Research Article

Received: 10 October 2012 Revised: 8 December 2012 Accepted: 12 December 2012 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2013, 27, 591602 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6489

Gas chromatography combined with mass spectrometry, ame ionization detection and elemental analyzer/isotope ratio mass spectrometry for characterizing and detecting the authenticity of commercial essential oils of Rosa damascena Mill.
Federica Pellati1*, Giulia Orlandini1, Katryna A. van Leeuwen2, Giulia Anesin1,2, Davide Bertelli1, Mauro Paolini2, Stefania Benvenuti1 and Federica Camin2**
1 2

Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 183, 41125 Modena, Italy Food Quality and Nutrition Department, IASMA Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele allAdige (TN), Italy

RATIONALE: The essential oil of Rosa damascena Mill. is known for its ne perfumery application, use in cosmetic preparations and for several pharmacological activities. Due to its high value, it can be easily adulterated with avors or cheaper oils. This study is aimed at a detailed phytochemical characterization of commercial samples of R. damascena essential oil and at their authenticity assessment. METHODS: Nineteen commercial samples of R. damascena essential oil of different geographic origin and an additional authentic one, directly extracted by hydro-distillation from fresh owers, were considered. GC/MS and GC/FID techniques were applied for the phytochemical analysis of the samples. EA/IRMS (Elemental Analyzer/Isotope Ratio Mass Spectrometry) and GC/C (Combustion)/IRMS were used to determine the d13C composition of bulk samples and of some specic components. RESULTS: Citronellol (28.755.3%), geraniol (13.527.3%) and nonadecane (2.618.9%) were the main constituents of Bulgarian and Turkish essential oils, while those from Iran were characterized by a high level of aliphatic hydrocarbons (nonadecane: 3.723.2%). The d13C values of bulk samples were between 28.1 and 26.9%, typical for C3 plants. The d13C values of specic components were in the usual range for natural aromatic substances from C3 plants, except for geranyl acetate, which displayed higher values (up to 18%). These unusual d13C values were explained by the addition of a natural cheaper oil from a C4 plant (Cymbopogon martinii, palmarosa), which was found to occur in most of the essential oils. CONCLUSION: GC/C/IRMS, in combination with GC/MS and GC/FID, can be considered as an effective and reliable tool for the authenticity control of R. damascena essential oil. Copyright 2013 John Wiley & Sons, Ltd.

Rosa damascena Mill. is one of the most important species belonging to the genus Rosa (Rosaceae family).[1] This plant is commonly called damask rose because it was originally brought to Europe from Damascus.[1] At present, R. damascena is widely cultivated in many countries, such as Turkey, Bulgaria, Iran, India and Morocco.[2] The techniques used to extract the essential oil from rose are mainly based on hydro- or steam distillation from fresh owers[2] or on solvent extraction.[3] Due to its low content in this species, rose essential oil is one of the most expensive in the world market.[1] In this context, the chemical characterization of this natural product is considered to be very important. * Correspondence to: F. Pellati, Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 183, 41125 Modena, Italy. E-mail: federica.pellati@unimore.it ** Correspondence to: F. Camin, Food Quality and Nutrition Department, IASMA Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach, 1, 38010 San Michele allAdige (TN), Italy. E-mail: federica.camin@iasma.it

The essential oil of R. damascena is known for its ne perfumery application and use in cosmetic preparations,[1] including perfumes, creams, soaps, and lotions. Several biological activities have been attributed to rose essential oil, including antimicrobial,[4] antioxidant,[5] analgesic,[6] anti-inammatory[6] and antispasmodic.[7] Several effects on the central nervous system have also been described,[8] such as a relaxing effect.[9] The composition of rose essential oil is known to be very complex,[10] including monoterpene alcohols, in particular citronellol, geraniol, nerol, as well as the aromatic phenylethyl alcohol, and long-chain hydrocarbons, such as nonadecane, nonacedene, eicosane, and heneicosane.[2] From the sensory point of view, various minor constituents, such as rose oxides, signicantly contribute to its characteristic aroma.[2] Adulteration is a well-known and serious problem in essential oils, especially for the most expensive ones, such as R. damascena. There are several avoring compounds available that could be added to an essential oil to increase its fragrance. The adulterants can be natural, obtained by physical, enzymatic or microbial processes from natural material, such as eugenol (from Syzygium aromaticum), geraniol (from Cymbopogon martinii (palmarosa) or Cymbopogon nardus (citronella)), geranyl acetate

