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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. Using a 3 mL Luer lock syringe with 0.22 µm filter, 3 mL of extract was filtered. For DI-SPME Arrow experiments, 1 mL of the filtered extract was added to 19 mL of LCMS grade water (i.e., 20 mL final volume) in a 20 mL headspace vial. In addition, 20 µL of 100 μg/mL internal standard (ISTD) solution was added. Subsequently, the headspace vial was capped and spun for 10 seconds. For LI syringe experiments, 500 µL of the filtered extract was added to a 2 mL autosampler vial. In addition, 5 µL of the 10 μg/mL ISTD solution was added. Subsequently, the autosampler vial was capped and spun for 10 seconds.
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. Using a 3 mL Luer lock syringe with 0.22 µm filter, 3 mL of extract was filtered. For DI-SPME Arrow experiments, 1 mL of the filtered extract was added to 19 mL of LCMS grade water (i.e., 20 mL final volume) in a 20 mL headspace vial. In addition, 20 µL of 100 μg/mL internal standard (ISTD) solution was added. Subsequently, the headspace vial was capped and spun for 10 seconds. For LI syringe experiments, 500 µL of the filtered extract was added to a 2 mL autosampler vial. In addition, 5 µL of the 10 μg/mL ISTD solution was added. Subsequently, the autosampler vial was capped and spun for 10 seconds.


====Terpenes standards and internal standards====
Differences in linear range between DI-SPME Arrow and LI syringe necessitated the use of the different intermediate and ISTD solutions. Intermediate concentrations of 1000 μg/mL and a 10 μg/mL were prepared from the 2,500 μg/mL Terpene Standards 1 and 2. To prepare the 1000 μg/mL intermediate, 400 µL of each terpene standard (i.e., 800 µL total) was added to 200 µL of IPA, then capped and vortexed. The 10 μg/mL intermediate was prepared from the 1000 μg/mL intermediate by adding 10 µL of the 1000 μg/mL intermediate to 990 µL of IPA, then capped and vortexed. A solution of naphthalene-d8 ISTD was made at 100 μg/mL for DI-SPME Arrow experiments and 10 μg/mL for LI syringe experiments.


