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36 February / March 2018


Acidic cannabinoids; CBD-A, THC-A, and CBG-A showed greater retention in acidic mobile phase (pH 3) than when separated under basic conditions (pH 10), while the retention of the neutral cannabinoids remained the same (Figure 2). The elution order of the unknown constituent was altered by the change in pH, therefore it was determined that the unknown constituent was susceptible to ionisation.


Figure 2: Elution order of biosynthetic cannabinoid precursor forms in hemp extract as a result of mobile phase pH. Separation at (A) pH 10 and (B) pH 3. Cannabinoid acids are tracked in colour; CBD-A (red), CBG-A (yellow) and THC-A (blue). The unknown constituent (green) is marked with an asterisk.


A separation of the hemp extract was performed at pH 10 using the ‘turn-key’ method with 10mM ammonium bicarbonate (mobile phase A) and acetonitrile (mobile phase B). As an orthogonal approach, a separation at pH 3 was performed using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). For efficiency purposes, separation conditions were the same for both pHs starting at 36% mobile phase B with a 0.5 minute hold. A linear ramp was made over 5 minutes to a maximum of 84% mobile phase B. The separation was followed by a rapid equilibration back to starting conditions.


The unknown constituent of interest was collected with the WFM-A using the separation at pH 10 and evaporated to dryness. After reconstitution of the collected fraction in 200 µL of 50:50 methanol / water (v/v), the solution was injected to confirm that the retention time of the collected peak matched the unknown constituent retention time in the extract. The fraction was heated at 120o


C for one hour in a heat block to induce decarboxylation.


Results and Discussion


The separation of neutral cannabinoid reference standards CBD, CBG, CBN, Δ9 THC, Δ8 THC and CBC, as well as biosynthetic precursor forms CBD-A, CBG-A and THC-A, and variants CBD-V and THC-V, was achieved using a potential ‘turn-key’ reversed-phase method at pH 10 (Figure 1). In raw hemp extract, a peak of significant area overlaid with the retention time of CBD-A, as expected in unheated, selectively bred high CBD hemp. Cannabinoids CBG-A, THC-A, CBD, CBG, Δ9 THC and CBC were also present in the extract in lower quantities by detection at 228 nm. An unknown constituent eluted at 2.1 minutes and exhibited an area response at the detection wavelength comparable to THC-A (Figure 1). The unknown constituent’s retention time and PDA-UV profile did not correspond to any of the cannabinoid reference standards, with a distinct lambda max at 246 nm, while the QDa-MS m/z of 357.2 Da was identical to isobaric cannabinoids CBD-A and THC-A (data not shown).


By reversed-phase, the elution order of analytes which can be ionised is influenced by mobile phase pH, while the retention of neutral compounds is not affected [18].


When the extract was heated to induce decarboxylation, the acidic cannabinoids CBD-A, THC-A, CBG-A, and the unknown constituent, were no longer present in the chromatography. There was an increase in the abundance of the neutral cannabinoids, as expected from the conversion of the acid form to the neutral form induced by the decarboxylation process (Figure 3) [19].


The unknown constituent was collected after separation at pH 10 via the WFM-A. The isolated fraction was subjected to heat to confirm that it, when present alone, was susceptible to decarboxylation. Two main peaks were observed after heat exposure (Figure 4). When separated at pH 10, the first eluting peak corresponded with the retention time of the unknown constituent, while an additional peak correlated to the retention time of the cannabinoid reference standard CBC. This information, including the vulnerability to ionisation, as shown when orthogonal chromatographic mobile phase pHs were employed, and isobaric similarity to CBD-A and THC-A, suggested the unknown constituent was a biosynthetic acid form of CBC, known as cannabichromenic acid (CBC-A).


To investigate the hypothesis, a certified CBC-A reference standard was obtained. The CBC-A reference standard showed a retention time and QDa-MS spectra that matched that of the unknown constituent. Since many cannabinoids are isobaric, the mass information was not enough alone to confirm the unknown constituent’s identity. The unique PDA-UV spectral profile of the CBD-A reference standard provided the most conclusive result. The PDA-UV spectral profile of CBC-A reference standard was identical to that of the unknown constituent with a major lambda maximum at 246.4 nm, and minor maxima at 293.1 nm and 326.4 nm (Figure 5). Combined with the retention time and MS results, the unknown constituent in the hemp extract was positively identified as the non-decarboxylated acid form of CBC, known as CBC-A.


Figure 3: UPLC separation in basic mobile phase of raw hemp extract (A) before decarboxylation and (B) after decarboxylation. The unknown peak of interest is marked with an asterisk.


CBC, a non-psychoactive cannabinoid, has been reported to have a wide range of therapeutic properties with anti-depressant and anti-inflammatory activity. Some studies on mice have shown that the administration of


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