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F. Pellati et al. (from Cymbopogon citratus (lemongrass)) and linalool (from Ocimum basilicum (basil)).[11] The avoring adulterants can also be nature-identical, i.e. chemically identical to those present in nature but obtained by chemical synthesis or isolated by chemical processes.[11] In the case of R. damascena, the most frequently employed adulterants are natural or nature-identical, including citronellol, geraniol and geranyl acetate.[12] Other common adulterants are much cheaper essential oils rich in geraniol, such as C. martinii essential oil, mainly produced in India.[13,14] Adulterants are usually added at low percentages (58%) to avoid detection by commonly used analytical methods.[15] Gas chromatography/mass spectrometry (GC/MS) is one of the most reliable techniques for the determination of the composition of essential oils. Through GC/MS it is possible to study the effects of different extraction methods and geographic origin on the essential oil composition. In addition to conventional GC techniques, reliable analytical methods based on isotope ratio mass spectrometry (IRMS) and on enantioselective analysis for origin assessment and quality assurance of essential oils are of fundamental interest.[16] In particular, the determination of the d13C values of target components of natural products, by means of GC/C/ IRMS, has become a rapid and convenient tool for authenticity assessment,[17] allowing both the determination of geographic origin and the evaluation of possible adulteration of essential oils with synthetic or natural compounds. Analysis of the essential oils of orange,[18] lemon, lemon grass, lemon balm, lemon gum, citronella,[1921] coriander,[22] mandarin,[23,24] dill,[25] anise, fennel,[26] tarragon, basil, Mexican marigold, West Indian Bay tree fruit,[27] cinnamon (cassia),[28] oregano, savory, thyme, fennel oil,[29] lavender,[30] neroli[31] and bergamot[32] by GC/C/IRMS to determine authenticity has been well documented. In the case of R. damascena, isotopic data of the essential oil have not been reported in the peer-reviewed scientic literature, with the exception of a technical paper concerning the d13C values of citronellol, nerol, geraniol and phenylethyl alcohol of two samples.[33] Due to the high commercial value of rose extracts in perfumery and cosmetics, this study is aimed at a detailed phytochemical characterization of commercial samples of R. damascena essential oil of different geographic origin by means of GC/MS and GC/FID techniques and at their authenticity assessment using elemental analysis EA/IRMS and GC/C/IRMS. To the best of our knowledge, this is the rst report of a comprehensive multi-component analysis of rose essential oil from different countries by the above-cited techniques. In particular, the d13C values of bulk R. damascena essential oils and of some specic components are described here for the rst time. additional authentic R. damascena essential oil, directly extracted by hydro-distillation from fresh owers in the laboratory of Agronatura (Spigno Monferrato, Alessandria, Italy), was analyzed. On the basis of what was reported in the labels, the Bulgarian samples had been extracted by hydro-distillation, with the exception of samples BULG 01 and 04, whose extraction method was not specied. Turkish samples labelled as TURK 0103 and 05 were extracted by hydro-distillation; for sample TURK 08 the extraction procedure was not specied. The Iranian samples, indicated as IRAN 0103, were produced by hydro-distillation: in particular, sample IRAN 02 is a rst rose oil, i.e. it was obtained by direct hydro-distillation of fresh rose petals, while sample IRAN 03 is a second rose oil, i.e. obtained by re-distillation of rose water. Two additional samples of C. martinii (Roxb.) Wats. var. motia Burk. (Poaceae family) (palmarosa) essential oil were purchased in local pharmacies in Fall 2010. These samples originated from Nepal and India. All samples were stored at low temperature (+4  C), protected from light and humidity, until required for chemical analysis. Chemicals and solvents All reference standards used for GC analysis were of chromatographic grade and were purchased from Sigma-Aldrich (Milan, Italy), Extrasynthese (Genay, France) and Roth (Karlsruhe, Germany). Chromatographic grade organic solvents were from Sigma-Aldrich and VWR (Milan, Italy). Sample preparation for GC/MS and GC/FID analysis The essential oils were diluted 1:2 (v/v) with n-hexane before GC/MS analysis. In the case of GC/FID, the samples were directly analyzed, without dilution. Three injections were performed for each sample. GC/MS conditions Analyses were performed on a 6890N gas chromatograph (Agilent Technologies, Waldbronn, Germany), coupled with a 5973 Network mass spectrometer (Agilent Technologies). Compounds were separated on a HP-5 MS cross-linked poly-5% diphenyl95% dimethyl polysiloxane (30 m 0.25 mm i.d., 1.00 mm lm thickness) capillary column (Agilent Technologies). The column was maintained at 60  C for 6 min after the injection, then programmed at 3  C/min to 230  C, at which temperature it was maintained for 7 min. The injection volume was 0.1 mL, with a split ratio 1:100. Helium was used as the carrier gas at a ow rate of 0.7 mL/min. The injector, transfer line and ion-source temperatures were set at 250, 280 and 230  C, respectively. MS detection was performed with electron ionization (EI) at 70 eV, operating in the full-scan acquisition mode in the m/z range 30350. GC/FID conditions Analyses were carried out on a model 8610 gas chromatograph (DANI Instruments, Milan, Italy) with ame ionization detection (FID). Compounds were separated on a HP-5 cross-linked poly-5% diphenyl95% dimethyl polysiloxane (25 m 0.2 mm
Rapid Commun. Mass Spectrom. 2013, 27, 591602

EXPERIMENTAL
Rose essential oil samples Nineteen commercial samples of R. damascena essential oil were kindly gifted by the manufacturers or purchased in Italian pharmacies and herbal shops in Winter 2009Spring 2010. Based on the label claims, these samples were grouped in agreement with their place of origin: eight samples were from Bulgaria (indicated in the text as BULG 0108), eight from Turkey (TURK 0108) and three from Iran (IRAN 0103). An

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GC/MS, GC/FID and GC/C/IRMS analysis of Rosa damascena essential oil i.d., 0.5 mm lm thickness) capillary column (Agilent Technologies). The temperature program and the chromatographic conditions were the same as described above. The injection volume was 0.2 mL, with a split ratio 1:20. Helium was used as the carrier gas at a pressure of 1.5 bar at the column head. The injector and detector temperatures were set at 250  C. A mixture of aliphatic hydrocarbons (C8C25) in n-hexane (Sigma) was injected under the above temperature program to calculate the linear retention index (LRI) of each compound. Qualitative and semi-quantitative analysis Compounds were identied by comparing the retention times of the chromatographic peaks with those of authentic reference compounds run under the same conditions and by comparing the LRIs with published data. Peak enrichment on co-injection with authentic reference compounds was also carried out. Comparison of the MS fragmentation pattern of the target analytes with those of pure compounds was also performed. A mass spectrum database search was performed using the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) mass spectral database (version 2.0d, 2005). The percentage relative amount of individual components was expressed as the percent peak area relative to the total peak area obtained by the GC/FID analysis. Semi-quantitative data were acquired from the mean of three analyses. Sample preparation for EA/IRMS and GC/C/IRMS analysis Aliquots of 0.3 mg of bulk essential oils and pure commercial compounds (used as reference standards) were weighed in tin capsules (Sntis Analytical AG, Teufen, Switzerland) for EA/IRMS analysis. In the case of GC/C/IRMS analysis, essential oils were diluted 1:3 (v/v) with n-hexane before injection. Each sample was measured at least twice in EA/IRMS and three times in GC/C/IRMS. EA/IRMS conditions The analyses were carried out using an elemental analyzer (Flash EA 1112, Thermo Scientic, Bremen, Germany), equipped with an autosampler (Finnigan AS 200, Thermo Scientic) and interfaced through a ConFlo III dilutor device (ThermoFinnigan, Bremen, Germany) with a DELTA V isotope ratio mass spectrometer Thermo Scientic). GC/C/IRMS conditions A model 6890A gas chromatograph (Agilent Technologies) equipped with an autosampler (GC-PAL, GC Analytics AG, Zwingen, Switzerland) and a DB-WAX capillary column (30 m 0.32 mm i.d., 0.25 mm lm thickness; Agilent Technologies) was used. The injector temperature was set at 250  C and the temperature program was as follows: the initial column temperature was 50  C, held for 4 min; then increased to 160  C at a rate of 5  C/min, to 180  C at 2  C/min, to 225.5  C at 5  C/min and to 250  C at 10  C/min, where it was held for 5 min. Helium was used as the carrier gas at a ow rate of 2 mL/min. The gas chromatograph was supplied with an oxidation reactor composed of a 32 cm long alumina tube in which three thin (0.125 mm diameter) braided wires were placed: one nickel oxide, one copper oxide and one platinum. The tube was kept at a temperature of 940  C. Water was eliminated by a water-removing trap, consisting of a NaonW membrane. The GC/C system was interfaced with an isotope ratio mass spectrometer (DELTA V) through an open split interface. C/12C analysis of bulk rose essential oils and pure commercial compounds The samples were quantitatively burnt to carbon dioxide and water (the second one was removed using a magnesium perchlorate lter) in the presence of oxygen and copper oxide in an elemental analyzer. The carbon dioxide was ushed into the isotope ratio mass spectrometer, where the content of the isotopomers at m/z 44 (12C16O2) and 45 (13C16O2) was determined.[34] The isotopic values were calculated by comparing the ratio of the sample with that of a working standard (oil) calibrated against international reference materials: fuel oil NBS-22 (International Atomic Energy Agency (IAEA), Vienna, Austria) and sugar IAEA-CH-6 (IAEA). The isotopic ratio values of the aforementioned reference materials and, therefore, also of the samples were expressed in d% versus the international standard V-PDB (Vienna-Pee Dee Belemnite), according to the following equation:
13

