Cannabinoid Profiling and Potency Testing of Cannabis and Cannabis Products

The last decade witnessed the legalization of recreational and medical use of cannabis across the US and many other countries. The plant (Cannabis sativa, Cannabis indica and Cannabis ruderalis) contains over 100 cannabinoids, only some have been characterized. ∆-9-tetrahydrocannabinol (THC) is the primary psychoactive constituent and cannabidiol (CBD) is the primary non-psychoactive constituent. Hemp was legalized in the US in 2018 with additional limits on total THC content in hemp derived CBD products. Regulatory requirements differ between countries and states; however, potency testing, and cannabinoid profiling requirements are mandated to varying degrees. 

Reliable and accurate profiling and quantitation of major and minor cannabinoids in cannabis/hemp plant materials, extracts and products ensures transparency, uniformity, and quality of the preparations.

High-throughput analysis of cannabinoids in a variety of matrices using Ascentis^® Express C18 Column

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Types of Cannabinoids

Cannabinoids are a group of C21 terpenophenolic compounds, found primarily in Cannabis indica, Cannabis sativa and Cannabis ruderalis. They interact with cannabinoid receptors in the body to produce psychoactive and non-psychoactive effects. Cannabinoids can be divided into 11 subclasses: cannabigerol (CBG-type), (–)-Δ9-tetrahydrocannabinol (Δ9-THC-type), cannabidiol (CBD-type), cannabichromene (CBC-type), cannabinol (CBN-type), (–)-Δ8-tetrahydrocannabinol (Δ8-THC-type), cannabicyclol (CBL-type), cannabinodiol (CBND-type), cannabielsoin (CBE-type), cannabitriol (CBT-type) and miscellaneous others. Cannabis plant cultivars have been developed to optimize production of either THC or CBD. Pharmacological effects of most other cannabinoids are not well studied but are of emerging interest.

Workflow for Cannabinoid Analysis

Cannabis is a challenging product to analyze as it contains numerous cannabinoids and terpenes and is available in various forms such as flowers and concentrates, food, and cosmetic products having different concentrations of different cannabinoids. A typical workflow for cannabinoid analysis involves:

• sample preparation by extraction, QuEChERS (quick, easy, cheap, effective, rugged, and safe) or SPE (solid-phase extraction)
• calibration using high-quality reference standards
• chromatographic separation and analysis by LC or GC methods

Cannabis Sample Preparation

Cannabis samples are extremely diverse, ranging from dried flowers, cannabis extracts, cosmetics, and various kinds of edibles. The cannabis plant itself is a complex matrix with numerous terpenes, cannabinoids, lipids, chlorophyl, and other constituents. Solvent based extraction, QuEChERS, and SPE are common extraction and clean-up methods used in the sample preparation of cannabis products.

Standards and Calibration

Accurate quantitation of cannabinoids requires the use of precise calibrators. Pure cannabinoid standards are often resinous, viscous, air sensitive and difficult to handle. They are therefore often provided in a solution format, individual or as mixtures. Cannabinoid standards are often provided in a solution format, individual or as mixtures. Certified Reference Materials (CRMs) manufactured according to ISO17034 with certified accuracy, uncertainty and traceability are required by many state regulators and by ISO17025 accredited testing laboratories. System suitability standards ensure method control and resolution of closely eluting cannabinoids. This is especially relevant for hemp product analysis where the amount of total THC (THC and THCA) allowed is limited by regulations.

HPLC Analysis of Cannabinoids

HPLC combined with UV or MS detection is the primary technique used for potency analysis of commercial cannabis and hemp. Superficially porous/Fused Core^® C18 and C8 columns provide rapid high-resolution separations of cannabinoids in under 10 min. Monolithic columns provide ruggedness for high throughput applications. Cost effective methods include the use of methanol as organic mobile phase. UV detection is cost effective and practical for high throughput cannabinoid analysis. MS detection differentiates cannabinoids based on their mass-to-charge (m/z) ratio to provide specificity and selectivity for profiling, characterization and quantitation. MS methods require the inclusion of isotopically labeled internal standards for quantitative analysis.  

