Identification of thermally transformed cannabinoids of CBDA and Cannabis extract
To identify the products of CBD and Cannabis extracts transformed under thermal reaction conditions, individual reactant residues were analyzed by UHPLC-Q/TOF–MS. The typical total ion chromatograms (TICs) of the conversion products of CBDA obtained after the thermal reaction of Cannabis extracts are shown in Fig. 3. A total of 7 transformed products were observed for CBDA after thermal reaction at 130 °C for 30 min. Transformed products in the CBDA reaction solution were mainly formed through decarboxylation, hydration, and cyclization of CBD. As shown in Fig. 3A, after the thermal reaction of isolated CBDA, CBDA (peak 5) and CBD (peak 6) were detected as major components. Minor components such as CBEA (peak 3), CBE (peak 4), △9-THC (peak 9), △8-THC (peak 10), and THCA (peak 12) were also detected. These transformed products including CBDA were clearly identified by matching retention times (RTs) and mass spectra of authentic standards, except for CBEA and CBE. These two were tentatively identified by LC retention behavior (Luca et al. 2021), exact mass measurement, and interpretation of MS/MS spectral patterns due to the lack of authentic standards. The exact mass values of CBEA and CBE were within ± 3.5 ppm of their corresponding theoretical mass values. The MS/MS spectra and fragmentation patterns of CBE and CBEA were intensively interpreted and will be described in “ESI mass fragmentations of thermal transformation cannabinoids of CBDA and Cannabis extract” Section. Interestingly, THC isomers, known psychotropic substances, were observed at trace levels. Therefore, the occurrence of THC isomers should be carefully monitored and controlled during the thermal reaction of Cannabis and its derived products.
To investigate the transformed cannabinoid profiles, Cannabis extracts were analyzed and compared by LC/MS scans before and after the thermal reaction [Fig. 3B, C]. After thermal reaction of Cannabis extract at 130 °C for 30 min, acid cannabinoids of CBDA, THCA, CBDVA, and CBGA were almost fully converted into their corresponding neutral cannabinoids of CBD, THC, CBDV, and CBG, respectively, through decarboxylation. These compounds were identified by matching the RTs and mass spectra of authentic standards. Most of the CBDA in Cannabis extract was converted into CBD and expected to be transformed to △9-THC, resulting in significant increase in its amount [peak 5 in Fig. 3B]. Decarboxylation of CBDA, THCA, CBDVA, and CBGA also readily converted these species into their corresponding neutral cannabinoids, exhibiting decarboxylation reaction yields of 97, 99, 93, and 98% in Cannabis extract, respectively. However, about 43% of the isolated CBDA was decarboxylated, as shown in the TICs in Fig. 3A, indicating less frequent decarboxylation of CBDA than for Cannabis extract. This is consistent with the literature (Wang et al. 2016), where decarboxylation of CBDA readily occurred under thermal reaction of Cannabis extract but did not readily occur for isolated CBDA. This suggests that the matrix components in Cannabis extract play an important role as catalysts during the thermal reaction. Also, the formation yields of THCA and THC were less than 0.1% for isolated CBDA, indicating that CBDA was rarely converted into THCA and THC. On the other hand, no CBEA and CBE were observed without thermal treatment of Cannabis extract. These compounds are known to be formed under oxidative conditions of CBDA (Hanuš et al. 2016). This is thought to be generated by the reaction of a trace amount of oxygen in the closed vial with CBDA.
ESI mass fragmentations of thermal transformation cannabinoids of CBDA and Cannabis extract
All ESI–MS/MS spectra of thermal transformation products, neutral cannabinoids, and acidic cannabinoids obtained in this study are presented in Additional file 1: Fig. S1 and Additional file 1: Fig. S2. ESI mass spectra of cannabinoids exhibited protonated molecular ions [M + H]+. The MS/MS spectra of acidic cannabinoids (CBDA, CBGA, CBCA, CBDVA, and THCA) can be characterized by the weak intensity of the [M + H]+ ion and their common of [M + H-H2O]+, m/z 285, 261, and 233 ions and abundant m/z 219 ion. Their MS/MS spectra and fragmentation pathways are depicted in Additional file 1: Fig. S1 and Additional file 1: Scheme S1. These common fragment ions at m/z 285 and 261 might be formed by losses of C4H8 (butene) and C6H8 (hexadiene) molecules from the [M + H-H2O]+ ion. Also, the abundant characteristic ion at m/z 219 may be produced by loss of C3H6 (propene) molecules accompanied by hydrogen migration at the unsaturated alkyl chain from the m/z 261 ion. Several fragment ions below m/z 177 may be formed by successive loss of CH2 radicals at the alkane chain (C5H11) of the benzene ring from the m/z 219 ion.
