Article pubs.acs.org/JAFC
Quality Evaluation of Terpinen-4-ol-Type Australian Tea Tree Oils and Commercial Products: An Integrated Approach Using Conventional and Chiral GC/MS Combined with Chemometrics Mei Wang,† Jianping Zhao,† Bharathi Avula,† Yan-Hong Wang,† Amar G. Chittiboyina,† Jon F. Parcher,† and Ikhlas A. Khan*,†,‡,§ †
National Center for Natural Products Research and ‡Division of Pharmacognosy, Department of BioMolecular Science, School of Pharmacy, University of Mississippi, University, Mississippi 38677, United States § Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia S Supporting Information *
ABSTRACT: GC/MS, chiral GC/MS, and chemometric techniques were used to evaluate a large set (n = 104) of tea tree oils (TTO) and commercial products purported to contain TTO. Twenty terpenoids were determined in each sample and compared with the standards specified by ISO-4730-2004. Several of the oil samples that were ISO compliant when distilled did not meet the ISO standards in this study primarily due to the presence of excessive p-cymene and/or depletion of terpinenes. Forty-nine percent of the commercial products did not meet the ISO specifications. Four terpenes, viz., α-pinene, limonene, terpinen-4-ol, and α-terpineol, present in TTOs with the (+)-isomer predominant were measured by chiral GC/MS. The results clearly indicated that 28 commercial products contained excessive (+)-isomer or contained the (+)-isomer in concentrations below the norm. Of the 28 outliers, 7 met the ISO standards. There was a substantial subset of commercial products that met ISO standards but displayed unusual enantiomeric +/− ratios. A class predictive model based on the oils that met ISO standards was constructed. The outliers identified by the class predictive model coincided with the samples that displayed an abnormal chiral ratio. Thus, chiral and chemometric analyses could be used to confirm the identification of abnormal commercial products including those that met all of the ISO standards. KEYWORDS: tea tree oil, conventional and chiral GC/MS, chemometrics, quality evaluation
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INTRODUCTION Tea tree oil is defined1 for commercial products as any essential oil obtained by steam distillation of the foliage and terminal branchlets of Melaleuca alternifolia, Melaleuca linariifolia, Melaleuca dissitif lora or other species of the genus Melaleuca that meets all of the physical and composition specifications given in ISO 4730-2004. This definition, however, currently does not specify a given plant species. Due to the high phylogenetic variations of Malaleuca species of tea tree, evaluation of such commercial TTO products can be problematic. GC/MS techniques are commonly used to determine the chemical composition of TTO products. However, verifying that a commercial product purported to contain TTO meets the ISO standards without substitution or adulteration can still be difficult. Due to the increasing market for TTO, there is a growing trend toward adulteration or substitution of these products.2,3 The primary components4 of TTOs are mono- and sesquiterpenes and their associated ethers and alcohols. TTOs have become popular because of their efficacy, history, and perceived “natural” label. Their medicinal properties, including cytotoxic, antimicrobial, anti-inflammatory, antioxidant, anticancer, and antiviral properties, have been extensively investigated in recent years.5−7 The chemical composition of compromised or unsatisfactory products to be sold as TTOs can be altered by producers to meet ISO standards by substitution with other plant species or adulteration with synthetic or natural chemical compounds. In © 2015 American Chemical Society