13 
12 C

sample 13 C
12 C

13 
C
12 C

std

1000

std

The uncertainty of measurement, calculated as 2 standard deviation (SD) of the intra-laboratory reproducibility, was 0.3%.
13

C/12C analysis of specic compounds in rose essential oils

To analyze the main constituents present in R. damascena essential oil (citronellol, nerol, geraniol), 1 mL of sample solution was injected using a 10 mL Hamilton syringe in the split mode (1:75). In the essential oils, the nonadecane peak was not completely resolved and, consequently, it was not considered for the analysis, with the exception of the Iranian samples, in which this compound was determined. After the chromatographic separation, each compound was quantitatively burnt to carbon dioxide and water in the oxidation reactor. The solvent was vented with a back-ush valve during the rst 200 s of analysis to avoid damage and inactivation of the oxidation column. Carbon dioxide, carried by a helium stream, was then introduced into the ion source of the isotope ratio mass spectrometer for the measurement of d13C values. The lower concentration compounds were analyzed using a 1:25 split ratio with 1 mL of solution injected and venting the main compounds with a back-ush valve. Under these conditions, satisfactory results were obtained for geranyl acetate, heneicosane and farnesol. The other compounds were not completely separated or their concentration was too low to be analyzed (<0.5% by GC/MS or GC/FID analysis). The peak of each constituent was identied by previously analyzing pure commercial reference compounds under the same experimental conditions. To calculate the d13C values, a reference mixture composed of pure compounds was used,

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F. Pellati et al. for which the d13C values had previously been measured using EA/IRMS. The analytical procedure followed a specic sequence: re-oxidation of the oxidation reactor through a ow of oxygen; injection of n-hexane (used in reference mix) to check for purity; four injections of the reference mixture (the rst one was not considered); two samples (three injections of each); and three injections of the reference mixture. The instrumental data for each constituent were corrected on the basis of the difference existing between the d13C value of the pure compound in GC/C/IRMS (mean of the six results, three before and three after the samples) and that in EA/IRMS. The SD obtained from three repeated analyses of one sample was 0.2% for citronellol, 0.4% for nerol and geraniol, 0.5% for geranyl acetate and 1% for the other compounds. Statistical analysis Analysis of variance (ANOVA) was used to evaluate the statistical signicance of the measured differences between essential oils of different origin. To evaluate the most important variables that discriminate between origins, a post hoc test was performed using the Tukey Honestly Signicant Difference (HSD) test. For all these tests, the P level was set at 0.05. The statistical analysis was performed using Statistica 6 for Windows (StatSoftW Italia, Vigonza, Italy). Other constituents were found to be nerol (1.73.3%), nonadecene (0.84.8%) and phenylethyl alcohol (0.33.4%). cis- and trans-Rose oxides were found at low levels (0.10.5%). These substances are very important for the characteristic rose scent and exhibit extremely low threshold values. The described composition was found to be in good agreement with published results where Gochev et al.[35] analyzed the chemical composition of a historical rose oil from Bulgaria and determined citronellol (23.4%), geraniol (19.0%), nonadecane (11.9%) and nerol (7.5%) as the main constituents. Table 2 shows the composition of Turkish rose essential oils extracted by hydro-distillation. The rose essential oils described in Table 2 have a chemical composition closely related to those of the above described samples of Bulgarian origin: the main constituents were found to be citronellol (41.755.3%), geraniol (15.827.3%) and nonadecane (3.87.1%). These results are also in good agreement with those of hydro-distilled essential oils from Turkey previously described: Bayrak and Akgl[36] reported the presence of citronellol in the range 24.542.9%, 2.118.0% for geraniol and 6.419.0% for nonadecane. In the essential oils analyzed by Chalchat and zcan[37] the levels of citronellol, geraniol and nonadecane were 43.048.0%, 12.2 19.9% and 9.811.0%, respectively. Table 3 shows the chemical composition of Iranian rose essential oils which were extracted by hydro-distillation. The samples labeled as IRAN 01 and 02 showed a chemical composition based on high levels of aliphatic hydrocarbons, such as nonadecane (16.123.2%), nonadecene (5.36.9%), heneicosane (4.36.9%) and heptadecane (3.74.2%), and quite low relative percentages of monoterpene alcohols, such as citronellol (25.225.5%) and geraniol (8.39.6%). This composition was found to be in agreement with the results obtained by the analysis of a historical rose oil sample from Iran,[2] which contained citronellol (25.1%), nonadecane (13.4%), geraniol (11.8%), nonadecene (6.9%), heneicosane (6.2%) and heptadecane (3.9%) as the main constituents. Other workers have reported similar compositions for Iranian essential oils.[38,39] Sample IRAN 03 displayed a chromatographic prole very similar to those oils of Bulgarian and Turkish origin, having citronellol (45.9%) and geraniol (28.5%) as the main constituents and a low percentage of nonadecane (3.7%). This sample has a chemical composition in

RESULTS AND DISCUSSION

Chemical composition of rose essential oils Figure 1 shows a representative GC/FID chromatogram obtained by the analysis of a R. damascena essential oil from Bulgaria (BULG 01). A total of 39 compounds were identied in this sample, representing 98.9% of the overall essential oil composition. Table 1 shows the chemical composition of the Bulgarian rose essential oils analyzed, all of them having been extracted by hydro-distillation. All these samples displayed a common phytochemical prole, based on the presence of citronellol (28.753.1%), geraniol (13.525.0%) and nonadecane (2.618.9%).

