Detailed characterisation of essential oils by flow-modulated GC GC TOF MS with Tandem Ionisation

Transcription

1 pplication Released: June 6 pplication Note 538 Detailed characterisation of essential oils by flow-modulated G G TOF MS with Tandem Ionisation Summary In this pplication Note, we describe the use of comprehensive two-dimensional gas chromatography (G G) with BenchTOF time-of-flight mass spectrometry (TOF MS) and Tandem Ionisation for characterisation and comparison of the composition of essential oils, aiding rigorous quality control. Nevertheless, the similar terpenes found in essential oils can be difficult (or impossible) to speciate when using conventional 7 ev electron ionisation, even with the added power of retention indices in two dimensions. Here we examine the use of novel Tandem Ionisation available with BenchTOF time-of-flight instruments to provide two complementary data sets from a single analysis one containing 7 ev data, and the other with soft I for enhancement of molecular ions and improved isomer speciation. This pplication Note shows the comparison of a range of essential oils by flow-modulated G G and BenchTOF detection, combined with multivariate statistical analysis for fast evaluation of compositional differences. Introduction Volatile oils of plants, also known as essential oils, have been used for many years for their flavour, fragrance and medicinal properties. However, their extreme complexity and diversity presents analysts with a significant challenge when it comes to quality control. They are generally composed of monoterpenes and sesquiterpenes, as well as their oxygenated derivatives, such as aliphatic aldehydes, alcohols and esters. In many cases, trace components can impart a distinctive fragrance character to the oil, meaning that rigorous quality control procedures are required to ensure that essential oils do not vary significantly from the initial reference batch, or contain any unwanted adulterants. Furthermore, because essential oils are raw materials for the perfume industry, analysts must now also ensure that they adhere to new legislation on fragrance allergens. Traditionally, gas chromatography coupled with mass spectrometry (G MS) has been the standard method for qualitative and quantitative analysis of such samples, relying on the combination of retention indices and mass spectra to confirm the identity of individual terpenes. However, in recent years, comprehensive two-dimensional G with time-of-flight MS (G G TOF MS) has become an attractive technique for characterisation of essential oils. The enhanced separation capacity offered by the coupling of two columns with different selectivities, combined with highly sensitive mass spectral identification, provides a highperformance solution for rapid screening of essential oils. Tandem Ionisation xclusive to Markes, Select-eV allows you to collect soft I spectra down to ev, with no inherent loss in sensitivity and full automation in TOF-DS software with no need for manual intervention. Tandem Ionisation now adds unparalleled productivity for soft ionisation. Soft and hard ionisation spectra are obtained across every peak, in both G and G G analyses, for comprehensive sample characterisation in a single run. aryophyllene ev One chromatographic peak, two complementary spectra. Tandem Ionisation using Select-eV enables 7 ev and soft I spectra to be collected across every peak in a single run. 7 ev T: +44 () F: +44 () : enquiries@markes.com

2 Page Background to BenchTOF systems BenchTOF instruments are time-of-flight mass spectrometers designed specifically for gas chromatography. They are particularly appropriate for robust and confident characterisation of essential oils, for the following reasons: Sensitivity: Highly efficient direct-extraction technology allows BenchTOF systems to acquire full-range spectra with SIM-like sensitivity, allowing them to reliably detect trace-level targets and unknowns in a single run, which would be difficult or impossible on a quadrupole system. Spectral quality: The reference-quality spectra produced by BenchTOF systems are a close match for those in commercial libraries such as NIST or Wiley. This enables quick and confident matching of analytes. Speed: The ability to record full-range mass spectral information to extremely high densities (, transient spectral accumulations per second) enables BenchTOF to handle the narrowest peaks encountered in well-optimised G G couplings. xperimental Sample preparation: % (v/v) dilutions of eight essential oils were prepared in ethyl acetate. G: Injector: Split/splitless Liner: Single taper with wool, 4 mm (i.d.) arrier gas: Helium, constant-flow at.6 ml/min Mode: Split : Temperature: 8 Septum purge: On, ml/min D column set: st dimension: BPX5, m.8 mm.8 µm nd dimension: DB7, m.5 mm.5 μm Temp. program: Main oven: 4 ( min), 3 /min to 6, /min to 8 ( min). Modulator: Insight flow modulator (SepSolve nalytical) Loop dimensions:.53 mm i.d. 3 mm (loop volume: 5 µl) Fill time: 36 ms Flush time: ms Modulation period (P M ): 3.8 s TOF MS: Instrument: BenchTOF-Select (Markes International) Filament voltage:.7 V Ion source: 3 Transfer line: 8 Mass range: m/z 45 4 Data rate: Hz in Tandem Ionisation mode at 7 ev and ev Software: Instrument control and data acquisition: TOF-DS (Markes International) Image processing: G Image (G Image, LL) Statistical analysis: MTLB (Ra, MathWorks) Results and discussion Screening of essential oils The G G TOF MS colour plots for eight essential oils (Figure ) display dramatic differences in monoterpene and seqsuiterpene content. Some, such as Sweet Birch (F), almost entirely comprise monoterpenes ( t R : 8 min), while others, like Patchouli (D), mostly contain sesquiterpenes ( t R > min). summary of the main characteristics of each oil is provided in Table. Bergamot B hamomile Second-dimension retention time (s) Himalayan edar D Patchouli First-dimension retention time (min) Figure : omparison of G G TOF MS colour plots for eight essential oils (continued on next page). T: +44 () F: +44 () : enquiries@markes.com

