Furthermore, the complexity of cannabis increases geometrically under the ‘entourage effect’, which postulates that cannabinoids interact to modulate their therapeutic effects. An experimental basis for the entourage effect is provided by murine studies, which have demonstrated that binary combinations with acidic cannabinoids increase bioavailability, potency and efficacy of neutral cannabinoids in epilepsy models.Clinical evidence is also mounting, with a recent meta-analysis on observational studies of epileptic patients concluding that crude cannabis extracts yielded a greater reduction in seizure frequency and had fewer side-effects than equivalent doses of purified CBD. However, as most extracts were only characterised to the extent of standardising the CBD dose, information about other cannabinoids was absent or based on inference. Consequently, the authors’ attribution of the differences between the extracts and purified CBD to the entourage effect was speculative. It was not possible to evaluate if the effects of the other cannabinoids added together, comparable to merely increasing the dose of CBD, or if they magnified the effect to surpass what CBD could achieve alone. Evidently, to progress beyond studies of binary combinations or poorly characterised extracts, routine analyses capable of quantifying panels of cannabinoids could help to better inform the design and interpretation of future studies that investigate the entourage effect. A clinical understanding of this effect might subsequently inform the extent to which cannabinoids are screened during cannabis product quality control. Several published methods are available for the separation and quantification of cannabinoids, with a variety of limitations which constrain their routine use. For the analysis of neutral cannabinoids, GC is simple, sensitive, and provides acceptable resolution. However, GC is not immediately suitable for acidic cannabinoids, weed trimming tray as they are poorly volatilised and rapidly undergo thermal decarboxylation into neutral cannabinoids.
Fortunately, this limitation can be surmounted by trimethylsilyl derivatisation of the labile acid group. Alternatively, some analysts have adopted LC for the separation of cannabinoids in medicinal cannabis. Following separation by LC, detection can be achieved by MS or by PDA. The MS detector enables the peak identity confirmation from their fragmentation patterns and relative ratios, and is sufficiently specific to recognise coeluting impurities in complex matrices. However, the required technical expertise, operation, and maintenance costs prohibit the use of MS for the routine analysis of cannabinoids. The UV-Vis PDA detectors are much cheaper, require less operator expertise, and are widely available. Since cannabinoids contain UV chromophores, they are amenable to PDA detection. Moreover, the UV spectra may assist with compound identity confirmation and the measurement of peak purity, which aids in quantification. Whichever detector is used, the elevated cost and limited availability of certified analytical reference standards for some cannabinoids remain impediments to their analysis. The cost can exceed $200 AUD per mg, and newly identified pharmacological leads in cannabis are possibly more expensive with significantly longer shipping times. To surmount this, some analysts have performed stereoselective microscales syntheses to obtain cannabinoids in a timelier manner, but this is beyond the remit of a typical QC lab. If the analysis of such cannabinoids is to become routine, the cost for their quantification must be mitigated. To this end, this study aimed to develop and validate a HPLC-PDA method for the determination of ten cannabinoids in medicinal cannabis inflorescence and oil and to explore the feasibility of using RRT for peak identification and RRF for their quantification. By this approach, an initial once-off purchase of all the standards was required to establish the RRT and RRF between the cannabinoids and the reference compounds: CBD as a reference for neutral cannabinoids, and CBDA as a reference for acidic cannabinoids; chosen as they are cheaper and available in many jurisdictions. Subsequently, the method may be routinely used in QC laboratories for the quantification of a panel of ten cannabinoids, requiring only sparing amounts of the reference compounds.
To optimise sample preparation, a variety of extraction solvents were tested with duplicate extractions. The solvents trailed were methanol, ethanol, acetonitrile, ethyl acetate, methanol:water , and acetonitrile:methanol . Cannabinoid peak areas were maximised by ethyl acetate and acetonitrile:methanol. However, due to markedly mismatching the initial mobile phase condition, ethyl acetate gave rise to significant band broadening. Thus, acetonitrile:methanol was selected as the extraction solvent, which is consistent with other extraction optimisation reports. The CV contribution of the method preparation procedure to the total uncertainty was determined by performing six replicate extractions and analysis of a single cannabis inflorescence sample. Grinding the inflorescence to pass through a < 710 µm sieve before sub-sampling achieved a CV range of 1.2 to 3.6%. When sub-sampling without grinding, the CV unacceptably ranged from 7.6 to 23.6%, thus indicating the importance of preparing a homogeneous sample. To optimise the chromatographic conditions, the method was iteratively developed. Baseline separation was achieved for eight of the ten cannabinoid standards , however, the CBD and CBG standard peaks overlapped slightly , as shown in Fig. 1A. Likewise, acceptable separation of cannabinoids in the extracts of cannabis inflorescence and cannabis oil were demonstrated in Fig. 1B and C, respectively. Whilst most matrix components eluted before the cannabinoids, a compound in inflorescence samples was observed to elute between CBDA and CBGA. This peak was identified to be tetrahydrocannabivarin by comparison with the UV spectrum and retention time obtained for the THCV standard. THCV was not included in the present method validation study as it was not part of the original selected set of analytes. When the analytes were sufficiently abundant in the sample, the UV spectra of their peaks were compared to that of the standard. As shown in Fig. 2, spectra superimposed closely, indicating good peak purity. To optimise PDA detection, wavelengths corresponding to the λmax of the different cannabinoids, specifically 210, 232, and 270 nm, were considered. Whilst 210 nm has been used in other studies, it produced a sloping baseline in the present study due to the use of methanol rather than exclusively using acetonitrile as the organic component of the mobile phase. Instead, it was found that visualising the chromatogram at 232 nm gave the best compromise between sensitivity and baseline noise. Some studies used 270 nm to improve sensitivity for the acidic cannabinoids, but this higher sensitivity is not required due to their relatively high abundance in the inflorescence samples. This high abundance was anticipated as acidic cannabinoids are the secondary metabolites synthesised in cannabis, whereas the neutral forms are produced by spontaneous decarboxylation. Retention times pooled from the three analysts are reported in Table 3. The CV in the retention times for each cannabinoid ranged from 0.18% to 0.56%, demonstrating an excellent inter-batch repeatability.
