- Email: [email protected]
GAS CHROMATOGRAPHY | Terpenoids☆
GAS CHROMATOGRAPHY | Terpenoids☆ GB Lockwood, University of Manchester, Manchester, UK ã 2013 Elsevier Inc. All rights reserved.
Analytical Techniques Terpene Extraction Chromatographic Separation of Constituents Detection and Characterisation of Terpenes Qualitative Techniques Applications Monoterpenes/Sesquiterpenes Essential Oils Cosmetics/Perfumes Forensic Science Cannabis Diterpenes Triterpenes Steroids Tetraterpenes Carotenoids Polyterpenes Rubber Problems and Perspectives Conclusions References
Glossary AEDA Aroma extract dilution analysis DCM Dichloromethane DHEA Dehydroepiandrosterone EI Electron impact FAB-MS Fast atom bombardment-MS FID Flame ionization detector GC Gas chromatography GC-DCCC GC-droplet counter centre chromatography GC-MS GC-mass spectroscopy GC-O GC-olfactometry GC-TOF-MS GC-time of flight-MS
3 3 4 4 5 5 5 6 6 7 7 8 9 10 10 10 10 10 11 11 11
HPLC High pressure liquid chromatography HS Head space IRMS Isotope ratio MS MDGC Multi-dimensional GC MSD Mass sensitive detector PTV Programmed temperature vaporiser RI Retention index SFE Supercritical fluid extraction SIM Selected (single) ion monitoring SPME Solid phase micro-extraction THC Tetrahydrocannabinol THCA Tetrahydrocannabinolic acid
Gas chromatography (GC) has long been used for separation of related terpenes in analysis of natural products and pharmaceuticals, specifically for characterisation of new entities, and quality control of known terpenes. The monoterpenoids and sesquiterpenoids, often co-occur in essential oils, which are important flavour and fragrance components in food and cosmetics. The diterpenes possess a number of historical applications, for example as components in paints and varnishes, triterpenes encompass the steroids and other commercially important compounds used in medicines, and the tetraterpenes such as the carotenoids are widely present in food plants, also having wide-ranging medical applications. These individual terpene groups, particularly the mono- and sesqui-terpenes, often occur both individually and together in large numbers of structurally closely related compounds, demanding separation systems capable of high resolution. Natural rubber, polyisoprene, is the terpene with the highest molecular weight, and GC applications for the analysis of this polymer are also possible. Specific drugs of abuse, namely cannabinoids and steroids have also been chromatographed using GC. Table 1 shows the range of structural types of terpenes analysed by gas chromatography (GC). ☆
Change History: March 2013. GB Lockwood updated text throughout.
Reference Module in Chemistry, Molecular Sciences and Chemical Engineering
http://dx.doi.org/10.1016/B978-0-12-409547-2.04768-5
1
2
GAS CHROMATOGRAPHY | Terpenoids
Table 1
Range of structural types of terpenoids widely analysed by GC
Structural type
Example
Monoterpene
a-Pinene
Diterpene
Abietic acid
Structure
H
O HO
Sesquiterpene
a-Phellandrene
Cannabinoid
11-nor-D9-tetrahydrocannabinol-9-carboxylic acid
O
OH
OH H H O
Triterpene
Lupeol
HO
Steroid
Dehydroepiandrosterone
CH3 CH3 H HO
H H
O
GAS CHROMATOGRAPHY | Terpenoids
Table 1
3
(Continued)
Structural type
Example
Tetraterpene
Lycopene
Structure
CH3
CH3
H3C
CH3
H3C
CH3 CH3
CH3
Polyterpene
Rubber (Polyisoprene)
CH2 C H3C
H2C
CH3
CH3
n
C H
Traditional terpene analyses involved identification of terpenes from plant, animal, and synthetic sources, but currently a larger range of matrices are also being widely analysed, namely biological fluids (containing also the metabolites of endogenous terpenes), microrganisms, historical artefacts, and even vapourised components of smoke. Technological developments in GC have allowed the necessary improvements in resolution required to separate large numbers of individual constituents. At the same time advances and improvements in detection, stationary phases, derivatisation, and hyphenated techniques, in particular linked to spectroscopy have allowed increasing ability to characterise compounds. Advances in extraction and concentration techniques have furthered applications for GC analysis of a wide range of terpenes in low levels. Increasingly GC is being used in combination with other types of chromatography, for example, High Pressure Liquid Chromatography (HPLC) which is suited to separation of non-volatile terpenes. A range of techniques have been used for terpene extraction, concentration, chromatographic separation of constituents, and detection and identification, both of the original compounds and their metabolites in biological matrices.
Analytical Techniques Terpene Extraction Extraction of the mono- and sesqui-terpenes of essential oils is still carried out using traditional methods. Cold expression yields the best oils, but is only suitable for certain fleshy materials eg. citrus peel oils. Extraction from plant material and other matrices is usually effected with steam distillation. Extraction from plant material for up to 30 min has been shown to produce no degradation products, although possible problems have been reported, where degradation of sabinene has been shown to occur, caused by acid catalysed hydration. Superheated water extraction, in which solutes are extracted by partitioning with hexane has also been employed. Solid phase microextraction (SPME) is used by many researchers. A number of adsorbents have been used for collection of volatile mono- and sesqui-terpenes in the gaseous environment, however decomposition of certain terpenes has been reported on Tenax TA and Carboxen 569. Procedures for thermal desorption and using non-polar solvent extraction of constituents have been carried out. Microwave assisted extraction has been used for mono- and sesqui-terpene extraction from a range of matrices. A comparison of the recoveries of terpenes obtained by steam distillation, simultaneous distillation and dichloromethane solvent extraction, microwave assisted extraction with dichloromethane, and supercritical fluid extraction (SFE) using CO2, revealed that more constituents were collected using an SFE system, less for the microwave assisted technique, fewer still for distillation/solvent extraction, and the lowest number using steam distillation. Cryotrap concentration of atmospheric extracts is often carried out after adsorption of solutes, and has been used for the quantitation of monoterpenes in humid and ozone-rich air. Low level constituents from plant materials, and complex matrices such as dosage forms, have been collected with the headspace technique, by volatilizing the terpenoids in the sample in a closely confined space, and then analyzing the constituents in this gaseous phase. Both analytical and commercial scale sample sizes of mono- and sesqui-terpenes have been extracted with liquid CO2, and SFE. SFE is thought to produce a superior product to steam distillation. SFE has been investigated for re-extraction of those compounds from foodstuffs and cosmetics. The problem with such matrices containing both volatile and non-volatile substances is their complexity and requirement of pre-fractionation. This has been resolved by developing GC inlet liners packed with polydimethylsiloxane which retains high boiling (non-volatile) solutes. This inlet has to be exchanged between injections by means of an automatic liner exchanger.
4
GAS CHROMATOGRAPHY | Terpenoids
Simultaneous steam distillation-solvent extraction has been found to produce a higher proportion of oxygenated compounds and monoterpenes than SFE. Monoterpenes may also exist as their glycosides in plant material, and this non-volatile form requires hydrolysis either by enzymic hydrolysis with b-glucosidase, or by acid hydrolysis.The free aglycone has been analysed by GC, and more than 10 aglycones have been identified in wine. GC of trimethylsilyl and trifluoroacetyl derivatives give complementary results to HPLC methods. Diterpenes are often extracted using solvents, or headspace trapping techniques, but many of these constituents may also occur in steam distilled extracts such as the essential oils. Triterpenes may also occur in solvent extracts containing diterpenes, but are widely collected by solvent extraction, or as such after glycosidic hydrolysis, and silylation and pyrolysis have also been employed. Tetraterpenes are also solvent extracted, but also silylated and pyrolyzed as for the diterpenes. Polyisoprenes such as natural rubber are usually prepared for GC by pyrolysis.