====DI-SPME Arrow calibration====
For the highest DI-SPME Arrow calibration point (level 7), 153.6 µL of 1000 μg/mL terpene solution was added to hops. Once extracted, the extract was brought to 12 mL with IPA and filtered, thereby reducing calibration level 7–12.8 μg/mL. Intermediate serial dilutions (Supplementary Table S5) were carried out on calibration level 7 to make the other six calibration points. For example, 1500 µL of calibration level 7 was added to 1500 µL of IPA to make calibration level 6. This process was then repeated for the other calibration points. However, the final calibration solutions required a secondary dilution into 20 mL headspace vials (Supplementary Table S6). For example, 1 mL of the calibration level 6 (i.e., 6.4 μg/mL) was added to 19 mL of water (i.e., 20 mL total volume) for a final concentration of 0.32 μg/mL and then spiked with ISTD. For a DI-SPME Arrow CCV equivalent to calibration level 3, calibration 7 filtered extract was diluted with IPA, spiked with ISTD, capped, and vortexed for 10 seconds.
====LI syringe calibration====
For the highest LI syringe calibration point (level 10), 61.4 µL of the 1000 μg/mL terpene solution was added to the hops. Once extracted, the extract was brought to 12 mL with IPA and filtered, thereby reducing calibration level 10 to 5.12 μg/mL. Serial dilutions (Supplementary Table S7) were carried out on calibration level 10 to make the other nine calibration points. For example, 500 µL of calibration level 10 was added to 500 µL of IPA to make calibration level 9. This process was then repeated for the other calibration points. Finally, 5 µL of the 10 μg/mL ISTD solution was added to each calibration vial at levels 2–10, and 10 µL of the 10 μg/mL ISTD solution was added to level 1 given the difference in final volume. After being spiked with ISTD, the vial was capped and spun for 10 seconds. See Supplementary Table S7 for the LI syringe calibration curve.
====Method validation and chemovar experiments====
This section addresses the following method validations: method detection limit (MDL)/limit of quantitation (LOQ), analytical precision, method precision, and % recovery. The DI-SPME Arrow and LI syringe MDLs/LOQs were determined from seven replicate low-level calibration points. In addition, LCSs were run to determine the analytical precision and % recovery of both methods. For a DI-SPME Arrow LCS, 76.8 µL of the 1000 μg/mL intermediate terpene solution was added to hops (equivalent to calibration level 6). For an LI syringe LCS, 384 µL of 10 μg/mL intermediate terpene solution was added to the hops (equivalent to calibration level 6). It is important to note that the LCS represents a separate hops spike and extraction. Furthermore, the DI-SPME Arrow and LI syringe method precisions were determined from seven different aliquots of cannabis shake. Finally, three different chemovars of cannabis flower were evaluated for terpenes with DI-SPME Arrow and LI syringe.
==Results and discussion==
===HS syringe vs. HS-SPME Arrow vs. DI-SPME Arrow===
Initial work compared three different types of sample preparation/introduction techniques for terpene analysis via GC-MS. Techniques were evaluated based on relative compound response using only reference terpene standards. First, the more traditional approach using a HS syringe was compared to HS-SPME Arrow (120 µm DVB/PDMS). As shown in Figure 1, 13 of 23 terpenes were identified using the HS syringe approach. However, this approach was unable to effectively pick up the later eluting and less volatile terpenes, which fall into the sesquiterpene category. When samples were analyzed via HS-SPME Arrow, 23 of 23 terpenes were able to be identified.
[[File:Fig1Act Myers FrontChem2021 9.jpg|782px]]
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{| border="0" cellpadding="5" cellspacing="0" width="782px"
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  | style="background-color:white; padding-left:10px; padding-right:10px;"| <blockquote>'''Figure 1.''' Analytical parameters for evaluating terpenes in cannabis with DI-SPME Arrow and LI-Syringe</blockquote>
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When comparing responses for the 13 terpenes that were able to be identified in both approaches, HS-SPME Arrow had much greater responses than the HS syringe approach. For the terpenes found in both HS techniques, the responses on the SPME Arrow were >10× that of the HS syringe. Both samples were prepared identically and analyzed with the suggested parameters for each technique. When first looking at the HS syringe results, it was unclear if the less volatile sesquiterpenes were partitioning into the HS of the 20 mL vial. However, after analyzing the results for the HS-SPME Arrow and detecting the less volatile compounds, it was confirmed that these compounds are partitioning into the HS of the vial. It is not clear as to where the terpenes were lost (i.e., not transferred efficiently) in the HS syringe process, and it was outside of the scope of this study to determine the root cause. 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. However, it was desired to see how HS-SPME Arrow compared to DI-SPME Arrow.
HS-SPME Arrow samples and DI-SPME Arrow samples were prepared according to their respective approach, but they were analyzed under the same instrument conditions. Both techniques were able to identify all terpenes within the reference standard samples. However, as shown in Figure 2, terpene samples analyzed via DI-SPME Arrow showed improved responses over HS-SPME Arrow, especially for the higher molecular weight terpenes and also proved to be more reproducible (i.e., provide better precision). Responses for the DI-SPME Arrow averaged 6× greater than that of the HS-SPME Arrow. %RSDs for the HS-SPME Arrow were as high as 76%, while all DI-SPME Arrow %RSDs were ≤15%. Potential limitations of DI-SPME Arrow include shortened fiber lifetime and/or increased matrix exposure; however, given the improved responses and reproducibility, it was selected as the technique to move forward for further method validation and compared against a traditional liquid injection (LI) syringe method.
[[File:Fig2 Myers FrontChem2021 9.jpg|782px]]
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{| border="0" cellpadding="5" cellspacing="0" width="782px"
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  | style="background-color:white; padding-left:10px; padding-right:10px;"| <blockquote>'''Figure 2.''' HS-SPME Arrow vs DI-SPME Arrow for terpenes</blockquote>
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===Hand shakeout vs. accelerated solvent extractor===
Several terpene extraction approaches were considered for the current study. The full evaporative technique (FET), which is popular within the cannabis testing industry, was not evaluated in the current study as this technique’s foundation is HS syringe. Additionally, the results discussed in HS syringe vs. HS-SPME Arrow vs. DI-SPME Arrow demonstrated that HS syringe did not perform as well as HS-SPME Arrow, which was also inferior to DI-SPME Arrow for the analysis of terpenes. Other industries already capitalize on the benefits of ASE 350<ref name="LigorComp14" /><ref name="ChiesaAccel17" /><ref name="HuSimult20" /><ref name="NingAccel20" /> Therefore, a simple hand shakeout solvent extraction method was compared to an ASE 350 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 350 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 350 approaches. With the exception of [[camphene]] and [[linalool]] (''p'' = 0.050), the Kruskal–Wallis tests indicate a general trend of no statistically significant difference between the hand shakeout and ASE 350 extraction techniques for the 13 detected terpenes.
[[File:Tab4 Myers FrontChem2021 9.jpg|1145px]]
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  | style="background-color:white; padding-left:10px; padding-right:10px;"| <blockquote>'''Table 4.''' Hand shakeout vs. accelerated solvent extraction (ASE 350) for extraction of terpenes from cannabis flower</blockquote>
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==References==
==References==

Revision as of 23:58, 26 April 2021

Full article title Accelerated solvent extraction of terpenes in cannabis coupled with various injection techniques for GC-MS analysis
Journal Frontiers in Chemistry
Author(s) Myers, Colton; Herrington, Jason S.; Hamrah, Paul; Anderson, Kelsey
Author affiliation(s) Restek Corporation, Verity Analytics
Primary contact colton dot myers at restek dot com
Year published 2021
Volume and issue 9
Article # 619770
DOI 10.3389/fchem.2021.619770
ISSN 2296-2646
Distribution license Creative Commons Attribution 4.0 International
Website https://www.frontiersin.org/articles/10.3389/fchem.2021.619770/full
Download https://www.frontiersin.org/articles/10.3389/fchem.2021.619770/pdf (PDF)
Emblem-important-yellow.svg This article should be considered a work in progress and incomplete. Consider this article incomplete until this notice is removed.

