Natural Cannabinoid and Cannflavin Profiling by HPLC-PDA

Caleb King, Director of Process Development; Reginald Gaudino, PhD, Chief Science Officer¹, Dominika Gruszecka², Katherine Stenerson, Product Manager³

¹Front Range Biosciences, ²Shimadzu Scientific, ³Merck

Article from Analytix Reporter - Issue 16

• Introduction
• Experimental
• Results and Discussion
• Conclusions

Introduction

Cannabinoids are a diverse group of diterpenoid compounds primarily observed in Cannabis and Rhododendron species. To date, over 120 phytocannabinoids have been identified and quantified in Cannabis extracts using analytical techniques such as High Performance Liquid Chromatography (HPLC). With the federal legalization of hemp, a type of Cannabis, and state-supported legalization measures for high-THC Cannabis, HPLC testing of dried plant material for psychotropic potency and therapeutic dosing has become part of nearly every piece of legislation. While numerous chromatographic methods have been developed for Cannabis Testing and the detection and quantification of THCA, CBDA, CBGA, CBNA, and their decarboxylated forms, many do not account for the possibility of coelutions with other secondary metabolites in plant samples such as cannabinoids, flavonoids, and terpenes. To complicate analyses further, the metabolomes of different Cannabis varieties can vary greatly, resulting in chromatographic coelutions that are present in some extracts but not in others.

The method presented in this application note attempts to resolve most of the significant coelutions common to different types of Cannabis and was designed for laboratories interested in the quantification of minor cannabinoid and cannflavin constituents. Using this method, a total of 34 unique Cannabis analytes were quantified in less than 32 minutes. (Table 1). The method described has been successfully applied to not only leaf and flower Cannabis tissue, but cannabis/hemp products such as concentrates, oils, and cosmetic products.

Table 1. Abbreviations used for cannabinoids included in this method

Compound	Abbreviation
Cannabidiorcin	CBDO
Cannabidivarinic acid	CBDVA
Cannabidivarin	CBDV
Cannabigerivarin	CBGV
Cannabigerovarinic acid	CBGVA
Cannabielsoin	CBE
Cannabidibutol	CBDB
Cannabichromeorcin	CBCO
Cannabidiolic acid	CBDA
Cannabigerolic acid	CBGA
Cannabigerol	CBG
Cannabidiol	CBD
Δ9-Tetrahydrocannabivarin	Δ9-THCV
Δ9-Tetrahydrocannabivarinic acid	Δ9-THCVA
Cannabichromevarin	CBCV
Cannabidiphorolic acid	CBDPA
Cannabichromevarinic acid	CBCVA
Cannabinol	CBN
Cannabinolic acid	CBNA
Cannabidiphorol	CBDP
Δ9-Tetrahydrocannabinol	Δ9-THC
Δ8-Tetrahydrocannabinol	Δ8-THC
Cannabicyclol	CBL
Δ9-Tetrahydrocannabinolic acid	Δ9-THCA
Cannabichromene	CBC
Cannabichromenic acid	CBCA
Cannabicyclolic acid	CBLA
Cannabidiol Monomethyl ether	CBDM
Cannabigerol monoethylether	CBGM
∆9-tetrahydrocannabiphorol	Δ9-THCP
Cannabicitran	CBT
∆9-tetrahydrocannabiphorolic acid	Δ9-THCPA

Experimental

Sample Preparation

Air dried samples were milled to a powder using stainless steel ball-bearings with stems and seeds mechanically removed after pulverization. Between 0.2 and 0.5 grams of powder aliquots were solvent extracted in 10 mL of HPLC-grade acetone using ultrasonication for a total of 30 minutes, at a water temperature no greater than 35 °C. Sample extracts were syringe-filtered with 0.22 µm PTFE filters, followed by either a 2-fold dilution for leaf extracts or a 5-fold dilution for floral extracts.

Method

A Shimadzu Prominence-i LC-2030C Plus system, equipped with an Ascentis^® Express C18 column and a photodiode array detector (PDA) was utilized to quantitate cannabinoid and cannflavin analytes in dried hemp tissues (Table 2).

Table 2. Instrument and mobile phase conditions

LC Conditions
Column:	Ascentis® Express C18, 2.7 μm,  150 x 3 mm I.D. (53816-U)
Mobile Phase:	[A] Water, 8% (v/v) methanol, 0.035% (v/v) formic acid, 1.8 mM ammonium formate; [B] acetonitrile
Gradient:	See Table 3
Flow rate:	0.45 mL/min
Column temp.:	24°C
Autosampler temp.:	15°C
Detector:	PDA (various wavelengths, see Table 4)
Injection:	2 µL
Samples:	leaf extracts and floral extracts

Table 3. Gradient conditions
(recommended equilibration time: 4 minutes)

Time (min)	% A	% B
0	59	41
1	58	42
10	37	63
16	32	68
26	19	81
28	13	87
29.5	0	100
30.5	0	100
31	59	41

Table 4. Photodiode array detector conditions

Analyte	Quantitative Wavelength	Analyte	Quantitative Wavelength	Analyte	Quantitative Wavelength
Cannflavin B	340 nm	CBG	230 nm	CBL	230 nm
CBDO	230 nm	CBD	230 nm	Δ9-THCA	270 nm
CBDVA	270 nm	THCV	230 nm	CBC	280 nm
CBDV	230 nm	Δ9- THCVA	270 nm	CBCA	258 nm
CBGV	230 nm	CBCV	280 nm	CBLA	270 nm
CBGVA	270 nm	CBDPA	270 nm	CBDM	230 nm
CBE	230 nm	CBCVA	258 nm	CBGM	230 nm
CBDB	230 nm	CBN	280 nm	Δ9-THCP	230 nm
CBCO	280 nm	CBNA	258 nm	CBT	230 nm
CBDA	270 nm	CBDP	230 nm	Δ9-THCPA	270 nm
Cannflavin A	340 nm	Δ9-THC	230 nm	PDA Conditions Lamp: D2 Cell Temperature: 40 °C Polarity: + Slit Width: 8nm
CBGA	270 nm	Δ8-THC	230 nm