Chromatographic Separation of Constituents Since the 1950s, GC has been the method of choice for the separation, identification, and quantification of both volatile and less volatile terpenoids. Short packed columns of large diameter were used for separation of constituents using a range of stationary phases until the 1980s. Since then stationary phases on capillary columns have been used for separation of mono-and sesquiterpenes include DB-1, Carbowax, OV-1, OV-101, BP5, DB-5, which cover a range of polarities, and are the most widely used. Column lengths are usually 30 m, but may reach 60 m or more, and stationary phase film thicknesses range from 0.2 to 0.7 mm. Programming temperatures are usually in the range of 50 (70)–280 , at 2–5 per minute. In the 1990s the discovery of chiral phases allowed resolution of enantiomers of volatile terpenoids, which removed the ambiguity of results when analysing isomeric terpenes on normal phases. A wide range of commercial cyclodextrin phases have been developed, and used for separation of enantiomeric mono- and sesqui-terpenes. Overall, stationary phase developments have allowed use of higher operating temperatures due to greater stability, and reproducible results are provided due to increased reproducibility of column. Currently the industry standard capillary column is a 25–50 m column, 0.2–0.3 mm id, and 0.25 mm phase film thickness. Tandem techniques have been developed, including GC-GC, which involves coupling two columns or two chromatographs and HPLC-GC, namely multi-dimensional (MD)-GC, which bring about an increase in resolutions obtainable. These techniques allow sequential use of techniques with different separating abilities, combining chiral and non-chiral columns, polar and non-polar, or reversed phase HPLC with temperature programmed GC. GC-GC analysis has been used to separate complex mixtures of enantiomeric monoterpene hydrocarbons and alcohols. GC-GC in comparison with single column separation, produces increased constituent resolution. Multidimensional heart-cutting gas chromatography involves the transfer of one or more unresolved fractions from the first dimension (column) to a second one where separation will be achieved. Normally, columns have different polarities, where the first one is non-polar and the second is the polar dimension. The heart-cut can undertake two pathways, either transferred to the second column directly, or entrapped on a cryogenic device. Accordingly, the trap can be augmented with many heart-cuts as a result of chronological injections, which can be transferred later. A GC oven, two columns, two detectors, and a switching system are required to perform this technique. Both columns can be kept within the same or separate ovens. The analytes can be transferred between columns by means of moving mechanical valves, pneumatic pressure controlled switch, or valvless pressure switch. The former are relatively cheap and easy to use. However, they have many disadvantages, for instance; dead volumes, resistance to high temperature and sample adsorption. The later valveless switch is time consuming, but eliminates most of the previously mentioned problems. Pressure changes direct the analytes flow through the three live T-pieces. A mass spectrometer can be linked to the above for the purpose of detection. Figure 1 outlines the two possible setups for multidimensional heart cutting. HPLC-GC has been shown to distinguish qualitative differences between terpene compositions from different extraction procedures. GC linked to Droplet Counter Current Chromatography (GC-DCCC) has been used for chromatography of monoterpenes, and identification of novel mono- and di-saccharide aglycones of Reisling wine. Diterpenes and triterpenes are often chromatographed following derivatisation, typically silylation. Tri- and tetra-terpenes are often prepared for by pyrolysis, but tetraterpenes have also been hydrogenated. Rubber has been chromatographed after solvent extraction, SPME or pyrolysis.
Detection and Characterisation of Terpenes The flame ionisation detector (FID) is the most widely used for detection and quantification of terpenoids, although the photoionisation detector and surface ionisation detectors, have been used for investigation of monoterpenes in the atmosphere, and detection of mixtures of terpenes. Separated constituents have traditionally been identified by co-chromatography with standards, but mono- and sesqui-terpene identification is carried out by comparison with Kovats retention indices (RI). This data from two GC columns of different polarity, allows reliable identification of large numbers of terpenes. The most widely used system, is GC-MS for component identification, but GC-IR, or GC-FTIR are also employed. The latter differs from GC-MS as it can provide solute identification in cases where the molecular ion is not apparent in the mass spectrum. GC can be coupled with either quodrupole MS or ion trap MS detector. GC-Isotope Ratio (IR)-MS involves use of the isotope ratio mass spectrometer, and measures ratios of stable carbon isotopes of 13C/12C for each eluted peak. This can be used to determine the origin of enantiomeric pairs.
GAS CHROMATOGRAPHY | Terpenoids
Injector
Detector 1
5
Detector 2
Column 1
Column 2 or
MS
MV or VS (a)
Injector
Detector 1 Detector 2
Column 2
CT
Column 1
MS MV or VS
(b)
Figure 1 A schematic representation of multidimensional heart-cutting gas chromatography. (a) Separate ovens and (b) Single oven. MV, mechanical valve; VS, valveless switch; MS, mass spectrometer.
Sniff testing, GC-Olfactometry (GC-O), is widely used to detect highly volatile terpenes with pronounced aroma. Aroma extract dilution analysis (AEDA), allows for identification of low levels of flavour constituents using sequential dilution until the point that the aroma can no longer be detected, gives further information concerning the qualities of specific terpenoids. Characterisation of the higher terpenes also involves the use of FID, plus a range of tandem techniques.
Qualitative Techniques A number of databases of RIs of the mono- and sesqui-terpenes have been published. Some list RIs for particular temperature programme ramps, and there is also a database which includes data derived from 3 commonly used temperature ramps. Principle component analysis has been carried out on a number of volatile terpenes in specific matrices.
Applications Techniques for GC analysis of a large number of volatile mono- and sesqui-terpenes have been previously reviewed, and new applications are routinely appearing. With increasing molecular weights and lower volatility of the higher terpenoids, it can be seen that the number of applications decreases, as they are less volatile and consequently less suited to analysis by GC. However, applications are appearing for the less-widely publicised use of GC for the higher terpenoids, with the increasing use of supplementary techniques.
Monoterpenes/Sesquiterpenes The range of these volatile terpenes includes; terpenoids in essential oils, free terpenoids in plant material and plant products, glycosidically bound terpenoids in plant material, terpenoids in plant tissue cultures, atmospheric/airborne terpenoids in natural habitats, terpenoids in urban and industrial environments, and terpenoids in microorganisms.
6
GAS CHROMATOGRAPHY | Terpenoids
Essential Oils The now wide scale use of GC-MS in this area enables scientists to collate vast amounts of chemical data on composition of essential oil containing plants, for example providing detailed information on the essential oils in Turkish flora. This detailed information allows new chemotaxonomic classifications of species, supplies information for possible future commercial exploitation, and provides necessary data for optimal harvesting times. A typical example of a project to evaluate a range of essential oils from the Pimpinella genus used a 60 m HP-Innowax FSC capillary column operated from 60 C for 10 min, then over two temperature ramps, firstly 4 C min 1 to 220 C, holding for 10 min, then further to 240 C at 1 C min 1. MS data were recorded for 140 different constituents. A detailed investigation of mastic gum terpenes and other volatile constituents was investigated. 34 components were identified in the volatile oil, and 26 from the aqueous ethanolic extract of the gum. The major components were shown to be a-pinene, b-myrcene, b-pinene, limonene and caryophyllene. A HS-SPME/GC/MS method was developed for identification of components of human urine after consumption of mastic derived food products. A fast separation was run in less than 10 min., and the 5 major components of the raw materials were clearly identified in the urine samples, in addition to r-methyl anisole, terpinene, carveol, and myrtenol. A GC fitted with a DB-5MS capillary column (30 m length, 0.25 mm i.d., 0.10 m film thickness), programmed from 50 C to 250 C, was operated in the EI mode. Constituents were identified using a mass spectral database, NIST 05, and by reference to n-alkanes. HSSPME conditions were optimised, extraction time was set at 30 min., and extraction temperature at 25 C.1 The advent of chiral phases for separation has allowed increased characterisation of volatile terpenes in essential oil samples, which also allows confirmation of provenance of highly valuable oils, previously subject to adulteration by natural or synthetic substitution. A typical example of “heart cutting” may use two GC ovens, one achiral column such as SE-52, and a chiral column such as diethyl-tert-butyl-silyl-b-cyclodextrin, both columns of 25 m length, and eluting to a quadrupole MS. This enables separation of specific groups of separated enantiomeric monoterpenes with clear resolution of the individual enantiomers from for example, 8 isomeric pairs of monoterpenes. Notable examples of new techniques, include more widespread use of short narrow bore columns, and increased use of chiral columns. The use of GC/GC gives particular advantages for resolution of volatile terpenoids, but does have cost implications. One major future development in the field is believed to be the use of low thermal mass columns, which would allow reduced cooling times, leading to even faster separations. Although the traditional area of interest in mono- and sesquiterpene analysis has been in commercial essential oils, increased interest is now shown in plant products containing low but commercially important levels, such as grapes and their products. More specific techniques are now widely used such as solvent extraction of free terpenes or SPME followed by hydrolysis for glycosidic forms, instead of distillation followed by GC-MS. New trends in the analysis of plant volatiles include the increased interest in simplified extraction procedures which are easier to interphase with automation, and solventless clean-up techniques such as solid phase extraction. Head space sampling techniques have increased, with notable examples such as high concentration capacity (HCC) headspace sorptive extraction, sorptive tape extraction, and solvent enhanced headspace sorptive extraction. Speeds of separations have increased with the use of narrow bore columns and modern GC instrumentation, and analytical runs can now be routinely carried out at one third or previous times. One typical example is that of the separation of Juniper volatiles. Using speed optimised flow HS-SPME-enantioselective (ES)-GC-MS resolution times for 6 terpenes has been reduced from 27 mins. Down to less than 8 mins. Compared with conventional HS-SPMEES-GC-MS. Figure 2 outlines comparative conventional and fast HS-SPME-ES-GC-MS of juniper volatiles.2 Hyphenated and MD-GC analysis (e.g. HPLC-GC, SFE-GC and MDGC-heart cut concept) are widely used to achieve enhanced separations, as well as GC-O, GC-MS, RI, and more recently GC-time of flight mass spectroscopy (GC-TOF-MS) and GC GC. An automated headspace solid phase microextraction combined with GC-ion trap MS has been devised specifically to separate a large number of volatile compounds present in wine. In addition to identification of 18 terpenes and sesquiterpene alcohols, and 5 norisoprenoids, numerous esters, alcohols, carbonyl compounds, an acid, a sulphur compound and a phenol were established out of a list of 66 compounds in total.3
Cosmetics/Perfumes GC is the most useful technique for analysis of volatile fragrance components of cosmetics and perfumes. Many examples of formulations have been analysed such as soaps, mascaras and other make up materials, and hair dyes, usually after extraction with a variety of organic solvents ranging in polarity from methanol to n-hexane. Some cosmetics are extremely complex matrices containing for example inorganics, dyes, fixed oils, waxes, paraffins, emulsifiers, water and antioxidants, in addition to essential oil components. Preferential solvent extraction may allow fractionation of the volatiles, followed by GC separation, or fractionation of the non-volatiles followed by FAB-MS identification, or alternatively a wide range of excipients may also be analysed in a GC system. GC-MS of non-polar components has been carried out and concomitant analysis of perfume components, preservatives, antioxidants, and even volatile silicon anti-foaming agents identified, typically a SE-54 capillary in a GC-MS system has been employed to separate and identify components of mascara. SPB-5 stationary phase has been used to separate perfume components, dye solvents, antioxidants, fatty acids and fatty alcohols, present in hair dyes. In addition the colouring dyes can be separated, revealing the presence of individual dye components plus their specific solvents. Many complex cosmetics may require prior silica gel column chromatography, using for example methanol as eluting solvent, prior to GC-MS.
