Terpene Analysis

Potency
Solvent
Microbial
Terpene

Potency Testing

At this time the Alaska’s Marijuana Control Board requires concentration testing of the first five cannabinoids listed below. CannTest, LLC will include the additional significant cannabinoids in the profile, and add new compounds as science evolves.

Potency Testing

At this time the Alaska’s Marijuana Control Board requires concentration testing of the first five cannabinoids listed below. CannTest, LLC will include the additional significant cannabinoids in the profile, and add new compounds as science evolves.

Chemical Compounds

Delta-9 Tetrahydrocannabinol Acid
Δ9-THC-A
Delta-9 Tetrahydrocannabinol Acid (Δ9-THC-A, THCA) an acid with the carboxylic group (COOH) attached. In its acid form, THC is not very active. It is only when the carboxyl group is removed through decarboxylation  by heating or exposure to air that THC becomes psychoactive.
Cannabidiolic Acid
CBD-A
Cannbidiolic Acid (CBD-A, CBDA) an acid with the carboxylic group (COOH) attached, is a precursor to Cannabidiol. When the carboxyl group is removed through decarboxylation by exposure to heat or air CBDA converts to CBD.
Delta-9 Tetrahydrocannabinol
Δ9-THC
Delta-9 Tetrahydrocannabinol (Δ9-THC, THC) is the primary psychoactive component of the plant.
Cannabidiol
CDB
Cannabidiol (CDB) is a non psychoactive cannabinoid. It is often associated with having a sedative effect thereby reducing anxiety.
Cannabinol
CBN
Cannabinol (CBN) is a break down product of THC. The amount increases with the amount of time that has passed since harvest. The substance is known for having a sedative effect.
Delta-8 Tetrahydrocannabinol
Δ8-THC
Delta-8 Tetrahydrocannabinol (Δ8-THC) is similar to Δ9-THC however it has lower psychotropic potency. It is associated with simulating appetite and analgesic effect.
Cannabigerol
CBG
Cannabigerol (CBG) is a non psychotropic cannabinoid. It has been associated with treatment of glaucoma and inflammatory bowel disease.
Cannabichromene
CBC
Cannabichromene (CBC) is a non psychotropic cannabinoid that has been associated with ant- inflammatory and anti-viral effects.

Compounds

Delta-9 Tetrahydrocannabinol Acid
Δ9-THC-A
Delta-9 Tetrahydrocannabinol Acid (Δ9-THC-A, THCA) an acid with the carboxylic group (COOH) attached. In its acid form, THC is not very active. It is only when the carboxyl group is removed through decarboxylation  by heating or exposure to air that THC becomes psychoactive.
Cannabidiolic Acid
CBD-A
Cannbidiolic Acid (CBD-A, CBDA) an acid with the carboxylic group (COOH) attached, is a precursor to Cannabidiol. When the carboxyl group is removed through decarboxylation by exposure to heat or air CBDA converts to CBD.
Delta-9 Tetrahydrocannabinol
Δ9-THC
Delta-9 Tetrahydrocannabinol (Δ9-THC, THC) is the primary psychoactive component of the plant.
Cannabidiol
CDB
Cannabidiol (CDB) is a non psychoactive cannabinoid. It is often associated with having a sedative effect thereby reducing anxiety.
Cannabinol
CBN
Cannabinol (CBN) is a break down product of THC. The amount increases with the amount of time that has passed since harvest. The substance is known for having a sedative effect.
Delta-8 Tetrahydrocannabinol
Δ8-THC
Delta-8 Tetrahydrocannabinol (Δ8-THC) is similar to Δ9-THC however it has lower psychotropic potency. It is associated with simulating appetite and analgesic effect.
Cannabigerol
CBG
Cannabigerol (CBG) is a non psychotropic cannabinoid. It has been associated with treatment of glaucoma and inflammatory bowel disease.
Cannabichromene
CBC
Cannabichromene (CBC) is a non psychotropic cannabinoid that has been associated with ant- inflammatory and anti-viral effects.