Abstract

The cannabis market is expanding exponentially in the United States. As state-wide legalization efforts increase, so also do demands for analytical testing methodologies. One of the main tests conducted on cannabis products is the analysis for terpenes. This research focused on implementation of accelerated solvent extraction (ASE), utilizing surrogate matrix matching, and evaluation of traditional vs. more modern sample introduction techniques for analyzing terpenes via gas chromatography–mass spectrometry (GC-MS). Introduction techniques included headspace syringe (HS syringe), HS-solid-phase microextraction Arrow (HS-SPME Arrow), direct immersion-SPME Arrow (DI-SPME Arrow), and liquid injection syringe (LI syringe). The LI syringe approach was deemed the most straightforward and robust method, with terpene working ranges of 0.04–5.12 μg/mL; r2 values of 0.988–0.996 (0.993 average); limit of quantitation values of 0.017–0.129 μg/mL (0.047 average); analytical precisions of 2.58–9.64% RSD (1.56 average); overall ASE-LI-syringe-GC-MS method precisions of 1.73–14.6% RSD (4.97 average); and % recoveries of 84.6–98.9% (90.2 average) for the 23 terpenes of interest. Sample workflows and results are discussed, with an evaluation of the advantages/limitations of each approach and opportunities for future work.

Keywords: accelerated solvent extraction (ASE), terpenes, solid-phase microextraction (SPME), solid-phase microextraction Arrow (SPME Arrow), gas chromatography–mass spectrometry (GC-MS)

Introduction

The legal cannabis market is one of the fastest growing markets across the globe. In 2019, cannabis use for medicinal purposes in the United States generated $4 billion to $4.9 billion in sales, compared to the adult-use estimates between $6.6 billion and $8.1 billion.[1] As the United States and additional countries continue to legalize the use of medicinal and recreational cannabis, analytical testing demands increase. A 2020 report by Market Data Forecast valued the global cannabis testing market at $1,218.0 million in 2019 and estimated it to be growing at a compound annual growth rate (CAGR) of 12.42%.[2] The market is projected to almost double at $2,187.3 million by 2024.[2] Of the examinations conducted in cannabis testing laboratories, terpene profiling is a popular analysis, regardless of state regulations.

Terpenes are a naturally occurring set of organic compounds, which are commonly found in plants, and are typically strong in odor.[3] Terpenes are made up of isoprene units and are classified by the number of their isoprene units.[3] The two types of terpenes that are commonly analyzed in the cannabis testing industry are monoterpenes, which have two isoprene units, and sesquiterpenes, which have three isoprene units. Over 100 terpenes have been identified in different cannabis chemical varieties (chemovars).[4] Each cannabis chemovar has its own unique terpene profile, giving consumers different aromas, flavors, and experiences depending on the chemovar they use. According to Russo et al., terpenes play a major role in the entourage effect, which is the synergistic interaction between phytocannabinoids and terpenoids with respect to treating numerous ailments (e.g., depression).[5] The desire to understand and capitalize on this entourage effect is the motivation for terpene testing in the cannabis industry.

Terpenes have been analyzed in numerous commodities within the food and beverage industry. Previous studies have looked at a variety of matrices (e.g., tequila) and have used different analytical techniques (e.g., solid-phase microextraction [SPME]) to conduct the analysis.[4][6][7][8][9][10][11][12][13][14][15][16][17][18][19] However, only a few studies have shown the analysis of terpenes in cannabis and hemp matrices (e.g., flower, gummy), and their robustness for compliance laboratories remains uncertain. Calvi et al., Ternelli et al., Gaggotti et al., and Stenerson et al. did not perform extractions on cannabis and hemp samples; rather, they added the samples directly to a headspace (HS) vial and demonstrated the analysis of terpenes using HS-SPME.[4][10][15][18] Nguyen et al. utilized a pseudo extraction by adding a solvent to dried material, followed by analysis via headspace gas chromatography–mass spectrometry (HS-GC-MS).[17] The five aforementioned studies appear to lack an exhaustive cannabis or hemp extraction, and therefore this calls into question the real-world applicability of these methods. Furthermore, Calvi et al., Ternelli et al., Gaggotti et al., and Stenerson et al. only focused on profiling the terpenes in the cannabis or hemp matrices studied and therefore only presented qualitative and semi-quantitative data.[4][10][15][18]

Bakro et al., Brown et al., Ibrahim et al., and Shapira et al. extracted cannabis flower with ethanol, hexane, ethyl acetate, and methanol, respectively, and provided quantitative results.[11][12][13][14] However, Bakro et al. only looked at hemp and used a nonspecific gas chromatography with flame-ionization detection (GC-FID) approach, which is cumbersome when attempting to differentiate between coeluting terpenes of interest and matrix interferences.[14] Brown et al. did not provide method accuracies for all targeted terpenes and reported less than desirable linearities, which fell below an r2 value of 0.960 for each terpene of interest.[11]

