Extraction of terpenes pdf

It is not clear as to where the terpenes were lost i. Because the HS-SPME Arrow method was able to identify all of the terpenes in the samples, this approach was chosen to move forward in the study. Both techniques were able to identify all terpenes within the reference standard samples. Several terpene extraction approaches were considered for the current study. Other industries already capitalize on the benefits of ASE Ligor et al.

Therefore, a simple hand shakeout solvent extraction method was compared to an ASE extraction method to evaluate the performance of each technique for extracting terpenes from cannabis flower.

Three different cannabis chemovars were extracted using both techniques and the average of their FID responses were determined Table 4. Both techniques extracted the same 13 terpenes from the cannabis flower. On average, the hand shakeout responses were better than the ASE responses for 11 of the 13 terpenes detected. Given the small sample size, a nonparametric Kruskal-Wallis test was completed to compare the averages and determine if there was a statistical difference between the hand shakeout and ASE approaches.

Hand shakeout vs. In addition, several factors were considered when selecting the extraction method. The hand shakeout required more use of nonreusable consumables e. In addition, the cannabis flower hand shakeout technique could potentially differ depending on the lab technician completing the manually intensive hand shakeout extraction. On the contrary, the ASE cells were reused by cleaning after extraction.

Furthermore, the extraction consistency that the ASE offers was not user dependent. The lack of statistical significance between the two extraction techniques, coupled with less consumable needs and user variability, leads to utilizing the ASE for all of the following DI-SPME Arrow vs.

LI-Syringe experiments. It is important to note that this was the first study to utilize an ASE for the extraction of terpenes from cannabis. Despite this novel development for the field of cannabis, future studies should consider the further development of the current ASE parameters to optimize extraction efficiency by changing solvent extraction ratios, extraction temperature, etc.

Of the 23 terpenes evaluated, 17 had positive MEs 7 average , as defined by Chambers et al. The signal suppression for the aforementioned alcohols is contradictory to the theory of matrix-induced chromatographic response and represents a testament to the complexities of matrix effects.

Regardless, these results indicated that cannabis flower MEs were present and therefore a matrix match calibration approach was deemed ideal. However, it was outside the scope of the current manuscript to fully dissect all of the current ME phenomenon associated with cannabis, especially considering the wide array of cannabis matrices. Future researchers are encouraged to expand upon the current start to understanding cannabis MEs. Due to the numerous types of matrices cannabis testing laboratories analyze, laboratories are forced to become creative when doing matrix matching for their calibrations.

Matrix matching for terpenes in cannabis flower represents a particularly tough issue, because similar plant species to cannabis also contain terpenes.

In this study, a novel method of cleaning hops was utilized to provide a terpene-free surrogate for matrix matched calibrations. Matrix blanks were run to demonstrate the cleaned hops were free of terpenes and did not contribute to compound responses for the terpenes of interest Figure 3. This was the first study to utilize cleaned hops as a clean surrogate for cannabis flower, and the following method validation results demonstrate not only that this was a viable technique, but also it produced a desirable outcome i.

Method validation was done in accordance with the California Bureau of Cannabis Control guidelines and regulations California Bureau of Cannabis Control, Single quad MS mode was used to be more representative of most cannabis laboratories, and SIM was used to minimize matrix interferences. Terpene method validation was completed for DI-SPME Arrow to evaluate performance and possible implementation into cannabis testing laboratories.

Method performance can be seen in Table 5. As shown in Table 5 , an average r 2 value of 0. While most compounds had a working range of 0.

When compound saturation occurs within the calibration, the calibration curve will plateau, resulting in poor r 2 values. This phenomenon can also be combated by adjusting the parameters e. An average LOQ of 0. MDLs were calculated as the standard deviation of the seven replicate measurements multiplied by 3. Analytical precision was evaluated using reference standards. In addition to analytical precision, overall method precision i.

Seven shake extractions were made and 1 sample from each extraction was analyzed to evaluate method precision. Method performance was evaluated for a LI-Syringe method and can be seen in Table 6. Over an average calibration working range of 0. It is important to note that the LI-Syringe technique was able to achieve a higher range for the terpenes.

