To find relevant articles for this narrative review paper, a combination of keywords such as medicinal cannabis, analytical, quantification and cannabinoids were searched for in PubMed, EMBASE, MEDLINE, Google Scholar and Cochrane Library (Wiley) databases.
However, unlike GC, HPLC allows for differentiation between acidic and neutral cannabinoids (Romano and Hazekamp 2013) because it does not require a derivatization step prior to analysis because high temperatures are not involved in the analysis (Aizpurua-Olaizola et al. 2014; Leghissa et al. 2018a). As a result, cannabinoids do not undergo decarboxylation in an HPLC column, so HPLC provides a more comprehensive chemical report of cannabis samples compared to GC. Table 4 shows LOD and LOQ values for some of the most well-known cannabinoids using HPLC and GC-MS.
LC and GC methods comparison
Grauwiler, Scholer (2007) developed a method to simultaneously detect five cannabinoids in human plasma and urine using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and atmospheric pressure chemical ionization (APCI). Their method had a 25-min runtime with a 0.2 ng/mL lower limit of quantification on samples following human oral administration of 20 mg of THC. Although APCI methods are less sensitive than ESI methods, APCI methods were chosen instead of ESI methods because they produced fewer matrix effects. Limits of detection and limits of quantification were found to be acceptable even with APCI methods (Grauwiler et al. 2007).
Figure 3 plots the effect of varying the percentage and composition of mobile phase B (MPB) on the isocratic resolution of 1:2 Δ9-THC:Δ8-THC using the same Evoke C18, 15 cm x 4.6 mm column. Consider the analysis when performed with H2O/MPB = 10/90. The resolution of Δ9-THC and Δ8-THC is 1.06 when MPB = 100% acetonitrile. When MPB = 100% methanol, the resolution is 2.84. Maximum resolution (Rs = 3.12) is observed when MPB = 15:85 acetonitrile:methanol. That relatively minor improvement in resolution afforded by the blended MPB might suggest pure methanol to be the preferred organic modifier for this analysis, especially given the convenience of using a single solvent over pre-mixing a blend of acetonitrile:methanol or investing in alternative pumping instrumentation (e.g. quaternary pumps). With complex samples, though, care must be taken to observe how a desired change in selectivity can affect other analytes in the separation.
Historically, gas chromatography/mass spectrometry (GC/MS) has been used for the separation and quantification of cannabinoids and other compounds of interest in cannabis analysis. With GC, though, care must be taken to avoid decarboxylation of acidic species during the heated injection. High pressure liquid chromatography (HPLC) methods permit analysts to eschew many sample preparation and derivatisation steps and have become the preferred approaches to cannabis potency analysis [3,4,5]. In general, all approaches to HPLC method development look to balance several elements, among which are the ultimate goals of the analysis, resolution of target compounds and potential interferences, speed, and assay robustness.
Upon evaluating the molecules of interest in terms of their charges, polarities, and other functionalities, chromatographic method developers turn their focus to column and solvent selection, pH conditions, buffer selection and concentration, temperature, etc. Specific approaches can differ depending upon the primary goals of a separation. For example, if comprehensive characterisation of a complex sample is desired, approaches to maximising overall separation at the expense of analysis time may be acceptable. If, on the other hand, resolution of only a particular critical pair is required, speed and selectivity (for the crucial pair) may be the primary focus. With these concerns in mind, we set out to develop an HPLC method capable of fully resolving 17 cannabinoids in a minimal amount of time. Additionally, a second objective concerning the improved resolution of a specific critical pair of THC isomers (Δ8-THC and Δ9-THC) was explored.
The interplay of buffer concentration and pH was further investigated with respect to the retention time of one of the carboxylated species, CBNA. The conditions are outlined in Table 3. In the first three cases, mobile phase A was prepared with 0.1% formic acid and ammonium formate concentration of 0 mM, 5 mM, and 10 mM. The unadjusted pH values were measured as 2.7, 3.1, and 3.5, respectively. As described above, retention of CBNA decreased with increased buffer concentration (8.31 min, 7.76 min, and 7.34 min). In the fourth case, 10 mM ammonium formate was used in mobile phase A and no formic acid was used in either mobile phase A or B. CBNA is ionised under these conditions, and its retention was reduced to 1.64 minutes. In the fifth case, 10 mM ammonium formate was used and pH was adjusted with formic acid to a value of 3.1 in order to match the pH of mobile phase A when prepared with 0.1% formic acid and 5 mM ammonium formate, and the retention time of CBNA was 7.36 minutes. In the final case, 10 mM ammonium formate was adjusted to a pH value of 2.8, and the retention time was 7.52 minutes. Thus, it can be seen that the retention of the carboxylated, ionisable cannabinoids is a complex function of eluotropic strength, pH (and the corresponding protonation state of the analyte), and buffer concentration/ionic strength.