Testing Method

There are presently two methods used for potency testing of cannabinoids (THC, THCA, CBD, CBDA, CBN and others). The method of Gas Chromatography (GC) requires that a cannabis sample first be ground to a fine powder. Addition of an organic solvent such as methanol followed by sonification and centrifuging  is then used to extract cannabinoids from the plant material.  A small amount of the sample is injected into a GC machine where it is heated up to 300 degrees Celsius thereby vaporizing the cannabinoids into a gaseous mixture. The gas is fed through a column that through its chemical makeup retains some compounds longer than others. As each cannabinoid leaves the column at a unique time a flame burns the gas and a detector determines the amount of each cannabinoid passing through the column. This is termed Flame Ionization Detection (FID). 

The problem with Gas Chromatography is that much of the natural cannabinoids in cannabis exist in an acid form such as THC-A and CBD-A then decarboxylate (release carbon dioxide) to the neutral forms of THC and CBD. THC has a psychoactive effect, but THC-A does not. Typically when cannabis is smoked or vaporized THC-A transforms to THC resulting in the “high”. However research has indicated that MS patients have had a therapeutic effect with intake of unheated cannabis due to anti-inflammatory effects of THC-A. Recent lab studies have shown that a GC machine does not decarboxlyate 100% of the THC-A and CBD-A. Roughly 60-70% was the average rate.

The second method of testing of cannabinoid potency is High Performance Liquid Chromatography (HPLC). In the HPLC method a cannabis sample is ground to a fine powder and cannabinoids are extracted using a solvent, sonification and centrifuging as in GC. However in HPLC the sample passes through a column in a liquid phase and cannabinoids are detected by an untraviolet light. Because HPLC does not add heat to the system it allows for the unique determination of THC-A and CBD-A as well as THC and CBD.

CannTest, LLC will use the LC method of testing for cannabinoid potency as it it the most accurate means of determining the Total THC and CBD that will be provided on delivery. Less than 100% decarboxylation during GC means that the total amount of THC and CBD can not be known. The GC system will only measure the THC available at the detector. In addition GC can not produce a measurement of THC-A and CBD-A which will be valuable information for many medical users.

Testing Method

There are presently two methods used for potency testing of cannabinoids (THC, THCA, CBD, CBDA, CBN and others). The method of Gas Chromatography (GC) requires that a cannabis sample first be ground to a fine powder. Addition of an organic solvent such as methanol followed by sonification and centrifuging  is then used to extract cannabinoids from the plant material.  A small amount of the sample is injected into a GC machine where it is heated up to 300 degrees Celsius thereby vaporizing the cannabinoids into a gaseous mixture. The gas is fed through a column that through its chemical makeup retains some compounds longer than others. As each cannabinoid leaves the column at a unique time a flame burns the gas and a detector determines the amount of each cannabinoid passing through the column. This is termed Flame Ionization Detection (FID). 

The problem with Gas Chromatography is that much of the natural cannabinoids in cannabis exist in an acid form such as THC-A and CBD-A then decarboxylate (release carbon dioxide) to the neutral forms of THC and CBD. THC has a psychoactive effect, but THC-A does not. Typically when cannabis is smoked or vaporized THC-A transforms to THC resulting in the “high”. However research has indicated that MS patients have had a therapeutic effect with intake of unheated cannabis due to anti-inflammatory effects of THC-A. Recent lab studies have shown that a GC machine does not decarboxlyate 100% of the THC-A and CBD-A. Roughly 60-70% was the average rate.

The second method of testing of cannabinoid potency is High Performance Liquid Chromatography (HPLC). In the HPLC method a cannabis sample is ground to a fine powder and cannabinoids are extracted using a solvent, sonification and centrifuging as in GC. However in HPLC the sample passes through a column in a liquid phase and cannabinoids are detected by an untraviolet light. Because HPLC does not add heat to the system it allows for the unique determination of THC-A and CBD-A as well as THC and CBD.

