Most of the traditional methodologies for the determination of cannabinoids are based on solvent extraction, filtration, and concentration. These techniques can be cumbersome, time-consuming, and suffer from analyst-to-analyst variability while producing data of limited value. This article explores how thermal desorption GC/MS analysis eliminates conventional sample preparation regimes and can be used as a good rapid screening technique.
As the demand for the analysis of cannabinoids increases, it is imperative that the day-to-day analytical protocols be reproducible, accurate and efficient. Many laboratories routinely “screen” each sample in order to quickly determine the potential for matrix interference and instrument contamination while providing an estimate of the target compound’s concentation. A good “screening” method is simple (i.e., minimal or no sample preparation), fast (analysis in less than 5 minutes) and semi-quantitative.
One of the most widely used analytical techniques for “screening” is Thermal Desorption-GC/MS (TD-GC/MS) [1,2]. This technique does not require any solvent extraction or sample pretreatment. Milligram quantities are put in an inert sample cup which is then ‘ready to analyze’. The multi-mode Pyrolyzer with a vertical micro-furnace design allows programmable and multiple thermal desorption analysis on a single sample. This process which refers to Evolved Gas Analysis (EGA) starts with the acquisition of a thermal profile (i.e., detector response as a function of sample temperature) of each sample type.
To perform EGA, a short, deactivated tube (2.5m, 0.15mm id.) is used to connect the injection port to the MS detector. The sample is dropped into the furnace at a relatively low temperature (40-100˚C). The furnace is then programmed to a much higher temperature (600-800˚C). Compounds “evolve” from the sample as the temperature increases. A plot of detector response versus furnace temperature is obtained. Extracted ion chromatograms (EIC) are used to identify the thermal zone over which specific compounds of interest evolve from the sample.
Now, these optimum TD temperatures can be used in subsequent TD-GC/MS experiments to introduce the key components of interest while minimizing introduction of the matrix. Only this portion of the sample is actually transferred (i.e., injected on) to the analytical column. Injecting only a small portion of the sample provides immediate benefits to the laboratory:
- The high boiling fraction of the sample remains in the sample cup. This eliminates the need for a high-temperature bake out. Thus, column lifetime increases, there is little to no system contamination and run-to-run cycle time decreases.
- More sample can be put in the sample cup which has the effect of lowering detection limits – without affecting instrument performance or cycle time.
With respect to the analysis of cannabinoids, it is important to keep in mind that TD-GC/MS is based on the volatilization of target compounds from the matrix. Those compounds that are thermally labile or easily converted to an alternative compound need to be identified. In these instances, it is the reformed compounds that are identified and monitored. Decarboxylation is forced to completion which could give the “screening determinations” higher values: the concentration range increases and dilution factors more accurately determined.
Three edible samples are analyzed using TD-GC/MS method for quantification of cannabinoids. The experiments are performed by a multi-mode pyrolyzer (Frontier EGA/PY 3030D multi-shot pyrolyzer) directly interfaced to a bench-top GC/MS system. To automate the sequence and reduce the workload by increasing reliability of analysis, the Auto-Shot Sampler (AS-1020E) is combined with the multi-mode pyrolyzer. The vent-free GC/MS adaptor that allows switching of a GC separation column without breaking vacuum on the MS detector is also used. The adaptor is utilized for switching the EGA tube to the separation column and vice versa without venting the MS detector which saves time and increase productivity.
A commercial package of cannabis-infused chocolate brownie containing 10 brownie bites with the total of 100 mg THC (Tetrahydrocannabinol) was used. According to the product label, each brownie bite contains 10 mg of THC. In this experiment, one of the brownie bites from the package is placed on an analytical balance, and the weight was recorded as 10.17 grams. So based on the label, there is 10mg/10g=1mg/g of THC present in that brownie bite. The same brownie bite with the recorded weight was used to perform the TD-GC/MS analysis to confirm the theoretical THC value according to the label.
To calculate and confirm the amount of THC, EGA was first performed on THC standard standard-Absolute Standard. The pyrolyzer furnace was programmed from 100 to 800°C (20°C/min), and the EGA thermogram as shown below was obtained.
From the EGA thermogram, the optimal thermal desorption zone of THC was identified as 100 to 300°C. In fact, using the MS interpretation library, the peak with the apex of 185°C (100 to 300°C temperature zone) was identified as THC. TD-GC/MS analysis was then performed on the brownie bite in triplicate as shown in Figure 2. To perform TD-GC/MS analysis, the pyrolyzer furnace was programmed from 100 to 300°C (100°C/min) after the EGA tube was replaced by a separation column (easily facilitated by using the vent-free GC/MS adaptor). The amount of sample used to obtain the TD chromatograms was 0.25-0.26 mg.
Figure 2: TD-GC/MS Chromatograms of the Brownie Bite in Triplicate
The peak shown in Figure 2 (*) between the retention time of 12 to 14 minutes was identified as THC using the MS interpretation library. The RSD% of 4.6% was calculated based on the area counts of the THC peak. The most intense peak around 6 minute was identified as 5-Hydroxymethylfurfural (dehydration of sugar).
To confirm the THC concentration in the brownie bite, standard addition calibration curve was created. The standard solution was a mixture of CBD (Cannabidiol), THC, and CBN (Cannabinol). Figure 3 shows the calibration curve and calculated amount of THC in the brownie bite.
Using the calibration curve, the THC concentration is calculated as 0.996mg/g while the product label indicates 1mg/g of THC.