Figure 1. GC/FID chromatogram obtained by the analysis of a R. damascena essential oil from Bulgaria (BULG 01). For peak identication, see Table 1.
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Table 1. Volatile aroma components identied in Bulgarian R. damascena essential oils by GC analysisa

Peak number 0.3e 0.1e 0.5e tr tr

GC/MS, GC/FID and GC/C/IRMS analysis of Rosa damascena essential oil

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Compoundb LRI

BULG 01c

BULG 02c

BULG 03c

BULG 04c

BULG 05c

BULG 06c

BULG 07c

BULG 08c

Identication methodd

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 0.1e 0.3 0.1 0.1e tr tr 2.9e 0.5e 1.0e 0.2e 0.5e 0.6e 53.1 0.3 1.9 0.1 0.2e 23.3 0.1 1.3 0.1 0.1e 0.5e 1.1 0.1 0.7e 1.4 0.1 0.6e 0.3e 0.2e

Hexanol Heptanal a-Pinene Benzaldehyde Sabinene b-Pinene Myrcene Limonene g-Terpinene Terpinolene Linalool cis-Rose oxide Phenylethyl alcohol trans-Rose oxide Terpinen-4-ol a-Terpineol Citronellol Nerol Neral Geraniol Geranial Methyl geranate Citronellyl acetate Eugenol Geranyl acetate Methyl eugenol b-Caryophyllene a-Guaiene a-Humulene

866 901 935 963 975 979 990 1030 1060 1091 1102 1113 1120 1130 1184 1198 1238 1239 1247 1263 1276 1323 1353 1367 1384 1408 1435 1447 1467

0.3 0.1 0.1e 1.1 0.2 0.1e 0.1e 0.3 0.1 0.5 0.1 0.1e tr 0.1e 1.8 0.2 0.5 0.1 1.1 0.1 0.2e 0.4 0.1 0.4e 49.5 0.9 1.7 0.3 0.8 0.5 20.1 0.7 1.5 0.1 0.1e 0.5e 1.2 0.1 1.0e 1.6 0.1 0.6 0.1 0.4e 0.3e 0.2e 0.1e 0.7 0.1 tr tr 0.2e e 0.4 0.1e tr tr 1.6 0.1 0.4e 2.5 0.2 0.2e 0.4e 0.4e 47.1 0.1 2.1 0.5 0.8 0.1 23.6 0.2 1.4e 0.1e 0.6 0.1 1.2e 1.1 0.1 1.7 0.1 0.6e 0.4e 0.3e 0.7 0.1 0.2e 1.2 0.1 0.1e 0.2 0.1 0.2 0.1 37.4 0.4 3.3 0.7 0.9 0.1 20.5 0.5 1.2 0.1 0.6 0.1 1.6 0.1 1.5 0.1 1.6 0.2 0.4 0.1 0.3 0.1 0.3 0.2 0.1e 0.1e 0.6 0.1 tr 0.1e 0.3 0.1 0.1e tr tr 2.3 0.1 0.4e 3.4 0.1 0.2e 0.3e 0.5e 44.1 1.1 3.1 0.3 0.8 0.1 24.0 0.4 1.0e tr 0.4e 1.4 0.1 1.3e 1.4 0.1 0.4e 0.2e 0.2e

0.2 0.1 tr 0.6e tr 0.1e 0.2 0.1 tr tr 1.8 0.1 0.3e 1.3 0.1 0.2 0.1 0.5 0.1 0.5 0.1 45.8 1.0 2.8 0.2 0.8e 25.0 0.5 1.5 0.1 tr 0.4e 1.3 0.1 0.8 0.1 1.7e 0.5e 0.3e 0.3e

0.4 0.1 0.1e 0.6e 0.1e 0.2e 0.3e 1.2 0.1 0.5e 0.3e 0.2e 0.4e 0.3e 51.9 0.9 2.8 0.2 1.0 0.1 22.3 0.5 1.3 0.1 0.1e 0.6 0.1 0.5e 0.9e 1.9 0.1 0.6e 0.4e 0.2 0.1

0.1e 0.3 0.1 0.3e 0.1e 0.2 0.1 tr 0.7 0.1 0.2e 0.4 0.1 0.1e 0.2e 0.1e 28.7 0.1 2.2 0.1 0.7 0.1 13.5 0.1 0.8e tr 0.5 0.1 0.6 0.1 0.8e 1.2 0.1 0.8e 0.7 0.1 0.5e

a,b,c,d b,d a,b,c,d b,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d b,d a,b,c,d b,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d b,d a,b,c,d a,b,c,d b,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d b,d a,b,c,d (Continues)