CannTest, LLC will use the LC method of testing for cannabinoid potency as it it the most accurate means of determining the Total THC and CBD that will be provided on delivery. Less than 100% decarboxylation during GC means that the total amount of THC and CBD can not be known. The GC system will only measure the THC available at the detector. In addition GC can not produce a measurement of THC-A and CBD-A which will be valuable information for many medical users.

Residual Solvent Testing

Current regulations allow for THC potency up to 76 percent. To achieve these high levels of THC marijuana production facilities produce solvent-based marijuana concentrate by using the hydrocarbons N-butane, isobutane, propane, or heptane to extract the cannabinoid from plant material. After extraction is complete the solvent must be purged from the product through evaporation. Often there is not complete evaporation of solvents leaving dangerous chemical in the concentrate.

The following solvents will be tested for in all marijuana concentrates. Values below are given in Parts Per Million (PPM). 1% is equivalent to 10,000PPM. Therefore if there is a 1 gram sample of concentrate the upper limit of butane allowed would be 800/10,000 x 0.01 or 0.08mg. Those with limits higher than the values below will be rejected and not sold in retail outlets.

Residual Solvent Testing

Current regulations allow for THC potency up to 76 percent. To achieve these high levels of THC marijuana production facilities produce solvent-based marijuana concentrate by using the hydrocarbons N-butane, isobutane, propane, or heptane to extract the cannabinoid from plant material. After extraction is complete the solvent must be purged from the product through evaporation. Often there is not complete evaporation of solvents leaving dangerous chemical in the concentrate.

The following solvents will be tested for in all marijuana concentrates. Values below are given in Parts Per Million (PPM). 1% is equivalent to 10,000PPM. Therefore if there is a 1 gram sample of concentrate the upper limit of butane allowed would be 800/10,000 x 0.01 or 0.08mg. Those with limits higher than the values below will be rejected and not sold in retail outlets.

Solvents

Butane
C4H10
Butanes must be less than 800 parts per million.
Heptane
C7H16
Heptane must be less than 500 parts per million.
Benzene
C6H6
Benzene must be less than 1 part per million.
Toluene
C7H8
Toluene must be less than 1 part per million.
Hexane
C6H14
Hexane must be less than 10 parts per million
Xylene
C8H10
Total Xylenes (m, p, o-xylenes) must be less than 1 part per million.

Solvents

Butane
C4H10
Butanes must be less than 800 parts per million.
Heptane
C7H16
Heptane must be less than 500 parts per million.
Benzene
C6H6
Benzene must be less than 1 part per million.
Toluene
C7H8
Toluene must be less than 1 part per million.
Hexane
C6H14
Hexane must be less than 10 parts per million
Xylene
C8H10
Total Xylenes (m, p, o-xylenes) must be less than 1 part per million.

Testing Method

Residual Solvent testing uses Gas Chromatography (GC) with Flame Ionization Detection (FID) and Headspace Injection.  To conduct this test first a very small sample (50mg) is heated to 140 degrees Celsius for 30 minutes in a sealed environment (the Headspace). This creates a gaseous mixture of residual solvents. The sample gas is fed into a Gas Chromatography (GC) column through the use of a carrier gas such as Helium. The GC column, due to the chemical properties of the column liner, reacts uniquely with each gas present in the mixture. 

Each compound takes a specific amount of time to pass through the column. As the compounds exit the column they are detected by the GC and sent to a computer processing system. The program produces a graph or chromatogram of residual solvents elicited from the column over a period of time. For the peaks of the chromatogram to have quantitative meaning a calibration must be done prior to testing. A residual solvent mixture of known concentration is procured from a company such as Restek, Inc. The sample is diluted to make mixtures of several different concentrations. Each concentration is tested as per the above description to create a calibration curve. The peaks of the sample can than be compared to the calibration curve to determine the actual concentration of each compound in the chromatogram.

Testing Method

Residual Solvent testing uses Gas Chromatography (GC) with Flame Ionization Detection (FID) and Headspace Injection.  To conduct this test first a very small sample (50mg) is heated to 140 degrees Celsius for 30 minutes in a sealed environment (the Headspace). This creates a gaseous mixture of residual solvents. The sample gas is fed into a Gas Chromatography (GC) column through the use of a carrier gas such as Helium. The GC column, due to the chemical properties of the column liner, reacts uniquely with each gas present in the mixture. 

