, M. R. Marshall, J. Food Sci. 2010, 75, M222 - M230 ; b) P. P. Legnani, E. Leoni, F. Righi, L. A. Zarabini, Ital. J. Food Sci. 2001, 13, 337 - 345 ; c) B. B. Mishra, S. Gautam, A. Sharma, J. FoodSci.2004, 69, M274-M279; d)C.K. 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J. Marriott, R. Shellie, C. Cornwell, J. Chromatogr. A 2001, 936, 1 - 22. [10] J. M. D. Dimandja, S. B. Stanfill, J. Grainger, D. G. Patterson, HRC-J. High Resolut. Chromatogr. 2000, 23, 208 - 214. [11] P. Q. Tranchida, L. Mondello, S. D. H. Poynter, R. A. Shellie, in Comprehen- sive Chromatography in Combination with Mass Spectrometry (Ed. : L. Mondello), Wiley, Hoboken, New Jersey, 2011, pp. 171 - 242. [12] a) M. Brokl, M. L. Fauconnier, C. Benini, G. Lognay, P. du Jardin, J. F. Focant, Molecules 2013, 18, 1783 - 1797; b) P. M. Harvey, S. D. H. Poynter, R. A. Shellie, LC GC Eur. 2011, 24, 548 - 555 ; c) R. Costa, P. Dugo, L. Santi, G. Dugo, L. Mondello, Curr. Org. Chem. 2010, 14, 1752 - 1768; d) P. D. Morrison, P. J. Marriott, S. D. H. Poynter, R. A. Shellie, LC GC Eur. 2010, 23, 76 - 80 ; e) P. J. Marriott, G. T. Eyres, J. P. Dufour, J. Agric. Food Chem. 2009, 57, 9962 - 9971. [13] C. Kirkin, B. Mitrevski, G. Gunes, P. J. Marriott, Food Chem. 2014, 154, 255-261. [14] B. Mitrevski, P. Wynne, P. J. Marriott, Anal. Bioanal. Chem. 2011, 401, 2361-2371. [15] P. J. Marriott, S.-T. Chin, B. Maikhunthod, H.-G. Schmarr, S. Bieri, TrAC Trends Anal. Chem. 2012, 34, 1 - 21. [16] a) N. Maltar-Strmecki, B. Ljubic' -Beer, R. Laskaj, J. Aadrovic' ,P.Dzaja, Food Control 2013, 34, 132-137;b)Y.P. Ma, X.G. Lu, X.H. Liu, H.L.Ma, Post- harvest Biol. Technol. 2013, 84, 36 - 42 ; c) M. Oraei, A. Motallebi, E. Hosei- ni, S. Javan, Int. J. Food Sci. Technol. 2012, 47, 977- 984. [17] J. Horv^thov^, M. Suhaj, M. Polovka, V. Brezov^, P. Simko, Czech. J. Food Sci. 2007, 25, 131 - 143. [18] O. A. Emam, S. A. Farag, N. H. Aziz, Z. Lebensm.-Unters. Forsch. 1995, 201, 557 - 561. Received : December 19, 2013 Revised : April 16, 2014 Published online on May 21, 2014 Celale Kirkin,[b] Blagoj Mitrevski,[a] Gurbuz Gunes,[b] and Philip J. Marriott*[a] [a] Dr. B. Mitrevski, Prof. P. J. Marriott Australian Centre for Research on Separation Science School of Chemistry, Monash University Wellington Rd, Clayton, VIC 3800 (Australia) Fax: (+ 61) 3-99058501 E-mail : [email protected] [b] C. Kirkin, Prof. G. Gunes Food Engineering Department Faculty of Chemical and Metallurgical Engineering Istanbul Technical University Maslak 34469, Istanbul (Turkey) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cplu.201300430. It contains the full GC ^ GC-FID data for each spice-oil extract in modified atmosphere without irradiation; data acquired for GC ^ GC-TOFMS for components 1-3; and a range of partial GC ^ GC-FID traces for each condition used for all spices. Part of a Cluster Issue on "Two-Dimensional Gas Chromatography". To view the complete issue, visit : http://onlinelibrary.wiley.com/doi/10.1002/cplu.v79.6/issuetoc. (c) 2014 Blackwell Publishing Ltd. |