addition, the chemical composition of a TTO product or oil that initially meets ISO standards may vary with time or storage conditions to yield a product that no longer meets the standards. In particular, terpinen-4-ol, α-terpinene, and γ-terpinene can oxidize to p-cymene.6,8,9 The lower limits specified by the ISO 4730-2004 for terpinen-4-ol, α-terpinene, and γ-terpinene are 30%, 5%, and 10%, respectively, while the upper limit for pcymene is 8%. Oxidation of commercial TTO products may result in low terpinene and high p-cymene concentrations outside the ISO specified limits. The “use by” dates for commercial TTO products specified by health authorities are designed to obviate this problem. However, the application of ISO standards alone to verify a commercial TTO product is often unsatisfactory. A secondary method of product quality evaluation involves the determination of enantiomeric ratios of chiral compounds. This type of analysis can also be carried out with GC/MS instrumentation with chiral, usually cyclodextrin-based, stationary phases. Unfortunately, the enantiomeric ratios published to date do not provide clear validation criteria of the identification of genuine TTO content. The range of reported enantiomeric compositions often included racemic mixtures. For Received: Revised: Accepted: Published: 2674
January 9, 2015 February 25, 2015 February 28, 2015 February 28, 2015 DOI: 10.1021/acs.jafc.5b00147 J. Agric. Food Chem. 2015, 63, 2674−2682
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Journal of Agricultural and Food Chemistry
phellandrene (6), α-terpinene (7), p-cymene (8), limonene (10), γterpinene (11), terpinolene (12), and terpinen-4-ol (13) (listed in Table 1 and Figure S1, Supporting Information) were purchased from
example, the following (peak area) percentages have been reported: sabinene (42−63%), α-phellandrene (37−63%), linalool (30−68%), and β-pinene (35−78%).9−12 On the other hand, at least four terpenes have been reported with consistently predominate (+)-enantiomers, viz., α-pinene (60−90%), terpinen-4-ol (65−70%), α-terpineol (70−78%), and limonene (58−62%).9−13 No clear standard has been developed primarily due to the limited number of investigations and samples. The measurement of chiral ratios of TTO components is demanding and often requires the use of multidimensional GC or NMR with lanthanide shift reagents. These techniques are labor intensive and require extensive operator expertise. Chemometrics has increasingly found applications in quality control of natural products. It can be used to extract chemically relevant information out of large quantities of chemical data to provide a better way to represent and display the information as well as a better understanding of the chemical system. Two chemometric approaches are usually applied: the targeted approach selectively analyzes a subset of compounds, while the nontargeted approach provides a holistic analysis of the entire chemical data set. Chemometrics has been used extensively14−18 for the analysis of infrared and mass spectra of essential oils. However, this data analysis tool has not been used to validate TTOs or commercial products based on GC/MS data with conventional or chiral columns. The inherent complexity and variability within plants and essential oils pose challenges in the identification and quantification of specific chemical compounds as quality control parameters. The need exists for the development of chemical analysis techniques and data analysis tools that can profile the whole chemical system including those components present at very low concentration levels. As part of an ongoing research program on authentication, safety, and quality control of phytochemicals, the present investigation was carried out employing the aforementioned conventional and chiral GC/ MS systems combined with chemometric analyses to assess the quality of tea tree oils and commercial products. The study involved a large number (n = 104) of samples divided into two categories, viz., known provenance essential oils and commercial products of unknown provenance. Thus, an independent norm from the chiral analysis for a given set of samples could be established for comparison purposes. The objectives of the current investigation were to employ the three previously discussed techniques in the investigation of an extensive set of TTOs and commercial products. This holistic approach will be used to identify TTO and commercial samples that do not represent the common terpinen-4-ol type of tea tree oil.
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Table 1. Twenty Most Abundant Compounds in the Tea Tree Oil Sample (A-22) by Conventional GC/MS Analysis no.