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596
LRI 1496 1511 1520 1696 1728 1796 1874 1898 1995 2110 1.1 0.1 0.2e 0.3e 1.1 0.2 1.0 0.2 0.1e 1.6 0.4 5.3 1.0 0.4 0.2 1.5 0.6 98.9 0.3 0.8e 0.1e 0.3e 1.0 0.1 1.4 0.1 0.1e 1.5 0.3 5.0 0.8 0.3 0.1 1.4 0.3 99.0 0.2 0.9e 0.1e 0.3e 0.8 0.1 1.0 0.1 0.1e 1.3 0.3 4.0 1.0 0.3 0.1 1.3 0.5 99.0 0.6 1.0e 0.1e 0.3e 1.6 0.1 1.2 0.2 0.2e 3.3 0.4 10.6 0.8 0.9 0.1 3.9 0.4 95.4 1.9 0.6e 0.1e 0.2e 1.1 0.1 0.7 0.1 0.1e 1.3 0.1 5.8 0.6 0.3e 1.0 0.1 98.1 0.4 0.7 0.1 0.2e 0.2 0.1 0.6e 0.9e 0.1e 0.8 0.1 2.6 0.1 0.2e 0.7 0.2 99.1 0.2 0.9 0.1 0.2e 0.3e 1.0 0.1 1.2 0.1 0.1e 1.3 0.1 4.0 0.1 0.2 0.1 0.8 0.2 98.2 0.6 1.2 0.1 0.3e 0.5e 2.1 0.1 2.2 0.1 4.8 0.1 18.9 0.1 1.9e 9.3 0.1 94.9 0.2 BULG 01c BULG 02c BULG 03c BULG 04c BULG 05c BULG 06c BULG 07c BULG 08c Identication methodd b,d b,d b,d a,b,c,d a,b,c,d b,d b,d a,b,c,d b,d a,b,c,d LRI 866 901 935 963 975 979 990 1030 1060 1091 1102 1113 1120 1130 1184 0.4e tr 0.5e tr tr 0.1e 0.2 0.1 tr 1.6e 0.5e 2.4 0.1 0.3e 0.5e 0.2e 2.0e 0.2e 0.4e 1.0e 0.1e 0.1e tr 2.8 0.1 0.3e 1.7e 0.2 0.1 0.6e 0.3e 0.1e 1.3e 0.1e 0.3e 0.6e 0.1e tr tr 0.7e 0.4e 1.9e 0.2e 0.5e TURK 01c TURK 02c TURK 03c TURK 04c 0.3 0.1 0.2e 1.4 0.2 0.2 0.1 0.4 0.1 0.7 0.1 0.1e 0.1e 1.1 0.1 0.4e 2.4 0.2 0.2e 0.6e TURK 05c 0.3e 0.2 0.1 1.8 0.1 0.2e 0.4e 0.8 0.1 0.1e tr tr 0.9 0.1 0.3e 1.6 0.1 0.2e 0.5e TURK 06c 0.3e 0.2e 1.0e 0.1e 0.2e 0.5e 0.1e tr tr 0.9 0.1 0.3e 2.2 0.1 0.2e 0.5e TURK 07c 0.4 0.1 tr 0.6 0.1 0.1e tr 0.2 0.1 0.2e tr tr 1.5 0.2 0.5e 2.1 0.2 0.3 0.1 0.4 0.1 TURK 08c 0.6 0.2 2.0 0.4 0.2 0.1 Identication methodd a,b,c,d b,d a,b,c,d b,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d b,d a,b,c,d b,d a,b,c,d (Continues) F. Pellati et al.

Table 1. (Continued)

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Peak number

Compoundb

30 31 32 33 34 35 36 37 38 39

Germacrene D Pentadecane d-Guaiene Heptadecane Farnesol Octadecane Nonadecene Nonadecane Eicosane Heneicosane Total

GC conditions as in Experimental section. Compounds are listed in order of elution time. c Data are expressed as mean (n = 3) of % relative peak area values SD. d a: retention time; b: LRI; c: peak enrichment; d: mass spectrum. e SD <0.05.

Table 2. Volatile aroma components identied in Turkish R. damascena essential oils by GC analysisa

Copyright 2013 John Wiley & Sons, Ltd.

Peak number

Compoundb

1 2 3 5 4 6 7 8 9 10 11 12 13 14 15

Hexanol Heptanal a-Pinene Benzaldehyde Sabinene b-Pinene Myrcene Limonene g-Terpinene Terpinolene Linalool cis-Rose oxide Phenylethyl alcohol trans-Rose oxide Terpinen-4-ol

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Table 2. (Continued)

GC/MS, GC/FID and GC/C/IRMS analysis of Rosa damascena essential oil

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Peak number LRI 1198 1238 1239 1247 1263 1276 1323 1353 1367 1384 1408 1435 1447 1467 1496 1511 1520 1696 1728 1796 1874 1898 1995 2110 0.3e 55.3 0.8 1.9 0.2 0.6 0.1 17.0 0.4 1.0 0.3 0.1e 0.8e 0.4e 1.3e 2.7 0.1 0.4e 0.3e 0.2 0.1 0.6e 0.1e 0.1e 1.0 0.1 0.4 0.1 0.1e 1.5 0.2 4.3 0.3 0.3e 1.0e 98.2 0.6 0.7 0.1 42.7 0.9 2.8 0.6 0.5 0.1 27.3 0.4 0.8e 0.1e 0.6e 1.0e 1.6e 1.6 0.1 0.5e 0.3e 0.3e 0.6e 0.1e 0.2e 1.0 0.1 0.7e 0.1e 1.1 0.2 3.8 0.5 0.3 0.1 0.8 0.1 99.1e 0.2e 50.1e 1.4e 0.1e 19.0e 0.9 0.1 0.1e 0.8e 1.7e 1.5e 2.6e 0.5e 0.4e 0.3e 0.8e 0.2 0.1 0.3 0.1 1.0e 0.6e 0.1e 1.6e 5.4 0.1 0.5 0.1 1.8e 98.3 0.1 0.2e 48.3 1.5 2.6 0.5 0.6 0.1 19.8 1.2 0.9e 0.8 0.1 1.3 0.1 2.1 0.1 2.4 0.1 0.5e 0.5 0.1 0.3e 1.1 0.2 0.2e 0.3 0.1 0.7 0.2 0.5 0.2 0.1e 1.2 0.6 4.2 1.0 0.3 0.2 1.4 0.8 98.6 0.9 0.2e 41.7 1.0 2.6 0.2 0.5e 23.6 0.8 0.8 0.1 tr 0.7e 0.8e 1.8 0.1 1.7 0.1 0.6e 0.5e 0.4e 1.1 0.1 0.2 0.1 0.3 0.1 1.0 0.2 0.9 0.1 0.1e 1.6 0.4 6.6 1.0 0.6 0.1 2.6 0.4 98.2 0.4 0.2e 47.4 1.2 2.5 0.4 0.7 0.1 20.5 0.9 0.9e tr 0.8e 1.4 0.1 2.1 0.1 2.4 0.1 0.5e 0.5e 0.3e 1.0e 0.2e 0.3 0.1 0.8 0.2 0.6 0.1 0.1e 1.5 0.6 4.1 1.2 0.4 0.2 1.9 1.0 97.8 0.5 0.3e 52.1 1.0 1.7 0.5 0.6 0.1 15.8 1.3 1.2 0.2 0.1e 0.8 0.1 0.4 0.1 1.3 0.1 2.7 0.1 0.4e 0.3e 0.2e 0.7 0.1 0.1e 0.2e 1.3 0.3 0.5 0.1 0.1e 2.0 0.6 7.1 0.7 0.5 0.2 1.8 0.8 98.4 0.2 0.2e 49.2 1.2 2.4 0.1 0.3 0.1 22.0 0.9 0.8e 0.5 0.1 0.4 0.2 0.9 0.1 1.8 0.2 0.2e 0.2 0.1 0.1e 0.4 0.1 tr 0.8 0.3 1.1 0.3 0.1e 2.3 0.2 7.1 0.4 0.5 0.2 2.6 0.7 96.6 0.1

Compoundb

TURK 01c

TURK 02c

TURK 03c

TURK 04c

TURK 05c

TURK 06c

TURK 07c

TURK 08c

Identication methodd a,b,c,d a,b,c,d a,b,c,d b,d a,b,c,d a,b,c,d b,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d b,d a,b,c,d b,d b,d b,d a,b,c,d a,b,c,d b,d b,d a,b,c,d b,d a,b,c,d

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

a-Terpineol Citronellol Nerol Neral Geraniol Geranial Methyl geranate Citronellyl acetate Eugenol Geranyl acetate Methyl eugenol b-Caryophyllene a-Guaiene a-Humulene Germacrene D Pentadecane d-Guaiene Heptadecane Farnesol Octadecane Nonadecene Nonadecane Eicosane Heneicosane Total

Copyright 2013 John Wiley & Sons, Ltd.

