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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.
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.
Chromatographic method development was performed on a Shimadzu Nexera (Kyoto, Japan) using an Evoke C18, 15 cm x 4.6 mm column packed with 3 µm fully porous particles from Regis Technologies, Inc (Morton Grove, IL, USA). Reversed-phase conditions were screened using different organic modifiers (methanol and acetonitrile) in both isocratic and gradient modes of operation. Acid additives (formic acid and trifluoroacetic acid) were also investigated and found important in achieving good peak shape for the carboxylated species (e.g. CBCA, CBDA, etc.). The conditions that resulted in the most baseline resolved peaks and served as the foundation for further method development are listed in Table 1.
Figure 2. Effect of the addition of ammonium formate to mobile phase A. a) No ammonium formate added. b) 5 mM ammonium formate added. c) 10 mM ammonium formate added. d) 7.5 mM ammonium formate added. Additional chromatographic conditions listed in Table 1.
Table 1. Chromatographic conditions used in the development of the method to separate 17 cannabinoid analytical reference standards.
Column: Instrument: Mobile phase A:
Mobile phase B: Flow:
Gradient:
Oven Temp: Inj. Vol:
Detection:
Evoke C18; 15 cm x 4.6 mm; 3 µm Shimadzu Nexera
Water + 0.1% formic acid (+ ammonium formate concentration specified with chromatogram) Acetonitrile + 0.1% formic acid 2.0 mL/min Time (min.) 0.00
15.00 30° C 5 µL
228 nm
Figure 2a shows the baseline-subtracted chromatogram for the separation of the 17 cannabinoid test mixture using the conditions listed in Table 1. Baseline resolution is achieved for each of the component peaks with the exceptions of CBGA and CBG (Rs = 1.40), THCVA and CBN (Rs = 1.42), and the coelution of Δ8-THC and CBNA at 8.20 minutes. In an effort to improve the resolution of these pairs, the effect of adding ammonium formate, the ammonium salt of formic acid, to mobile phase A in concentrations ranging between 5 and 10 mM was investigated. The addition of ammonium formate to formic acid
mobile phases increases the ionic strength as well as slightly raises the pH [6,7]. With 0.1% formic acid and ammonium formate concentrations of 0 mM, 5 mM, and 10 mM, the pH values of mobile phase A were measured to be 2.7, 3.1, and 3.5, respectively.
As shown in Figure 2, the addition of ammonium formate to mobile phase A results in reduced retention of the carboxylated cannabinoids while the decarboxylated species remain unaffected, thus baseline-resolving CBGA/CBG and THCVA/CBN. With 5 mM ammonium
%B 75 90
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