GAS CHROMATOGRAPHY | Terpenoids
7
(x 100 000) 6.0
TIC
3a
Juniper headspace analysis HS-SPME-Conv-ES-GC-MS.
5.0 4.0 3.0 1a 1b
2.0
4a 5b 5a 4b
2b 2a 3b
1.0
6a 6b
0.0 (a)
6.0 5.0 4.0 3.0 2.0 1.0 0.0 (b)
7.5
10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 min
1a 1b 2a
Juniperus chiral marker standard mixture Conv.ES-GC-MS
5a 5b
3a 3b
4a
4b
2b 6b 6a
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 min (x 1 000 000) TIC
Juniper headspace analysis HS-SPME-Fast-SOF-ES-GC-MS.
3a
1.25 1.00 0.75 0.50 0.25 (c)
1a 1b
3.5
4a 2a 2b
4.0
3b
4.5
5.0
5b 5a 4b
5.5
6a 6b
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
min
Figure 2 HS-SPME-ES-GC-MS profiles of juniper volatiles. Techniques (a), (b), and (c). as specified. (Reproduced from Bicchi, C.; Cagliero, C.; Rubiolo, P. New Trends in the Analysis of the Volatile Fraction of Matrices of Vegetable Origin: A Short Overview. A Review. Flavour Frag. J. 2011, 26, 321–325, with permission).
In addition to routine analysis of volatile terpene constituents of formulated products in quality control, GC-MS systems are becoming widely used in the search for allergenic and potentially allergenic components of fragrances used to enhance their acceptability. Patch testing for allergenicity is routinely followed by GC-MS of the range of materials shown to elicit positive effects; the aim being to identify common components, confirming patch test allergenic activity, then to remove or reduce levels of specific causative agents in the formulations.
Forensic Science Cannabis The major active constituents, the cannabinoids such as tetrahydrocannabinol (THC), are of terpenoid biogenetic origin, being derived from geranyl pyrophosphate. In addition to these constituents, cannabis mono- and sesquiterpene aroma volatiles have often been the subject of investigation. A fairly simple extraction of cannabis resin or herb, is usually carried out for forensic purposes. Being non-polar, similar polarity solvents are employed for plant material, but cannabinoids have also been extracted from hair using prior alkaline digestion, and final extraction from a solid phase cartridge. Headspace SPME has also been used. Headspace volatiles have been collected both after a pre-heating and at ambient temperature, and absorbed onto a range of adsorbents including methylsiloxane and divinylbenzene solid phase fibres. Solute extraction or heat desorption may later be used prior to chromatography, but adsorbent fibres have also been used which can later be inserted directly into the GC injection port, and the volatiles chromatographed. Volatile oils of cannabis have forensic significance and are extracted by both traditional steam distillation and via pyrolysis. Figure 3 shows a chromatogram of the essential oil revealing separation of 48 components. Cannabis smoke has been collected, and fractionated with acid/base partition, which is followed by chromatography.
8
GAS CHROMATOGRAPHY | Terpenoids
Figure 3 Gas chromatogram of the essential oil of a plant of accession from Afganistan assigned to the wide-leaflet drug biotype of Cannabis indica. i.s., internal standard; Column, DB-5MS. (Reproduced from Hillig, K. W. A Chemotaxonomic Analysis of Terpenoid Variation in Cannabis. Biochem. Syst. Ecol. 2004, 32, 875–891, with permission).
GC separations have been carried out on a number of stationary phases, including columns typically packed with 3% OV-17, 6% OV-101, and 30 m capillary columns such as DB-5, HP-5MS, DB-5MS. Detection has traditionally involved FID, but GC-MS is now routinely used. Quantification has been carried out by both internal normalization, and via use of calibration curves prepared using an internal standard, which is the norm in forensic work. Cannabis essential oil is usually quantified using peak normalisation, due to the large number of constituents similarly to other essential oils. Results obtained with both packed columns and capillary columns have separated 50–60 components. Identifications of the major metabolite, THC acid (THCA), in hair and urine following consumption of cannabis, have been carried out. THCA has been analyzed in hair at very low concentrations, after a digestion process, followed by acidification and solid phase extraction. The resultant extract has been derivatized with pentafluoropropionic anhydride/hexafluoroisopropanol, and chromatographed by GC-MS in the negative chemical ionisation mode. Single ion monitoring of THCA extracted from urine has been carried out on GC-MS, and automated solid phase extraction has also been carried out; revealing a detection limit of 1 ng ml 1, with a quantitation limit of 4 ng ml 1. Forensic analyses of cannabinoids using GC continues to be developed. A GC-NCI-MS/MS method has been developed for the determination of 11-nor-D9-tetrahydrocannabinol-9-carboxylic acid in human hair. This is a particularly useful development, as it would previously have been possible using only HPLC. 25 mg hair samples were powdered, digested with1.0 M sodium hydroxide, and extracted with hexane: ethyl acetate (9:1). Derivatisation of the dried extract with pentafluoropropanol and pentafluoropropionic anhydride was followed by GC-MS/MS in the negative ion chemical ionisation mode. The limits of detection and quantification were 0.015 and 0.05 pg mg 1, respectively, and recoveries of THC acid were in the range of 79.4–87.1%.4 Principle component analysis has been carried out, which allows assignment of plants of diverse geographical origins, into specific biotypes. Overall, the major cannabinoids are readily separated, even using packed columns, and capillary columns and GC-MS has allowed identification of a greater range of components.
Diterpenes Diterpenes are less volatile and less numerous than the mono- and sesquiterpenes, consequently they rarely appear in distilled essential oils. However, traditional extraction using distillation allows separation and identification of diterpenes present in essential oils. The diterpenes in frankincense have been analysed using GC-MS after headspace trapping on adsorbent fibres.
GAS CHROMATOGRAPHY | Terpenoids
9
100% = 96.765
Total
Sesquiterpenes
Monoterpenes Diterpenes
Isoincensole acetate Cembrene A
(a)
10:00 Total
15:00
20:00
25:00
30:00
35:00
40:00
45:00
50:00
100% = 104.122
Diterpenes
Monoterpenes
55:00 01:00:00
Isoincensole acetate Sesquiterpenes
Cembrene A
(b)
10:00
15:00
20:00
25:00
30:00
35:00
40:00
45:00
50:00
55:00 01:00:00
Figure 4 Comparison between SPME and dichloromethane extraction. (Reproduced from Hamm, S.; Lesellier, E.; Bleton, J.;Tchapla, A. Optimization of Headspace Solid Phase Microextraction for Gas Chromatography/Mass Spectrometry Analysis of Widely Different Volatility and Polarity Terpenoids in Olibanum. J. Chromatogr. A. 2003, 1018, 73–83, with permission).
Figure 4 shows the two chromatograms obtained from these extraction techniques, and also demonstrates the separation of mono-, sesqui- and diterpene fractions and their constituent entities. The resolution of this particular system allowed for identification of 66 mono- and sesqui-terpenes along with diterpenes. The analysis of small amounts of diterpenes in archaeological and historical samples requires specialised techniques. Diterpenes in canvas paintings have been analysed either after formation of ethyl or silyl ethers of the hydroxylated diterpenes of resins present or pyrolysis GC-MS, with prior silylation and typically a range of 20 different diterpenes have been identified. A further interesting matrix has been investigated using GC-MS and two dimensional Time of Flight (TOF) MS. Cannabis cigarette smoke condensate was collected on glass fibre pads and extracted by simultaneous distillation and SDE. The DCM extract was washed with 5% sodium hydroxide and 5% hydrochloric acid, and then chromatographed in two systems; a 50 m DB Petro capillary linked to electron impact MS, and GC positive ion Chemical Ionisation MS, and the same column also fitted to TOF MS. Over 100 compounds may be identified, including phytaene, phytadienes, cembranes, and labdanane diterpenes. Recently, increased interest has been shown in microbial diterpene metabolites. Solvent and headspace extracts, have been used to extract diterpenes, which can then be identified by GC-MS.
Triterpenes On occasions the chromatographic system used for diterpenes present in particular matrices is also able to separate and identify triterpenes from the same source, using silylation and GC-MS of a DCM extract. Triterpene dialcohols, have been analysed using reversed phase HPLC and GC employing a programmed temperature vapourizer at the interface. This procedure allows analysis of small samples of aqueous methanolic extracts to be chromatographed via transfer of the triterpenes directly from HPLC into the programmed temperature vapourizer (PTV) injector attached to a GC.