compound
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
α-thujene α-pinene sabinene β-pinene myrcene α-phellandrene α-terpinene p-cymene 1,8-cineole limonene γ-terpinene terpinolene terpinen-4-ol α-terpineol aromadendrene alloaromadendrene ledene δ-cadinene globulol viridiflorol
17 18 19 20
retention index
literature value17 (methyl silicone column)
925 931 965 969 983 996 1009 1011 1019 1021 1052 1083 1172 1182 1452 1471
931 939 972 978 986 1002 1016 1016 1025 1025 1056 1081 1175 1185 1475
1503 1526 1568 1577
1484 1524 1588
% peak area (ISO) 1−6 trace−3.5
5−13 0.5−8 trace−15 0.5−1.5 10−28 1.5−5 30−48 1.5−8 trace−3
trace−3 trace−3 trace−1 trace−1
Sigma-Aldrich. A mixture of a series of n-alkanes (C9H20 to C22H46) was used for the determination of the retention indices, and the alkane standards were purchased from PolyScience Corp. The analytical standard n-tridecane (C13H28) was selected as the internal standard and obtained from PolyScience Corp. The enantiomeric compounds of αpinene, limonene, terpinen-4-ol, and α-terpineol were purchased from Sigma-Aldrich. Sample Preparation. Five microliter samples were diluted in 1 mL of n-hexane. The selected internal standard (C13H28) was added to each sample solution at a constant concentration. Chromatography Instruments and Conditions. Conventional GC/MS Analyses. Gas chromatographic analysis was performed on an Agilent 7890 GC instrument equipped with an Agilent 5975C massspecific detector and an Agilent 7693 autosampler. An Agilent J&W HP1 fused silica capillary column (60 m × 0.25 mm i.d.) coated with a 0.25 μm film of cross-linked 100% dimethyl-polysiloxane was used with helium as the carrier gas at a constant pressure of 24 psi. In a typical analysis, the oven was held for 2 min at 50 °C and then programmed at 2.0 °C/min to 180 °C. The post run was set at 250 °C for 5 min to clean up the column. The injector temperature was 250 °C. The split ratio was set to 25:1. Duplicate injections were made for each sample. For the scan measurements, the spectra were recorded at 70 eV from m/z 35 to 450. Compound identification involved comparison of the spectra with the databases (Wiley and NIST) using a probability-based matching algorithm. Further identification was based on the relative retention indices compared with the literature and the reference standards purchased from commercial sources. Chiral GC/MS Analyses. The same instrument setting used for the conventional GC/MS analysis was applied for the chiral GC/MS analysis. The chiral GC/MS column was an Agilent J&W HP-Chiral-20β (30 m × 0.25 mm, i.d.) coated with a 0.25 μm film of 20% β-cyclodextrin in (35%-phenyl)-methylpolysiloxane. Helium was used as a carrier gas. The flow rate was 1 mL/min for the first 27 min and then programmed at 0.2−3 mL/min for the rest of the experiment. In a typical chiral analysis, the oven was held for 10 min at 50 °C and programmed at 3.0 °C/min to 100 °C, 2.0 °C/min to 140 °C, and then 4.0 °C/min to 180 °C in the last step. The injector temperature was 250 °C. The split ratio
MATERIALS AND METHODS
Sample Information. Fifty-seven known provenance tea tree oil samples (A1−14, A17−40, A48−64, A84, and A90) were obtained by steam distillation of Melaleuca alternifolia. Oils A15 and 16 were obtained from the steam distillation of M. linariifolia and M. dissitif lora, respectively. Forty-three commercial samples that claimed to be tea tree oil samples (A41−44, A46, A47, A65−83, A85−89, A92, and A100− 110) were purchased from various foreign sources. The provenance of these samples was unknown. Sample A45 was sold as synthetic tea tree oil, and A99 was sold as synthetic terpinen-4-ol. All TTOs and commercial products were collected by the Australian Tea Tree Industry Association (ATTIA, Casino NSW, Australia). Specimens of all samples are deposited at the botanical repository of the National Center for Natural Products Research (NCNPR), University of Mississippi. Reagents and Standards. GC-grade n-hexane and the reference standards α-thujene (1), α-pinene (2), β-pinene (4), myrcene (5), α2675
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Figure 1. GC/MS chromatograms of sample A-22. (A) Total ion chromatogram (m/z 35−450). (B) Extracted ion chromatogram (m/z 136) for monoterpenes. (C) Extracted ion chromatogram (m/z 204) for sesquiterpenes. was set to 40:1. Duplicate injections were made for each sample. The mass spectra were recorded at 70 eV from m/z 40 to 550. Chemometric Analysis. The GC/MS data were acquired by Agilent MSD Productivity ChemStation software (E.02.02). Extraction of the GC/MS data was performed using the NIST Automated Mass Spectral Deconvolution and Identification Software (AMDIS). Ions were extracted as entities characterized by retention time (tR), peak intensity, and mass to charge ratio (m/z). The ELU file format required for the chemometric analysis was created by AMDIS for each sample and then exported into the Mass Profiler Professional software (MPP, version B.12.05, Agilent Technologies). A holistic nontargeted approach was used for the preprocessing of GC/MS data using the full spectra which allows for unbiased analysis. The initial stage involved baseline correction, data smoothing, and peak alignment. Various minimum abundance settings and compound quality score filter parameters were examined; 5000 counts and a score of 20 were finally selected for the entities extraction in the entire experiment time window for the minimum absolute abundance and minimum quality score, respectively. Alignment of retention time with a tolerance retention time window of 0.15 min and similarity of spectral pattern was carried out across the entire sample set. The internal standard selected for GC/MS analysis was applied for normalization of the peak intensity
to account for the differences in the abundances of each compound. A list of 634 entities obtained from the MPP software was exported to SIMCA (version 12) for chemometric analysis. Principal component analysis (PCA) was performed for a quick overview of the data. A sample class predictive model (PCA-class) based on the TTO samples that met the ISO standards was constructed. The class predictive model was cross-validated and then applied to evaluate the remainder of the samples in the TTO data set.