GC conditions as in Experimental section. Compounds are listed in order of elution time. c Data are expressed as mean (n = 3) of % relative peak area values SD. d a: retention time; b: LRI; c: peak enrichment; d: mass spectrum. e SD <0.05.

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F. Pellati et al. Table 3. Volatile aroma components identied in Iranian R. damascena essential oils by GC analysisa Identication methodd a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d b,d a,b,c,d b,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d b,d a,b,c,d a,b,c,d b,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d a,b,c,d b,d a,b,c,d b,d b,d b,d a,b,c,d a,b,c,d b,d b,d a,b,c,d b,d a,b,c,d

Peak number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
a

Compoundb Hexanol a-Pinene Sabinene b-Pinene Myrcene Limonene g-Terpinene Terpinolene Linalool cis-Rose oxide Phenylethyl alcohol trans-Rose oxide Terpinen-4-ol a-Terpineol Citronellol Nerol Neral Geraniol Geranial Methyl geranate Citronellyl acetate Eugenol Geranyl acetate Methyl eugenol b-Caryophyllene a-Guaiene a-Humulene Germacrene D Pentadecane d-Guaiene Heptadecane Farnesol Octadecane Nonadecene Nonadecane Eicosane Heneicosane Total

LRI 866 935 975 979 990 1030 1060 1091 1102 1113 1120 1130 1184 1198 1238 1239 1247 1263 1276 1323 1353 1367 1384 1408 1435 1447 1467 1496 1511 1520 1696 1728 1796 1874 1898 1995 2110

IRAN 01c 2.0 0.2 0.2 0.1 0.5 0.2 0.8 0.1 0.1e 0.1e tr 1.8e 0.3 0.2 1.0 0.3 0.1e 0.2e 0.4e 25.5 0.6 0.9 0.2 0.1e 8.3 0.3 0.2e tr 0.8e 0.6e 1.4 0.1 0.8e 0.7e 0.8e 0.6 0.1 2.8 0.1 0.4e 0.6e 4.2 0.1 0.8e 0.4e 6.9 0.1 23.2 0.4 1.7 0.1 6.9 0.1 96.1 1.5

IRAN 02c 5.9 0.3 0.5 0.1 1.1 0.2 2.3 0.2 0.3 0.1 0.3 0.1 0.1e 0.5 0.1 0.2 0.1 0.5 0.2 0.1e 0.1e 0.1e 25.2 0.2 0.9 0.1 0.1e 9.6 0.2 0.2 0.1 0.3e 1.3 0.2 0.5 0.1 3.3 0.3 0.4 0.1 1.1 0.1 1.4 0.2 1.0 0.1 4.2 0.2 0.7 0.1 1.0 0.1 3.7 0.1 1.1 0.2 0.3 0.1 5.3 0.9 16.1 0.4 1.3 0.5 4.3 0.4 95.1 0.2

IRAN 03c 0.1e 0.5e tr 0.1e 0.3e 0.1e tr tr 2.6e 0.2e 1.6 0.1 0.1e 0.5e 0.6e 45.9 0.5 2.4 0.3 0.5e 28.5 0.1 0.6 0.1 0.5e 1.2 0.1 1.7e 1.3 0.1 0.3e 0.4e 0.3e 1.3e 0.2e 0.3e 1.0 0.1 0.4 0.1 0.1e 1.1e 3.7 0.1 0.2e 0.6 0.1 99.3e

GC conditions as in Experimental section. Compounds are listed in order of elution time. c Data are expressed as mean (n = 3) of % relative peak area values SD. d a: retention time; b: LRI; c: peak enrichment; d: mass spectrum. e SD <0.05.
b

agreement with that of a so-called Iranian second rose oil,[39] which was produced by re-distillation of rose water, and contained 53.4% of citronellol, 22.7% of geraniol and 3.4% of nonadecane. A statistical analysis was performed on the GC data of the Bulgarian, Turkish and Iranian essential oils and the results are shown in Table 4. Considering the data reported in Tables 13, some minor compounds, such as benzaldehyde, g-terpinene, terpinolene, heptanal and methyl geranate, were excluded from the statistical analysis. It is evident from the data of Table 4 that all the volatile compounds present a very high variability. Signicant differences were observed for 20 of the 35 considered compounds and these differences are more frequent for compounds with

high retention times. The HSD test did not allow identication of any particular trend in the samples related to the geographic origin of the essential oils. d13C composition of rose essential oils The d13C values of bulk samples of R. damascena essential oil ranged between 28.1 and 26.9%, with a mean value of 27.5% and a SD of 0.4%. No signicant differences (P <0.05) are evident between geographic origins. These values are typical for C3 plants, such as R. damascena, that assimilate atmospheric carbon dioxide according to the so-called C3 pathway described by Calvin and are characterized by d13C values between 34 and 22%.[40] Plants
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Copyright 2013 John Wiley & Sons, Ltd.