10
GAS CHROMATOGRAPHY | Terpenoids
Steroids There are a vast number of steroids obtained from a wide variety of matrices, including plant, animal, and synthetic sources, and their metabolites such as the urinary metabolites. GC has been used from the early days of instrumentation, and typically has been involved in analysis on both packed and capillary columns, using a variety of detectors, and recently a number of tandem techniques. This is due to the increased forensic significance of steroid metabolites, for instance in sport and workplace drug testing, GC has to some extent been supplanted by use of large scale immunoassays, but this technique does not supply the wealth of data, either qualitative or quantitative, that GC systems are capable of. The forte of GC and hyphenated techniques, is the range and extent of information available from single injection or auto sampler-fed systems. Dehydroepiandrosterone (DHEA) is an example of a steroid used both medically, and for abuse, and its analysis covers the whole range of available techniques. DHEA is both an endogenous androgen and a synthetic product, with wide applications in the areas of medicinal product formulations and extraction and analyses in varied materials. DHEA has been analysed in GC systems capable of screening a number of related steroids. Both solid and liquid dosage forms have been analysed by basifying and extracting in non-polar solvent. Derivatization by silylation prior to chromatographing allows resolution and identification of a large number of related steroids using Selected Ion Mode (SIM)- MS. SPME, followed by on-fibre silylation has been used to analyse steroids present in river water and fish blood serum at detection levels of 0.002–0.378 mg l 1 and 0.004–0.475 mg l 1 respectively, and quantification levels of 0.008–1.26 mg l 1 and 0.013–1.579 mg l 1 respectively. Human urinary levels of DHEA and metabolites are often analysed after oral ingestion. Also GC/Combustion/ Isotope Ratio Mass Spectroscopy (IRMS) has been employed for urine samples, and conjugated steroids hydrolysed with glucuronidase, and purified on a standard reverse phase HPLC column. Acetylated derivatives were re-chromatographed on HPLC and collected, before chromatography and detection with Mass sensitive Detector (MSD). IRMS was carried out after a combustion stage at 940 C, and isotope ratio values were shown to be unchanged following ingestion. This procedure is now routinely carried out in urine samples containing concentrations greater than 100 ng ml 1 of DHEA. A novel method for analysis of anabolic steroids was devised using GC-microchip atmospheric pressure photoionisation-MS. Atmospheric pressure ion MS is usually used with HPLC, but researchers established a system of linking to GC. A heated nebuliser microchip was successfully interfaced with atmospheric pressure photoionisation-tandem MS and used for the analysis of underivatised anabolic steroids present in urine. Seven anabolic steroids including nandrolone and methyltestosterone were resolved and identified. The method showed good sensitivity and repeatability, and was compared with conventional GC-EI-MS following derivatisation. The results were found to be in good agreement, and demonstrated the simplicity, avoiding the derivatisation stage in analysis.5 It is evident that many examples of GC analysis of a wide range of steroids have been carried out, but there is a trend towards use of HPLC for determination of conjugated steroids in biological fluids.
Tetraterpenes Carotenoids Early work on carotenoids often employed packed columns such as 2% SE-50, 2% High Vacuum Grease, or 3% OV-17, either under isothermal conditions or temperature programming. Carotenoids have been hydrogenated prior to analysis, in order to prevent thermal degradation. Carotenoids have a number of medicinal and other applications and natural product mixtures have been derivatised by silylation or methylation, then pyrolyzed at temperatures ranging from 400 to 750 C before chromatographing. Single carotenoids such as lycopene and its degradation products have been studied. Direct solvent extraction has been used to collect the less volatile components and GC resolved both partially isomerised degradation products and the more volatile degradation products.
Polyterpenes Rubber Rubber is a polyisoprene polymer composed of isoprene units, with a molecular weight ranging from 100 000 to 1 000 000. Due to the non-volatility of rubbers, pyrolysis followed by GC-MS is a routine technique for analysis. After pyrolisis, volatile degradation products may be chromatographed on a methylsiloxane capillary column, which allows routine identification of constituent monomers such as butadiene, isoprene, styrene and dipentene. Low molecular weight components of commercial, natural and derivatised rubbers used in vehicle tires and in pharmaceutical packaging have been investigated using both Soxhlet and headspace extractions followed by GC-MS. A range of solvents have been tested for Soxhlet extractability, ranging from cyclohexane to isopropanol. Alternatively, constituents have been analysed by GC-MS of their pyrolysis products. Pyrolysis temperatures up to 500 C have been employed, using a coupling connected to the GC operated at 300 C to prevent condensation of the products. GC-MS-MS has been used to separate the dimer products of isoprene.
GAS CHROMATOGRAPHY | Terpenoids
11
Problems and Perspectives One possible problem with modern GC analysis is the reliance on computerized structure prediction based upon data libraries. Structural identification of mono- and sesqui-terpenes is routinely corroborated with RIs, and confirmation is more secure, although reproducibility of RIs has been questioned when using temperature programming. New databases are appearing which may reduce possible errors of identification. This problem may occur with any class of terpenes, but the large groups of individual mono- and sesqui-terpenes are potentially more prone to mis-identification. The increasing use of MS to elucidate structures of coeluted solutes has caused a few doubts to be raised concerning the validity of the procedure. Other terpene groups are more likely to be identified via a number of techniques, due to comparatively reduced numbers of related compounds, and consequently, will be less liable to misinterpretation. The possible degradation of terpenes under GC conditions has been previously questioned. There is however little hard evidence to substantiate this possibility. Although the degradation of monoterpenes on Tenax adsorbent during sample storage, even under extreme non-standard conditions, resulted in 88% recovery of the undegraded terpene. Limited work on thermal degradation of monoterpenes has been investigated; treatment in conditions simulating the commercial wood drying process, which involves the presence of air which is not encountered in GC systems has been found to yield a large range of degradation products. Consequently, there is little evidence of degradation during the GC stage of analyses.
Conclusions The high volatility of the mono- and sesqui-terpenes has allowed GC to become the preferred analytical tool. Improved concentration techniques, sensitive detectors and hyphenated systems, allow quantification and characterization of large numbers of low levels of volatile terpenes. The less volatile higher terpenes often exist in lesser numbers in particular matrices, but GC is still a major technique for their analysis. Higher terpene analysis has also benefited from developments in extraction and concentration techniques. Again, development of ancillary techniques has dramatically improved their characterisation and quantification. When compared with other available techniques, the use of basic FID detection plus linking to MS, allows complete characterization of many natural materials and their derivatives, currently not available using other chromatographic systems.
References 1. Zachariadis, G. A.; Langioli, A. V. Headspace Solid Phase Microextraction for Terpenes and Volatile Compounds Determination in Mastic Gum Extracts, Mastic Oil and Human Urine by GC-MS. Anal. Lett. 2012, 45, 993–1003. 2. Bicchi, C.; Cagliero, C.; Rubiolo, P. New Trends in the Analysis of the Volatile Fraction of Matrices of Vegetable Origin: A Short Overview. A Review. Flavour Frag. J. 2011, 26, 321–325. 3. Barros, P. E.; Moreira, N.; Pereira, G. E.; Leite, S. G. F.; Rezende, C. M.; Pinho, P. G. Development and Validation of Automatic HS-SPME with a Gas Chromatography Ion Trap/ Mass Spectrometry Method for Analysis of Volatiles in Wines. Talanta 2012, 101, 177–186. 4. Kim, J. Y.; Cheong, J. C.; Lee, J. I.; In, M. K. Improved Gas Chromatography-Negative ion Chemical Ionization Tandem Mass Spectrometric Method for Determination of 11-nor-D 9-Tetrahydrocannabinol-9-Carboxylic Acid in Hair Using Mechanical Pulverization and Bead-Assisted Liquid-Liquid Extraction. Forensic Sci. Int. 2011, 206, e99–e102. 5. Hintikka, L.; Haapala, M.; Franssila, S.; Kuuranne, T.; Leinonen, A.; Kostiainen, R. Feasibility of Gas Chromatography-Microchip Atmospheric Pressure Photoionization-Mass Spectrometry in Analysis of Anabolic Steroids. J. Chromatogr. A 2010, 1217, 8290–8297.
Further Reading 1. Adams, R. P. Identification of Essential Oil Components by Gas Chromatography-Mass Spectrometry; Allured: Illinois, 1995. 2. Bicchi, C.; Cagliero, C.; Rubiolo, P. New Trends in the Analysis of the Volatile Fraction of Matrices of Vegetable Origin: A Short Overview. A Review. Flavour Frag. J. 2011, 26, 321–325. 3. Davies, N. W. Gas Chromatographic Retention Indices of Monoterpenes and Sesquiterpenes on Methyl Silicone and Carbowax 20M Phases. J. Chromatogr. 1990, 503, 1–24. 4. Hamm, S.; Lesellier, E.; Bleton, J.; Tchapla, A. Optimization of Headspace Solid Phase Microextraction for Gas Chromatography/Mass Spectrometry Analysis of Widely Different Volatility and Polarity Terpenoids in Olibanum. J. Chromatogr. A 2003, 1018, 73–83. 5. Lockwood, G. B. Techniques for Gas Chromatography of Volatile Terpenoids from a Range of Matrices. J. Chromatogr. A 2001, 936, 23–31. 6. Marriott, P. J.; Shellie, R.; Cornwell, C. Gas Chromatographic Technologies for the Analysis of Essential Oils. J. Chromatogr. A 2001, 936, 1–22.