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RESULTS AND DISCUSSION Typical chromatograms of a TTO sample obtained from GC/ MS with a conventional stationary phase (HP-1) and a chiral stationary phase (HP-Chiral-20B) are shown in Figures 1 and 2, respectively. The assignment of the 20 components and their corresponding retention indices in the conventional chromatogram as well as the concentration limits regulated by ISO standards are listed in Table 1. Table S1, Supporting Information, gives the (peak area percentage) analysis results for the 20 compounds listed in Table 1 with the conventional GC stationary phase. Table S2, Supporting Information, gives the results from chiral analyses. 2676
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Figure 2. Chromatograms of chiral GC/MS analysis for the major enantiomers in TTOs. (A) Mixture of standards. (B) Sample A-1. (C) Extracted ion chromatograms. Dashed line (EIC at m/z 68) illustrates the detection of (+)/(−) limonene. Solid line (EIC at m/z 134) illustrates the detection of pcymene.
Figure 3. Comparison of upper and lower limits of the major terpenes in tea tree oils, commercial products, and ISO standards. Synthetic samples A45, A99, and the samples of M. linariifolia (A15) and M. dissitif lora (A16) were not included.
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Figure 4. (A) Chiral analysis results for α-pinene. Gap in the data represents the omission of 15 points with the average enantiomeric ratio. Samples that displayed an enantiomeric ratio greater/less than the average of TTOs ± three times the standard deviation (SD) are shown as ▲/▼ in the figure. (B) Chiral analysis results for limonene. Data points shown with open symbols represent samples that did not meet ISO standards. (C) Chiral analysis results for terpinen-4-ol. (D) Chiral analysis results for α-terpineol.
Conventional GC/MS Analyses: Comparison with ISO Standards. Known Provenance Tea Tree Oil Samples. Figure 3 shows the concentration ranges of the eight major ISO-
specified terpenes and related alcohols and ethers along with the ranges of concentration specified by ISO 4730-2004. The measured concentration ranges for the same components in the 2678
DOI: 10.1021/acs.jafc.5b00147 J. Agric. Food Chem. 2015, 63, 2674−2682
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Journal of Agricultural and Food Chemistry ATTIA TTO samples are also shown in the figure. The compounds that were outside the ISO standards are marked in gray in Table S1, Supporting Information. Fourteen of 57 TTO samples did not meet at least one of the ISO standards, even though all of the oils were ISO compliant when distilled as evidenced by certificates of analysis obtained by ATTIA immediately after distillation. The primary problems were excess p-cymene and diminished α- and γ-terpinene as well as terpinolene concentrations. Auto-oxidation was probably responsible for most of the observed deviations.19 Aside from the four probe solutes involved in the generation of p-cymene, all of the TTOs were within ISO standards for the other 10 solutes. The analytical results for the five sesquiterpenes specified in the ISO standards are included in the tables; however, their concentrations were always less than 3%. Their low concentrations and relatively high variability and uncertainty limited their use as verification probes. Commercial Samples. In Table S1, Supporting Information, the TTO and commercial samples that did not meet the ISO standards are marked in gray. Samples A45 and A99 were labeled as synthetic TTO and synthetic terpinen-4-ol, respectively. These synthetic samples both contained about 95% terpinen-4-ol along with low concentrations of α- and γ-terpinene and pcymene. Twenty-three other commercial samples did not meet ISO standards. The major problems were an excess of limonene (16 samples), α-terpineol (7 samples), or p-cymene (11 samples). A lower number of samples was deficient in αterpinene (7 samples), γ-terpinene (4 samples), and terpinen-4ol (3 samples). On the basis of solely the ISO standards, 49% of the commercial TTO samples were not qualified to be sold as “tea tree oils” on the commercial market. This raises the