GC/MS, GC/FID and GC/C/IRMS analysis of Rosa damascena essential oil Table 4. Statistical analysis of Bulgarian, Turkish and Iranian essential oils Compound Hexanol a-Pinene Sabinene b-Pinene Myrcene Limonene Linalool cis-Rose oxide Phenylethyl alcohol trans-Rose oxide Terpinen-4-ol a-Terpineol Citronellol Nerol Neral Geraniol Geranial Methyl geranate Citronellyl acetate Eugenol Geranyl acetate Methyl eugenol b-Caryophyllene a-Guaiene a-Humulene Germacrene D Pentadecane d-Guaiene Heptadecane Farnesol Octadecane Nonadecene Nonadecane Eicosane Hexanol
a b

Overall mean 0.20 0.14 1.13 1.31 0.08 0.12 0.25 0.24 0.51 0.50 0.07 0.07 1.48 0.76 0.34 0.14 1.61 0.81 0.17 0.07 0.40 0.14 0.34 0.18 44.2 9.00 2.20 0.68 0.57 0.28 20.2 5.42 0.96 0.37 0.06 0.08 0.66 0.22 0.99 0.44 1.43 0.62 1.73 0.63 0.54 0.19 0.45 0.27 0.34 0.20 1.15 0.89 0.19 0.17 0.32 0.20 1.36 0.97 0.90 0.42 0.12 0.09 2.21 1.66 7.46 5.72 0.58 0.50 2.40 2.29

Bulgarian 0.20 0.12 0.56 0.32 0.02 0.05 0.14 0.08 0.28 0.14 0.05 0.05 1.62 0.75 0.37 0.12 1.41 1.05 0.17 0.04 0.35 0.11 0.37 0.17 44.7 8.11 2.48 0.59 0.77 0.25 21.5 3.66 1.24 0.25 0.05 0.05 0.51 0.08 1.13 0.38 1.01 0.27 1.54 0.22 0.57 0.13 0.38 0.15 0.29 0.09 0.91 0.21 0.21 0.16 0.30 0.09 1.16 0.47 1.19 0.44 0.10 0.05 2.00 1.34 7.02 5.33 0.58 0.58 2.49 2.94

Turkish 0.28 0.12 1.07 0.68 0.10 0.09 0.24 0.15 0.50 0.35 0.06 0.05 1.28 0.72 0.34 0.16 2.03 0.30 0.19 0.09 0.48 0.16 0.29 0.18 48.3 4.52 2.23 0.49 0.50 0.17 20.6 3.70 0.91 0.14 0.05 0.05 0.73 0.13 0.92 0.51 1.59 0.42 2.24 0.46 0.45 0.12 0.37 0.12 0.27 0.09 0.78 0.27 0.27 0.13 0.22 0.12 0.95 0.17 0.65 0.24 0.09 0.02 1.59 0.39 5.31 1.43 0.42 0.10 1.74 0.65

Iranian 0.03 0.06 2.81 2.80 0.21 0.24 0.57 0.48 1.11 1.03 0.16 0.10 1.64 1.04 0.23 0.09 1.01 0.53 0.10c 0.27 0.21 0.37 0.25 32.2 11.8 1.41 0.88 0.24 0.22 15.5 11.3 0.36 0.24 0.10 0.17 0.87 0.40 0.78 0.40 2.12 1.03 0.83 0.48 0.70 0.40 0.86 0.48 0.66 0.37 2.78 1.45 1.45 0.42 0.62 0.33 2.96 1.74 0.78 0.33 0.28 0.16 4.43 2.99 14.35 9.87 1.04 0.77 3.92 3.20

ANOVA Pa <0.05 <0.05 NS <0.05 <0.05 NS NS NS NS NS <0.05 NS <0.05 NS <0.05 NS <0.05 NS <0.05 NS <0.05 <0.05 NS <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 NS NS NS AB A A A A A A A A A A A AB A A A A A A A A AC A A A A A AB A A A AB A A A

HSD testb A AB A AB A A A A A A A A A A AB A B A B A AB B A AB AB AB AB A AB B AB A A A A B B A B A A A A A A A A B A B A C A B A B C A C C C C B C AB C B A A A

NS = not signicant. Results of the HSD test: the same letter in the same row indicates no signicant differences (P <0.05). c SD <0.005.

belonging to the genus Cymbopogon (monocotyledon), such as C. martinii, one of the common adulterants of R. damascena essential oil, C. nardus and C. citratus, from which natural geraniol and geranyl acetate can be extracted, assimilate carbon dioxide according to the C4 or Hatch-Slack cycle, and are characterized by signicantly higher d13C values, ranging from 14 to 10%.[40] Since any potential addition of natural avors from C4 plants at the usual level of 58%[15] cannot be easily identied by measuring the d13C values of bulk samples, the d13C values of some single constituents of R. damascena samples using GC/C/IRMS were determined. A DB-WAX capillary column was selected for this analysis, since it allowed a good degree of separation of citronellol, geraniol and nerol under the applied chromatographic conditions. With regard to the application of GC/C/IRMS to essential oils, it has been reported that monoterpenes, including citronellol, from the essential oils of C3 plants, such as peppermint, mandarin, mixed camphor, cedar-wood, turpentine and peony, have d13C values in the range of 34 to 26%, mostly
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between 29 and 27%.[41] Linalool and linalyl acetate from lavender, spike lavender, bois de rose, bergamot, geranium, clary sage, petit grain and coriander have shown d13C values from 32 to 25%.[42] The characteristic avor components of mandarin essential oil have displayed d13C values from 34.5 to 25.5%.[23,24] d13C values between 33 and 24% have been described for bergamot essential oil (containing geranyl acetate),[32] between 31 and 23% for neroli essential oil (containing nerol, geranyl acetate, farnesol)[31] and from 31 to 25% for lemon essential oil (containing nerol, geraniol, geranyl acetate).[19] The d13C values of citronellol, nerol and geraniol of two samples of R. damascena essential oil ranged between 28.9 and 24.7%.[33] The d13C values of the aldehydes neral, geranial (and of their combination) and citral from lemon balm, Listea cubeba, lemon myrtle, lemon and lemon tea tree oil have been found to be from 29 to 24%,[20] and those from Lippia citriodora and some commercial lemon oils have shown d13C values between 23 and 21%,[20] at the limit of the variability eld for C3 plant components.