For the cannabinoids detected in the available inflorescence samples, the retention times observed for the sample peaks deviated by <1% from the standard retention times. To formalise the peak identification, and to demonstrate further gains in the inter-batch repeatability, the RRT were also pooled from the three analysts and were appended to Table 3. RRT should correct for inter-batch variabilities in retention times, provided that the variation in conditions proportionally affected all of the closely related analytes being studied. As anticipated, the pooled RRT values for each cannabinoid had CV which ranged from 0.04 to 0.34%. This represents a modest gain in repeatability, which should be maintained even if the retention times start to shift by >1%. Critically, it was also shown that the range of RRT values for each cannabinoid did not overlap. This means that analysts reported comparable values for the RRT, and that these values were unique for each cannabinoid. Thus, cannabinoid peaks in samples may be identified from their RRT values relative to the retention time of the CBD or CBDA from the working standard tested in the same batch of analysis. Detection and quantification limits for the cannabinoids are presented in Table 4. The LoD ranged from 20 to 78 µg/g and the LoQ ranged from 60 to 238 µg/g, relative to the inflorescence sample preparation. These limits are sufficiently low to enable the quantification of the studied cannabinoids in cannabis biomass and, observing that even relatively small amounts in crude biomass can be extracted and concentrated to therapeutically relevant concentrations in final products, these limits are suitable for quality control throughout the supply chain. However, with the quantification limits in the determined order of magnitude, it is unlikely that this method could be adapted for the analysis of the recently identified trace cannabinoids with heptyl sidechains. This includes THCP, which, by a published MS method, was identified in the inflorescences of THC dominant chemovars at concentrations routinely less than 140 µg/g and was undetected in CBD dominant chemovars. Accuracy of the method was evaluated from the recoveries of analytes spiked onto surrogate matrices, as presented in Table 7. For the cannabis inflorescence and oil, the spike recoveries from the surrogate matrices ranged from 90.1 to 109.3% and from 95.4 to 103.1% , respectively. Most recoveries were within 5% of the nominal concentration and the only two recoveries which were outside of this criterion had been spiked at the quantification limit, so their recoveries within 10% were acceptable. The precision of the recoveries was also acceptable, except at the LoQ of Δ8 -THC and CBDV which were only precise to 12%. Therefore, the method for the quantification of cannabinoids has acceptable accuracy. In this study, chamomile was selected as surrogate matrix for cannabis inflorescence as it was floral, available at little cost and, vertical grow system with the exception of the cannabinoids, shared phytochemical classes such as fragrant terpenes and flavonoids. Other published articles have used Urtica dioica or Humulus lupulus, with justifications based on tracing their phylogenies relative to Cannabis sativa.
Whilst sharing botanical orders or even families does not necessarily provide better matrix matching, it may be a reasonable approximation. Likewise, for cannabis oil, the choice of olive oil as a surrogate matrix had precedent from previous publications. Indeed, some cannabis oil products contain refined resins or even crude inflorescence extracted into an olive oil base, making its choice as the surrogate matric reasonable for such products. The appearance of publications employing surrogate matrices is being increasingly accepted as a cost-reduction strategy during method development, which is a clear advantage over articles which did not conduct recovery studies at all. Analysts in some jurisdictions may also find it pertinent to consider the use of surrogate matrices if licencing requirements preclude the use of the amount of cannabis material which would be required for the complete spike-recovery protocol on the true matrices. Cannabinoid concentrations in six different inflorescence samples were determined by conventional multipoint calibrations and the RRF method, as reported in Fig. 3. For cannabinoids above the order of magnitude of the LoQ, concentrations determined by the two methods agreed satisfactorily . The only cannabinoid above the LoQ which differed between quantifications by more than 5% was CBC but, relative to its low concentrations, the absolute differences was always acceptably less than 80 µg/g. The good agreement between the results obtained using the two different quantification methods applied to real samples demonstrates that the use of RRF for quantification is a valid alternative with its concomitant cost saving. Considering the cannabinoid profiles of the inflorescence samples, the high ratios of acidic to neutral cannabinoids were indicative of good drying and storage conditions. Furthermore, samples A and B were classified as having moderately high total THC and low total CBD , whilst samples C to F had moderate amounts of both . Beyond these observed concentrations, the proposed method is appropriate to analyse most samples with even greater levels of cannabinoids, as very few inflorescences exceed 200 mg/g total THC. Other cannabinoids such as CBC and CBN were also quantifiable, but Δ8 -THC was not detected in any sample. However, other authors have reportedly identified inflorescence samples with Δ8 -THC concentrations up to 4.9 mg/g, well above the LoQ of the present method. Accordingly, the present method has sufficient dynamic range to quantify cannabinoids at their various native concentrations. Public opinion toward cannabis, particularly for medicinal uses, has shifted in a more positive direction since the 1990’s. The perception of cannabis from the public is informed by a number of factors, and each individual may have a different view based on personal needs or experience.