Green Supreme
12-23-2011, 07:20 PM
Ok I have started a thread on ICmag about standards and testing. It is going off with some pretty good info from a few educated folks. Looks like it could go deeper too. Gonna cut and paste some Chimera posts then will link the thread. Peace GS
Allow me to contribute, with the first statement being I am far from an expert in the field of analytical Chemistry. I am however beginning to work with a American PhD Chemist, who I consider to be one of the finest minds in our industry, and we have spoken at great detail about the issue so I can offer a little bit of insight.
This is a complicated issue, so I'll probably end up trying to address it in a few posts. Any experts in Analytical Chemistry please correct my mistakes, there are sure to be some since my understanding of the matter is not altogether as deep as it should be... gimme a break I fell under the Biology umbrella, and didn't put as much emphasis into Chemistry as I now realize I should have.
All excellent questions, and a smart and relevant topic GS.
We should all agree and understand that ALL cannabinoids, in the USofA and Canada, are restricted chemicals or controlled substances. People or entities (private labs) simply cannot possess the chemicals without a proper licence. This means if you want to order them from a chemical supply company, or a scientific supply company like Sigma Aldrich, you need a specific licence which they will request a copy of prior to processing your order. They must fulfill this obligation, or the DEA would be all over them. Any breach would cost them the ability to provide these chemicals for any client, so they are not going to jeopardize their business by illegally shipping you cannabinoid standards.
GW has hit the nail on the head with a few things here, one of them being that the labs he tried to contract with were not able to obtain standards of THCA, and only had THC, CBD and CBN. Tip 'o the iceberg, as it were.
Is this the end of the world? Well in a way yes it is really... because it makes all of the results questionable, and as he stated so eloquently it eliminates the ability to compare results from one lab to another- there is simply no external validity to these tests, for a whole variety of reasons (more on that later).
So without pure chemical standards of each cannabinoid or chemical compound, how do we accurately identify each peak on the chromatogram as corresponding to a single identifiable compound? Can we?
Groups of scientists have formed electronic databases, with electronic GC chromatograms of thousands of unique chemical compounds, such as the NIST 11 Mass Spectral Library http://www.sisweb.com/software/ms/nist.htm . Molecules (standards) are run through a known GC and known column, and the results are placed into an electronic database against which further GC results can be compared. Some GC computers have modems that automatically compare the result against this type of database, which 'eliminates' the need for labs to purchase thousands and thousands of standards.
However, this is problematic because different GCs operate a little bit differently, and there are many types of columns- and this variation in equipment results in slightly different retention times for varying compounds. Also, many compounds, especially in 'new' areas of interest such as cannabis, the chemicals are simply not commonly known, are unavailable (it costs too much too fabricate a compound that only 2 people will buy), or are simply not listed in the database.
For this reason, it's really important (moreover, necessary) to have a pure sample of the known chemical to run through your GC and column with the internal standard (usually squalene), to identify the unique signature of your compound, and create a literal 'standard' against which future runs through your GC/column setup can be compared. Also the NIST-like databases are a good approximation, and may in many cases lead to proper identification of the compounds of interest, but they are not entirely %100 correct and can mis-identify molecules that have similar retention times in most columns. I suspect that this is really common in cannabis analytics, because CBD and CBC have incredibly similar retention times with most columns, the peaks are almost indistinguishable and one is often mis-identified as the other. Put that in your pipe and smoke it.
to be continued....
-Chimera
next post
Let's knock these questions out and then we'll get back to more details.
Quote:
Originally Posted by Gray Wolf View Post
1. Where are the standards for cannabinoids and turpenoids coming from? I didn't see them listed in the standard source offered.
a) Terpenes, not turpenes.
b) Cannabinoids and terpene standards are available from scientific supply firms like Sigma Aldrich, Fisher Scientific etc. You can look around and you'll find firms that supply most of the cannabinoids, but very few firms are selling pure cannabinoid standards for the entire series of major cannabinoids, let alone the more rare minor cannabinoids. Terpenes are available from scientific supply companies, and are not controlled substances- that is not to say placing an order for them might not place your name on a flagged list... who knows.
I checked out the link posted above by highonmt, and the only cannabinoid they have a standard for is THC.
This THC standard, if you read the description, is a 99.9% methanol 0.1% THC standard. This is problematic, for reasons we will adress later.
Quote:
Originally Posted by Gray Wolf View Post
2. How is the test equipment certified, so as to be precisely duplicate-able lab to lab?
This one is easy! It's not certified. There is no certification body regulate or to standardize the protocols amongst the different labs, although an association has been formed in California between some of the labs to address some of these issues. Currently, however, there is no standard protocol that labs follow, no requirements for cannabinoid standardization, no internal or external validity within or between the different labs. Your results are only as good as your lab. So in order to qualify your results, you have to know how accurate your lab is.