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F. Pellati et al. The d13C values of neral, geranial and citral from C4 plants, such as lemongrass and citronella, have been described in the range from 14 to 7%.[20,21] For each sample analyzed in these studies, these three aldehydes were characterized by very similar d13C values. Within a closed system such as a plant, all the secondary metabolites are isotopically correlated with each other and with the primary metabolites (carbohydrates), with the exception of moderate shifts due to isotope effects in the course of their biosynthesis, i.e. in the case of metabolic branching.[43] Intermolecular isotopic correlations have been shown in several authentic essential oils, thus becoming indicative of the authenticity of natural avors.[19,2224,31,32] This relationship is particularly true for structurally related compounds, such as geraniol and geranyl acetate, nerol and neryl acetate or linalool and linalyl acetate. It is evident that the three above-mentioned esters have d13C values (around 1%) that are similar to or slightly lower than those of the corresponding alcohols in the essential oil.[19,22,3032,42] Table 5 shows the d13C values of geranyl acetate, citronellol, nerol, geraniol, nonadecane, heneicosane and farnesol of R. damascena essential oil samples. As explained in the Experimental section, we took into account the main compounds characterized by baseline separation, and also the secondary compounds with good resolution and % relative peak area higher than 0.5 in GC/MS or GC/FID analysis. It is evident that for the analyzed essential oils the d13C values for most of the compounds are in the usual range for natural aromatic substances from C3 plants, except for geranyl acetate with higher values (up to 18%). No signicant differences (P <0.05) are evident between the three geographic origins. The comparison of the d13C values of geranyl acetate with those of geraniol indicated that the ester has values more than 4% higher than its parent compound, except for the sample BULG 07. This is in disagreement with what has previously been described, where it has been shown that an ester has a similar or slightly lower d13C value than the corresponding alcohol.[19,22,3032,42] As a conrmation of what has been reported for other essential oils, the GC/C/IRMS analysis of an authentic R. damascena essential oil, directly obtained in the laboratory by hydro-distillation from fresh petals, was carried out. In the authentic sample, the d13C values of geraniol (27.5%) and geranyl acetate (27%) were found to be similar, as

Table 5. d13C values (%) of specic compounds of commercial essential oils of R. damascenaa Compoundb Retention time (s) BULG 01 BULG 02 BULG 03 BULG 04 BULG 05 BULG 06 BULG 07 BULG 08 Mean SD Min Max TURK 01 TURK 02 TURK 03 TURK 04 TURK 05 TURK 06 TURK 07 TURK 08 Mean SD Min Max IRAN 01 IRAN 02 IRAN 03 Mean SD Min Max
a b

Geranyl acetate 1368 20.3 21.1 20.9 18.3 20.3 20.1 25.6 20.7 20.9 2.1 25.6 18.3 20.7 20.4 19.4 19.7 20.3 20.5 21.6 21.3 20.5 0.7 21.6 19.4 19.9 18.2 19.6 19.2 0.9 19.9 18.2

Citronellol 1392 27.8 28.2 28.4 27.5 28.0 27.3 28.1 27.0 27.8 0.5 28.4 27.0 27.0 27.9 27.2 26.4 27.7 27.8 27.5 30.0 27.7 1.1 30.0 26.4 27.8 27.9 27.2 27.6 0.4 27.9 27.2

Nerol 1426 24.0 25.4 25.2 25.9 24.2 24.7 25.2 24.2 24.9 0.7 25.9 24.0 25.1 25.2 24.4 23.6 24.7 25.5 23.6 22.0 24.3 1.2 25.5 22.0 23.0 23.8 23.2 23.3 0.4 23.8 23.0

Geraniol 1485 26.2 26.0 27.1 26.0 24.0 26.0 25.8 25.0 25.8 0.9 27.1 24.0 25.1 24.5 26.0 24.9 24.0 25.7 25.1 24.0 24.9 0.7 26.0 24.0 25.1 25.9 24.1 25.0 0.9 25.9 24.1

Nonadecane 1567 * * * * * * * *

Heneicosane 1828 31 30 29 29 30 29 30 29 30 1 31 29 30 31 29 29 30 30 28 28 29 1 31 28 28 29 29 29 1 29 28

Farnesol 2223 27 31 32 29 30 31 32 32 31 2 32 27 29 30 30 27 31 29 31 28 29 1 31 27 30 30 * 30 0 30 30

* * * * * * * *

31 30 30

GC/C/IRMS conditions as in Experimental section. Compounds are listed in order of elution time. *Data not shown for co-eluting peaks or % relative peak area < 0.5 in GC/MS or GC/FID analysis.

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GC/MS, GC/FID and GC/C/IRMS analysis of Rosa damascena essential oil expected for a natural product. In the light of this, the high d13C values of geranyl acetate in the commercial rose essential oils can be explained by the addition of avors or essential oils from C4 plants, such as C. martinii (palmarosa). To deeper investigate this hypothesis, two commercially available samples of C. martinii essential oil were fully characterized by GC/MS, GC/FID and GC/C/IRMS. In agreement with literature results,[13,14] the main volatile compounds of these samples were found to be geraniol (79.882.1%) and geranyl acetate (8.610.4%). The d13C values of geraniol and geranyl acetate in the two C. martinii essential oils were found to be 10.2 and 11.5% and 10.8 and 9.9%, respectively, which are typical for a C4 plant. Therefore, it can be hypothesized that the commercial rose essential oils are made up of a mixture containing 95% R. damascena essential oil, e.g. with 20% geraniol (d13C: 27.5%) and 0.5% geranyl acetate (d13C: 27%), and 5% C. martinii, e.g. with 80% geraniol (d13C: 10%) and 10% geranyl acetate (d13C: 11%), resulting in 23% geraniol (d13C: 24.5%) and 1% geranyl acetate (d13C: 19%). Among the R. damascena essential oils, only BULG 07 showed an isotopic prole that could indicate the absence of the addition of an essential oil from a C4 plant. The addition of natural and/or synthetic components to essential oils is a fairly common practice in the essential oil industry. Enantioselective capillary GC and GC/C/IRMS are important tools in the authenticity control of avors and essential oils.[16,44] The adulteration of a commercially available rose oil sample by the addition of the unnatural enantiomers, (+)-cis-rose oxide, (+)-trans-rose oxide and (R)(+)-citronellol, has been previously described.[45] In relation to GC/C/IRMS, this study demonstrated for the rst time that geranyl acetate can be considered as a suitable endogenous reference compound for the authenticity assessment of rose essential oil. While reliable criteria for authenticity are reected by the enantiomeric composition of major chiral constituents in some essential oils,[46] enantioselective GC is less conclusive for those oils with varying enantiomeric composition of the chiral constituents.[46] GC/C/IRMS did not show these limitations and it is therefore a highly recommended technique for detecting possible adulteration of essential oils.

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CONCLUSIONS
The described results demonstrated that stable isotope ratio analysis, using GC/C/IRMS, represents a reliable technique, in combination with GC/MS and GC/FID, for the complete phytochemical characterization of rose essential oil. The addition of a natural cheaper oil from a C4 plant (C. martinii) was observed in most of the samples analyzed. Since the reported adulteration was undetected by GC/FID and GC/MS analysis of rose essential oils, the d13C isotopic ngerprint represents an effective tool for the authenticity assessment of these economically important natural products.

Acknowledgement
The authors are grateful to Dr Carlo Dappino of Agronatura (Spigno Monferrato, Alessandria, Italy) for providing an authentic sample of R. damascena essential oil.

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