Each compound takes a specific amount of time to pass through the column. As the compounds exit the column they are detected by the GC and sent to a computer processing system. The program produces a graph or chromatogram of residual solvents elicited from the column over a period of time. For the peaks of the chromatogram to have quantitative meaning a calibration must be done prior to testing. A residual solvent mixture of known concentration is procured from a company such as Restek, Inc. The sample is diluted to make mixtures of several different concentrations. Each concentration is tested as per the above description to create a calibration curve. The peaks of the sample can than be compared to the calibration curve to determine the actual concentration of each compound in the chromatogram.

Microbial Testing

Cannabis that is grown outside ideal temperature and humidity conditions can develop mold during growth. Improper conditions during the drying process can also result in mold growth. Common molds found in improperly handled cannabis include Cladosporium, Penicillium, Alternaria, Aspergillus and Mucur. Bacteria associated with cannabis include E.coli, Salmonella and Listeria. Molds produce toxins that can result in serious health concerns including cancer, bronchpulmonary complications, sinusitis and gastrointestinal problems. CannTest, LLC will use a process developed by the U.S. Pharmacopeial Convention and detailed in their US 2021 and USP 61 documents. The U.S. Pharmacopeial Convention (USP) is a scientific nonprofit organization that since 1820 has set standards for the identity, strength, quality, and purity of medicines, food ingredients, and dietary supplements manufactured, distributed and consumed worldwide. Testing utilizing this process will allow ensure that cannabis that has been produced without using sound agricultural practices to prevent the development of mold and bacteria will not make it to the shelves of Alaska’s dispensaries.

The Marijuana Control Board currently requires testing for the following bacteria and fungi.

Microbes

Escherichia coli
E. coli
Escherichia coli (E. coli) bacteria normally live in the intestines of people and animals. Most E. coli are harmless and actually are an important part of a healthy human intestinal tract. However, some E. coli are pathogenic, meaning they can cause illness, either diarrhea or illness outside of the intestinal tract. STEC is a type of E. Coli associated with contaminated cannabis plant. Less than one colony forming unit per gram will be allowed to pass this test. Cannabis with a higher level will not be allowed to be sold in a retail outlet.
Salmonella
Salmonella is of concern because it is able to survive the drying process of cannabis. Salmonella would not survive the heat of smoking, but it has the potential to sicken people through edibles.  Salmonella would be introduced onto cannabis through poor handling processes similar to the outbreaks of salmonella associated with other foods. Less than one colony forming unit per gram will be allowed to pass this test. Cannabis with a higher level will not be allowed to be sold in a retail outlet.
Aspergillus
fumigatus, flavus, niger
These are three species of the fungus Aspergillus. They are extraordinarily resistant to heat, and could survive the heat of smoking or decarboxylation. They are not known to cause disease through the oral route, but the spores can enter the lungs, germinate, and cause invasive lung disease in susceptible individuals. Healthy people have extremely high innate immunity to Aspergillus, however people with compromised immune systems, such as those using cannabis for medical reasons, can be severely effected by this mold. Less than one colony forming unit per gram will be allowed to pass this test. Cannabis with a higher level will not be allowed to be sold in a retail outlet.