Analytical Techniques Terpene Extraction Chromatographic Separation of Constituents Detection and Characterisation of Terpenes Qualitative Techniques Applications Monoterpenes/Sesquiterpenes Essential Oils Cosmetics/Perfumes Forensic Science Cannabis Diterpenes Triterpenes Steroids Tetraterpenes Carotenoids Polyterpenes Rubber Problems and Perspectives Conclusions References
Glossary AEDA Aroma extract dilution analysis DCM Dichloromethane DHEA Dehydroepiandrosterone EI Electron impact FAB-MS Fast atom bombardment-MS FID Flame ionization detector GC Gas chromatography GC-DCCC GC-droplet counter centre chromatography GC-MS GC-mass spectroscopy GC-O GC-olfactometry GC-TOF-MS GC-time of flight-MS
3 3 4 4 5 5 5 6 6 7 7 8 9 10 10 10 10 10 11 11 11
HPLC High pressure liquid chromatography HS Head space IRMS Isotope ratio MS MDGC Multi-dimensional GC MSD Mass sensitive detector PTV Programmed temperature vaporiser RI Retention index SFE Supercritical fluid extraction SIM Selected (single) ion monitoring SPME Solid phase micro-extraction THC Tetrahydrocannabinol THCA Tetrahydrocannabinolic acid
Gas chromatography (GC) has long been used for separation of related terpenes in analysis of natural products and pharmaceuticals, specifically for characterisation of new entities, and quality control of known terpenes. The monoterpenoids and sesquiterpenoids, often co-occur in essential oils, which are important flavour and fragrance components in food and cosmetics. The diterpenes possess a number of historical applications, for example as components in paints and varnishes, triterpenes encompass the steroids and other commercially important compounds used in medicines, and the tetraterpenes such as the carotenoids are widely present in food plants, also having wide-ranging medical applications. These individual terpene groups, particularly the mono- and sesqui-terpenes, often occur both individually and together in large numbers of structurally closely related compounds, demanding separation systems capable of high resolution. Natural rubber, polyisoprene, is the terpene with the highest molecular weight, and GC applications for the analysis of this polymer are also possible. Specific drugs of abuse, namely cannabinoids and steroids have also been chromatographed using GC. Table 1 shows the range of structural types of terpenes analysed by gas chromatography (GC). ☆
Change History: March 2013. GB Lockwood updated text throughout.
Reference Module in Chemistry, Molecular Sciences and Chemical Engineering
http://dx.doi.org/10.1016/B978-0-12-409547-2.04768-5
1
2
GAS CHROMATOGRAPHY | Terpenoids
Table 1
Range of structural types of terpenoids widely analysed by GC
Structural type
Example
Monoterpene
a-Pinene
Diterpene
Abietic acid
Structure
H
O HO
Sesquiterpene
a-Phellandrene
Cannabinoid
11-nor-D9-tetrahydrocannabinol-9-carboxylic acid
O
OH
OH H H O
Triterpene
Lupeol
HO
Steroid
Dehydroepiandrosterone
CH3 CH3 H HO
H H
O
GAS CHROMATOGRAPHY | Terpenoids
Table 1
3
(Continued)
Structural type
Example
Tetraterpene
Lycopene
Structure
CH3
CH3
H3C
CH3
H3C
CH3 CH3
CH3
Polyterpene
Rubber (Polyisoprene)
CH2 C H3C
H2C
CH3
CH3
n
C H
Traditional terpene analyses involved identification of terpenes from plant, animal, and synthetic sources, but currently a larger range of matrices are also being widely analysed, namely biological fluids (containing also the metabolites of endogenous terpenes), microrganisms, historical artefacts, and even vapourised components of smoke. Technological developments in GC have allowed the necessary improvements in resolution required to separate large numbers of individual constituents. At the same time advances and improvements in detection, stationary phases, derivatisation, and hyphenated techniques, in particular linked to spectroscopy have allowed increasing ability to characterise compounds. Advances in extraction and concentration techniques have furthered applications for GC analysis of a wide range of terpenes in low levels. Increasingly GC is being used in combination with other types of chromatography, for example, High Pressure Liquid Chromatography (HPLC) which is suited to separation of non-volatile terpenes. A range of techniques have been used for terpene extraction, concentration, chromatographic separation of constituents, and detection and identification, both of the original compounds and their metabolites in biological matrices.
Analytical Techniques Terpene Extraction Extraction of the mono- and sesqui-terpenes of essential oils is still carried out using traditional methods. Cold expression yields the best oils, but is only suitable for certain fleshy materials eg. citrus peel oils. Extraction from plant material and other matrices is usually effected with steam distillation. Extraction from plant material for up to 30 min has been shown to produce no degradation products, although possible problems have been reported, where degradation of sabinene has been shown to occur, caused by acid catalysed hydration. Superheated water extraction, in which solutes are extracted by partitioning with hexane has also been employed. Solid phase microextraction (SPME) is used by many researchers. A number of adsorbents have been used for collection of volatile mono- and sesqui-terpenes in the gaseous environment, however decomposition of certain terpenes has been reported on Tenax TA and Carboxen 569. Procedures for thermal desorption and using non-polar solvent extraction of constituents have been carried out. Microwave assisted extraction has been used for mono- and sesqui-terpene extraction from a range of matrices. A comparison of the recoveries of terpenes obtained by steam distillation, simultaneous distillation and dichloromethane solvent extraction, microwave assisted extraction with dichloromethane, and supercritical fluid extraction (SFE) using CO2, revealed that more constituents were collected using an SFE system, less for the microwave assisted technique, fewer still for distillation/solvent extraction, and the lowest number using steam distillation. Cryotrap concentration of atmospheric extracts is often carried out after adsorption of solutes, and has been used for the quantitation of monoterpenes in humid and ozone-rich air. Low level constituents from plant materials, and complex matrices such as dosage forms, have been collected with the headspace technique, by volatilizing the terpenoids in the sample in a closely confined space, and then analyzing the constituents in this gaseous phase. Both analytical and commercial scale sample sizes of mono- and sesqui-terpenes have been extracted with liquid CO2, and SFE. SFE is thought to produce a superior product to steam distillation. SFE has been investigated for re-extraction of those compounds from foodstuffs and cosmetics. The problem with such matrices containing both volatile and non-volatile substances is their complexity and requirement of pre-fractionation. This has been resolved by developing GC inlet liners packed with polydimethylsiloxane which retains high boiling (non-volatile) solutes. This inlet has to be exchanged between injections by means of an automatic liner exchanger.
4
GAS CHROMATOGRAPHY | Terpenoids
Simultaneous steam distillation-solvent extraction has been found to produce a higher proportion of oxygenated compounds and monoterpenes than SFE. Monoterpenes may also exist as their glycosides in plant material, and this non-volatile form requires hydrolysis either by enzymic hydrolysis with b-glucosidase, or by acid hydrolysis.The free aglycone has been analysed by GC, and more than 10 aglycones have been identified in wine. GC of trimethylsilyl and trifluoroacetyl derivatives give complementary results to HPLC methods. Diterpenes are often extracted using solvents, or headspace trapping techniques, but many of these constituents may also occur in steam distilled extracts such as the essential oils. Triterpenes may also occur in solvent extracts containing diterpenes, but are widely collected by solvent extraction, or as such after glycosidic hydrolysis, and silylation and pyrolysis have also been employed. Tetraterpenes are also solvent extracted, but also silylated and pyrolyzed as for the diterpenes. Polyisoprenes such as natural rubber are usually prepared for GC by pyrolysis.
Chromatographic Separation of Constituents Since the 1950s, GC has been the method of choice for the separation, identification, and quantification of both volatile and less volatile terpenoids. Short packed columns of large diameter were used for separation of constituents using a range of stationary phases until the 1980s. Since then stationary phases on capillary columns have been used for separation of mono-and sesquiterpenes include DB-1, Carbowax, OV-1, OV-101, BP5, DB-5, which cover a range of polarities, and are the most widely used. Column lengths are usually 30 m, but may reach 60 m or more, and stationary phase film thicknesses range from 0.2 to 0.7 mm. Programming temperatures are usually in the range of 50 (70)–280 , at 2–5 per minute. In the 1990s the discovery of chiral phases allowed resolution of enantiomers of volatile terpenoids, which removed the ambiguity of results when analysing isomeric terpenes on normal phases. A wide range of commercial cyclodextrin phases have been developed, and used for separation of enantiomeric mono- and sesqui-terpenes. Overall, stationary phase developments have allowed use of higher operating temperatures due to greater stability, and reproducible results are provided due to increased reproducibility of column. Currently the industry standard capillary column is a 25–50 m column, 0.2–0.3 mm id, and 0.25 mm phase film thickness. Tandem techniques have been developed, including GC-GC, which involves coupling two columns or two chromatographs and HPLC-GC, namely multi-dimensional (MD)-GC, which bring about an increase in resolutions obtainable. These techniques allow sequential use of techniques with different separating abilities, combining chiral and non-chiral columns, polar and non-polar, or reversed phase HPLC with temperature programmed GC. GC-GC analysis has been used to separate complex mixtures of enantiomeric monoterpene hydrocarbons and alcohols. GC-GC in comparison with single column separation, produces increased constituent resolution. Multidimensional heart-cutting gas chromatography involves the transfer of one or more unresolved fractions from the first dimension (column) to a second one where separation will be achieved. Normally, columns have different polarities, where the first one is non-polar and the second is the polar dimension. The heart-cut can undertake two pathways, either transferred to the second column directly, or entrapped on a cryogenic device. Accordingly, the trap can be augmented with many heart-cuts as a result of chronological injections, which can be transferred later. A GC oven, two columns, two detectors, and a switching system are required to perform this technique. Both columns can be kept within the same or separate ovens. The analytes can be transferred between columns by means of moving mechanical valves, pneumatic pressure controlled switch, or valvless pressure switch. The former are relatively cheap and easy to use. However, they have many disadvantages, for instance; dead volumes, resistance to high temperature and sample adsorption. The later valveless switch is time consuming, but eliminates most of the previously mentioned problems. Pressure changes direct the analytes flow through the three live T-pieces. A mass spectrometer can be linked to the above for the purpose of detection. Figure 1 outlines the two possible setups for multidimensional heart cutting. HPLC-GC has been shown to distinguish qualitative differences between terpene compositions from different extraction procedures. GC linked to Droplet Counter Current Chromatography (GC-DCCC) has been used for chromatography of monoterpenes, and identification of novel mono- and di-saccharide aglycones of Reisling wine. Diterpenes and triterpenes are often chromatographed following derivatisation, typically silylation. Tri- and tetra-terpenes are often prepared for by pyrolysis, but tetraterpenes have also been hydrogenated. Rubber has been chromatographed after solvent extraction, SPME or pyrolysis.
