Standards Used in Testing

[Green Supreme]

So I see all these people so firm in their stance for one testing facility or the other. My questions would be, where did these places acquire the standards to test against? Especially those in the US. Can we really believe these tests? Just wondering how so many folks can put faith in such things. Standards are not easy to come by. Peace GS

ps. apologies if this is in the wrong forum, if so feel free to move

 

[Gray Wolf]

I just glimpsed this post and not only applaud the question, but have some related questions to add. Please excuse my ignorance on any really dumb ones!

As a manufacturing engineer, I selected and installed automatic GC analysis equipment, and without exception, the automated equipment I installed, required a standard signature programmed into them, to be able to sort out the results from the test.

In keeping with that limited experience, my negotiations with a local lab for shared research fell through as the details unfolded, because they also need standards. When I asked for it to be tested for THC, CBD, and CBN, there was no problem and the owner proudly noted that he belonged to an association and they compared and shared standards.

The problem began when I asked if he could also do THCA and he said he would need another standard and then I started talking about the other cannabinoids, turpenoids and residual solvent, and contaminants in the solvent, and pesticides, and very quickly all my shared research plans fell apart from unattractive costs and lack of standards. He was willing to do anything I wanted, but it required another test and or standard, that I specifically had to specify, if I wanted it done.

I am especially interested in their standards for all the turpenoids known to be present in cannabis, as well as what contaminants they automatically check for.

I would also be interested in how many GC samples they run to positively identify and measure constitutes in the low PPM and PPB range?

Any of ya'll experts on GC and HPLC care to share your thoughts on the above?

 

[Rusty420]

sam skunkman will know..

 

[Green Supreme]

Ya GW, you see where I am going. Peace GS

 

[Green Supreme]

Well I seriously thought this would inspire more debate. Where are all those test braggers now? Peace GS

 

[highonmt]

Well I seriously thought this would inspire more debate. Where are all those test braggers now? Peace GS

Here
http://www.usp.org/referenceStandards/
Probably checking out the reference standards provided by the dorky chick in the hair net. Seriously you just purchase standards and run a standard then run your tests followed by another run of the standard to verify. Without a standard of known concentration quantification is very difficult. With a standard you just calculate the area under the peak of the standards and then compare it to the peak of your analyte. Retention times of the standards tell you what is coming off the column...easy as pie no need for debate..HM

 

[Green Supreme]

Still wondering where these places everyone banks on, like Steephill get their standards. Peace GS

 

[Gray Wolf]

Here
http://www.usp.org/referenceStandards/
Probably checking out the reference standards provided by the dorky chick in the hair net. Seriously you just purchase standards and run a standard then run your tests followed by another run of the standard to verify. Without a standard of known concentration quantification is very difficult. With a standard you just calculate the area under the peak of the standards and then compare it to the peak of your analyte. Retention times of the standards tell you what is coming off the column...easy as pie no need for debate..HM

Thanks for the link brother HOM; I will check their product line out.

Do you have any insight into the rest of the above questions?

 

[Gray Wolf]

Yup, OK I did; have you read their catalog? It doesn't appear to include cannabinoids or any of the principal turpenoids?

 

[legalizeDK]

GW i know i litle bit about ms maybe i can help but i am unsure of waht you want to know

 

[Gray Wolf]

GW i know i litle bit about ms maybe i can help but i am unsure of waht you want to know

Thanks bro!! I was hoping for a champion!

Hope this doesn't come across as asking you to take a drink from a fire hose, held by an inquisitive, but dumb six year old!

My rambling questions are in more detail in post #2 of this thread, but mostly relate to what standards are available and are actually being used by the different labs analyzing and comparing numbers.

In summary, they are as follows:

1. Where are the standards for cannabinoids and turpenoids coming from? I didn't see them listed in the standard source offered.

2. How is the test equipment certified, so as to be precisely duplicate-able lab to lab?

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?

4. Where would those standards come from?

5. Do all labs have the full arsenal of standards?

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?

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?

8. What is the next winning mega bucks lottery ticket number?

 

[GreenintheThumb]

Full Spectrum Labs admitted to me their calibration standards were invalid and recalled. They chose to continue to use them. And we wonder why everything's testing near 30% THC in Colorado?

 

[GreenintheThumb]

sam skunkman will know..

I'm pretty sure sam publicly said he had to make his own standards...but that was a long time ago, perhaps it's easier to get accurate standards these days...

 

[Gray Wolf]

Sooo, don't know how busy the clever lad is, but sent him a PM drawing this thread to his attention.

 

[Skinny Leaf]

In the catalog of standards by USP it has a standard for THC. The catalog number is 1651621. I didn't look for any other stuff. They are expensive for their products. This would have to be the place where some labs get there standards if possible. I am just interested in the topic and really have no knowledge of GC. The catalog lists thc for 1ml at $232. If USP is sending a standard sample of thc to someone wouldn't that require the DEA getting involved? Does the lab have to have a certification by some narco policing agent to get this standard or any standard listed as a controlled substance? I don't think I could buy the standard and have it sent to the house. Or am I misinterpeting the standards catalog?

 

[Chimera]

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

 

[Chimera]

Let's knock these questions out and then we'll get back to more details.

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.

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.

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-techniques/mass-spectrometry-ms/headspace-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.

4. Where would those standards come from?

>> See above, scientific supply companies, or a pharmaceutical firm.

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.

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.

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.

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

 

[Chimera]

I'll take this one too thanks

 

[northstate]

Very interesting topic I have wondered how standards in this industry were made and just how accurate the labs really are. IC is so great for de-bunking the hype with proof and knowledge! NS

 

[Chimera]

I'm going to gobble another few posts up in this thread, and will delete them later if they are not used... thanks for your patience.