Quote:
Originally Posted by Gray Wolf View Post
3. If I want to know what all of the individual cannabinoids, turpenoids, residual solvents, residual solvent impurities, aflatoxins, pesticides, et al that are in a sample down to the low parts per millionth or high parts per billionth level, how many test would I have to run to positively indentify their presence and precise levels?
Once your lab has received all the necessary standards and properly calibrated the equipment, they would need to run your sample(s) to determine the quantities all of the cannabinoids present, and then disassemble the column from the suitable GC apparatus, recalibrate it with a new column for the terpene tests (it requires a different column) and run a duplicate sample of the same cannabis for the terpene assay. Again, the equipment should be calibrated against all the known terpenes that are found in cannabis. The terpene setup is known as Headspace GC/MS. As far as I understand it (and I could be wrong) some types of GC/MS are not able to perform Headspace GC/MS. This whole recalibration/ column change is not a small process, so labs are going to be hesitant to change it over, and if they do they'll charge you a necessary fee to compensate for their time.
GW if you are thinking what I am thinking, it might be possible to determine if hydrocarbons are still present in oils/extracts via headspace GC/MS... but that is really nothing more than an uneducated guess, and may or may not be possible. I will confer with my colleague and get back to you on that one. It seems plausible however.
On Headspace:
"Headspace gas chromatography/mass spectrometry (Headspace GC/MS) is a specific GC/MS technique used to analyze volatile compounds. A sample is placed in a closed sampling vessel; heated using a known temperature profile, and the vapor in the vessel is sampled for analysis." http://www.polymersolutions.com/hgcms.html
"Headspace GC-MS (HS/GC-MS) is used for the analysis of volatile organic compounds in solid and liquid samples, such as residual solvents and hydrocarbons."
http://www.m-scan.com/analytical-tec...adspace-gc-ms/
I know of only one lab in the country that has the equipment to run cannabinoid and terpene analysis simultaneously, but they do not use a standard $30- 50,000 GC/MS equipment, and are not a public lab. It is 'possible' that I could arrange for your samples to be run, but they would have to be grown in and for medicinal use in California. Even then, it would likely not be cheap.
~~~~~
On Pesticides:
Pesticides, fungicides etc are a HUGE class of compounds and are very difficult to identify by GC/MS. There are literally hundreds if not thousands of pesticides- so without knowing what you are screening for, it is near impossible to conclusively say that there are no pesticides present. You may be able to rule out a specific pesticide, but the reality is no testing facility is able to determine that the herbs they test are in fact pesticide free.
The presence of fungi, and thus the potential for aflotoxins, are typically detected by visual inspection under a scope, and not analyzed by GC. Labs may also use agar plating techniques for microbiological screening.
Quote:
Originally Posted by Gray Wolf View Post
4. Where would those standards come from?
>> See above, scientific supply companies, or a pharmaceutical firm.
Quote:
Originally Posted by Gray Wolf View Post
5. Do all labs have the full arsenal of standards?
>> Nope. I'd be surprised if to hear that more than a couple of the labs currently operating in the US have a full complement of standards. I know they are available via a firm in Europe, however importation of these compounds is a federal offense, so the only way to get them would be to smuggle them. I have heard through the cannabinoid grapevine that some standards have been smuggled in clandestinely- not via my colleague, but by an individual from another lab. I do not know if said lab has a full complement of the cannabinoid standards however.
Quote:
Originally Posted by Gray Wolf View Post
6. How many verification tests are typically run to verify trace elements in the low parts per millionth, or billionth range, and do you test for those items routinely in every sample?
>> That I simply do not know, sorry.
Quote:
Originally Posted by Gray Wolf View Post
7. How are the more complex alkenes and aromatic benzene ring molecules interpreted by the GS software, if it has no programmed standard for it?
>>Again, beyond my level of comprehension of the subject, being trained in genetics and neuroscience, not chemistry. I would assume the more common chemicals could be potentially identified through a database such as NIST. Can't offer anything else on the matter other than that though.
Quote:
Originally Posted by Gray Wolf View Post
8. What is the next winning mega bucks lottery ticket number?
>> That one is easy, but I'm going to keep that secret and harvest the funds myself to fund future research. Look for the guy with a big phatty in his mouth and an oversized cheque.
