Analysis of Terpenes in Cannabis Using Headspace Solid-Phase Microextraction and GC–MS

February 28, 2018
Table I
Click to enlarge, Table I: HS-SPME and GC–MS parameters used for quantitative determination of terpenes from cannabis
Abstract / Synopsis: 

As the legalization of medicinal cannabis continues to sweep across the United States, an urgent need has developed for fast, accurate, and efficient analytical testing. In addition to testing for contaminants and potency, there is also interest in the determination of terpene identity and concentration levels present in different strains of cannabis. Terpenes have been shown to have therapeutic uses for treatment of different medical conditions ranging from cancer and inflammation to anxiety and sleeplessness. It is believed that the combination of terpenes and cannabinoids in cannabis produce a synergistic effect with regards to medical benefits. The traditional testing method for terpenes in plant materials involves a solvent-based extraction followed by gas chromatography (GC) analysis. In this work, headspace solid-phase microextraction (HS-SPME) was used to identify and quantify terpene content in cannabis. The HS-SPME method offered several advantages compared to solvent extraction in that it provided a cleaner analysis, free of interferences from coextracted matrix, and was nondestructive to the sample. A cannabis sample of unknown origin was first analyzed qualitatively by HS-SPME and GC–mass spectrometry (MS). Spectral library matching and retention indices were used to identify 42 terpenes. Quantitative analysis was then performed for several selected terpenes using spiked samples. Method accuracy was >90%, with reproducibility of <5% relative standard deviation (RSD) for analysis of spiked replicates. The HS-SPME results were then compared to an analysis using a conventional solvent extraction method, and the two approaches were found to produce comparable results.

Terpenes are small molecules synthesized by some plants. The name terpene is derived from turpentine, which contains high concentrations of these compounds. Terpene molecules are constructed from the joining of isoprene units in a “head-to-tail” configuration. Classification is then done according to the number of these isoprene units in the structure. For example, monoterpenes contain two units, and sesquiterpenes contain three. The structures of terpenes can be cyclic or open, and can include double bonds, and hydroxyl, carbonyl, or other functional groups. If the terpene contains elements other than carbon and hydrogen, it is referred to as a terpenoid (1). Well known for their characteristic flavors and fragrances, terpenes are contained in the derived essential oils of cannabis. Cannabis contains more than 100 different terpenes and terpenoids, including mono-, sesqui-, di-, and triterpenes, as well as other miscellaneous compounds of terpenoid origin (2). Different cannabis strains have been developed that contain distinct aromas and flavors, which is a result of the differing amounts of specific terpenes present (3). The analysis of cannabis for terpene concentrations can be applied to strain identification, referred to as fingerprinting, and for concentration accuracy when applied to medicinal treatments.

With the growing legalization of medicinal cannabis worldwide, methods for qualifying and quantifying terpene concentrations has risen to the forefront in the analytical industry. As the analytical world tries to keep up with this rapid pace, a need exists for development of faster and more efficient methods that will produce rapid and accurate results at a low cost. Terpenes have high vapor pressures, are extremely volatile, and thus are excellent candidates for static headspace gas chromatography (GC) analysis. Headspace solid-phase microextraction (HS-SPME) offers a low cost and easy alternative to traditional headspace methods. It has been used for both qualitative and quantitative determination of terpenes in a wide variety of matrices, including various foods, beverages, and plant materials (4–7). However, in the analytical testing of cannabis for terpenes, rather than headspace, a common approach has been to use a solvent-based extraction of the plant material followed by GC–flame ionization detection (FID) analysis. In this work, HS-SPME was combined with GC–mass spectrometry (MS) for the qualitative and quantitative analysis of terpenes in cannabis. This approach offers several advantages compared to solvent extraction and GC–FID. It does not require the use of organic solvents, does not coextract matrix (which could potentially interfere with the chromatographic analysis or contaminate the GC system), and provides additional means of peak identification and purity using spectral data. In the first portion of this application, HS-SPME was used to profile the predominant terpenes in an unknown variety of cannabis. A quantitative HS-SPME method was then developed for the determination of several selected terpenes from spiked cannabis samples. The results of this method were then compared to the analysis of the same terpenes using solvent extraction followed by GC–FID.

Experimental

Dried cannabis sample was obtained courtesy of Dr. Hari H. Singh, Program Director at the Chemistry & Physiological Systems Research Branch of the National Institute on Drug Abuse at the National Institute of Health. The extract strain and composition of the sample were not known. The SPME parameters and GC–MS conditions used for quantitative analysis are listed in Table I. (See upper right for Table I, click to enlarge.) All SPME analyses were conducted on a Gerstel MPS 2 autosampler with heated agitator, and an Agilent 6890/5973N GC–MS system operated in scan mode. The qualitative terpene profiling was performed on 0.5 g of dried cannabis weighed into a 10-mL headspace vial, capped, and analyzed directly without any further modification. Terpene identification was done using retention indices and spectral searching against National Institute of Standards and Technology (NIST) and Wiley libraries.