Improvements were made when looking at method precision via LI-Syringe vs. The aforementioned results are comparable to a previously published study by Ibrahim et al. The current study was able to build off of the foundation from Ibrahim et al. However, this study excelled with lower working calibration range and an order of magnitude lower LOQs 0. In addition, this study employed the use of accelerated solvent extraction and matrix matched calibrations.

Results shown in Table 7 represent the concentration in the sample for Mint Chocolate Chip i. From the data collected, several points should be discussed when comparing the two methods. It should be noted that 1 compound p -Cymene was not detected ND by either technique and should be considered as not present in this chemovar. Regardless, the LI-Syringe appeared to provide better results when analyzing a true sample.

Nearly all compounds fell within the LI-Syringe calibration working ranges and saturation was not an issue. Comparing the average concentrations specifically, both techniques showed similar concentrations for half of the compounds and divergent concentrations for the other half. More importantly, when comparing the overall terpene profiles of each injection technique, the profiles showed similar results Figure 5.

A novel method for cleaning hops was developed to provide terpene-free hops utilized as a surrogate for matrix matched calibrations. For the first time, both hops and cannabis flower were extracted with ASE and then utilized for method validation. Both methods proved to be viable options for the analysis of terpenes in cannabis flower. When comparing the average LOQs of the terpenes of interest, both techniques were near identical.

However, results suggest that LI-Syringe would be the preferred approach for this analysis based on several observations. In addition, better analytical and method precision was achieved by LI-Syringe. Furthermore, LI-Syringe appeared to provide a more complete chemovar profile of cannabis flower and at higher concentrations. While results indicate that LI-Syringe is the preferred technique, other factors should be considered for future work.

This includes inlet consumable changes, analytical column trimming, and MS maintenance, which will come as a result of the matrix being injected into the system. It is hypothesized that less matrix may be exposed to the GC when running the DI-SPME Arrow method, which in turn may lead to longer instrument uptime and less time and money spent on maintenance.

It is recommended that the scientific cannabis community reconsider utilizing HS-Syringe for the analysis of terpenes in cannabis products, as the current results suggest it is inferior to all of the approaches discussed, especially LI-Syringe. Furthermore, the FET approach is not amenable to splitting samples for other cannabis test methods e.

In addition, it is recommended that future cannabis work continue to evaluate the use of terpene-free hops for surrogate matrix matching of flower. Furthermore, it is recommended that additional cannabis research further the development of ASE methods for the extraction and analysis of terpenes.

Under the appropriate conditions, the ASE has the potential to improve laboratory workflows by using one extraction and splitting that extract between multiple analyses in addition to terpene profiling e. Lastly, it is recommended that this work should be expanded to additional matrices that cannabis testing laboratories frequently analyze e.

These additional matrices bring new challenges and will need to be addressed to improve the science of cannabis testing. CM started this study by investigating different approaches for analyzing terpenes. CM and JH outlined the research plan, analyzed data, and wrote the manuscript.

PH facilitated the research by providing a licensed cannabis testing laboratory to continue the research. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. National Center for Biotechnology Information , U.

Journal List Front Chem v. Front Chem. Published online Apr 1. Herrington , 1 Paul Hamrah , 2 and Kelsey Anderson 2. Jason S. Author information Article notes Copyright and License information Disclaimer. This article was submitted to Analytical Chemistry, a section of the journal Frontiers in Chemistry. Received Oct 21; Accepted Jan 4. The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.

No use, distribution or reproduction is permitted which does not comply with these terms. This article has been cited by other articles in PMC. Associated Data Supplementary Materials datasheet1. Abstract The cannabis market is expanding exponentially in the United States.

Introduction The legal cannabis market is one of the fastest growing markets across the globe. Experimental The following experimental sections describe the detailed procedures utilized during the three main parts of this manuscript: 1.

