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HomeCannabinoid Profiling and Potency Testing of Cannabis and Cannabis ProductsNatural Cannabinoid and Cannflavin Profiling by HPLC-PDA

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

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.

CompoundAbbreviation
CannabidiorcinCBDO
Cannabidivarinic acidCBDVA
CannabidivarinCBDV
CannabigerivarinCBGV
Cannabigerovarinic acidCBGVA
CannabielsoinCBE
CannabidibutolCBDB
CannabichromeorcinCBCO
Cannabidiolic acidCBDA
Cannabigerolic acidCBGA
CannabigerolCBG
CannabidiolCBD
Δ9-TetrahydrocannabivarinΔ9-THCV
Δ9-Tetrahydrocannabivarinic acidΔ9-THCVA
CannabichromevarinCBCV
Cannabidiphorolic acidCBDPA
Cannabichromevarinic acidCBCVA
CannabinolCBN
Cannabinolic acidCBNA
CannabidiphorolCBDP
Δ9-TetrahydrocannabinolΔ9-THC
Δ8-TetrahydrocannabinolΔ8-THC
CannabicyclolCBL
Δ9-Tetrahydrocannabinolic acidΔ9-THCA
CannabichromeneCBC
Cannabichromenic acidCBCA
Cannabicyclolic acidCBLA
Cannabidiol Monomethyl etherCBDM
Cannabigerol monoethyletherCBGM
9-tetrahydrocannabiphorolΔ9-THCP
CannabicitranCBT
9-tetrahydrocannabiphorolic acidΔ9-THCPA
Table 1.Abbreviations used for cannabinoids included in this method

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).

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 2.Instrument and mobile phase conditions
Time
(min)
%
A
%
B
05941
15842
103763
163268
261981
281387
29.50100
30.50100
315941
Table 3.Gradient conditions
(recommended equilibration time: 4 minutes)
AnalyteQuantitative WavelengthAnalyteQuantitative WavelengthAnalyteQuantitative Wavelength
Cannflavin B340 nmCBG230 nmCBL230 nm
CBDO230 nmCBD230 nmΔ9-THCA270 nm
CBDVA270 nmTHCV230 nmCBC280 nm
CBDV230 nmΔ9- THCVA270 nmCBCA258 nm
CBGV230 nmCBCV280 nmCBLA270 nm
CBGVA270 nmCBDPA270 nmCBDM230 nm
CBE230 nmCBCVA258 nmCBGM230 nm
CBDB230 nmCBN280 nmΔ9-THCP230 nm
CBCO280 nmCBNA258 nmCBT230 nm
CBDA270 nmCBDP230 nmΔ9-THCPA270 nm
Cannflavin A340 nmΔ9-THC230 nmPDA Conditions
Lamp: D2
Cell Temperature: 40 °C
Polarity: +
Slit Width: 8nm
CBGA270 nmΔ8-THC230 nm
Table 4.Photodiode array detector conditions

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.

A chromatogram plotted between absorbance (measured in mAU) on y-axis and retention time (measured in minutes) on x-axis, depicts distinct peaks for 34 different cannabinoids and cannflavins in a standard, recorded at concentrations of around 1 µg/mL, observed at a wavelength of 230 nm

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

A chromatogram plotted between absorbance (measured in mAU) on y-axis and retention time (measured in minutes) on x-axis, depicting clear labeled peaks of cannabinoids and cannflavins derived from a mix of hemp flower acetone extracts at a dilution of 50X, captured at a wavelength of 230 nm

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).

AnalyteAvg.
Recovery
(%)
RSD
(%)
Cannflavin B1013.5
CBDO1207.5
CBDVA97.62.5
CBDV1122.8
CBGV1082.0
CBGVA1083.2
CBE1112.9
CBDB99.51.8
CBCO1012.8
CBDA1032.3
Cannflavin A1117.5
CBGA1001.5
CBG89.73.7
CBD1033.7
Δ9-THCV94.23.3
Δ9-THCVA91.50.6
CBCV1022.0
CBDPA1022.4
CBCVA97.42.3
CBN95.32.8
CBNA972.4
CBDP1143.3
Δ9-THC99.81.9
Δ8-THC1031.3
CBL1055.0
Δ9-THCA99.72.1
CBC85.64.9
CBCA98.42.4
CBLA98.72.6
CBDM1011.4
CBGM98.92.1
Δ9-THCP1032.6
CBT1012.1
Δ9-THCPA960.7
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)
AnalyteAvg.
Recovery
(%)
RSD
(%)
CBDVA1062.0
CBDV1031.3
CBDA1231.8
CBGA1132.0
CBG1131.1
CBD1021.5
Δ9-THCV1041.4
Δ9-THCVA98.81.9
CBN1011.3
Δ9-THC1111.2
Δ8-THC1071.4
Δ9-THCA1081.9
CBC1061.3
CBCA1052.1
Table 6.Average percent recoveries and percent relative standard deviations of PT test samples. N=3 replicates per performance test

Limits of Detection

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

AnalyteLOD*
(Wt%)
Cannflavin B0.002
CBDO0.003
CBDVA0.006
CBDV0.003
CBGV0.003
CBGVA0.006
CBE0.003
CBDB0.001
CBCO0.003
CBDA0.005
Cannflavin A0.003
CBGA0.005
CBG0.003
CBD0.003
Δ9-THCV0.003
Δ9-THCVA0.006
CBCV0.003
CBDPA0.006
CBCVA0.006
CBN0.003
CBNA0.006
CBDP0.003
Δ9-THC0.003
Δ8-THC0.003
CBL0.003
Δ9-THCA0.005
CBC0.003
CBCA0.006
CBLA0.006
CBDM0.006
CBGM0.006
Δ9-THCP0.003
CBT0.003
Δ9-THCPA0.006
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

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%.

HPLC, Solvents & Reagents, Sample Preparation
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Reference Materials (Neutrals & Cannflavin)
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Reference Materials (Acids)
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