A commercial cannabis-infused dark chocolate bar with the total of 100 mg THC and net weight of 50g (1.7oz) is used. According to the product label, there are 20 pieces of chocolates and each piece of chocolate contains 5 mg of THC, so there is 100mg/50g=2mg/g of THC present in each piece. To demonstrate the accuracy and precision of the methodology, the analysis was performed in triplicate. The weights of each piece are recorded using an analytical balance as 0.099 mg, 0.097mg, and 0.105mg.
The same methodology was used for analyzing sample 2. First, the EGA was performed as the fast rapid screening technique. Then the optimal thermal desorption zone of THC was identified. The pyrolyzer furnace was programmed from 100 to 300°C (100°C/min) to obtain the thermal desorption chromatograms for all three pieces of the chocolate. Figure 4 shows the TD chromatograms in triplicate.
The peak shown (*) between the retention time of 12 to 14 minutes was identified as THC using the MS interpretation library. The RSD% of 4.6% was calculated based on the area counts of the THC peak.
To confirm the THC concentration in the chocolate pieces, standard addition calibration curve was created. The standard solution was a mixture of CBD (Cannabidiol), THC, and CBN (Cannabinol). Figure 5 shows the calibration curve and calculated amount of THC in the pieces.
Based on the calibration curve, the THC concentration is calculated as 2.1mg/g while the product label indicates 2mg/g of THC.
A commercial cannabis-infused gummy package with the net weight of 45g (1.59 oz) is used. The package contains 10 gummies with THC and CBD (2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol). According to the product label, each gummy has 10 mg CBD and 5mg THC. The label also indicates that THCA, CBDA, and CBN (other required cannabinoids) are 0.0mg/package.
To perform the TD-GC/MS analysis, one of the gummies was weighted on an analytical balance The weight was recorded as 0.109mg. The sample was then placed in MeOH and sonicated for one hour. 1 microliter of MeOH solution was spiked into the sample cup.
It is important to mention that the gummy sample could be analyzed as is without dissolving in any solvent. The experiment on sample 3 is performed based on external standard calibration to demonstrate the reliability and flexibility of this technique.
Figure 6 shows the TD-GC/MS chromatograms of the gummy samples in triplicate. The EGA thermogram of the gummy confirmed the thermal zone of 100 to 300°C for both THC and CBD. Note the detection peaks of both CBD and THC in the chromatograms. The RSD% of 2.5% for CBD and 2.3% for THC are calculated based on the area counts of the THC peak.
To confirm the THC and CBD concentrations in the gummy sample, an external standard calibration curve was created. Figure 7 shows the calibration curves and the calculated concentrations which are in excellent agreement with the package label. CBD concentration is calculated as 8.5mg/gummy compared to the label value of 10mg/gummy. The calculated THC is 6.1 mg/gummy while the label indicates the value of 5mg/gummy.
Table 1 shows the calculated and label values of cannabinoids concentration in all three commercial editable samples. Using TD-GC/MS the cannabinoids are thermally extracted from the mixture and the calculated concentrations are in excellent agreement with the package labels. Standard addition calibration curves were used for the brownie bite and chocolate piece while an external standard calibration curve was created to calculate THC and CBD in the gummy sample.
In conclusion, thermal desorption analysis eliminates conventional sample prep regimes; the sample is heated to the point that the cannabinoids desorb from the edible matrix (thermal extraction). It is fast, uses minimal or no solvent, and eliminates the need for expensive glassware. TD-GC/MS is a “volatiles only” analysis; high boiling sample constituents remain in the sample cup which eliminates system contamination, increases system stability and reduces run-to-run analysis time.
The Multi-Mode Pyrolyzer provides the users with a clear picture of the sample’s composition by identifying the thermal zones and the compounds in each zone. Using the EGA thermogram, one can simply determine the suitable temperature program and program the Pyrolyzer’s furnace appropriately.
Using Pyrolysis GC/MS, solid samples can be analyzed. There is no solvent required when using Pyrolysis-thermal desorption GC/MS as opposed to traditional GC/MS techniques. In other words, the solid and liquid samples can be injected (using an inert sample cup) into the Pyrolyzer without any solvent and sample pretreatment. This advantage, as well as rapid screening capability, is one of the primary reasons many laboratories integrate the multi-mode Frontier Pyrolyzer into both their day-to-day QC and analytical research protocols.
Pyrolysis GC/MS techniques also enable material characterization of virtually any organic material by providing detailed information about the composition of the samples. This technique is used for identification of complex polymers, copolymers, volatiles, and additives as well as failure, contamination, deformulation, and degradation analyses. As opposed to GC/MS alone, Pyrolysis GC/MS allows multiple and customized analysis on the same sample while the MS libraries offer a broad range of references: from low b.p. compounds to pyrolyzates and heavier polymeric fragmentations. In terms of the ability to run samples at higher temperature, the Frontier Multi-Shot Pyrolyzer provides the user with an expanded temperature range from ambient + 10 to 1050°C (± 0.1°C precision) as well as a high Interface temperature (maximum 450°C).
- ASTM D7823-16 “Determination of Low Level, Regulated Phthalates in Poly (Vinyl Chloride) Plastics by Thermal Desorption—Gas Chromatography/Mass Spectrometry.
- IEC 62321-8: Determination of certain substances in electrotechnical products – Part 8: Phthalates in polymers by gas chromatography-mass spectrometry (GC-MS), gas chromatography-mass spectrometry using a pyrolyzer/thermal desorption accessory (PY/TD-GC-MS).