Terpene Extraction Evaluation An evaluation of terpene extraction processes was conducted to understand advantages and limitations of certain techniques. Open in a separate window. After ASE Processing After ASE extraction, all extracts, which were typically between 10 and 11 mL, were brought to a final volume of 12 mL in order to consistently evaluate extracts of the same volume. Results and Discussion HS-Syringe vs.

Hand Shakeout vs. Accelerated Solvent Extractor Several terpene extraction approaches were considered for the current study. NA, not applicable; ND, not detected. Cleaned hops blank LI-Syringe demonstrating terpene-free surrogate matrix. Author Contributions CM started this study by investigating different approaches for analyzing terpenes.

Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Click here for additional data file. References Abilleira E. An accurate quantitative method for the analysis of terpenes in milk fat by headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry.

Food Chem. Simultaneous determination of terpenes and cannabidiol in hemp Cannabis sativa L. Validated quantitative cannabis profiling for Canadian regulatory compliance—cannabinoids, aflatoxins, and terpenes. Acta , 79— Evaluation of three headspace sorptive extraction coatings for the determination of volatile terpenes in honey using gas chromatography-mass spectrometry.

A , 18— Laws and regulations: California Bureau of Cannabis Control. Calvi L. Comprehensive quality evaluation of medical Cannabis sativa L. B , 22— Food Addit. Piezoelectric peptide-hpDNA based electronic nose for the detection of terpenes; evaluation of the aroma profile in different Cannabis sativa L. Actuators B Chem. Thus, at least 50 mg of tobacco leaf material should be used for suitable downstream detection. Grind plant material into a powder in liquid nitrogen in a glass vial or using a mortar and pestle.

The crude extract should be transferred to a glass vial or flasks for shaking for at least 3—4 hours or overnight. The ratio of hexane: ethyl acetate can be changed depending on the polarity of the terpene sought. Usually a higher proportion of hexane will extract more non-polar terpenes, whereas, higher proportion of ethyl acetate will extract more polar terpenes. Ideally, the internal standard will be of similar chemical composition to the target terpene.

The amount of internal standard used will depend on the final concentration desired. Then, load the concentrated sample onto a small silica column Figure 2. Allow the sample to be absorbed into the column, then add 1—3 mL of hexane and collect the column eluate.

Transfer this to an appropriate vial for GC analysis. If the terpene is volatile, then all concentration and drying steps under a nitrogen stream should be conducted with the sample vial sitting in ice. Also, the small chromatographic step may not be enough to separate and purify all compounds in the mixture, and the eluate will contain a mixture of compounds.

However, this chromatographic step can exclude pigments large size molecules and other chemical constituents, which may dirty and reduce the performance of the injector port or the injector syringe of the analytical equipment. B For purifying large quantities of the target terpene from a larger amount of plant material: concentrate the extract to as small a volume as possible, but keeping enough volume so that all chemical components in the extract remain solublized including some leaf or tissue debris and can be easily loaded into the column in the next step.

Prepare the silica column as shown in Figure 2 and load solvent onto the column using at least two times the volume of silica within the column, ensuring that the silica is completely saturated with solvent. It is very convenient to fill half the volume of the column with silica, so that an equal amount of solvent can be added to the top without the need to measure each time. Load the concentrated extracts onto the top of column. Then wait for the extract to fully enter the column.

Concentrating the extract to as small a volume as possible ensures that the sample will enter the column as a sharp band and will prevent spreading of the compound over more fractions than necessary. Add a volume of hexane equal to the silica volume half column volume in the set-up recommended above and allow this to enter the column.

Repeat multiple times. Do not add the hexane until all the extract has fully entered the column, otherwise the introduced hexane will dilute the concentrated sample. How long the entire elution takes will depend on the affinity of target terpene molecules to the silica gel. Usually the molecules with less polarity and smaller size will take less time to elute.

For example, squalene may require multiple column volumes to elute fully. It is best to check empirically using a purified standard if possible. If a standard cannot be obtained, a compound that is of similar chemical composition can substitute as a starting point. In this step, if the plant has a relatively high concentration of the desired terpene, Thin Layer Chromatography, TLC see protocol 2 can be used to directly show which fraction has the highest amount of the desired terpene.