3 Page 3 Scots Pine F Sweet Birch Second-dimension retention time (s) G Tea Tree H Vetiver First-dimension retention time (min) Figure : omparison of G G TOF MS colour plots for eight essential oils (continued from previous page). ssential oil haracteristics Bergamot Mainly monoterpenes including camphene, linalool, limonene, γ-terpinene and β-pinene. B hamomile Mainly sesquiterpenes including farnesenes, bisabolone oxide and bisabolol oxide. Himalayan edar Mainly sesquiterpenes including α- and β-himachalene. D Patchouli Mainly sesquiterpenes and oxygenated derivatives, e.g. patchouli alcohol. Scots Pine Mix of mono- and sesquiterpenes, as well as benzyl alcohol (not observed in significant quantity in any other oil) and several oxygenated terpenes. F Sweet Birch Major peak for methyl salicylate. G Tea Tree Mainly monoterpenes including α- and γ-terpinene and α-pinene. H Vetiver Mainly sesquiterpenes including β- and γ-vetivenene. information into the key differences in sample composition. It is worth pointing out that the high repeatability of t R and t R using flow modulation is due to a dedicated P (electronic pressure control) module for each column. This makes flow modulation well-suited to large-scale projects and comparisons across multiple data sets, because of the minimal retention-time differences for any given peak. The P score plot (Figure 3) shows that hamomile (B), Himalayan edar (), Patchouli (D) and Vetiver (H) all cluster closely, while the other oils are spread out across the plot. The first three principal components described 8.8% of the sample variation. The major contributors to P are terpinen 4-ol and α-terpinene, while for P they are monoterpenes including linalool, limonene, linalyl acetate and β-pinene. Finally, methyl salicylate contributes most to the variation on P3, explaining why Sweet Birch (F, which has a high content of this compound) is separated from the other oils on this axis. Table : Summary of the major characteristics of essential oils analysed in this study. The flow-modulated G G TOF MS method effectively separated multiple chemical classes that would co-elute in a one-dimensional separation. It is useful to have retention times in both dimensions, as well as mass spectra for confirming compound identity. This is even more important when investigating final fragrance mixtures or cosmetics, which contain higher matrix levels that can hinder identifications in a D separation. Fast comparisons of composition template of over different target compounds was prepared and applied to each sample, in order to collate the peak area data (using I chromatograms for a specified quant ion for each target). Principal components analysis (P) was then applied to the peak lists to distil the P3 (.7%) P (6.7%) 7 F H D B.5.5 G.5.5 Figure : P score plot comparing the eight essential oils. P (33.4%) 7.5 T: +44 () F: +44 () : enquiries@markes.com