Detection and Characterisation of Terpenes The flame ionisation detector (FID) is the most widely used for detection and quantification of terpenoids, although the photoionisation detector and surface ionisation detectors, have been used for investigation of monoterpenes in the atmosphere, and detection of mixtures of terpenes. Separated constituents have traditionally been identified by co-chromatography with standards, but mono- and sesqui-terpene identification is carried out by comparison with Kovats retention indices (RI). This data from two GC columns of different polarity, allows reliable identification of large numbers of terpenes. The most widely used system, is GC-MS for component identification, but GC-IR, or GC-FTIR are also employed. The latter differs from GC-MS as it can provide solute identification in cases where the molecular ion is not apparent in the mass spectrum. GC can be coupled with either quodrupole MS or ion trap MS detector. GC-Isotope Ratio (IR)-MS involves use of the isotope ratio mass spectrometer, and measures ratios of stable carbon isotopes of 13C/12C for each eluted peak. This can be used to determine the origin of enantiomeric pairs.
GAS CHROMATOGRAPHY | Terpenoids
Injector
Detector 1
5
Detector 2
Column 1
Column 2 or
MS
MV or VS (a)
Injector
Detector 1 Detector 2
Column 2
CT
Column 1
MS MV or VS
(b)
Figure 1 A schematic representation of multidimensional heart-cutting gas chromatography. (a) Separate ovens and (b) Single oven. MV, mechanical valve; VS, valveless switch; MS, mass spectrometer.
Sniff testing, GC-Olfactometry (GC-O), is widely used to detect highly volatile terpenes with pronounced aroma. Aroma extract dilution analysis (AEDA), allows for identification of low levels of flavour constituents using sequential dilution until the point that the aroma can no longer be detected, gives further information concerning the qualities of specific terpenoids. Characterisation of the higher terpenes also involves the use of FID, plus a range of tandem techniques.
Qualitative Techniques A number of databases of RIs of the mono- and sesqui-terpenes have been published. Some list RIs for particular temperature programme ramps, and there is also a database which includes data derived from 3 commonly used temperature ramps. Principle component analysis has been carried out on a number of volatile terpenes in specific matrices.
Applications Techniques for GC analysis of a large number of volatile mono- and sesqui-terpenes have been previously reviewed, and new applications are routinely appearing. With increasing molecular weights and lower volatility of the higher terpenoids, it can be seen that the number of applications decreases, as they are less volatile and consequently less suited to analysis by GC. However, applications are appearing for the less-widely publicised use of GC for the higher terpenoids, with the increasing use of supplementary techniques.
Monoterpenes/Sesquiterpenes The range of these volatile terpenes includes; terpenoids in essential oils, free terpenoids in plant material and plant products, glycosidically bound terpenoids in plant material, terpenoids in plant tissue cultures, atmospheric/airborne terpenoids in natural habitats, terpenoids in urban and industrial environments, and terpenoids in microorganisms.
6
GAS CHROMATOGRAPHY | Terpenoids
Essential Oils The now wide scale use of GC-MS in this area enables scientists to collate vast amounts of chemical data on composition of essential oil containing plants, for example providing detailed information on the essential oils in Turkish flora. This detailed information allows new chemotaxonomic classifications of species, supplies information for possible future commercial exploitation, and provides necessary data for optimal harvesting times. A typical example of a project to evaluate a range of essential oils from the Pimpinella genus used a 60 m HP-Innowax FSC capillary column operated from 60 C for 10 min, then over two temperature ramps, firstly 4 C min 1 to 220 C, holding for 10 min, then further to 240 C at 1 C min 1. MS data were recorded for 140 different constituents. A detailed investigation of mastic gum terpenes and other volatile constituents was investigated. 34 components were identified in the volatile oil, and 26 from the aqueous ethanolic extract of the gum. The major components were shown to be a-pinene, b-myrcene, b-pinene, limonene and caryophyllene. A HS-SPME/GC/MS method was developed for identification of components of human urine after consumption of mastic derived food products. A fast separation was run in less than 10 min., and the 5 major components of the raw materials were clearly identified in the urine samples, in addition to r-methyl anisole, terpinene, carveol, and myrtenol. A GC fitted with a DB-5MS capillary column (30 m length, 0.25 mm i.d., 0.10 m film thickness), programmed from 50 C to 250 C, was operated in the EI mode. Constituents were identified using a mass spectral database, NIST 05, and by reference to n-alkanes. HSSPME conditions were optimised, extraction time was set at 30 min., and extraction temperature at 25 C.1 The advent of chiral phases for separation has allowed increased characterisation of volatile terpenes in essential oil samples, which also allows confirmation of provenance of highly valuable oils, previously subject to adulteration by natural or synthetic substitution. A typical example of “heart cutting” may use two GC ovens, one achiral column such as SE-52, and a chiral column such as diethyl-tert-butyl-silyl-b-cyclodextrin, both columns of 25 m length, and eluting to a quadrupole MS. This enables separation of specific groups of separated enantiomeric monoterpenes with clear resolution of the individual enantiomers from for example, 8 isomeric pairs of monoterpenes. Notable examples of new techniques, include more widespread use of short narrow bore columns, and increased use of chiral columns. The use of GC/GC gives particular advantages for resolution of volatile terpenoids, but does have cost implications. One major future development in the field is believed to be the use of low thermal mass columns, which would allow reduced cooling times, leading to even faster separations. Although the traditional area of interest in mono- and sesquiterpene analysis has been in commercial essential oils, increased interest is now shown in plant products containing low but commercially important levels, such as grapes and their products. More specific techniques are now widely used such as solvent extraction of free terpenes or SPME followed by hydrolysis for glycosidic forms, instead of distillation followed by GC-MS. New trends in the analysis of plant volatiles include the increased interest in simplified extraction procedures which are easier to interphase with automation, and solventless clean-up techniques such as solid phase extraction. Head space sampling techniques have increased, with notable examples such as high concentration capacity (HCC) headspace sorptive extraction, sorptive tape extraction, and solvent enhanced headspace sorptive extraction. Speeds of separations have increased with the use of narrow bore columns and modern GC instrumentation, and analytical runs can now be routinely carried out at one third or previous times. One typical example is that of the separation of Juniper volatiles. Using speed optimised flow HS-SPME-enantioselective (ES)-GC-MS resolution times for 6 terpenes has been reduced from 27 mins. Down to less than 8 mins. Compared with conventional HS-SPMEES-GC-MS. Figure 2 outlines comparative conventional and fast HS-SPME-ES-GC-MS of juniper volatiles.2 Hyphenated and MD-GC analysis (e.g. HPLC-GC, SFE-GC and MDGC-heart cut concept) are widely used to achieve enhanced separations, as well as GC-O, GC-MS, RI, and more recently GC-time of flight mass spectroscopy (GC-TOF-MS) and GC GC. An automated headspace solid phase microextraction combined with GC-ion trap MS has been devised specifically to separate a large number of volatile compounds present in wine. In addition to identification of 18 terpenes and sesquiterpene alcohols, and 5 norisoprenoids, numerous esters, alcohols, carbonyl compounds, an acid, a sulphur compound and a phenol were established out of a list of 66 compounds in total.3
Cosmetics/Perfumes GC is the most useful technique for analysis of volatile fragrance components of cosmetics and perfumes. Many examples of formulations have been analysed such as soaps, mascaras and other make up materials, and hair dyes, usually after extraction with a variety of organic solvents ranging in polarity from methanol to n-hexane. Some cosmetics are extremely complex matrices containing for example inorganics, dyes, fixed oils, waxes, paraffins, emulsifiers, water and antioxidants, in addition to essential oil components. Preferential solvent extraction may allow fractionation of the volatiles, followed by GC separation, or fractionation of the non-volatiles followed by FAB-MS identification, or alternatively a wide range of excipients may also be analysed in a GC system. GC-MS of non-polar components has been carried out and concomitant analysis of perfume components, preservatives, antioxidants, and even volatile silicon anti-foaming agents identified, typically a SE-54 capillary in a GC-MS system has been employed to separate and identify components of mascara. SPB-5 stationary phase has been used to separate perfume components, dye solvents, antioxidants, fatty acids and fatty alcohols, present in hair dyes. In addition the colouring dyes can be separated, revealing the presence of individual dye components plus their specific solvents. Many complex cosmetics may require prior silica gel column chromatography, using for example methanol as eluting solvent, prior to GC-MS.