However, in the example presented here, the squalene levels in plants are usually low and TLC is not sensitive enough to detect it. Combine all the fractions that contain squalene or the terpenoid of interest. Then concentrate the fractions under a nitrogen stream. The concentrated sample should contain most of squalene. The quantity and purity of the squalene can be evaluated by analyzing a small fraction of the sample by Gas Chromatography GC.

The sample may contain other molecules which co-elute with squalene and these will affect the purity of the sample. HPLC coupled to a photo diode array is generally not a very sensitive method for the detection of terpenes.

This is due to the lack of specific UV wavelengths that they absorb. However, in larger-scale purification where the concentration of the terpene will be present in large amounts, using a wavelength of nm will suffice. Using wavelengths of to nm is not very specific for any chemical feature, so it can also be used to track impurities. Collect the eluate fractions, concentrate under a stream of nitrogen and evaluate the individual fractions for those containing the highest amount of squalene.

GC analysis is the preferred method for this, but TLC may also be used. Repetitive chromatographic runs can be used to enhance and increase the purity of the desired terpenoid compound, in this case squalene. Many of the monoterpenes and sesquiterpenes can be volatile, and while these serve important roles in plant interactions with their environments, they require special methods for their analysis. The volatile terpenes present in specific plant tissues can be studied directly through traditional extraction techniques e.

However, the terpenes that are emitted to the atmosphere accumulate only temporarily in leaf aqueous and lipid phases, and thus are present in only small concentrations in tissue samples Wu et al.

Hence, studying the biosynthesis and emission of terpene emissions requires molecular trapping techniques. In this alternative protocol, we describe how to trap the volatile sesquiterpenes, like patchoulol, emitted from transgenic tobacco plants generated by Wu et al. Put the plant in a sealable chamber with two ports see Figure 3. Set up a glass Pasteur pipette packed with mg of the tenax resin similar to the set up used for the silica column presented in Figure 2 and plumb in-line prior to the air entering the chamber.

This will pre-filter the air entering the chamber. Use simple connectors to allow easy exchange of the traps. Plumb the input gas line into the bottom of the chamber. Be sure to wrap the plant pot in plastic wrap, covering all the soil and wrapping around the plant stem. This should leave only the aerial portions of the plant exposed to the circulating air. Head space gas analysis for volatile terpenes. To efficiently collect the volatile terpenes emitted from the aerial portions of a plant, the pot and soil portion of the plant are tightly wrapped in cellophane and placed in a gas tight, glass chamber like a desiccation chamber.

The tenax trapped compounds are subsequently eluted from the resin using appropriate organic solvents and concentrated as need prior to GC analysis. Ensure that a balance of air is pumped into and out of the chamber, because the small tubing used can create resistance.

Collect the volatile compounds by plumbing a glass Pasteur pipette packed with mg tenax resin the same as that filtering the input air entering the chamber connected in-line with the output gas line via simple connectors to allow for easy exchange of the traps. Exchange the traps every 1—4 hours, and elute the trapped terpenes with 2 or more washes of 0. It is common for terpenes to be modified by the addition of substituent groups that increases their polarity, for instance by the addition of hydroxyl groups or oxidation of a methyl group to the corresponding carboxylic acidic function.

However, it is important to remember that the number and type of modifications can increase or decrease the polarity of the final terpene product. Furthermore, while the addition of a small number of polar modifications may allow for analysis by GC, addition of a large number of such modification or a very polar group e. This protocol will use the extraction of a sesquiterpenoid, capsidiol, from a tobacco cell culture as a demonstration of how to extract a moderately polar terpenoid.

These compounds can be extracted from the cell culture medium and methanol extracts of cells using a chloroform partitioning method. Add 20 mL chloroform, secure glass stopper, invert funnel, open stopcock, and gently shake for 15 sec. Close stopcock, invert funnel, remove glass stopper and let stand for 1 to 2 min, or until the phases separate and sharp interface forms. Repeat extraction steps 3—5 and collect the extract into same flask as the first extraction.