Some additional background reading on Gas Chromatography for those interested in understanding the background concepts in a little more detail.
http://en.wikipedia.org/wiki/Gas_chromatography
-Chimera
https://www.icmag.com/ic/showthread.php?t=227711
Allow me to contribute, with the first statement being I am far from an expert in the field of analytical Chemistry. I am however beginning to work with a American PhD Chemist, who I consider to be one of the finest minds in our industry, and we have spoken at great detail about the issue so I can offer a little bit of insight.
This is a complicated issue, so I'll probably end up trying to address it in a few posts. Any experts in Analytical Chemistry please correct my mistakes, there are sure to be some since my understanding of the matter is not altogether as deep as it should be... gimme a break I fell under the Biology umbrella, and didn't put as much emphasis into Chemistry as I now realize I should have.
All excellent questions, and a smart and relevant topic GS.
We should all agree and understand that ALL cannabinoids, in the USofA and Canada, are restricted chemicals or controlled substances. People or entities (private labs) simply cannot possess the chemicals without a proper licence. This means if you want to order them from a chemical supply company, or a scientific supply company like Sigma Aldrich, you need a specific licence which they will request a copy of prior to processing your order. They must fulfill this obligation, or the DEA would be all over them. Any breach would cost them the ability to provide these chemicals for any client, so they are not going to jeopardize their business by illegally shipping you cannabinoid standards.
GW has hit the nail on the head with a few things here, one of them being that the labs he tried to contract with were not able to obtain standards of THCA, and only had THC, CBD and CBN. Tip 'o the iceberg, as it were.
Is this the end of the world? Well in a way yes it is really... because it makes all of the results questionable, and as he stated so eloquently it eliminates the ability to compare results from one lab to another- there is simply no external validity to these tests, for a whole variety of reasons (more on that later).
So without pure chemical standards of each cannabinoid or chemical compound, how do we accurately identify each peak on the chromatogram as corresponding to a single identifiable compound? Can we?
Groups of scientists have formed electronic databases, with electronic GC chromatograms of thousands of unique chemical compounds, such as the NIST 11 Mass Spectral Library http://www.sisweb.com/software/ms/nist.htm . Molecules (standards) are run through a known GC and known column, and the results are placed into an electronic database against which further GC results can be compared. Some GC computers have modems that automatically compare the result against this type of database, which 'eliminates' the need for labs to purchase thousands and thousands of standards.
However, this is problematic because different GCs operate a little bit differently, and there are many types of columns- and this variation in equipment results in slightly different retention times for varying compounds. Also, many compounds, especially in 'new' areas of interest such as cannabis, the chemicals are simply not commonly known, are unavailable (it costs too much too fabricate a compound that only 2 people will buy), or are simply not listed in the database.
For this reason, it's really important (moreover, necessary) to have a pure sample of the known chemical to run through your GC and column with the internal standard (usually squalene), to identify the unique signature of your compound, and create a literal 'standard' against which future runs through your GC/column setup can be compared. Also the NIST-like databases are a good approximation, and may in many cases lead to proper identification of the compounds of interest, but they are not entirely %100 correct and can mis-identify molecules that have similar retention times in most columns. I suspect that this is really common in cannabis analytics, because CBD and CBC have incredibly similar retention times with most columns, the peaks are almost indistinguishable and one is often mis-identified as the other. Put that in your pipe and smoke it.
to be continued....
-Chimera
next post
Let's knock these questions out and then we'll get back to more details.
Quote:
Originally Posted by Gray Wolf View Post
1. Where are the standards for cannabinoids and turpenoids coming from? I didn't see them listed in the standard source offered.
a) Terpenes, not turpenes.
b) Cannabinoids and terpene standards are available from scientific supply firms like Sigma Aldrich, Fisher Scientific etc. You can look around and you'll find firms that supply most of the cannabinoids, but very few firms are selling pure cannabinoid standards for the entire series of major cannabinoids, let alone the more rare minor cannabinoids. Terpenes are available from scientific supply companies, and are not controlled substances- that is not to say placing an order for them might not place your name on a flagged list... who knows.
I checked out the link posted above by highonmt, and the only cannabinoid they have a standard for is THC.
This THC standard, if you read the description, is a 99.9% methanol 0.1% THC standard. This is problematic, for reasons we will adress later.
Quote:
Originally Posted by Gray Wolf View Post
2. How is the test equipment certified, so as to be precisely duplicate-able lab to lab?
This one is easy! It's not certified. There is no certification body regulate or to standardize the protocols amongst the different labs, although an association has been formed in California between some of the labs to address some of these issues. Currently, however, there is no standard protocol that labs follow, no requirements for cannabinoid standardization, no internal or external validity within or between the different labs. Your results are only as good as your lab. So in order to qualify your results, you have to know how accurate your lab is.
