36 August / September 2019
formate, the retention time of CBNA is shifted to 7.63 minutes and coelutes with exo-THC, an impurity formed in the synthesis of Δ9-THC (Figure 2b). By increasing the concentration to 10 mM ammonium formate, the retention of CBNA is further shifted, causing it to elute earlier than the THC isomers, but THCA-A is shifted into coeluting with CBC (Figure 2c). An intermediate concentration of 7.5 mM ammonium formate was found to provide baseline resolution of all 17 cannabinoids in the test mixture (Figure 2d). Table 2 shows how the retention times of the acidic cannabinoids change when the concentration of ammonium formate buffer in mobile phase A is varied.
Figure 3. The effect of the percentage and composition of mobile phase B (MPB) on the resolution of Δ9-THC and Δ8-THC. A blended organic modifier results in better resolution than pure methanol or pure acetonitrile. Evoke C18, 15 cm x 4.6 mm, 3 µm, 1.5 mL/min.
Table 2: Retention time of each acidic cannabinoid as a function of the ammonium formate concentration in MPA. Additional chromatographic conditions listed in Table 1.
Retention time (min)
CBDVA CBDA CBGA
THCVA CBNA
THCA-A CBCA
0 mM 2.52 3.69 3.89 6.27 8.20
10.00 10.92
5 mM 2.49 3.65 3.86 6.11 7.61 9.79
10.50
7.5 mM 2.46 3.61 3.82 6.02 7.38 9.68
10.38
10 mM 2.45 3.59 3.80 5.94 7.11 9.57
10.09
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.
Table 3: Conditions used to investigate the retention of CBNA as a function of pH and ammonium formate concentration. Additional chromatographic conditions (flow, gradient, etc.) listed in Table 1.
Condition # [ HCOONH4 ] pH of mobile phase A Rt of CBNA 1 2 3 4 5 6
0 mM 5 mM
10 mM 10 mM 10 mM 10 mM
2.7 (0.1% formic acid) 3.1 (0.1% formic acid) 3.5 (0.1% formic acid) 6.6 (no formic acid) 3.1 (pH adjusted) 2.8 (pH adjusted)
8.31 7.76 7.34 1.64 7.36 7.52
It should be noted that since ammonium formate is added only to the aqueous component of the mobile phase, the total ionic strength changes throughout the gradient runtime. For example, when 7.5 mM ammonium formate in mobile phase A is used in the gradient listed in Table 1, the total concentration on the column changes from 1.875 mM to 0.75 mM over the course of the 15 minute run. Nevertheless, with approximately 5 minute re-equilibration, run-to-run results were found to be reproducible. With real world samples, such as plant extracts, matrix effects may prove to be a concern. Although not determined
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