To date, the most promising methods—presented by Ibrahim et al. and Shapira et al.—utilize exhaustive cannabis and hemp extraction approaches, followed by GC-MS and reported desirable quantitative results.[12][13] Ibrahim et al. and Shapira et al. used sample introduction techniques like liquid injection without filtration and static headspace GC-MS (SHS-GC-MS), respectively. More importantly, none of the aforementioned studies accounted for matrix effects, as they all used solvent-based calibrations and, due to the complexity and dirtiness of cannabis matrices, this could lead to inaccurate reporting.[20] In addition, these studies did not evaluate more modern sample extraction approaches [e.g., accelerated solvent extraction (ASE)] or sample introduction techniques (e.g., direct immersion-SPME Arrow [DI-SPME Arrow]).

The following study was conducted to evaluate more modern sample preparation and introduction techniques and demonstrate their potential value to cannabis compliance testing laboratories in need of guidance for qualitative and quantitative terpenes analysis. In addition, this study evaluated accelerated solvent extraction (ASE 350) of terpenes in cannabis samples, which is commonly used in other markets within the analytical testing industry.[21][22][23][24] Furthermore, to avoid potentially inaccurate reporting, matrix-matched standards were used for calibration. Finally, the more traditional headspace syringe (HS syringe) and liquid injection syringe (LI syringe) approaches were compared to the more modern HS-solid-phase microextraction Arrow (HS-SPME Arrow) and DI-SPME Arrow, which have recently demonstrated enhanced robustness and improved sensitivity over traditional SPME fibers.[25]

Experimental

The following experimental sections describe the detailed procedures utilized during the three main parts of this manuscript:

1. HS syringe vs. HS-SPME Arrow vs. DI-SPME Arrow for the determination of the preferred sample introduction approach with the use of terpenes in solution; 2. Terpene extraction evaluation for the evaluation of an ideal terpene extraction method for cannabis flower; and 3. Combining the information gathered from the prior two procedures for a final comparison with an existing validated LI syringe technique (i.e., validated with the California Bureau of Cannabis Control [BCC]), outlined in DI-SPME Arrow vs. LI syringe.

Materials and reagents

Hop pellets were obtained from the Beverage Factory (San Diego, CA, United States) and stored at −10 °C for one hour. Dried cannabis material was obtained from Cream of the Crop (San Diego, CA, United States) and stored at −10 °C for one hour. Cannabis Terpene Standards #1 and #2 (cat# 34095 and 34096) were purchased from Restek Corporation (Bellefonte, PA, United States). Napthalene-d8 was purchased from Restek Corporation (Bellefonte, PA, United States). Isopropanol (IPA) was purchased from Filtrous (LCMS Grade) and 1.1 mm, 120 µm DVB/PDMS SPME Arrows were purchased from Restek Corporation (Bellefonte, PA, United States).

HS syringe vs. HS-SPME Arrow vs. DI-SPME Arrow

The following experiments were conducted to evaluate the differences between HS syringe, HS-SPME Arrow, and DI-SPME Arrow as sample introduction techniques. Each technique was evaluated using Cannabis Terpene Standard #1 and #2 (cat# 34095 and 34096) from Restek Corporation (Bellefonte, PA, United States). Samples were evaluated using the same GC-MS conditions shown in Supplementary Table S1. For the HS syringe and HS-SPME Arrow samples, a standard stock solution was made by diluting both standards into one solution for a final concentration of 5 μg/mL in IPA. Samples were prepared by adding 1.5 g of NaCl to a 20 mL HS vial, followed by 1 mL of 5 μg/mL stock solution and 4 mL of water for a final concentration of 1 μg/mL (see sampling conditions in Supplementary Tables S2, S3). Previous work (results not shown) demonstrated that HS-SPME Arrow analyte responses were higher and more reproducible when using an incubation temperature of 40 °C or less, hence having lower incubation temperature compared to the HS syringe method. The DI-SPME Arrow samples were prepared by diluting both standards into one stock solution for a final concentration of 20 μg/mL. To a 20 mL HS vial, 1 mL of the 20 μg/mL stock solution was added, followed by 19 mL of water for a final concentration of 1 μg/mL (same concentration for HS syringe and HS-SPME Arrow; see sampling conditions in Supplementary Table S3). Each technique was run in triplicate for the initial evaluation of sample introduction techniques.