question whether or not the ISO standards alone are sufficient to positively identify oils and commercial products that actually contain Australian “terpinen-4-ol type” tree oil. The most common secondary test for TTO quality evaluation involves the analysis of chiral components. Chiral GC/MS Analyses. A complete chiral analysis was carried out for the entire data set for four terpenes with predominately (+)-enantiomer for which standards were available, viz., α-pinene, limonene, terpinen-4-ol, and αterpineol. The results for all of the chiral solutes are given in Table S2, Supporting Information. Resolution of limonene enantiomers from p-cymene has proven to be a difficult problem in many investigations of the chiral components of TTOs.10,12,13 p-Cymene has been reported to coelute with (+)-limonene10,12 or (−)-limonene.13 Twodimensional GC has been used to overcome this problem.11,12 In the current study, p-cymene (8) was found to elute between (−)and (+)-limonene (10) as shown in Figure 2. The mass spectrum for limonene shows a base peak at m/z 68 (isoprenyl), while the spectrum for p-cymene is barren in this mass region. Thus, selected ion monitoring at m/z 68 was used to differentiate limonene from p-cymene (m/z 134) as shown in the inset (C) of Figure 2. Known Provenance Tea Tree Oil Samples. The enantiomeric ratios +/− for all of the known provenance essential oil samples were calculated and used as the norm for evaluation of the commercial samples. The tea tree oil samples showed consistent enantiomeric ratios. The averages and standard deviations were 13.0 (±0.8), 1.83 (±0.08), 1.72 (±0.03), and 3.86 (±0.03) for αpinene, limonene, terpinen-4-ol, and α-terpineol, respectively. The chiral ratios of α-pinene, limonene, terpinen-4-ol, and α-
terpineol for all of tea tree oils and commercial products are shown in Figure 4A−D. The lines in the plots are the averages for the enantiomeric ratios of just the known provenance oil samples. Incomplete resolution of enantiomers skewed the results toward the later eluting enantiomer, i.e., the (+)-enantiomer for all compounds except α-terpineol. The effect was especially obvious for the components at the highest concentration and lowest resolution, viz., the alcohols. Better resolution could be obtained with dilute samples or higher split ratio but with an unacceptable loss of sensitivity. The analyses of a large number of samples allowed the establishment of a characteristic +/− chiral ratio, i.e., the average of the TTO data, without regard to previously published data. Outliers could be identified by their “distance” from the average. Thus, the absolute value of the chiral ratio was less important than the deviation of a sample from the norm of the TTO data set. Commercial Samples. Except for the synthetic samples, all of the ATTIA samples met the ISO standards (1−6%) for α-pinene. Figure 4A shows the chiral results and outliers for α-pinene. The data are given as the ratio (+/−)-enantiomers rather than percentages because it was easier to identify outliers with ratios rather than percentages. Mosandl11 reported ratios of 6−10, Southwell reported a ratio of 9.3,6 and Leach13 reported a ratio of 9. The average value for α-pinene in Table S2, Supporting Information, was 13.0. Outliers were identified as samples that displayed an enantiomeric ratio greater than the average plus three times the standard deviation (SD). These points are shown as ▲ in the figure. The same criterion was used to determine outliers below the average minus three times the SD (▼). The chiral results did not show a clear plateau. This compound is ubiquitous in many natural products, and the concentration range was low, so this probe solute is not satisfactory for quality control of TTO products. The chiral results for limonene are illustrated in Figure 4B. The ratio of (+/−)-isomers in TTO has been reported as 1.4−1.6 by Mosandl11 and 1.7 by Shellie.12 In the present study, the average value for the limonene data was 1.83 for the 57 TTO samples, all of which met the ISO standards (0.5−1.5%). Four commercial samples, viz., A44, A76, A77, and A109, contained almost exclusively (+)-limonene. Samples A16, A85, and 101 contained a