Accurate Quantification of Cannabinoid Acids by GC – Is it Possible?

I think by now we’ve all heard that GC potency testing for cannabis or hemp has some drawbacks. That being said, GC is a popular, rugged, and cost-effective laboratory workhorse and is still employed in many cannabis laboratories. The major drawback of GC versus HPLC cannabinoid testing is the fact that the acidic cannabinoids convert to their neutral form in the GC inlet and cannot be separately quantified. This means that the amount of acidic cannabinoids in a sample cannot be reported by GC, and this result is becoming more and more important with the growing popularity of non-smoked cannabis products.


Cannabinoid Acids Convert to Their Neutral Forms in a Hot GC Inlet

Derivatization Intro Figure 1


While HPLC would be the more straightforward choice for cannabis or hemp potency analysis, some labs only have GC instruments. Joan Serdar at ARRO Laboratories, Inc. is one such analyst. Her lab is equipped with GC instruments, but she wants to accurately quantify both the neutral and acidic forms of cannabinoids in hemp and cannabis – good for you, Joan! In working with Joan, we were able to take a page from the toxicologists’ handbook and derivatize our cannabinoids. What is derivatization, you ask? In a nutshell, derivatization prior to GC analysis involves ‘sticking’ functional groups onto a molecule in order to make it more volatile, more stable, or to improve detectability.

In the case of our cannabinoids, we want to make them more stable so the acidic forms don’t convert to their neutral forms in our GC inlet (see figure above). The derivatization technique we decided to try for this analysis involves N,O-Bis(trimethylsilyl)trifluoracetamide + 1% trimethylchlorosilane (BSTFA + 1% TMCS). This derivatization reagent targets –OH groups and replaces the hydrogen in that group with a trimethylsilyl group, creating an ether that is easier to gas chromatograph than an acid or alcohol.


Hydroxyl (-OH) Groups on Cannabinoids are Derivatized Using BSTFA + 1% TMCS, Making Them Stable in the GC Injection Port

Derivatization Intro Figure 2

So did it work? The preliminary work performed by ARRO Laboratories and here at Restek indicates that this derivatization technique works for all of the cannabinoids of interest for most scientists, resulting in stable, chromatographically-resolved derivatization products. The chromatogram below is a derivatized high-level standard analyzed by my colleague Jack Cochran via GC-FID using the same 15m x 0.25mm x 0.25µm Rxi-35Sil MS column we recommend for underivatized cannabis potency work. The Rxi-35Sil MS had the selectivity needed to separate all of the derivatized cannabinoids. Jack also ran the sample by GC-TOFMS to verify peak IDs and ensure we were getting the derivatization products we hoped to get.


GC-FID Chromatogram of Derivatized Cannabinoid Acids and Neutrals – All Compounds are Resolved on Rxi-35Sil MS

Derivatization Intro Figure 3

So these results look pretty promising! While HPLC is a more straightforward way to measure cannabinoids, the derivatization used here was very simple and shows really good preliminary results. Stay tuned for more discussion of this method – does it work in the presence of matrix? Find out in our next installment (spoiler alert: it does).

5 Responses to “Accurate Quantification of Cannabinoid Acids by GC – Is it Possible?”

  1. Brendon says:

    Would be interesting to get an idea of the derivatization efficiencies. If, for example, the trimethylsilylation step does not go to completion, the non-derivatized cannabinoid acid would decarboxylate, causing error in the neutral cannabinoids. Was there any evidence of incomplete reaction? This is likely to be of particularly concern in the matrix samples, where there may be more competition, especially in sugar-rich edibles.

  2. Hi Brendon,

    Excellent question! You’re absolutely right that derivatization efficiencies will be affected by the presence of matrix since there are plenty of compounds in plant (and edible) matrices that can be derivatized. We have looked into the derivatization efficiency in the presence of plant matrix, and that is going to be the topic of my next blog. I don’t want to throw out any spoilers, but preliminarily, the efficiency is looking good. You should see the next installment of the blog sometime next week for more details.

    Our next step for this work is to explore derivatization efficiency in the presence of food matrix. I was thinking of doing a simple QuEChERS extraction and dSPE cleanup on a vending machine brownie, then spiking and derivatizing that extract. I figure brownie matrix has plenty of both sugar and fat to compete for derivatization reagent. If you have any ideas for further experiments or alternate matrices, I’d love to hear them!

    Thanks for commenting!

  3. Brendon says:

    Hi Amanda,

    Great to hear that derivatization efficiency will be covered! I’ll look forward to the post.

    One idea for derivatization efficiency that I’ve seen applied is using a diol to estimate the completion of derivatization. Basically, the proportion of 2TMS to 1TMS indicates whether derivatization was complete, or whether the reagent was quenched by matrix, etc., especially relevant for hindered groups which react more slowly in the SN2 reaction. The brownie sounds like it should be an interesting matrix, good thing for QuEChERS to help clean it up! Another matrix that may be interesting is lipid products, such as cooking oils and butters, in which the degree of decarboxylation would be good to know, and which may have some interesting matrix effects.

    Thanks for the great posts!

  4. Hi Brendon,

    Thank you for your feedback! Outside opinions are always wonderful for the thought process in setting up an experiment.

    The diol is a good idea. I was thinking that since THCA and CBDA (not to mention the neutral analytes) have multiple derivatization sites, I’d see something similar, especially since CBDA has three derivatization sites, two of which are hindered due to the position of one of the hydroxyl groups in relation to the carboxylic acid. That being said, your point that it might be hard to differentiate underivatized acids due to decarboxylation in the inlet is very salient. You’ll see in my next post that I measured the effect of matrix on derivatization efficiency by performing a linearity experiment in cannabis matrix using multiple dilutions of the sample, therefore if matrix were quenching the reaction, linearity would be lost. That being said, if the reaction is going almost to completion at high matrix levels, the linearity might not suffer too much, although I think we’d start seeing CBDA-2TMS starting to pop up first. Perhaps the most thorough test would be to look at the analytes separately – do an experiment with only acids, an experiment with only neutrals, then finally a linearity experiment with the whole mix, being sure to keep the molar concentration of analytes the same (meaning high) in all three experiments. It would be more work, but I think it would give a definitive answer. What do you think?

    Thanks again for jump-starting my brain!

  5. Brendon says:

    Hi Amanda,

    Thanks, it’s always fun to have other applications to think about. I’m mostly a petroleum fuels experimentalist, so switching gears over to other complex biological samples is enjoyable.

    I like the idea of the linearity experiment. It would be particularly interesting to see the slope of the neutrals. Basically, the idea being that as the reaction quenches as the dilution factor decreases, the underivatized cannabinoid acid will decarboxylate in the inlet and bias the cannabinoid neutral concentration. If for example, the original sample had a 50:50 ratio of THC-A and THC, an 80% derivatization efficiency of the THC-A’s carboxylic acid moiety, assuming complete decarboxylation in the inlet, would bias the THC measurement by 20%, and the ratio would instead be determined to be 40:60. Analyzing only the acids would be a surefire way to determine the ultimate amount of neutral cannabinoids produced either from incompletely-derivatized acid cannabinoids, or from the (unlikely?) breakdown of TMS-ester. Hooray for natural product testing; I like to think the field’s challenges helps keep us chromatographers employed!

    Have a great weekend!

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