Calibrations

Calibration standards were prepared gravimetrically from certified reference materials (CRMs) or research grade isolates for 34 unique cannabinoids and cannflavins at concentrations ranging from 0.1 to 800 µg/mL. The linearity for all compounds was R² ≥ 0.99 using linear correlations and a best-fit weighting of 1/concentration. The UV spectra of each analyte was recorded in a spectral library to assist in positive identification of cannabinoids and cannflavins in plant tissue extracts.

Results and Discussion

Separations of a cannabinoids/cannflavin standard and a hemp flower extract are presented in Figures 2A and B, showing the signal at 230 nm. By monitoring the multiple wavelengths described in Table 4, sufficient resolution was obtained for all peaks to allow for adequate identification and consistent integration. A solvent containing no analytes was applied to all standards and samples for consistent baseline identification.

Figure 2a. Chromatogram of 34 cannabinoids/cannflavins at approximately 1 µg/mL. (Trace at 230 nm only shown)

Figure 2b. Chromatogram of mix of hemp flower acetone extracts at a total dilution of 50X. (Trace at 230 nm only shown)

Accuracy and Precision

Accuracy and precision were evaluated by spiking all 34 analytes onto homogenized low-cannabinoid producing Cannabis plant material (Table 5). The concentration of cannabinoids and cannaflavins present in non- spiked Cannabis plant material was subtracted from the observed concentrations in the spiked samples. To further evaluate the method’s accuracy and precision, performance test (PT) samples provided by Merck were diluted by 5X and analyzed (Table 6).

Table 5. Average percent recoveries and percent relative standard deviations at approximately 5 µg/mL on-column or approximately 0.05 Weight %. (N=3 replicates)

Analyte	Avg. Recovery (%)	RSD (%)
Cannflavin B	101	3.5
CBDO	120	7.5
CBDVA	97.6	2.5
CBDV	112	2.8
CBGV	108	2.0
CBGVA	108	3.2
CBE	111	2.9
CBDB	99.5	1.8
CBCO	101	2.8
CBDA	103	2.3
Cannflavin A	111	7.5
CBGA	100	1.5
CBG	89.7	3.7
CBD	103	3.7
Δ9-THCV	94.2	3.3
Δ9-THCVA	91.5	0.6
CBCV	102	2.0
CBDPA	102	2.4
CBCVA	97.4	2.3
CBN	95.3	2.8
CBNA	97	2.4
CBDP	114	3.3
Δ9-THC	99.8	1.9
Δ8-THC	103	1.3
CBL	105	5.0
Δ9-THCA	99.7	2.1
CBC	85.6	4.9
CBCA	98.4	2.4
CBLA	98.7	2.6
CBDM	101	1.4
CBGM	98.9	2.1
Δ9-THCP	103	2.6
CBT	101	2.1
Δ9-THCPA	96	0.7

Table 6. Average percent recoveries and percent relative standard deviations of PT test samples. N=3 replicates per performance test

Analyte	Avg. Recovery (%)	RSD (%)
CBDVA	106	2.0
CBDV	103	1.3
CBDA	123	1.8
CBGA	113	2.0
CBG	113	1.1
CBD	102	1.5
Δ9-THCV	104	1.4
Δ9-THCVA	98.8	1.9
CBN	101	1.3
Δ9-THC	111	1.2
Δ8-THC	107	1.4
Δ9-THCA	108	1.9
CBC	106	1.3
CBCA	105	2.1

Limits of Detection

Determined Limits of Detection (LOD) as S/N > 3 by weight percent are shown in Table 7.

Table 7. Calculated method Limits of Detection (LOD*) at 50X total dilution factor and 0.2 g of sample
*LOD as S/N > 3:1

Analyte	LOD* (Wt%)
Cannflavin B	0.002
CBDO	0.003
CBDVA	0.006
CBDV	0.003
CBGV	0.003
CBGVA	0.006
CBE	0.003
CBDB	0.001
CBCO	0.003
CBDA	0.005
Cannflavin A	0.003
CBGA	0.005
CBG	0.003
CBD	0.003
Δ9-THCV	0.003
Δ9-THCVA	0.006
CBCV	0.003
CBDPA	0.006
CBCVA	0.006
CBN	0.003
CBNA	0.006
CBDP	0.003
Δ9-THC	0.003
Δ8-THC	0.003
CBL	0.003
Δ9-THCA	0.005
CBC	0.003
CBCA	0.006
CBLA	0.006
CBDM	0.006
CBGM	0.006
Δ9-THCP	0.003
CBT	0.003
Δ9-THCPA	0.006

Conclusions

A gradient HPLC method was developed for the quantification of 34 unique compounds in Cannabis within a single injection. Solvent consumption per injection was less than 16 mL with an injection-to- injection runtime of 35 minutes. The method described allows for the quantitation of major and minor phytocannabinoids in Cannabis with minimal coelutions from flavonoids or terpenes; thus, reducing limits of detection while maintaining accuracy at ≤ ±20% and precision at ≤ ±10%.