Terpene extraction evaluation

An evaluation of the terpene extraction processes was conducted to understand the advantages and limitations of certain techniques. Extractions using the Dionex Accelerated Solvent Extractor (ASE 350) were compared to a hand-solvent extraction for terpene analysis. Three chemical varieties (chemovars) were used to evaluate both extraction techniques. Cannabis flower was frozen at −10 °C for one hour, then homogenized on a sheet pan with a rolling pin. For ASE 350 extractions, 0.5 g of homogenized cannabis flower was weighed and added to a 10 mL stainless steel ASE 350 cell, and the remaining cell volume was lightly packed with diatomaceous earth. The cell was then extracted using the parameters in Table 1. The extract was then diluted to 12 mL in a graduated cylinder due to both convenience and the fact that this approach achieved the desired data quality objectives of this study (e.g., method precision RSDs <15%). However, future researchers may consider the use of volumetric flasks to achieve better precision. One mL of the cannabis flower extract was added to a 2.5 mL autosampler vial and then analyzed. For hand extractions, 0.5 g of homogenized cannabis flower was weighed and added to a 50 mL centrifuge tube, followed by 12 mL of IPA. Samples were vortexed for three minutes and sonicated at 40 °C for five minutes. Samples were then centrifuged in a Sorvall RT7 Plus centrifuge for three minutes. One mL of the supernatant was added to a 2.5 mL autosampler vial and then analyzed. ASE 350 and hand extractions were analyzed via GC-FID.


Fig1 Myers FrontChem2021 9.jpg

Table 1. Accelerated solvent extractor (ASE) parameters for extracting terpenes from hops pellets and cannabis flower

DI-SPME Arrow vs. LI syringe

Results from the experiments outlined in the prior two steps indicated DI-SPME Arrow was the preferred sample introduction approach, and ASE was the ideal terpene extraction technique for cannabis. This information was then utilized for a comparison to an existing validated LI syringe method. However, the experiments conducted in HS syringe vs. HS-SPME Arrow vs. DI-SPME Arrow were carried out in Pennsylvania, and the use of cannabis flower necessitated a fully licensed laboratory, which was located in California. The DI-SPME Arrow parameters, outlined in Table 2, had been further optimized for terpenes analysis in cannabis and used in the California laboratory; however, they are only slightly different from the initial DI-SPME Arrow parameters, as outlined in Supplementary Table S3. In addition, a gas chromatography–tandem mass spectrometry (GC-MS/MS) method was used in single quad MS mode in the California laboratory, since single quad MS is more common for this analysis (see parameters in Table 3). Furthermore, a selected ion monitoring (SIM) method (Supplementary Table S4) was utilized to help eliminate background noise and provide better sensitivity. LI syringe was only evaluated in California using the parameters listed in Table 3.


Tab2 Myers FrontChem2021 9.jpg

Table 2. Optimized and final DI-SPME Arrow parameters

Tab3 Myers FrontChem2021 9.jpg

Table 3. Analytical parameters for evaluating terpenes in cannabis with DI-SPME Arrow and LI-Syringe.

Hops pellets and cannabis flower preparation

Hops pellets were utilized as a terpene-free surrogate to matrix match cannabis flower for the following DI-SPME Arrow vs. LI syringe data: calibration curves, laboratory control samples (LCS), continuing calibration verification (CCV) samples, detection limit, and analytical precision samples. Hops were crushed and homogenized on a sheet pan with a rolling pin. The crushed hops were then cleaned with a proprietary solvent cleaning process to eliminate the presence of terpenes. Following solvent cleaning, the hops were dried in an oven. For the DI-SPME Arrow and LI syringe method precision experiments, cannabis shake (small pieces of cannabis flower that break off of larger buds) was homogenized and utilized. For the cannabis chemovar experiments, the flower was crushed and homogenized on a sheet pan using a rolling pin.

Accelerated solvent extractor

The following DI-SPME Arrow vs. LI syringe experiments were conducted utilizing hops pellets and cannabis flowers, which were extracted using an ASE 350 with the parameters previously shown in Table 1. For all of the following DI-SPME Arrow and LI syringe experiments, either 0.5 g of cleaned hops or 0.5 g of cannabis flower was weighed out and placed into a 10 mL ASE 350 stainless steel extraction cell. Diatomaceous earth was then slowly added and lightly packed to fill the remaining volume in the cell. Samples were then extracted using IPA. Other work has shown that extracting with IPA can lead to poor peak shape for the terpenes of interest.[26] However, IPA gave desirable peak shape for this study and was used because of its cost, convenience, and toxicity relative to other solvents demonstrated for cannabis extractions.

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. Using a 3 mL Luer lock syringe with 0.22 µm filter, 3 mL of extract was filtered. For DI-SPME Arrow experiments, 1 mL of the filtered extract was added to 19 mL of LCMS grade water (i.e., 20 mL final volume) in a 20 mL headspace vial. In addition, 20 µL of 100 μg/mL internal standard (ISTD) solution was added. Subsequently, the headspace vial was capped and spun for 10 seconds. For LI syringe experiments, 500 µL of the filtered extract was added to a 2 mL autosampler vial. In addition, 5 µL of the 10 μg/mL ISTD solution was added. Subsequently, the autosampler vial was capped and spun for 10 seconds.