Quote:
Originally Posted by Gray Wolf View Post
3. If I want to know what all of the individual cannabinoids, turpenoids, residual solvents, residual solvent impurities, aflatoxins, pesticides, et al that are in a sample down to the low parts per millionth or high parts per billionth level, how many test would I have to run to positively indentify their presence and precise levels?
Once your lab has received all the necessary standards and properly calibrated the equipment, they would need to run your sample(s) to determine the quantities all of the cannabinoids present, and then disassemble the column from the suitable GC apparatus, recalibrate it with a new column for the terpene tests (it requires a different column) and run a duplicate sample of the same cannabis for the terpene assay. Again, the equipment should be calibrated against all the known terpenes that are found in cannabis. The terpene setup is known as Headspace GC/MS. As far as I understand it (and I could be wrong) some types of GC/MS are not able to perform Headspace GC/MS. This whole recalibration/ column change is not a small process, so labs are going to be hesitant to change it over, and if they do they'll charge you a necessary fee to compensate for their time.
GW if you are thinking what I am thinking, it might be possible to determine if hydrocarbons are still present in oils/extracts via headspace GC/MS... but that is really nothing more than an uneducated guess, and may or may not be possible. I will confer with my colleague and get back to you on that one. It seems plausible however.
On Headspace:
"Headspace gas chromatography/mass spectrometry (Headspace GC/MS) is a specific GC/MS technique used to analyze volatile compounds. A sample is placed in a closed sampling vessel; heated using a known temperature profile, and the vapor in the vessel is sampled for analysis." http://www.polymersolutions.com/hgcms.html
"Headspace GC-MS (HS/GC-MS) is used for the analysis of volatile organic compounds in solid and liquid samples, such as residual solvents and hydrocarbons."
http://www.m-scan.com/analytical-tec...adspace-gc-ms/
I know of only one lab in the country that has the equipment to run cannabinoid and terpene analysis simultaneously, but they do not use a standard $30- 50,000 GC/MS equipment, and are not a public lab. It is 'possible' that I could arrange for your samples to be run, but they would have to be grown in and for medicinal use in California. Even then, it would likely not be cheap.
~~~~~
On Pesticides:
Pesticides, fungicides etc are a HUGE class of compounds and are very difficult to identify by GC/MS. There are literally hundreds if not thousands of pesticides- so without knowing what you are screening for, it is near impossible to conclusively say that there are no pesticides present. You may be able to rule out a specific pesticide, but the reality is no testing facility is able to determine that the herbs they test are in fact pesticide free.
The presence of fungi, and thus the potential for aflotoxins, are typically detected by visual inspection under a scope, and not analyzed by GC. Labs may also use agar plating techniques for microbiological screening.
Quote:
Originally Posted by Gray Wolf View Post
4. Where would those standards come from?
>> See above, scientific supply companies, or a pharmaceutical firm.
Quote:
Originally Posted by Gray Wolf View Post
5. Do all labs have the full arsenal of standards?
>> Nope. I'd be surprised if to hear that more than a couple of the labs currently operating in the US have a full complement of standards. I know they are available via a firm in Europe, however importation of these compounds is a federal offense, so the only way to get them would be to smuggle them. I have heard through the cannabinoid grapevine that some standards have been smuggled in clandestinely- not via my colleague, but by an individual from another lab. I do not know if said lab has a full complement of the cannabinoid standards however.
Quote:
Originally Posted by Gray Wolf View Post
6. How many verification tests are typically run to verify trace elements in the low parts per millionth, or billionth range, and do you test for those items routinely in every sample?
>> That I simply do not know, sorry.
Quote:
Originally Posted by Gray Wolf View Post
7. How are the more complex alkenes and aromatic benzene ring molecules interpreted by the GS software, if it has no programmed standard for it?
>>Again, beyond my level of comprehension of the subject, being trained in genetics and neuroscience, not chemistry. I would assume the more common chemicals could be potentially identified through a database such as NIST. Can't offer anything else on the matter other than that though.
Quote:
Originally Posted by Gray Wolf View Post
8. What is the next winning mega bucks lottery ticket number?
>> That one is easy, but I'm going to keep that secret and harvest the funds myself to fund future research. Look for the guy with a big phatty in his mouth and an oversized cheque.
Some additional background reading on Gas Chromatography for those interested in understanding the background concepts in a little more detail.
http://en.wikipedia.org/wiki/Gas_chromatography
-Chimera
https://www.icmag.com/ic/showthread.php?t=227711