Quantitation was applied to three selected terpenes: α-pinene, d-limonene, and linalool. The strain of the cannabis sample was unknown and HS-SPME analysis of this sample showed it to have a much lower terpene content than what is typically reported for many strains sold for medical and recreational use (8). Thus, to evaluate the HS-SPME method in relevant concentration ranges, the sample had to be spiked with additional amounts of terpenes. The terpene spiking solution was prepared, by weight, from individual neat materials and then diluted into hexane before use. Cannabis samples were prepared for HS-SPME by first grinding the material with a mortar and pestle, and weighing 0.1 g into a 20-mL head space vial. Varying volumes of terpene spiking solution (always less than 10 µL) were added to the dry cannabis to achieve the desired concentrations. After a 10-min equilibration time, 8 mL of deionized water was added to the cannabis, and the samples were placed on the autosampler for HS-SPME analysis. For method evaluation, three spiked samples, two unspiked blanks, and five matrix-matched standards were prepared. Quantitation using extracted ion chromatograms of each terpene was done for spiked samples by external standard against a five-point matrix-matched calibration curve. Matrix spiking was possible because of the low terpene content found to be present in the cannabis used for this study. Consequently, unspiked cannabis was included in the calibration as a zero point.

The solvent extraction procedure used was based on that described by Giese and colleagues (9). Samples were prepared using 0.25 g of ground cannabis weighed into 15-mL polypropylene centrifuge tubes. Spiking solution was added in a similar manner to that described for HS-SPME. After spiking and equilibration, 3 mL of 200-proof ethanol was added, and the samples were vortexed at 2500 rpm for 6 min. They were then centrifuged at 3000 rpm for 10 min, and the resulting supernatant was transferred to a GC autosampler vial for analysis. Three spiked and one unspiked cannabis sample were prepared. Quantitation was done against a five-point calibration curve prepared in ethanol. Samples were analyzed on an Agilent 6890 GC–FID system equipped with an Equity-1 capillary column (60 m x 0.25 mm, 0.25-µm df, MilliporeSigma). The injection and detector temperatures were 270 °C and 300 °C respectively, and the oven was programmed at 60 °C (2-min hold) to 140 °C at 5 °C/min, and to 250 °C at 15 °C/min. Helium carrier gas was used at a linear velocity of 20 cm/s (measured at 40 °C). A 1-µL sample was injected into a 4-mm i.d. tapered FocusLiner (SGE Analytical Science), at a 10:1 split ratio. The terpenes were identified by retention time and quantitated by external standard analysis using peak area.

References: 
  1. R.J. Fessenden and J.S. Fessenden, Organic Chemistry, 2nd Edition (Willard Grant Press, Boston, Massachusetts, 1979), pp. 897–904.
  2. C.E. Turner, E.G. Elsohly, and E.G. Boeren, J. Nat. Prod. 43, 169–234 (1980).
  3. B. Rahn, “Terpenes: The Flavors of Cannabis Aromatherapy,” 2014, www.leafly.com/news, accessed 9/18/2015.
  4. A. Peña-Alvarez, S. Capella, R. Juárez, and C. Labastida, J. Chrom. A 1134, 291–297 (2006).
  5. J.I. Cacho, C.P. Viñas, and M. Hernández-Córdoba, J. Chrom. A 1399, 18–24 (2015).
  6. M.L. Ruiz del Castillom, G.P. Blanch, and M. Herraiz, J. Chrom. A 1054, 87–93 (2004).
  7. A. Sartoratto and F. Augusto, Chromatographia 57, 351–356 (2003).
  8. K. Williams, “Cannabinoids and Terpenoids: The Future of Cannabis Connoisseurship,” 2014, www.leafly.com/news/tags/connoisseur (accessed June 2016).
  9. M.W. Giese, M.A. Lewis, L. Giese, and K.M. Smith, J. AOAC Int. 98(6), 1503–1522 (2015).
  10. M. Marchini, C. Charvoz, L. Dujourdy, and N. Baldovini, J. Chrom. A 1370, 200–215 (2014).
  11. W. Jennings and T. Shibamoto, Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography (Academic Press, New York, New York, 1980).
  12. National Institute of Standards and Technology (NIST) Chemistry WebBook, www.webbook.nist.gov/chemistry.
  13. L.V.S. Hood and G.T. Barry, J. Chrom. A. 166, 499–506 (1978).
  14. Terpene profiling services, www.thewercshop.com/services/terpene-profiling-services, accessed June, 2016.
  15. www.sclabs.com, view tested samples, accessed 1/13/2017.
  16. H.H. Jeleń and A. Gracka, J. Chrom. A 1418, 200–209 (2015).