Characterization of Δ⁸-THC Distillates using Non-Targeted Screening with High Resolution Mass Spectrometry 

The use of delta-8 tetrahydrocannabinol (Δ⁸-THC) has caused consumer safety concerns in the US. Delta-9 tetrahydrocannabinol (Δ⁹-THC) is the main intoxicating component in the cannabis plant. Its isomer, Δ⁸-THC, is also intoxicating and naturally occurs in the cannabis plant at trace levels. Products that contain Δ⁸-THC are formulated using Δ⁸-THC made from the chemical conversion of hemp-derived cannabidiol (CBD), which was defined and legalized under the 2018 US Farm Bill. Regulations governing the use of synthetic components derived from hemp are not clearly addressed which has created a growing market for Δ⁸-THC. Conversion of CBD to Δ⁸-THC often requires harsh conditions leading to reaction byproducts. This study demonstrates a workflow to characterize Δ⁸-THC distillates using non targeted screening and high-resolution mass spectrometry (HRMS). Cannabinoid libraries were generated with available authentic standards, which included accurate mass fragments and additional properties such as retention times and were used to identify target components. The purity of the distillate samples measured by UV ranged from 79.0–93.6%. Several unidentified peaks were detected in the UV data. The UV spectra indicated that there may be structural similarities between the unknown components and the primary compound in the distillates, Δ⁸-THC. During HRMS analysis, the software highlighted m/z 315.2318 as a base peak for several unknowns with proposed elemental compositions of C₂₁H₃₀O₂. Fragmentation data suggest that the components share structural characteristics with the C₂₁ neutral cannabinoids including Δ⁹-THC. A chlorinated component with a proposed elemental composition of C₂₁H₃₁ClO₂ and common fragments with many isomers of Δ⁹-THC was also observed in a purified distillate.

Benefits

• Enhanced confidence in compound identifications using an analytical solution that simultaneously collects accurate mass precursor and product ion data combined with compound libraries increasing confidence in the results for known and emerging components
• Structural elucidation tools such as common fragment searching can be useful in identifying structurally related components

The rapidly increasing use of Δ⁸-THC in consumer products has caused safety concerns in the US.^1–4 Δ⁹-THC is the main compound responsible for inducing an intoxicating effect in the cannabis plant and is regulated at levels exceeding 0.3% which is the legal definition of marijuana under the federal Controlled Substances Act. Delta-8 tetrahydrocannabinol is a double bond isomer of Δ⁹-THC, which also elicits an intoxicating effect, and naturally occurs in the cannabis plant at trace levels.^5–7 However recently, bulk Δ⁸-THC is being manufactured from hemp derived CBD (<0.3% Δ⁹-THC US/Canada) which many producers consider legal under the Agriculture Improvement Act of 2018 (commonly referred to as the 2018 Farm Bill).⁸ The regulations governing the use of synthetic components derived from hemp are not clearly addressed which has created a growing market for Δ⁸-THC production and use. The increased use of Δ⁸-THC has led both the CDA and the FDA to issue warnings due to reports of multiple adverse reactions directly linked to Δ⁸-THC.^3,4 The conversion of CBD to Δ⁸-THC requires conditions that can lead to multiple reaction byproducts which need to be characterized to enhance the chemical understanding of the components produced.^9–13

In this study, a workflow for the determination of components in Δ⁸-THC distillates will be demonstrated using data from non-targeted analysis by ultra-performance liquid chromatography (UPLC) and high-resolution time-of-flight mass spectrometry (ToF-MS) in addition to PDA detection. In-house cannabinoid reference libraries were used to assign identities to the compounds detected. These libraries were generated in-silico and from experimental data. Mass spectral information is predicted from chemical structures of known components supported by experimental data generated through the analysis of available authentic reference standards. The libraries consisted of accurate masses of molecular ions ([M+H]⁺), fragment ions, isotope patterns, and in cases where reference standards are available, additional chromatographic properties such as retention times, were used to identify components. Then, data from remaining components was evaluated using additional software tools including common fragment searching. 

Sample Preparation

A mixture of authentic cannabinoid standards was sequentially diluted in acetonitrile to a concentration of 100 µg/mL. Distillate samples were weighed out in glass scintillation vials, dissolved, and diluted with acetonitrile to a concentration of 1 mg/mL in preparation for injection.

A data independent acquisition mode, known as MS^E, was used to collect accurate mass measurements from precursor and product ions in a single injection.¹⁴ The incidence of false positives is significantly reduced when using multiple attributes (i.e., retention time, accurate mass, expected fragments) to search entries in a compound library, greatly increasing confidence in the identifications. The processed data files from the analysis of available authentic standards in combination with the relevant structural .mol files, were used to create a custom library of compounds of interest. Data import is available which will then be converted to the library within the software. Additional structures of compounds found in the literature, for which no reference standards were available, were also added to the library.^9–12 The library entry for Δ⁸-THC is shown in Figure 1.¹⁵

Analysis of Delta-8-THC Distillate Sample A

The chromatographic separation of an authentic standard mixture of eight isomeric cannabinoids (Figure 2) using PDA detection at 228 nm is shown in Figure 3A.

In the PDA data, the presence of Δ⁹-THC, exo-THC, and Δ⁸-THC were detected in a Δ⁸-THC distillate sample using retention time (t_R) and spectral matching. Several unknown components were detected in the UV with Area% values significantly exceeding 0.1% (Figure 3A and 3B). Investigation of the unknown components detected in the PDA using the HRMS data showed multiple components with a base peak of m/z of 315.2318 eluting in the region preceding the main Δ⁸-THC peak at 3.28 minutes (Figure 3B).