For neutral cannabinoids [Additional file 1: Fig. S2], the weak [M + H]+ ion and common characteristic ions [M + H-C4H8]+ (A) and [M + H-C9H14]+ (B) formed by cleavage of the substituted unsaturated group at the benzene ring were observed. Fragments A and B ions have a relatively stable benzyl cation. From alkane chain cleavage of A and B ions, other common ions such as [A-C2H4]+, [A-C3H6]+, [B-C2H4]+, and [B-C5H10]+ were also observed, as depicted in Additional file 1: Scheme S2. Below m/z 190, structures of common characteristic ions such as m/z 181, 153, 135, and 93 were suggested. ESI–MS/MS spectra of neutral cannabinoids can be characterized by the presence of these common ions. These fragmentation pathways can help to structurally elucidate new emerging cannabinoids in plants or transformed cannabinoids produced from thermal or chemical reactions of Cannabis species.
The MS/MS spectra of tentatively identified CBE and CBEA in this study are depicted in Fig. 4A, B. Structures of these substances with furan and cyclohexane rings are slightly different from those of other cannabinoids. Their MS/MS spectral patterns were complicated compared to those of other cannabinoids. For CBE, some of the common ions such as m/z 193, 165, 123, and 93 were the same as those of neutral cannabinoids. For CBEA, characteristic ions at m/z 357 and 339 formed by successive loss of H2O molecules from the [M + H]+ ion were observed, and the ions at m/z 231 and 219 were formed by the loss of C8H12 and C9H12 from the ion at m/z 339. The common ion of m/z 109 for CBEA and CBE was also observed. Based on the suggested fragmentation pathways of other acidic and neutral cannabinoids, fragmentation pathways of CBE and CBEA are suggested in Fig. 4C.
For further identification of CBE and CBEA, reactant residue was trimethylsilylated (TMS) with BSTFA and analyzed in GC/MS scan mode. As shown in Fig. 4D, E, CBE and CBEA were clearly detected as TMS derivatives in TIC, and their electron ionization (EI) mass spectra were obtained. Their molecular ions [M+] appeared at m/z 474 and 590, respectively, with weak intensities. The base peaks and abundant ions of the CBE-(OTMS)2 and CBEA-(OTMS)3 derivatives were commonly observed at m/z 108 [C8H12]+ and m/z 130 [C3H5OTMS]+, respectively, and these ions were formed by two bond cleavages of cyclohexane. Other characteristic ions were structurally assigned in their mass spectra, and the mass fragmentations are summarized in Fig. 4F. The ESI–MS/MS and GC/MS spectral data obtained in this study were complementary and useful for clear identification of acidic and neutral cannabinoids without authentic standards.
Time profiles of decarboxylation of CBDA during thermal reaction
In this study, the decarboxylation profiles of CBDA for Cannabis inflorescence extract and isolated CBDA were obtained at various thermal reaction temperatures. For quantification of the thermally transformed cannabinoids, isotope-labeled CBD-d3 and CBDA-d3 were used as internal standards. According to previous reports (Moreno et al. 2020; Ryu et al. 2021), CBDA was easily converted into CBD above 100 °C, but it was difficult to achieve sufficient decarboxylation of CBDA below 100 °C, even for 60 min. In this study, the temperature was adjusted among 110 °C, 120 °C, and 130 °C with various reaction times ranging from 5 to 60 min. Also, the thermal reaction was performed in a closed reactor to reduce the loss of neutral cannabinoids such as CBD and THC with relatively low boiling points. The time profiles of CBDA decarboxylation and CBD formation in the extract and isolated CBDA are shown in Fig. 5.