Microbes

Escherichia coli
E. coli
Escherichia coli (E. coli) bacteria normally live in the intestines of people and animals. Most E. coli are harmless and actually are an important part of a healthy human intestinal tract. However, some E. coli are pathogenic, meaning they can cause illness, either diarrhea or illness outside of the intestinal tract. STEC is a type of E. Coli associated with contaminated cannabis plant. Less than one colony forming unit per gram will be allowed to pass this test. Cannabis with a higher level will not be allowed to be sold in a retail outlet.
Salmonella
Salmonella is of concern because it is able to survive the drying process of cannabis. Salmonella would not survive the heat of smoking, but it has the potential to sicken people through edibles.  Salmonella would be introduced onto cannabis through poor handling processes similar to the outbreaks of salmonella associated with other foods. Less than one colony forming unit per gram will be allowed to pass this test. Cannabis with a higher level will not be allowed to be sold in a retail outlet.
Aspergillus
fumigatus, flavus, niger
These are three species of the fungus Aspergillus. They are extraordinarily resistant to heat, and could survive the heat of smoking or decarboxylation. They are not known to cause disease through the oral route, but the spores can enter the lungs, germinate, and cause invasive lung disease in susceptible individuals. Healthy people have extremely high innate immunity to Aspergillus, however people with compromised immune systems, such as those using cannabis for medical reasons, can be severely effected by this mold. Less than one colony forming unit per gram will be allowed to pass this test. Cannabis with a higher level will not be allowed to be sold in a retail outlet.

Testing Method

CannTest LLC will use quantitative real-time polymerase chain reaction (qPCR) to conduct testing for bacteria and molds. qPCR is a relatively new method which provides significant advantages over plate culture techniques which had previously been the standard for microbiological testing. Plate culture techniques take up to seven days to produce results, and those results are subject to human interpretation. qPCR produces results in  few hours through using a thermostable enzyme to double the quantity of a short specific part of the target microbe (E-Coli, Salmonella, etc.) DNA in successive heating/cooling cycles. In every cycle the number of short specific sections of DNA is doubled, leading to an exponential amplification of targets. The amplified DNA is fluorescently labeled and the amount of the fluorescence released during amplification can be measured in direct proportion to the amount of amplified DNA. The higher the initial number of DNA molecules in the sample, the faster the fluorescence will increase during the successive qPCR cycles. In other words, if a sample contains more DNA targets, the fluorescence will be detected in earlier cycles.

The number of cycles required to produce a given amount of flourescence is termed quantitation cycle (Ct for short) and is the basic data output unit of qPCR. The lower Ct values mean higher initial copy numbers of the target DNA. CannTest, LLC will use this fast and accurate method of qPCR to perform all required microbiological testing.

Testing Method

CannTest LLC will use quantitative real-time polymerase chain reaction (qPCR) to conduct testing for bacteria and molds. qPCR is a relatively new method which provides significant advantages over plate culture techniques which had previously been the standard for microbiological testing. Plate culture techniques take up to seven days to produce results, and those results are subject to human interpretation. qPCR produces results in  few hours through using a thermostable enzyme to double the quantity of a short specific part of the target microbe (E-Coli, Salmonella, etc.) DNA in successive heating/cooling cycles. In every cycle the number of short specific sections of DNA is doubled, leading to an exponential amplification of targets. The amplified DNA is fluorescently labeled and the amount of the fluorescence released during amplification can be measured in direct proportion to the amount of amplified DNA. The higher the initial number of DNA molecules in the sample, the faster the fluorescence will increase during the successive qPCR cycles. In other words, if a sample contains more DNA targets, the fluorescence will be detected in earlier cycles.

The number of cycles required to produce a given amount of flourescence is termed quantitation cycle (Ct for short) and is the basic data output unit of qPCR. The lower Ct values mean higher initial copy numbers of the target DNA. CannTest, LLC will use this fast and accurate method of qPCR to perform all required microbiological testing.

Terpene Testing

Terpenes are found in a wide variety of plants. They are responsible for the aroma and flavor of the plant. In cannabis they are also responsible for modulating the effect of cannabinoids on receptors within the body. Researchers have dubbed this the “Entourage Effect”. There is still much research that needs to be done to clearly identify the methods of synergistic interaction of these compounds, but for an introduction to this subject please visit this link to a paper in the British Journal of Pharmacology.  Research studies have identified the effects of individual terpenes on human physiology. 

Although testing for terpenes is not currently required by the Marijuana Control Board, CannTest, LLC will offer a test for concentrations of prominent cannabis terpenes listed below. The stand alone effect and where, other than cannabis, the substance is commonly found are also listed. CannTest, LLC will closely monitor research into the “Entourage Effect” to identify additional impacts on the human system so that consumers can maximize understanding of their purchases.