4 Page 4 This type of G G TOF MS screening approach, coupled with automated template-based compound identification in G Image, can allow differences in target compounds across multiple essential oil batches to be determined quickly and effectively in a quality control laboratory. Tandem Ionisation for confident identification of terpenes The screening approach described works well in cases where target compounds are known and have distinctive mass spectra for confident identification using automated templates. However, in many situations characterisation of the entire sample is required to provide the final fragrance formulation, including unknown compounds that may not be present in spectral libraries. This is a challenging prospect when the dominant constituents are terpenes with very similar I (7 ev) mass spectra, which are consequently difficult to speciate confidently. Tandem Ionisation is of great value in such cases, by allowing fast switching between conventional 7 ev ionisation and Select-eV soft I. This approach allows two complementary datasets to be acquired simultaneously without impacting laboratory workflow, and with perfectly aligned peaks for easy navigation of the soft I data. In fact, laboratory workflows can even be enhanced by applying a checklist (Table ) for identifying constituents of essential oils. Flow-modulated G G provides repeatable retention times in two dimensions ( t R and t R ), while Tandem Ionisation delivers confirmatory ion ratios (IRs) from both the 7 ev and soft I datasets, for robust quality control. ompound assignment t R t R Ion ratio 7 ev ev 7 ev/ ev Identity confirmed? β-pinene Y Linalyl acetate N (,6)-Farnesol Y (,6Z)-Farnesol Y B D amphene,5,5-trimethyl- 3-methylenecyclohexene ev Δ 3 -arene Terpinen-4-yl acetate 43 Oc ev 7 ev 7 7 ev ev 5 ev 5 7 ev 7 5 ev Table : Improved Q checks using G G TOF MS with Tandem Ionisation. Figure 3 provides spectral comparisons of two sets of terpenes cyclic monoterpenes (Figure 3 ) and oxygenated terpenes (Figure 3D ). The 7 ev spectra within each group of compounds are similar to each other, with identical ions present for each compound and in comparable ratios making it difficult to identify the individual compounds. The ev spectra, however, have distinct differences, meaning that the use of a library of ev spectra can add confidence to essential oil characterisation Isobornyl formate OHO ev 5 ev 5 Figure 3: Spectral comparisons at 7 and ev from Tandem Ionisation G G TOF MS for three cyclic monoterpenes (, m/z ) and two oxygenated terpenes (D, m/z 5) found in essential oils. T: +44 () F: +44 () : enquiries@markes.com

5 Page 5 onclusions In this pplication Note, we have shown that the use of flow-modulated G G with BenchTOF detection can be successfully applied for routine and repeatable screening of essential oils. This approach ensures that confident identification is achieved for these complex mixtures which is particularly important for quality control within the fragrance industry. The ability of G G TOF MS to provide structured chromatograms, with chemical classes eluting in bands, can aid data review, enabling swift tentative identifications to be made. The use of Tandem Ionisation then adds an additional level of confidence in situations where 7 ev data alone cannot speciate similar terpenes, but with no inherent loss in sensitivity or inconvenience to the operator. Tandem Ionisation can also fit seamlessly into laboratory workflows, providing independent ion ratio checks at both 7 ev and low ev for robust Q, alongside t R and t R. Importantly, application of Tandem Ionisation is fully controlled by TOF DS software, with two complementary datasets generated automatically, meaning that there is no inconvenience to the operator, and that no additional analysis time is required. Moreover, the peaks in both hard and soft ionisation data files are perfectly aligned, simplifying data exploration. References. B. d campora Zellner, P. Dugo, G. Dugo and L. Mondello, nalysis of essential oils (ch. 7), in Handbook of ssential Oils: Science, Technology and pplications (nd edition), K.H.. Baser and G. Buchbauer (eds.), R Press, 5, pp R.. Shellie, Volatile components of plants, essential oils and fragrances, in omprehensive Two Dimensional Gas hromatography, L. Ramos (ed.), lsevier, 9, pp Trademarks BenchTOF and BenchTOF Select are trademarks of Markes International. Select-eV and Tandem Ionisation are registered trademarks of Markes International. BPX5 is a trademark of SG nalytical Science (Trajan Scientific). G Image is a trademark of G Image, LL. Insight is a trademark of SepSolve nalytical. MTLB is a registered trademark of The MathWorks, Inc. pplications were performed under the stated analytical conditions. Operation under different conditions, or with incompatible sample matrices, may impact the performance shown. T: +44 () F: +44 () : enquiries@markes.com N