GAS CHROMATOGRAPHY | Terpenoids
7
(x 100 000) 6.0
TIC
3a
Juniper headspace analysis HS-SPME-Conv-ES-GC-MS.
5.0 4.0 3.0 1a 1b
2.0
4a 5b 5a 4b
2b 2a 3b
1.0
6a 6b
0.0 (a)
6.0 5.0 4.0 3.0 2.0 1.0 0.0 (b)
7.5
10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 min
1a 1b 2a
Juniperus chiral marker standard mixture Conv.ES-GC-MS
5a 5b
3a 3b
4a
4b
2b 6b 6a
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 min (x 1 000 000) TIC
Juniper headspace analysis HS-SPME-Fast-SOF-ES-GC-MS.
3a
1.25 1.00 0.75 0.50 0.25 (c)
1a 1b
3.5
4a 2a 2b
4.0
3b
4.5
5.0
5b 5a 4b
5.5
6a 6b
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
min
Figure 2 HS-SPME-ES-GC-MS profiles of juniper volatiles. Techniques (a), (b), and (c). as specified. (Reproduced from Bicchi, C.; Cagliero, C.; Rubiolo, P. New Trends in the Analysis of the Volatile Fraction of Matrices of Vegetable Origin: A Short Overview. A Review. Flavour Frag. J. 2011, 26, 321–325, with permission).
In addition to routine analysis of volatile terpene constituents of formulated products in quality control, GC-MS systems are becoming widely used in the search for allergenic and potentially allergenic components of fragrances used to enhance their acceptability. Patch testing for allergenicity is routinely followed by GC-MS of the range of materials shown to elicit positive effects; the aim being to identify common components, confirming patch test allergenic activity, then to remove or reduce levels of specific causative agents in the formulations.
Forensic Science Cannabis The major active constituents, the cannabinoids such as tetrahydrocannabinol (THC), are of terpenoid biogenetic origin, being derived from geranyl pyrophosphate. In addition to these constituents, cannabis mono- and sesquiterpene aroma volatiles have often been the subject of investigation. A fairly simple extraction of cannabis resin or herb, is usually carried out for forensic purposes. Being non-polar, similar polarity solvents are employed for plant material, but cannabinoids have also been extracted from hair using prior alkaline digestion, and final extraction from a solid phase cartridge. Headspace SPME has also been used. Headspace volatiles have been collected both after a pre-heating and at ambient temperature, and absorbed onto a range of adsorbents including methylsiloxane and divinylbenzene solid phase fibres. Solute extraction or heat desorption may later be used prior to chromatography, but adsorbent fibres have also been used which can later be inserted directly into the GC injection port, and the volatiles chromatographed. Volatile oils of cannabis have forensic significance and are extracted by both traditional steam distillation and via pyrolysis. Figure 3 shows a chromatogram of the essential oil revealing separation of 48 components. Cannabis smoke has been collected, and fractionated with acid/base partition, which is followed by chromatography.
8
GAS CHROMATOGRAPHY | Terpenoids
Figure 3 Gas chromatogram of the essential oil of a plant of accession from Afganistan assigned to the wide-leaflet drug biotype of Cannabis indica. i.s., internal standard; Column, DB-5MS. (Reproduced from Hillig, K. W. A Chemotaxonomic Analysis of Terpenoid Variation in Cannabis. Biochem. Syst. Ecol. 2004, 32, 875–891, with permission).
GC separations have been carried out on a number of stationary phases, including columns typically packed with 3% OV-17, 6% OV-101, and 30 m capillary columns such as DB-5, HP-5MS, DB-5MS. Detection has traditionally involved FID, but GC-MS is now routinely used. Quantification has been carried out by both internal normalization, and via use of calibration curves prepared using an internal standard, which is the norm in forensic work. Cannabis essential oil is usually quantified using peak normalisation, due to the large number of constituents similarly to other essential oils. Results obtained with both packed columns and capillary columns have separated 50–60 components. Identifications of the major metabolite, THC acid (THCA), in hair and urine following consumption of cannabis, have been carried out. THCA has been analyzed in hair at very low concentrations, after a digestion process, followed by acidification and solid phase extraction. The resultant extract has been derivatized with pentafluoropropionic anhydride/hexafluoroisopropanol, and chromatographed by GC-MS in the negative chemical ionisation mode. Single ion monitoring of THCA extracted from urine has been carried out on GC-MS, and automated solid phase extraction has also been carried out; revealing a detection limit of 1 ng ml 1, with a quantitation limit of 4 ng ml 1. Forensic analyses of cannabinoids using GC continues to be developed. A GC-NCI-MS/MS method has been developed for the determination of 11-nor-D9-tetrahydrocannabinol-9-carboxylic acid in human hair. This is a particularly useful development, as it would previously have been possible using only HPLC. 25 mg hair samples were powdered, digested with1.0 M sodium hydroxide, and extracted with hexane: ethyl acetate (9:1). Derivatisation of the dried extract with pentafluoropropanol and pentafluoropropionic anhydride was followed by GC-MS/MS in the negative ion chemical ionisation mode. The limits of detection and quantification were 0.015 and 0.05 pg mg 1, respectively, and recoveries of THC acid were in the range of 79.4–87.1%.4 Principle component analysis has been carried out, which allows assignment of plants of diverse geographical origins, into specific biotypes. Overall, the major cannabinoids are readily separated, even using packed columns, and capillary columns and GC-MS has allowed identification of a greater range of components.
Diterpenes Diterpenes are less volatile and less numerous than the mono- and sesquiterpenes, consequently they rarely appear in distilled essential oils. However, traditional extraction using distillation allows separation and identification of diterpenes present in essential oils. The diterpenes in frankincense have been analysed using GC-MS after headspace trapping on adsorbent fibres.
GAS CHROMATOGRAPHY | Terpenoids
9
100% = 96.765
Total
Sesquiterpenes
Monoterpenes Diterpenes
Isoincensole acetate Cembrene A
(a)
10:00 Total
15:00
20:00
25:00
30:00
35:00
40:00
45:00
50:00
100% = 104.122
Diterpenes
Monoterpenes
55:00 01:00:00
Isoincensole acetate Sesquiterpenes
Cembrene A
(b)
10:00
15:00
20:00
25:00
30:00
35:00
40:00
45:00
50:00
55:00 01:00:00
Figure 4 Comparison between SPME and dichloromethane extraction. (Reproduced from Hamm, S.; Lesellier, E.; Bleton, J.;Tchapla, A. Optimization of Headspace Solid Phase Microextraction for Gas Chromatography/Mass Spectrometry Analysis of Widely Different Volatility and Polarity Terpenoids in Olibanum. J. Chromatogr. A. 2003, 1018, 73–83, with permission).
Figure 4 shows the two chromatograms obtained from these extraction techniques, and also demonstrates the separation of mono-, sesqui- and diterpene fractions and their constituent entities. The resolution of this particular system allowed for identification of 66 mono- and sesqui-terpenes along with diterpenes. The analysis of small amounts of diterpenes in archaeological and historical samples requires specialised techniques. Diterpenes in canvas paintings have been analysed either after formation of ethyl or silyl ethers of the hydroxylated diterpenes of resins present or pyrolysis GC-MS, with prior silylation and typically a range of 20 different diterpenes have been identified. A further interesting matrix has been investigated using GC-MS and two dimensional Time of Flight (TOF) MS. Cannabis cigarette smoke condensate was collected on glass fibre pads and extracted by simultaneous distillation and SDE. The DCM extract was washed with 5% sodium hydroxide and 5% hydrochloric acid, and then chromatographed in two systems; a 50 m DB Petro capillary linked to electron impact MS, and GC positive ion Chemical Ionisation MS, and the same column also fitted to TOF MS. Over 100 compounds may be identified, including phytaene, phytadienes, cembranes, and labdanane diterpenes. Recently, increased interest has been shown in microbial diterpene metabolites. Solvent and headspace extracts, have been used to extract diterpenes, which can then be identified by GC-MS.
Triterpenes On occasions the chromatographic system used for diterpenes present in particular matrices is also able to separate and identify triterpenes from the same source, using silylation and GC-MS of a DCM extract. Triterpene dialcohols, have been analysed using reversed phase HPLC and GC employing a programmed temperature vapourizer at the interface. This procedure allows analysis of small samples of aqueous methanolic extracts to be chromatographed via transfer of the triterpenes directly from HPLC into the programmed temperature vapourizer (PTV) injector attached to a GC.