Terpenes standards and internal standards

Differences in linear range between DI-SPME Arrow and LI syringe necessitated the use of the different intermediate and ISTD solutions. Intermediate concentrations of 1000 μg/mL and a 10 μg/mL were prepared from the 2,500 μg/mL Terpene Standards 1 and 2. To prepare the 1000 μg/mL intermediate, 400 µL of each terpene standard (i.e., 800 µL total) was added to 200 µL of IPA, then capped and vortexed. The 10 μg/mL intermediate was prepared from the 1000 μg/mL intermediate by adding 10 µL of the 1000 μg/mL intermediate to 990 µL of IPA, then capped and vortexed. A solution of naphthalene-d8 ISTD was made at 100 μg/mL for DI-SPME Arrow experiments and 10 μg/mL for LI syringe experiments.

DI-SPME Arrow calibration

For the highest DI-SPME Arrow calibration point (level 7), 153.6 µL of 1000 μg/mL terpene solution was added to hops. Once extracted, the extract was brought to 12 mL with IPA and filtered, thereby reducing calibration level 7–12.8 μg/mL. Intermediate serial dilutions (Supplementary Table S5) were carried out on calibration level 7 to make the other six calibration points. For example, 1500 µL of calibration level 7 was added to 1500 µL of IPA to make calibration level 6. This process was then repeated for the other calibration points. However, the final calibration solutions required a secondary dilution into 20 mL headspace vials (Supplementary Table S6). For example, 1 mL of the calibration level 6 (i.e., 6.4 μg/mL) was added to 19 mL of water (i.e., 20 mL total volume) for a final concentration of 0.32 μg/mL and then spiked with ISTD. For a DI-SPME Arrow CCV equivalent to calibration level 3, calibration 7 filtered extract was diluted with IPA, spiked with ISTD, capped, and vortexed for 10 seconds.

LI syringe calibration

For the highest LI syringe calibration point (level 10), 61.4 µL of the 1000 μg/mL terpene solution was added to the hops. Once extracted, the extract was brought to 12 mL with IPA and filtered, thereby reducing calibration level 10 to 5.12 μg/mL. Serial dilutions (Supplementary Table S7) were carried out on calibration level 10 to make the other nine calibration points. For example, 500 µL of calibration level 10 was added to 500 µL of IPA to make calibration level 9. This process was then repeated for the other calibration points. Finally, 5 µL of the 10 μg/mL ISTD solution was added to each calibration vial at levels 2–10, and 10 µL of the 10 μg/mL ISTD solution was added to level 1 given the difference in final volume. After being spiked with ISTD, the vial was capped and spun for 10 seconds. See Supplementary Table S7 for the LI syringe calibration curve.

Method validation and chemovar experiments

This section addresses the following method validations: method detection limit (MDL)/limit of quantitation (LOQ), analytical precision, method precision, and % recovery. The DI-SPME Arrow and LI syringe MDLs/LOQs were determined from seven replicate low-level calibration points. In addition, LCSs were run to determine the analytical precision and % recovery of both methods. For a DI-SPME Arrow LCS, 76.8 µL of the 1000 μg/mL intermediate terpene solution was added to hops (equivalent to calibration level 6). For an LI syringe LCS, 384 µL of 10 μg/mL intermediate terpene solution was added to the hops (equivalent to calibration level 6). It is important to note that the LCS represents a separate hops spike and extraction. Furthermore, the DI-SPME Arrow and LI syringe method precisions were determined from seven different aliquots of cannabis shake. Finally, three different chemovars of cannabis flower were evaluated for terpenes with DI-SPME Arrow and LI syringe.

Results and discussion

HS syringe vs. HS-SPME Arrow vs. DI-SPME Arrow

Initial work compared three different types of sample preparation/introduction techniques for terpene analysis via GC-MS. Techniques were evaluated based on relative compound response using only reference terpene standards. First, the more traditional approach using a HS syringe was compared to HS-SPME Arrow (120 µm DVB/PDMS). As shown in Figure 1, 13 of 23 terpenes were identified using the HS syringe approach. However, this approach was unable to effectively pick up the later eluting and less volatile terpenes, which fall into the sesquiterpene category. When samples were analyzed via HS-SPME Arrow, 23 of 23 terpenes were able to be identified.


Fig1Act Myers FrontChem2021 9.jpg

Figure 1. Analytical parameters for evaluating terpenes in cannabis with DI-SPME Arrow and LI-Syringe

When comparing responses for the 13 terpenes that were able to be identified in both approaches, HS-SPME Arrow had much greater responses than the HS syringe approach. For the terpenes found in both HS techniques, the responses on the SPME Arrow were >10× that of the HS syringe. Both samples were prepared identically and analyzed with the suggested parameters for each technique. When first looking at the HS syringe results, it was unclear if the less volatile sesquiterpenes were partitioning into the HS of the 20 mL vial. However, after analyzing the results for the HS-SPME Arrow and detecting the less volatile compounds, it was confirmed that these compounds are partitioning into the HS of the vial. It is not clear as to where the terpenes were lost (i.e., not transferred efficiently) in the HS syringe process, and it was outside of the scope of this study to determine the root cause. 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. However, it was desired to see how HS-SPME Arrow compared to DI-SPME Arrow.