In-house cannabinoid reference libraries were used to assign putative identities to the compounds detected in the distillate samples. Unidentified major components visible in both PDA and mass spectrometry (MS) data were evaluated using the structural elucidation tools.

In addition to Δ⁹-THC, exo-THC, and Δ⁸-THC, CBN and CBD were identified in the distillate sample and could be verified using the t_R, precursor and fragment ion information from the MS library (Figure 4, table). A customizable workflow is shown on the left side of the figure. The workflow items can be arranged in a sequence to aid with data review. The Identified Components step is selected, automatically displaying all library components that have been identified by the software in the component summary table. The component summary table shows information pertaining to the identified analyte including, m/z, mass error, retention time and detected adduct. The entry for Δ⁸-THC is highlighted, displaying the precursor and fragment extracted ion chromatograms (XIC), associated mass and UV spectral data and structural assignments. 

UNIFI Software has a suite of tools that can aid in identifying and elucidating components present in samples, that are absent from in-house libraries. The tools include common fragments, neutral loss, and mass defect searching. Detected unknown components that share common structural features can be flagged automatically in the component summary table. In the case of common fragment searching, known fragments can be entered in the search criteria of the processing method for automated searching of these shared structural features. In Figure 5 several components have been flagged as having common fragments (highlighted in blue) with Δ⁹-THC and its isomers.

Selected high CE spectra for unknown components with an exact m/z 315.2318 at high intensity are shown in Figure 6. The proposed elemental composition for each of these unknown component precursors was C₂₁H₃₀O₂ (mass error -0.3 mDa). In addition, many common fragments were observed when the spectra were compared to that of an authentic standard of Δ⁸-THC, indicating the likelihood that these unknown components are structurally related and possibly isomers. The detection of isomers presents an analytical challenge which requires chromatographic resolution.

Structural .mol files based on components reported in recent publications on CBD conversion products were included in the library to aid in tentative assignment of unknown components detected in the distillate samples.^9–12 The proposed elemental compositions for several of the other high intensity unknown peaks eluting in the region between 0.8 and 3.0 minutes could be matched with the elemental compositions and fragments of components reported in the recent journal articles. Alternative assignments for each of the components are proposed by the library based on the analytical data, however, authentic standards were not available for confirmation (Figure 7).

Analysis of Delta-8-THC Distillate Sample B

In distillate sample B, two major components were identified by PDA detection at 228 nm. The primary component in the distillate sample was identified as Δ⁸-THC with a t_R of 3.27 min and a purity of 79%. An additional component was observed in the PDA data with a t_R of 3.10 minutes and an Area% of 19.4% (Figure 8). The UV spectrum for the unknown component matched that of Δ⁸-THC and eluted close to the t_R of an authentic standard of Δ⁹-THC.

In the MS data, the base peak of the unknown component had a m/z 351.2080 (Figure 9). Notably, the observed isotopic pattern indicated the presence of chlorine in the chemical structure. The software also flagged the presence of fragments common to Δ⁹-THC and its isomers. The proposed elemental composition of the unknown was C₂₁H₃₁ClO₂ (mass error –0.49 mDa) with an iFit confidence of 99.7%, indicating the agreement between the measured and the theoretical isotopic patterns. (Figure 10).

Confirmatory targeted tandem mass spectrometry (MS/MS) experiments using a 1 Da quadrupole window were performed to ensure that the fragments were not isotopes and originated from m/z 351 (Figure 11A). In addition, a second quadrupole resolution set to pass Cl-37 was used (Figure 11B). Both MS/MS experiments confirmed that the fragments observed in the initial high CE fragmentation analysis were the same (Figure 9).

These data suggest that the unknown is monochlorinated with structural similarity to Δ⁹-THC isomers. Mono- and dihalogenated derivatives of CBD and Δ⁹-THC have been reported previously, though the observed m/z and proposed elemental composition does not support the detection of monochlorinated CBD or Δ⁹-THC, which would have an elemental composition of C₂₁H₂₉ClO₂.^16–18

Several known cannabinoids were identified using a compound library which greatly aided with compound assignment as it was based on multiple characteristics including t_R, precursor mass, fragment ions, and isotopic patterns.

Several unknown components with a significant m/z 315.2318 and proposed elemental composition of C₂₁H₃₀O₂ were detected in the distillate samples using a non-targeted screening approach. Analytical results including common fragment ions suggest that they are potential structural isomers of Δ⁹-THC.

The Area% ranged from 0.13–4.9% and were derived from UV signals at 228 nm. UNIFI aids in the identification of unknown components that are not present in a library through the structural elucidation toolkit, which includes common fragments, neutral loss, and mass defect searching. These search features allow for easy filtering of components that exhibit structural similarity.

An unknown component discovered in the PDA with an Area% of 19.4% and a proposed elemental composition of C₂₁H₃₁ClO₂ was detected in distillate sample B, and the fragmentation data suggests structural similarity with Δ⁹-THC and its isomers. The purity of distillate B was less than 80%.

Multiple unknown components were detected in the distillates using a non-targeted screening approach. The discovery and characterization of unknown components is important to enable enhanced understanding of the complex chemistry and to ensure consumer product safety.

Analysis of distillate samples using UPLC-QTof-MS provides insights into elemental composition and other structural information that can aid in improving understanding of the structural relationships between unidentified components and knowns.

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