10
GAS CHROMATOGRAPHY | Terpenoids
Steroids There are a vast number of steroids obtained from a wide variety of matrices, including plant, animal, and synthetic sources, and their metabolites such as the urinary metabolites. GC has been used from the early days of instrumentation, and typically has been involved in analysis on both packed and capillary columns, using a variety of detectors, and recently a number of tandem techniques. This is due to the increased forensic significance of steroid metabolites, for instance in sport and workplace drug testing, GC has to some extent been supplanted by use of large scale immunoassays, but this technique does not supply the wealth of data, either qualitative or quantitative, that GC systems are capable of. The forte of GC and hyphenated techniques, is the range and extent of information available from single injection or auto sampler-fed systems. Dehydroepiandrosterone (DHEA) is an example of a steroid used both medically, and for abuse, and its analysis covers the whole range of available techniques. DHEA is both an endogenous androgen and a synthetic product, with wide applications in the areas of medicinal product formulations and extraction and analyses in varied materials. DHEA has been analysed in GC systems capable of screening a number of related steroids. Both solid and liquid dosage forms have been analysed by basifying and extracting in non-polar solvent. Derivatization by silylation prior to chromatographing allows resolution and identification of a large number of related steroids using Selected Ion Mode (SIM)- MS. SPME, followed by on-fibre silylation has been used to analyse steroids present in river water and fish blood serum at detection levels of 0.002–0.378 mg l 1 and 0.004–0.475 mg l 1 respectively, and quantification levels of 0.008–1.26 mg l 1 and 0.013–1.579 mg l 1 respectively. Human urinary levels of DHEA and metabolites are often analysed after oral ingestion. Also GC/Combustion/ Isotope Ratio Mass Spectroscopy (IRMS) has been employed for urine samples, and conjugated steroids hydrolysed with glucuronidase, and purified on a standard reverse phase HPLC column. Acetylated derivatives were re-chromatographed on HPLC and collected, before chromatography and detection with Mass sensitive Detector (MSD). IRMS was carried out after a combustion stage at 940 C, and isotope ratio values were shown to be unchanged following ingestion. This procedure is now routinely carried out in urine samples containing concentrations greater than 100 ng ml 1 of DHEA. A novel method for analysis of anabolic steroids was devised using GC-microchip atmospheric pressure photoionisation-MS. Atmospheric pressure ion MS is usually used with HPLC, but researchers established a system of linking to GC. A heated nebuliser microchip was successfully interfaced with atmospheric pressure photoionisation-tandem MS and used for the analysis of underivatised anabolic steroids present in urine. Seven anabolic steroids including nandrolone and methyltestosterone were resolved and identified. The method showed good sensitivity and repeatability, and was compared with conventional GC-EI-MS following derivatisation. The results were found to be in good agreement, and demonstrated the simplicity, avoiding the derivatisation stage in analysis.5 It is evident that many examples of GC analysis of a wide range of steroids have been carried out, but there is a trend towards use of HPLC for determination of conjugated steroids in biological fluids.
Tetraterpenes Carotenoids Early work on carotenoids often employed packed columns such as 2% SE-50, 2% High Vacuum Grease, or 3% OV-17, either under isothermal conditions or temperature programming. Carotenoids have been hydrogenated prior to analysis, in order to prevent thermal degradation. Carotenoids have a number of medicinal and other applications and natural product mixtures have been derivatised by silylation or methylation, then pyrolyzed at temperatures ranging from 400 to 750 C before chromatographing. Single carotenoids such as lycopene and its degradation products have been studied. Direct solvent extraction has been used to collect the less volatile components and GC resolved both partially isomerised degradation products and the more volatile degradation products.
Polyterpenes Rubber Rubber is a polyisoprene polymer composed of isoprene units, with a molecular weight ranging from 100 000 to 1 000 000. Due to the non-volatility of rubbers, pyrolysis followed by GC-MS is a routine technique for analysis. After pyrolisis, volatile degradation products may be chromatographed on a methylsiloxane capillary column, which allows routine identification of constituent monomers such as butadiene, isoprene, styrene and dipentene. Low molecular weight components of commercial, natural and derivatised rubbers used in vehicle tires and in pharmaceutical packaging have been investigated using both Soxhlet and headspace extractions followed by GC-MS. A range of solvents have been tested for Soxhlet extractability, ranging from cyclohexane to isopropanol. Alternatively, constituents have been analysed by GC-MS of their pyrolysis products. Pyrolysis temperatures up to 500 C have been employed, using a coupling connected to the GC operated at 300 C to prevent condensation of the products. GC-MS-MS has been used to separate the dimer products of isoprene.
GAS CHROMATOGRAPHY | Terpenoids
11
Problems and Perspectives One possible problem with modern GC analysis is the reliance on computerized structure prediction based upon data libraries. Structural identification of mono- and sesqui-terpenes is routinely corroborated with RIs, and confirmation is more secure, although reproducibility of RIs has been questioned when using temperature programming. New databases are appearing which may reduce possible errors of identification. This problem may occur with any class of terpenes, but the large groups of individual mono- and sesqui-terpenes are potentially more prone to mis-identification. The increasing use of MS to elucidate structures of coeluted solutes has caused a few doubts to be raised concerning the validity of the procedure. Other terpene groups are more likely to be identified via a number of techniques, due to comparatively reduced numbers of related compounds, and consequently, will be less liable to misinterpretation. The possible degradation of terpenes under GC conditions has been previously questioned. There is however little hard evidence to substantiate this possibility. Although the degradation of monoterpenes on Tenax adsorbent during sample storage, even under extreme non-standard conditions, resulted in 88% recovery of the undegraded terpene. Limited work on thermal degradation of monoterpenes has been investigated; treatment in conditions simulating the commercial wood drying process, which involves the presence of air which is not encountered in GC systems has been found to yield a large range of degradation products. Consequently, there is little evidence of degradation during the GC stage of analyses.
Conclusions The high volatility of the mono- and sesqui-terpenes has allowed GC to become the preferred analytical tool. Improved concentration techniques, sensitive detectors and hyphenated systems, allow quantification and characterization of large numbers of low levels of volatile terpenes. The less volatile higher terpenes often exist in lesser numbers in particular matrices, but GC is still a major technique for their analysis. Higher terpene analysis has also benefited from developments in extraction and concentration techniques. Again, development of ancillary techniques has dramatically improved their characterisation and quantification. When compared with other available techniques, the use of basic FID detection plus linking to MS, allows complete characterization of many natural materials and their derivatives, currently not available using other chromatographic systems.
References 1. Zachariadis, G. A.; Langioli, A. V. Headspace Solid Phase Microextraction for Terpenes and Volatile Compounds Determination in Mastic Gum Extracts, Mastic Oil and Human Urine by GC-MS. Anal. Lett. 2012, 45, 993–1003. 2. Bicchi, C.; Cagliero, C.; Rubiolo, P. New Trends in the Analysis of the Volatile Fraction of Matrices of Vegetable Origin: A Short Overview. A Review. Flavour Frag. J. 2011, 26, 321–325. 3. Barros, P. E.; Moreira, N.; Pereira, G. E.; Leite, S. G. F.; Rezende, C. M.; Pinho, P. G. Development and Validation of Automatic HS-SPME with a Gas Chromatography Ion Trap/ Mass Spectrometry Method for Analysis of Volatiles in Wines. Talanta 2012, 101, 177–186. 4. Kim, J. Y.; Cheong, J. C.; Lee, J. I.; In, M. K. Improved Gas Chromatography-Negative ion Chemical Ionization Tandem Mass Spectrometric Method for Determination of 11-nor-D 9-Tetrahydrocannabinol-9-Carboxylic Acid in Hair Using Mechanical Pulverization and Bead-Assisted Liquid-Liquid Extraction. Forensic Sci. Int. 2011, 206, e99–e102. 5. Hintikka, L.; Haapala, M.; Franssila, S.; Kuuranne, T.; Leinonen, A.; Kostiainen, R. Feasibility of Gas Chromatography-Microchip Atmospheric Pressure Photoionization-Mass Spectrometry in Analysis of Anabolic Steroids. J. Chromatogr. A 2010, 1217, 8290–8297.
Further Reading 1. Adams, R. P. Identification of Essential Oil Components by Gas Chromatography-Mass Spectrometry; Allured: Illinois, 1995. 2. Bicchi, C.; Cagliero, C.; Rubiolo, P. New Trends in the Analysis of the Volatile Fraction of Matrices of Vegetable Origin: A Short Overview. A Review. Flavour Frag. J. 2011, 26, 321–325. 3. Davies, N. W. Gas Chromatographic Retention Indices of Monoterpenes and Sesquiterpenes on Methyl Silicone and Carbowax 20M Phases. J. Chromatogr. 1990, 503, 1–24. 4. Hamm, S.; Lesellier, E.; Bleton, J.; Tchapla, A. Optimization of Headspace Solid Phase Microextraction for Gas Chromatography/Mass Spectrometry Analysis of Widely Different Volatility and Polarity Terpenoids in Olibanum. J. Chromatogr. A 2003, 1018, 73–83. 5. Lockwood, G. B. Techniques for Gas Chromatography of Volatile Terpenoids from a Range of Matrices. J. Chromatogr. A 2001, 936, 23–31. 6. Marriott, P. J.; Shellie, R.; Cornwell, C. Gas Chromatographic Technologies for the Analysis of Essential Oils. J. Chromatogr. A 2001, 936, 1–22.