HS-SPME Arrow samples and DI-SPME Arrow samples were prepared according to their respective approach, but they were analyzed under the same instrument conditions. Both techniques were able to identify all terpenes within the reference standard samples. However, as shown in Figure 2, terpene samples analyzed via DI-SPME Arrow showed improved responses over HS-SPME Arrow, especially for the higher molecular weight terpenes and also proved to be more reproducible (i.e., provide better precision). Responses for the DI-SPME Arrow averaged 6× greater than that of the HS-SPME Arrow. %RSDs for the HS-SPME Arrow were as high as 76%, while all DI-SPME Arrow %RSDs were ≤15%. Potential limitations of DI-SPME Arrow include shortened fiber lifetime and/or increased matrix exposure; however, given the improved responses and reproducibility, it was selected as the technique to move forward for further method validation and compared against a traditional liquid injection (LI) syringe method.


Fig2 Myers FrontChem2021 9.jpg

Figure 2. HS-SPME Arrow vs DI-SPME Arrow for terpenes

Hand shakeout vs. accelerated solvent extractor

Several terpene extraction approaches were considered for the current study. The full evaporative technique (FET), which is popular within the cannabis testing industry, was not evaluated in the current study as this technique’s foundation is HS syringe. Additionally, the results discussed in HS syringe vs. HS-SPME Arrow vs. DI-SPME Arrow demonstrated that HS syringe did not perform as well as HS-SPME Arrow, which was also inferior to DI-SPME Arrow for the analysis of terpenes. Other industries already capitalize on the benefits of ASE 350[21][22][23][24] Therefore, a simple hand shakeout solvent extraction method was compared to an ASE 350 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 350 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 350 approaches. With the exception of camphene and linalool (p = 0.050), the Kruskal–Wallis tests indicate a general trend of no statistically significant difference between the hand shakeout and ASE 350 extraction techniques for the 13 detected terpenes.


Tab4 Myers FrontChem2021 9.jpg

Table 4. Hand shakeout vs. accelerated solvent extraction (ASE 350) for extraction of terpenes from cannabis flower