As shown in Fig. 5A, the amount of CBDA in Cannabis extract decreased radically when the temperature was increased. The reaction time needed to obtain maximum decarboxylation of CBDA decreased as the temperature increased. At 130 °C, the amount of CBDA in the extract decreased exponentially to 10 min, and no significant decrease was observed after 20 min. The maximum decarboxylation rate of CBDA in the extract was approximately 95% at 110 °C for 60 min, 97% at 120 °C for 50 min, and 97% at 130 °C for 20 min. This phenomenon clearly demonstrates that the decarboxylation reaction of CBDA is temperature dependent. On the other hand, the maximum formation rate of CBD was approximately 63% at 110 °C for 60 min, 64% at 120 °C for 50 min, and 65% at 130 °C for 20 min [Fig. 5A]. Compared to the decarboxylation efficiency of CBDA, the formation rate of CBD was low. This may be because some of the CBD was lost due to its relatively low boiling point even in the closed reaction vial, and CBDA was partly converted into other transformed products such THC isomers, THCA, CBEA, and CBE during the thermal reaction.
As shown in Fig. 5A, B, compared to the decarboxylation efficiency of CBDA in Cannabis extract, that of isolated CBDA was significantly lower. The decarboxylation efficiency of CBDA gradually increased as temperature and reaction time increased. At 130 °C for 60 min, the decarboxylation yield of isolated CBDA was about 52%. Also, the formation efficiency of CBD increased when temperature and reaction time increased. At 130 °C for 60 min, the formation yield of CBD was about 25%. This implies that the matrix of the extract significantly influences the decarboxylation of CBDA. In other words, various components in the matrix of the extract can significantly influence and accelerate the decarboxylation reaction of CBDA and other acidic cannabinoids. This phenomenon produced similar results in a previous study (Wang et al. 2016).
According to previous studies on the kinetics of CBDA decarboxylation (Citti et al. 2018), an Arrhenius plot for CBDA decarboxylation against 1/T (T: absolute temperature, Kelvin) was generated and is shown in Additional file 1: Fig. S3A. The linear equations and correlation coefficients were calculated using the least squares method (Nielsen et al. 2001). As expected, the kinetic rate constant (k) increased when the reaction temperature increased. Also, the reaction rates of CBDA decarboxylation with various reaction temperatures were determined in the graph of concentration (C) against reaction time, as illustrated in Additional file 1: Fig. S3B, C. The correlation coefficients of CBDA decarboxylation in extract are 0.9681, 9737, and 0.9829 at 110 °C, 120 °C, and 130 °C, respectively, indicating good linearity. Regarding CBDA decarboxylation, the reaction exhibited first-order kinetics within the reaction time range. Based on rate constants according to variation of reaction temperatures, the decarboxylation rate constants of the isolated CBDA were about tenfold less than those of CBDA in extract. It can explain that matrix components of Cannabis extract are expected to be acted as catalyzers in decarboxylation of CBDA, resulting in increasing rate constants. However, the decarboxylation rate constant of isolated CBDA mainly depends on reaction temperature. Thus, the reaction temperature above the threshold temperature (100 °C) is a key factor for decarboxylation of CBDA.
Based on the identified transformed products and time profile of CBDA decarboxylation, the transformation pathways during the thermal reaction of CBDA are suggested in Fig. 6. The formation of the major product, CBD, can be simply explained by loss of CO2 molecules due to heat treatment. CBD can be partly converted into △9-THC via intramolecular cyclization and then partly isomerized into △8-THC (Hanuš et al. 2016). Although THC isomers were detected as minor components, they are potent psychoactive substances (Livne et al. 2022). Therefore, the formation of these compounds should be carefully monitored and controlled during processing of Cannabis-derived products.
Under thermal reaction of CBDA, CBEA and CBE were initially formed through epoxydation at the double bond of the cyclohexene ring to form an epoxy ring, followed by epoxy ring opening and intramolecular cyclization to form a furan ring. The formation mechanism of CBEA and CBE from CBDA is indicated in Scheme S3. It was reported that the thermal reaction of CBD in the presence of oxygen led to formation of CBE (Czégény et al. 2021). In this study, the amounts of CBEA and CBE significantly increased when the thermal reaction of CBDA was performed in an open reaction vial. Thus, conversion of CBDA into CBEA and CBE could be greatly affected by both above threshold temperature and presence of oxygen.