racemic mixture of limonene enantiomers. Ten other samples contained primarily the (+)-isomer but in concentrations higher than the average of all samples. Of the 17 samples that were outliers for the limonene chirality, 9 were also outliers for the ISO standards. The samples that did not meet ISO standards are shown as open data points in the figures. Terpinen-4-ol or 4-terpineol is a major, characteristic component of TTO (30−48%) and the basis for the so-called terpinen-4-ol type of TTO. Previous literature reports the chiral ratios for terpinen-4-ol in TTO range from 2.17,2 1.96,9 2.13− 2.57,10 1.78−2.23,11 2.22,12 and 1.88.13 The average of the data for terpinen-4-ol in Table S2, Supporting Information, was 1.72. The results are shown in Figure 4C. Only two samples (A16 and A89) displayed an excess of the (+)-enantiomer. However, 22 samples had measured ratios significantly less than the average of the full data set. Because of the broad range of concentration (30−48%) for terpinen-4-ol specified in the ISO document, only three samples (A16, A47, and A85), other than the two synthetic samples, did not meet the ISO standards for terpinen-4-ol. The two synthetic samples (A45 and A99) both gave measured +/− ratios of ∼0.3 compared to the average of 1.72 for the natural essential oil samples. Thus, a low enantiomeric ratio for 2679
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Figure 5. PCA score plot of the TTOs and commercial products. CP: Commercial samples met ISO standards. CF: Commercial samples failed to meet ISO standards. AF: TTOs failed to meet ISO standards. AP: TTOs met ISO standards. DS: Different species (A15 and A16).
utilizing information concerning all of the components in a TTO or commercial sample. However, three-dimensional GC/MS data sets are difficult to interpret because of their sheer size. For example, in the present study with 104 samples, ca. 50 components with a mass range of 500 amu, the data set contained over 2 million points. Chemometric pattern recognition techniques, such as principal component analysis, are valuable tools for reducing the complexity of such data sets. The GC/MS results normalized to the internal standard, C13H28, were subjected to PCA, in which all observations and variables are present. The scores plot for the entire data set is shown in Figure 5. The summary of the fit of the model is displayed with R2X (cum) [R2X shows the percentage of variance in the data that is explained by a particular component, R2X(cum) sums up the R2X as they accumulate with an increase in the number of components] and Q2 (cum) [indicates the predictive ability of the model]. In total the PCA describes 93.1% of the variation (R2X) in the data with a Q2 of 93.0% for the first component indicating good prediction properties of the model. The first two components explain 96.0% of the variables. The R2 and Q2 plot for the PCA is given in Figure S2, Supporting Information. In this case, PCA provides an overview of the data and identifies the trends, groups, and strong outliers. Class prediction analysis has proven to be a valuable technique in characterization and authentication of traditional medicinal
this component may be a clear indication of substitution or adulteration in commercial products. The results for the chiral analysis for α-terpineol are shown in Figure 4D. The previously measured chiral ratios for α-terpineol in TTOs were 2.92,2 3.31,9 2.85−3.35,10 2.65,12 and 2.94.13 The average for all of the TTO samples in Table S2, Supporting Information, was 3.86. Sixteen samples had excessively high +/− ratios, while nine displayed excessively low ratios. With this probe solute, there were seven samples (shown in Figure 4D as open data points) that did not meet the ISO composition standards and were outliers from the average value in the chiral analyses. Thus, this solute may provide a method for the detection of unacceptable commercial products that would not be detected with the ISO standards alone. Chiral analysis can be used to identify commercial samples that are unacceptable in chiral distribution but satisfactory in chemical composition (chemotype). However, chiral GC is difficult. Baseline resolution between enantiomers is hard to achieve and even more difficult to maintain especially with complex samples such as natural products. Thus, chiral analysis alone is possibly not the optimum method for evaluating or validating commercial TTO products. Chemometric Analyses. Chiral analysis is based only on limited probe solutes such as α-pinene, limonene, terpinen-4-ol, and α-terpineol. GC/MS analysis is a more holistic approach 2680