References

  1. ↑ Stelton-Holtmeier, J. (25 August 2020). "Chart: Nationwide sales of adult-use cannabis further eclipse those of medical marijuana". Marijuana Business Daily. https://mjbizdaily.com/chart-nationwide-sales-of-adult-use-cannabis-further-eclipse-those-of-medical-marijuana/. Retrieved 31 August 2020. 
  2. ↑ 2.0 2.1 "Chart: Nationwide sales of adult-use cannabis further eclipse those of medical marijuana". February 2020. https://www.marketdataforecast.com/market-reports/cannabis-testing-market. Retrieved 31 August 2020. 
  3. ↑ 3.0 3.1 Nuutinen, T. (2018). "Medicinal properties of terpenes found in Cannabis sativa and Humulus lupulus". European Journal of Medicinal Chemistry 157: 198–228. doi:10.1016/j.ejmech.2018.07.076. PMID 30096653. 
  4. ↑ 4.0 4.1 4.2 4.3 Calvi, L.; Pentimalli, D.; Panseri, S. et al. (2018). "Comprehensive quality evaluation of medical Cannabis sativa L. inflorescence and macerated oils based on HS-SPME coupled to GC–MS and LC-HRMS (q-exactive orbitrap) approach". Journal of Pharmaceutical and Biomedical Analysis 150: 208–19. doi:10.1016/j.jpba.2017.11.073. 
  5. ↑ Russo, E.B. (2011). "Taming THC: Potential cannabis synergy and phytocannabinoid-terpenoid entourage effects". British Journal of Pharmacology 163 (7): 1344-64. doi:10.1111/j.1476-5381.2011.01238.x. PMC PMC3165946. PMID 21749363. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=PMC3165946. 
  6. ↑ Abilleira, E.; de Renobales, M.; Nájera, A.I. et al. (2010). "An accurate quantitative method for the analysis of terpenes in milk fat by headspace solid-phase microextraction coupled to gas chromatography–mass spectrometry". Food Chemistry 120 (4): 1162-1169. doi:10.1016/j.foodchem.2009.11.050. 
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  8. ↑ Cacho, J.I.; Campillo, N.; Viñas, P. et al. (2015). "Evaluation of three headspace sorptive extraction coatings for the determination of volatile terpenes in honey using gas chromatography-mass spectrometry". Journal of Chromatography A 1399: 18–24. doi:10.1016/j.chroma.2015.04.041. PMID 25958092. 
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  11. ↑ 11.0 11.1 11.2 Brown, A.K.; Xia, Z.; Bulloch, P. et al. (2019). "Validated quantitative cannabis profiling for Canadian regulatory compliance - Cannabinoids, aflatoxins, and terpenes". Analytica Chimica Acta 1088: 79–88. doi:10.1016/j.aca.2019.08.042. PMID 31623719. 
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  13. ↑ 13.0 13.1 13.2 Shapira, A.; Berman, P. Futoran, K. et al. (2019). "Tandem Mass Spectrometric Quantification of 93 Terpenoids in Cannabis Using Static Headspace Injections". Analytical Chemistry 91 (17): 11425–32. doi:10.1021/acs.analchem.9b02844. PMID 31369251. 
  14. ↑ 14.0 14.1 14.2 Bakro, F.; Jedryczka, M.; Wielgusz, K. et al. (2020). "Simultaneous determination of terpenes and cannabidiol in hemp (Cannabis sativa L.) by fast gas chromatography with flame ionization detection". Journal of Separation Science 43 (14): 2817–26. doi:10.1002/jssc.201900822. 
  15. ↑ 15.0 15.1 15.2 Gaggiotti, S.; Palmieri, S.; Pelle, F.D. et al. (2020). "Piezoelectric peptide-hpDNA based electronic nose for the detection of terpenes; Evaluation of the aroma profile in different Cannabis sativa L. (hemp) samples". Sensors and Actuators B: Chemical 308: 127697. doi:10.1016/j.snb.2020.127697. 
  16. ↑ {{cite journal |title=Multivariate optimization of headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry for the analysis of terpenoids in sparkling wines |journal=Talanta |author=Muñoz-Redondo, J.M.; Ruiz-Moreno, M.J.; Puertas, B. et al. |volume=208 |at=120483 |year=2020 |doi=110.1016/j.talanta.2019.120483}
  17. ↑ 17.0 17.1 Nguyen, T.-D.; Riordan-Short, S.; Dang, T.-T.T. et al. (2020). "Quantitation of Select Terpenes/Terpenoids and Nicotine Using Gas Chromatography-Mass Spectrometry with High-Temperature Headspace Sampling". ACS Omega 5 (10): 5565-5573. doi:10.1021/acsomega.0c00384. PMC PMC7081649. PMID 32201850. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=PMC7081649. 
  18. ↑ 18.0 18.1 18.2 Ternelli, M.; Brighenti, V.; Anceschi, L. et al. (2020). "Innovative methods for the preparation of medical Cannabis oils with a high content of both cannabinoids and terpenes". Journal of Pharmaceutical and Biomedical Analysis 186: 113296. doi:10.1016/j.jpba.2020.113296. PMID 32334134. 
  19. ↑ Zhang, X.; Wang, C.; Wang, L. et al. (2020). "Optimization and validation of a head space solid-phase microextraction-arrow gas chromatography-mass spectrometry method using central composite design for determination of aroma compounds in Chinese liquor (Baijiu)". Journal of Chromatography A 1610: 460584. doi:10.1016/j.chroma.2019.460584. PMID 31607446. 
  20. ↑ Raposo, F.; Barceló, D. (2021). "Challenges and strategies of matrix effects using chromatography-mass spectrometry: An overview from research versus regulatory viewpoints". TrAC Trends in Analytical Chemistry 134: 116068. doi:10.1016/j.trac.2020.116068. 
  21. ↑ 21.0 21.1 Ligor, M.; Stankevičius, M.; Wenda-Piesik, A. et al. (2014). "Comparative Gas Chromatographic–Mass Spectrometric Evaluation of Hop (Humulus lupulus L.) Essential Oils and Extracts Obtained Using Different Sample Preparation Methods". Food Analytical Methods 7: 1433–42. doi:10.1007/s12161-013-9767-5. 
  22. ↑ 22.0 22.1 Chiesa, L.M.; Labella, G.F.; Panseri, S. et al. (2017). "Accelerated solvent extraction by using an 'in-line' clean-up approach for multiresidue analysis of pesticides in organic honey". Food Additives & Contaminants: Part A 34 (5): 809–18. doi:10.1080/19440049.2017.1292558. PMID 28277176. 
  23. ↑ 23.0 23.1 Hu, A.; Qiu, M.; Liu, H. et al. (2020). "Simultaneous determination of phthalate diesters and monoesters in soil using accelerated solvent extraction and ultra-performance liquid chromatography coupled with tandem mass spectrometry". Journal of Chromatography A 1626: 104366. doi:10.1016/j.microc.2019.104366. 
  24. ↑ 24.0 24.1 Min, N.; Yao, J.; Amde, M. et al. (2020). "Accelerated solvent extraction combined with GC–MS: A convenient technique for the determination and compound-specific stable isotope analysis of phthalates in mine tailings". Microchemical Journal 153: 461347. doi:10.1016/j.chroma.2020.461347. PMID 32797827. 
  25. ↑ Herrington, J.S.; Gómez-Ríos, G.A.; Myers, C. et al. (2020). "Hunting Molecules in Complex Matrices with SPME Arrows: A Review". Separations 7 (1): 12. doi:10.3390/separations7010012. 
  26. ↑ Krill, C.; Rochfort, S.; Spangenberg, G. (2020). "A High-Throughput Method for the Comprehensive Analysis of Terpenes and Terpenoids in Medicinal Cannabis Biomass". Metabolites 10 (7): 276. doi:10.3390/metabo10070276. 

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added. The original article lists references in alphabetical order; this wiki organizes them by order of appearance, by design.

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