35
Figure 1: Overlay of cannabinoid reference standard mixture and hemp extract separated using mobile phase at pH 10. The unknown constituent is marked by an asterisk.
Chromatographic orthogonality is the use of multiple separation mechanisms in order to gain additional analyte information. It is recognised that many cannabis laboratories may not have access to high-end scientific instrumentation therefore a simple approach to chromatographic orthogonality is most useful. Orthogonality was accomplished in this study using basic and acidic pH mobile phases on the same chromatographic platform (i.e. column and instrument) to identify an unknown constituent in cannabis extract.
Experimental Hemp Extract Preparation
At a partnering cannabis testing laboratory, hemp extract was generated from a 20 lb feed stock of Vermont grown hemp (Cannabis sativa L.) with seeds and stems removed. The buds
Table 1: Empirical formula of major cannabinoids and monoisotopic mass [16].
Cannabinoid Empirical Formula
Δ9 THC CBD CBC
Δ8 THC
THC-A CBD-A
THC-V CBD-V
CBG-A CBG
C22H30 C19H26
C22H32 C21
H32 CBN C21 H32 O4 O2
O4 O2
O2 358 286
360 316
310 C21H30 O2
Monoisotopic Mass
314
and leaves were ground, homogenised, and divided into five 4 lb bags. A sample was analysed from each bag with the average total cannabinoid content determined as 5.04 wt%. Six extractions were performed at the 5 L scale via a solvent- free Bio-Botanical Extraction System (SFE- BBES) (Waters, Milford, MA. USA).
Table 2: SFE – BBES extraction and collection conditions Extraction
Flow Rate Pressure
Temperature Time
Collection CS1 Pressure
CS1 Temperature CS2 Pressure
CS2 Temperature CS3 Pressure
CS3 Temperature
The cyclone separator 1 (CS 1) extracts were combined and homogenised. Ethanol was used to clean CS 1 post extraction. Approximately 85 g raw extract were added to 1.2 L of the ethanol wash, and the solution stored at -20o
C until analysis
and purification. Plant waxes were removed from the homogenised solution by vacuum filtration, and pigments were removed through a patent pending clarification strategy (Table 2).
Orthogonal Reversed-Phase Chromatography
Reversed-phase separations were performed using an ACQUITY H-Class UPLC System (Waters, Milford, MA. USA) equipped with a PDA (UV) detector at 228 nm, with a 4.8 nm resolution, and a 3D data λ range at 200-400 nm. The UHPLC was also equipped with a single quadrupole QDa (MS) detector with a programmed ESI (-) mass scan range of 100-600 Da, cone voltage at 15 V, capillary temperature of 500o
C and capillary voltage
Condition 170 g/min
344 bar 50o
C 210 nm
Condition 158 bar 45o
C
75 bar 40o
C
53 bar 35o
C
of 0.80 kV. An accessory fraction collector (Waters Fraction Manager – Analytical (WFM-A)) (Waters, Milford, MA. USA) was added for automated collection of the peak of interest. Separations were achieved using an ACQUITY CSH C18
, 130Å, 1.7µm, 2.1 mm x 50
mm column (Waters, Milford, MA. USA) at a temperature of 30o
C and a flow rate of 1.0 mL/
min. All data was collected and processed by Empower®
3 Chromatography Data Software.
A ‘turn-key’ chromatographic separation of the major cannabinoid reference standards (Cerilliant, Round Rock, TX. USA) cannabidivarian (CBD-V, C-140), tetrahydrocannabivarian (THC-V), cannabigerol (CBG, C-141), Δ9 tetrahydrocannabinol (Δ9
–trans- -THC), Δ8
–trans-tetrahydrocannabinol (Δ8 T-032), cannabinol (CBN), Δ9
-THC, –trans-
tetrahydrocannabinolic acid (THC-A), cannabidiol (CBD), cannabidiolic acid (CBD-A), cannabigerolic acid (CBG-A) and cannabichromene (CBC) prepared at 1.0 mg/mL in methanol was performed at pH 10 using 10mM ammonium bicarbonate (mobile phase A) and acetonitrile (mobile phase B).
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