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plants. The class prediction model allows assigning new samples into previously determined groups in an unbiased fashion. This workflow would be very useful for the QC of natural products and dietary supplements because the batch samples can be automatically acquired, processed, and class predicted. In the current investigation, all known provenance TTOs that met the ISO standards cluster in a single group (green) in the PCA score plot (Figure 5). The chiral ratios of α-pinene, limonene, terpinen-4-ol, and α-terpineol for the tea tree oil samples in this group were also within the ranges of norms. Thus, this group of samples was used to construct a class predictive model (PCAclass). The PCA-class model was cross-validated and used to evaluate all commercial samples and the TTO samples that did not meet the ISO standards. The R2 and Q2 values for the PCA class model (Figure S3, Supporting Information) were 95.5% and 95.4%, respectively. The results given in Table S3, Supporting Information, provide a confidence measure to the prediction, PModXPS+. This value shows the probability of the predicted observations belonging to the model. Observations with a probability of belonging less than 5% (PModXPS+ < 0.05) are considered to be outliers of the class predictive model and dissimilar to the observations used to build the model. In particular, there were several commercial products that met the ISO specifications but displayed unusual enantiomeric ratios (A100, A101, A104−106, and A110); those commercial products were also identified as outliers by the class prediction model. Additionally, there were TTOs and commercial products (A9, A17, A21−23, A48, A-50, and A-79) that met the chiral specifications but did not meet the ISO standards; they were also outliers identified by the PCA class prediction model. The results from the class prediction model and the outliers identified by different methods are summarized in Table S3, Supporting Information. In conclusion, both chiral and conventional GC/MS analysis can be used to identify abnormal commercial tea tree oil products. Four chiral components, viz., α-pinene, limonene, terpinen-4-ol, and α-terpineol, were utilized for chiral analysis of the TTO samples. These compounds are major components of terpinen-4-ol type oils. Chiral analysis based on these four probes could be used to identify unsatisfactory, adulterated, substituted, or otherwise unusual commercial TTO products. Analysis of three-dimensional GC/MS data along with chemometrics can also be used to isolate outliers in commercial samples. In this investigation, the outliers identified by chiral analysis coincided with the outliers identified by the class predictive model for all of the TTO components.
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This research was supported in part by “Science-Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 1U01FD004246-04 and the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement number 586408-1-603 amendment 04. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Agilent Technologies for provision of the instrumentation and MPP software. Appreciation is also extended to Tony Larkman and the Australian Tea Tree Industry Association (ATTIA) for provision of the tea tree oils and commercial samples.