The rotoevaporator temperature should be adjusted given the relative heat lability of your target terpene. Develop TLC plate using cyclohexane:acetone until solvent has moved 7 cm past application zone. Remove plate and dry. One of the advantages of using the vanillin indicator reagent is that it will produce a specific colored band depending on the terpene it interacts with, thus it adds a layer of specificity to determining the presence of your compound.

Other indicator dyes are possible as well. Analysis of terpenes conjugated to fatty acids or other acyl derivatives using an ester bond can be broken using saponification. These ester bonds leave a hydroxyl group present on the terpenoid backbone which can be silylated using a derivatization agent to allow for better volatility and analysis using GC. The protocol presented below demonstrates how to extract and analyze phytosterols. It is important to note that saponification is not required to analyze free sterols, as they will partition into the organic phase using methods such as those described in protocol 1.

Derivatization at the end can still be used even if saponification is not, as it will allow for better sensitivity in detection using GC. Saponification will allow extraction and analysis of the total phytosterol population of the analyzed tissue.

This protocol includes elements modified from that presented by Du and Ahn Kimble Chase, catalog number: A Use glass pipettes for transferring all liquids to prevent leaching of plasticizers into solution. Vigorously shake the mixture for minimally 30 seconds, then allow all the phases to separate.

Transfer the upper hexane phase to a clean vial. Repeat the extraction of the water phase with another 7. Dry sample under a stream of nitrogen. It is only important the derivatization agent be kept in excess, otherwise all of the compounds will not be silylated. In nature, many interesting terpenoids will be decorated with one or multiple polar groups or molecules, which increase significantly the size and polarity of the terpenoid molecule.

Extraction using a non-polar solvent i. Therefore, a more polar solvent should be used for extraction e. In this protocol, we will briefly describe a simplified method for extraction and analysis of artemisinin, which is one of the most important medicinal terpenoids. Lapkin et al.

These authors deduced that some emerging technologies e. Waters Alliance equipped with two pumps [pump 1: 1 mM ammonium acetate buffer, pH 5. Immerse one gram of leaf material in 6 mL of chloroform for 1 min.

The methanol:ammonium acetate solution can contain 0. This brief extraction is believed to be possible due to the fact that the majority of artemisinin in A. Note that tissue homogenization before extraction will probably be needed for most other extraction methods.

The initial composition of 1 mM ammonium acetate 1 mM, adjusted to pH 5. The column was allowed to equilibrate for 10 min between samples. Terpenoids comprise a large group of distinct natural metabolites, many of which were discovered in plants. Terpenes discovered and isolated from plants have been utilized widely in foods, cosmetics, pharmaceuticals and in various biotechnological applications. The ability to isolate and purify these valuable molecules from plants is key to elucidating their potential applications.

For example, the anti-cancer drug, paclitaxel, which was initially extracted from bark from the Pacific yew tree, continues to have efforts developed to improve its extraction efficiency from plant cell cultures Theodoridis et al.

This reiterates the points made in the introduction, that the more complex the target terpene becomes, the more complex the extraction procedure can be to optimize recovery, fully. In nature, a terpenoid may contain multiple cyclized structures and various types of groups e.

These decorations will increase significantly the polarity of the molecule. The polarity of the molecule is the most important feature to consider when determining how to purify the desired terpenoid. In addition, volatility and size are critical factors, which will dictate important aspects of the method as well as the type of analytical equipment which should be used.

The methods described in basic protocols 1 and 2 will be suitable for most non-polar terpenoids, whereas the methods described in basic protocol 3 and alternative protocol 2 may be used for polar terpenoids.

Again, Figure 1 may be used as a general guide—you can see which terpene most closely matches with your anticipated structure, and the shaded region in which it falls should serve as a general guide as to which protocol can be usedfor extraction: molecules best extracted and analyzed using a protocol similar to those presented in 1, 2, or 3 are highlighted in green, red, or blue, respectively.