GC Analysis of Cannabinoids

Gas chromatography (GC) coupled with either a flame ionization detector (FID) or a mass spectrometer (MS) is also utilized in cannabis testing. It uses run times of 20 minutes on low-polarity stationary phases, such as 5% diphenyl / 95% dimethyl polysiloxane stationary phases. The high temperatures used in GC cause the decarboxylation of acidic cannabinoids (THCA and CBDA). Cannabinoid content reported for an analyte is the sum of its respective acidic and neutral species. Derivatization is required to separately analyze acidic and neutral cannabinoids by GC.

Types of Cannabinoids

Cannabinoids are a group of C21 terpenophenolic compounds, found primarily in Cannabis indica, Cannabis sativa and Cannabis ruderalis. They interact with cannabinoid receptors in the body to produce psychoactive and non-psychoactive effects. Cannabinoids can be divided into 11 subclasses: cannabigerol (CBG-type), (–)-Δ9-tetrahydrocannabinol (Δ9-THC-type), cannabidiol (CBD-type), cannabichromene (CBC-type), cannabinol (CBN-type), (–)-Δ8-tetrahydrocannabinol (Δ8-THC-type), cannabicyclol (CBL-type), cannabinodiol (CBND-type), cannabielsoin (CBE-type), cannabitriol (CBT-type) and miscellaneous others. Cannabis plant cultivars have been developed to optimize production of either THC or CBD. Pharmacological effects of most other cannabinoids are not well studied but are of emerging interest.

Workflow for Cannabinoid Analysis

Cannabis is a challenging product to analyze as it contains numerous cannabinoids and terpenes and is available in various forms such as flowers and concentrates, food, and cosmetic products having different concentrations of different cannabinoids. A typical workflow for cannabinoid analysis involves:

• sample preparation by extraction, QuEChERS (quick, easy, cheap, effective, rugged, and safe) or SPE (solid-phase extraction)
• calibration using high-quality reference standards
• chromatographic separation and analysis by LC or GC methods

Cannabis Sample Preparation

Cannabis samples are extremely diverse, ranging from dried flowers, cannabis extracts, cosmetics, and various kinds of edibles. The cannabis plant itself is a complex matrix with numerous terpenes, cannabinoids, lipids, chlorophyl, and other constituents. Solvent based extraction, QuEChERS, and SPE are common extraction and clean-up methods used in the sample preparation of cannabis products.

Standards and Calibration

Accurate quantitation of cannabinoids requires the use of precise calibrators. Pure cannabinoid standards are often resinous, viscous, air sensitive and difficult to handle. They are therefore often provided in a solution format, individual or as mixtures. Cannabinoid standards are often provided in a solution format, individual or as mixtures. Certified Reference Materials (CRMs) manufactured according to ISO17034 with certified accuracy, uncertainty and traceability are required by many state regulators and by ISO17025 accredited testing laboratories. System suitability standards ensure method control and resolution of closely eluting cannabinoids. This is especially relevant for hemp product analysis where the amount of total THC (THC and THCA) allowed is limited by regulations.

HPLC Analysis of Cannabinoids

HPLC combined with UV or MS detection is the primary technique used for potency analysis of commercial cannabis and hemp. Superficially porous/Fused Core^® C18 and C8 columns provide rapid high-resolution separations of cannabinoids in under 10 min. Monolithic columns provide ruggedness for high throughput applications. Cost effective methods include the use of methanol as organic mobile phase. UV detection is cost effective and practical for high throughput cannabinoid analysis. MS detection differentiates cannabinoids based on their mass-to-charge (m/z) ratio to provide specificity and selectivity for profiling, characterization and quantitation. MS methods require the inclusion of isotopically labeled internal standards for quantitative analysis.  

GC Analysis of Cannabinoids

Gas chromatography (GC) coupled with either a flame ionization detector (FID) or a mass spectrometer (MS) is also utilized in cannabis testing. It uses run times of 20 minutes on low-polarity stationary phases, such as 5% diphenyl / 95% dimethyl polysiloxane stationary phases. The high temperatures used in GC cause the decarboxylation of acidic cannabinoids (THCA and CBDA). Cannabinoid content reported for an analyte is the sum of its respective acidic and neutral species. Derivatization is required to separately analyze acidic and neutral cannabinoids by GC.

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