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(1) European Commission, Scientific Committee on Consumer Products. Opinion on tea tree oil. Adopted Dec 16, 2008; p 34; http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/ sccp_o_160.pdf (Feb, 23, 2015). (2) Wong, Y. F.; Davies, N. W.; Chin, S.-T.; Larkman, T.; Marriott, P. J. Enantiomeric distribution of selected terpenes for authenticity assessment of Australian Melaleuca alternifolia oil. Ind. Crops Prod. 2015, 67, 475−483. (3) Chudleigh, P.; Simpson, S. Economic evaluation of investment in the tea tree oil R&D program, 2010; https://rirdc.infoservices.com.au/ items/10-212 (Feb 23, 2015). (4) International Standard ISO-4730. Oil of Melaleuca, terpinen-4-ol type (tea tree oil), 2004. (5) Hammer, K. A.; Carson, C. F.; Riley, T. V.; Nielsen, J. B. A review of the toxicity of Melaleuca alternifolia (tea tree) oil. Food Chem. Toxicol. 2006, 44, 616−625. (6) Southwell, I. Tea tree constituents. Tea tree-the genus Melaleuca; CRC Press: Boca Raton, FL, 1999; pp 29−62. (7) Carson, C. F.; Riley, T. V. Safety, efficacy and provenance of tea tree (Melaleuca alternifolia) oil. Contact Dermatitis 2001, 45, 65−7. (8) Brophy, J. J.; Davies, N. W.; Southwell, I. A.; Stiff, I. A.; Williams, L. R. Gas chromatographic quality control for oil of Melaleuca terpinen-4ol type (Australian tea tree). J. Agric. Food Chem. 1989, 37, 1330−5. (9) Sciarrone, D.; Ragonese, C.; Carnovale, C.; Piperno, A.; Dugo, P.; Dugo, G.; Mondello, L. Evaluation of tea tree oil quality and ascaridole: A deep study by means of chiral and multi heart-cuts multidimensional gas chromatography system coupled to mass spectrometry detection. J. Chromatogr. A 2010, 1217, 6422−6427. (10) Shellie, R.; Mondello, L.; Dugo, G.; Marriott, P. Enantioselective gas chromatographic analysis of monoterpenes in essential oils of the family Myrtaceae. Flavour Fragrance J. 2004, 19, 582−585. (11) Kreck, M.; Scharrer, A.; Bilke, S.; Mosandl, A. Enantioselective analysis of monoterpene compounds in essential oils by stir bar sorptive extraction (SBSE)-enantio-MDGC-MS. Flavour Fragrance J. 2002, 17, 32−40. (12) Shellie, R.; Marriott, P.; Cornwell, C. Application of comprehensive two-dimensional gas chromatography (GC × GC) to the enantioselective analysis of essential oils. J. Sep. Sci. 2001, 24, 823− 830. (13) Leach, D. N.; Wyllie, S. G.; Hall, J. G.; Kyratzis, I. Enantiomeric composition of the principal components of the oil of Melaleuca alternifolia. J. Agric. Food Chem. 1993, 41, 1627−32. (14) Tankeu, S.; Vermaak, I.; Kamatou, G.; Viljoen, A. Vibrational spectroscopy as a rapid quality control method for Melaleuca alternifolia Cheel (tea tree oil). Phytochem. Anal 2014, 25, 81−88. (15) Sandasi, M.; Kamatou, G. P. P.; Baranska, M.; Viljoen, A. M. Application of vibrational spectroscopy in the quality assessment of Buchu oil obtained from two commercially important Agathosma species (Rutaceae). S. Afr. J. Bot. 2010, 76, 692−700.
ASSOCIATED CONTENT
S Supporting Information *
Percentage of major compounds in tea tree oil samples and commercial products; enantiomeric percentage of four terpenes; results from PCA-class model prediction and outliers identified by ISO specifications, chiral analysis, and class prediction model; chemical structures of 20 major compounds present in tea tree oil (M. alternifolia); summary of R2X and Q2 for the PCA; summary of R2X and Q2 for the PCA-class model. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Tel.: +1-662-915-7821. Fax: +1-662-915-7989. E-mail: ikhan@ olemiss.edu. 2681
DOI: 10.1021/acs.jafc.5b00147 J. Agric. Food Chem. 2015, 63, 2674−2682
Article
Journal of Agricultural and Food Chemistry (16) Juliani, H. R.; Kapteyn, J.; Jones, D.; Koroch, A. R.; Wang, M.; Charles, D.; Simon, J. E. Application of near-infrared spectroscopy in quality control and determination of adulteration of African essential oils. Phytochem. Anal. 2006, 17, 121−128. (17) Jalali-Heravi, M.; Zekavat, B.; Sereshti, H. Characterization of essential oil components of Iranian geranium oil using gas chromatography-mass spectrometry combined with chemometric resolution techniques. J. Chromatogr. A 2006, 1114, 154−163. (18) Sandasia, M.; Kamatoua, G. P. P.; Gavaghanb, C.; Baranskac, M.; Viljoena, A. M. A quality control method for geranium oil based on vibrational spectroscopy and chemometric data analysis. Vib. Spectrosc. 2011, 57, 242−247. (19) Davies, N. W. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. J. Chromatogr. 1990, 503, 1−24.
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DOI: 10.1021/acs.jafc.5b00147 J. Agric. Food Chem. 2015, 63, 2674−2682