Compounds

Limonene
C10H16
Also found in lemons – Potent immunostimulant, active against acne bacteria, kills breast cancer cells, helps control gastro-oesophageal reflux.
α – Pinene
C10H16
Also found in pine needles – anti-inflammatory, bronchodilatory, aids memory
β – Myrcene
C10H16
Also found in hops – blocks inflammation, analgesic, muscle relaxant.
Linalool
C10H18O
Also found in lavender – anti-anxiety, local anesthetic, anticonvulsant, sedative.
β – Caryophyllene
C15H24
Also found in black pepper – gastric cytoprotective, anti-malarial.
Caryophyllene Oxide
C15H24
Also found in lemon balm – decreases platelet aggregation, antifungal.
Nerolidol
C15H26O
Also found in oranges – sedative, anti-malarial, aids with skin penetration.
Phytol
C20H40O
Also found in green tea – relaxing effect, prevents negative effect of excessive Vitamin A intake.

Terpenes

Limonene
C10H16
Also found in lemons – Potent immunostimulant, active against acne bacteria, kills breast cancer cells, helps control gastro-oesophageal reflux.
α – Pinene
C10H16
Also found in pine needles – anti-inflammatory, bronchodilatory, aids memory
β – Myrcene
C10H16
Also found in hops – blocks inflammation, analgesic, muscle relaxant.
Linalool
C10H18O
Also found in lavender – anti-anxiety, local anesthetic, anticonvulsant, sedative.
β – Caryophyllene
C15H24
Also found in black pepper – gastric cytoprotective, anti-malarial.
Caryophyllene Oxide
C15H24
Also found in lemon balm – decreases platelet aggregation, antifungal.
Nerolidol
C15H26O
Also found in oranges – sedative, anti-malarial, aids with skin penetration.
Phytol
C20H40O
Also found in green tea – relaxing effect, prevents negative effect of excessive Vitamin A intake.

Testing Method

Terpene testing uses Gas Chromatography (GC) with Flame Ionization Detection (FID) and Headspace Injection. To conduct this test first a very small sample (50mg) is heated to 140 degrees Celsius for 30 minutes in a sealed environment (the Headspace). This creates a gaseous mixture of terpenes. The sample gas is fed into a Gas Chromatography (GC) column through the use of a carrier gas such as Helium. The GC column, due to the chemical properties of the column liner, reacts uniquely with each gas present in the mixture. 

Each compound takes a specific amount of time to pass through the column. As the compounds exit the column they are detected by the GC and sent to a computer processing system. The program produces a graph or chromatogram of terpenes elicited from the column over a period of time. For the peaks of the chromatogram to have quantitative meaning a calibration must be done prior to testing. A terpene mixture of known concentration is procured from a company such as Restek, Inc. The sample is diluted to make mixtures of several different concentrations. Each concentration is tested as per the above description to create a calibration curve. The peaks of the sample can than be compared to the calibration curve to determine the actual concentration of each compound in the chromatogram.

Testing Method

Terpene testing uses Gas Chromatography (GC) with Flame Ionization Detection (FID) and Headspace Injection. To conduct this test first a very small sample (50mg) is heated to 140 degrees Celsius for 30 minutes in a sealed environment (the Headspace). This creates a gaseous mixture of terpenes. The sample gas is fed into a Gas Chromatography (GC) column through the use of a carrier gas such as Helium. The GC column, due to the chemical properties of the column liner, reacts uniquely with each gas present in the mixture. 

Each compound takes a specific amount of time to pass through the column. As the compounds exit the column they are detected by the GC and sent to a computer processing system. The program produces a graph or chromatogram of terpenes elicited from the column over a period of time. For the peaks of the chromatogram to have quantitative meaning a calibration must be done prior to testing. A terpene mixture of known concentration is procured from a company such as Restek, Inc. The sample is diluted to make mixtures of several different concentrations. Each concentration is tested as per the above description to create a calibration curve. The peaks of the sample can than be compared to the calibration curve to determine the actual concentration of each compound in the chromatogram.

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