Seventeen analytical reference cannabinoid standards (1 mg/mL) were acquired from Cerilliant (Round Rock, TX, USA) and combined to a final component concentration of approximately 59 µg/mL in 53:47 methanol:acetonitrile. The mixture was composed of Δ8-tetrahydrocannabinol (Δ8-THC), Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabicyclol (CBL), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), cannabigerol (CBG), cannabigerolic acid (CBGA), cannabinol (CBN), cannabinolic acid (CBNA), exo-tetrahydrocannabinol (exo-THC), tetrahydrocannabinolic acid A (THCA-A), tetrahydrocannabivarin (THCV), and tetrahydrocannabivarinic acid (THCVA). The molecular structures of these cannabinoids are shown in Figure 1.
Acetonitrile and methanol are two of the most common organic modifiers used in reversed-phase HPLC, and many studies have detailed the differing and often complementary selectivities that they provide. Fundamental understandings of the solute-mobile phase, solute-stationary phase, and stationary phase-mobile phase molecular interactions can inform the strategies used in HPLC method development . For instance, it has been noted that, depending on the modifier used and how it has partitioned or adsorbed into the stationary phase, differences in hydrophobicity, hydrogen-bonding, and dipole-type interactions can be observed [9,10]. When developing methods and selecting appropriate mobile phases, it can be useful to consult Snyder’s solvent selectivity triangle, which plots solvents according to their acidic, basic, and dipolar properties [11,12,13]. Solvents that feature one of those properties more prominently than the other two can be readily identified from the plot. For example, methanol has acidic properties, and acetonitrile has dipole properties. Since they are miscible, they can be mixed in any ratio to achieve intermediate or new solvent properties.
The global cannabis industry is growing rapidly, with many countries and US states adding regulatory frameworks for medical and recreational cannabis programs [1,2]. Quality control is an essential component in protecting the health and safety of the consumer in this emerging market, and there is increasing demand upon cannabis testing laboratories for analytical determination of multiple cannabinoids along with potential contaminants such as pesticides, mycotoxins, heavy metals, etc. Current regulations surrounding potency vary by jurisdiction, but usually require testing for the active forms of tetrahydrocannabinol (THC) and cannabidiol (CBD). In addition, many require testing for the acid forms, tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), along with other cannabinoids like cannabigerol (CBG), cannabigerolic acid (CBGA), tetrahydrocannabivarin (THCV), cannabichromene (CBC), cannabicyclol (CBL), and cannabinol (CBN). As regulations evolve, and as research interests in minor cannabinoids expand, it is important to have robust analytical methods in place that are capable of meeting those needs.
A brief example serves to illustrate that several parameters should be considered when developing a chromatographic method for the resolution of complex samples involving key critical pairs. Consider again the separation of 1:2 Δ9-THC:Δ8-THC in the presence of cannabicyclol (CBL). In Figure 3 it can be seen that the resolution of the THC isomers is superior with pure methanol than with pure acetonitrile as the organic modifier. As shown in Figure 4, though, if CBL is present, it coelutes with Δ8-THC in H2O/methanol = 10/90. CBL elutes well away from the critical pair if pure acetonitrile is used, but the THC isomers are insufficiently resolved (Rs = 1.06). A 50:50 blend of acetonitrile:methanol provides good resolution, with Rs > 2.5 for both pairs. So, while binary mobile phase systems are very common in reversed-phase HPLC separations, ternary mobile phases can provide access to unique selectivities based on the combination of acidic, basic, and dipolar properties of the mobile phases used.
In some assays, analysts are concerned with improving the resolution of certain critical pairs. This may be especially true in cases where one component is far more abundant than the other. In the gradient separations shown in Figure 2, the resolutions between Δ9-THC and Δ8-THC are approximately 1.50. These isomers are neutral, and their retentions are largely unaffected by changes in mobile phase pH or ionic strength. Often, it is possible to improve resolution by running an isocratic analysis and by reducing eluent strength. In the case of Δ9-THC and Δ8-THC, the greatest effect is observed by changing the composition of mobile phase B with various ratios of acetonitrile and methanol.