Accurate Quantification of Cannabinoid Acids and Neutrals by GC – Derivatives without Calculus

Derivatization is a widely-used technique for GC sample preparation across many industries and in widely varied matrices from soil to plastics to blood that is used to make polar and active compounds more amenable to good GC analysis. If you’re careful about testing your derivatization procedure during method development, you can be confident that you’ll have a reproducible method that can vastly improve the quality of your GC results. While derivatization does require some extra sample handling, the procedure I developed for cannabis plant matrix is very straightforward and easy to perform:

Derivatization Procedure for Cannabis and Hemp Plant Matrices:

  • Place 100µL of plant extract into a 1mL Micro-Vial
  • Evaporate to dryness at 50°C under a gentle stream of nitrogen
  • Add 50µL ethyl acetate and 50µL BSTFA + 1% TMCS
  • Incubate at 70°C for 30 minutes
  • Cool and dilute with ethyl acetate if desired

In my last blog, I introduced the concept of derivatization for use in cannabis or hemp cannabinoid testing. Once acidic cannabinoids are derivatized, they no longer break down in the GC inlet and can be quantified separately from the neutral cannabinoids. I demonstrated this through derivatization of high-level solvent standards, but work with solvent standards is a far cry from matrix work, which means the procedure needed to be tested in matrix. To kick off the matrix test, I spiked an extract with the most common cannabinoids of interest, derivatized it using the procedure listed above, and my colleague, Jack Cochran, analyzed it via GC-FID with our Rxi-35Sil MS GC column. We can see that we have a beautiful chromatogram with all of the derivatized cannabinoids separated, and very little matrix interference.

Big Mix

In addition to confirming that all derivatization sites are indeed derivatized by analyzing the standards with GC-MS (this is shown in my last blog), we also tested derivatization efficiency using a cannabis extract previously generated at Penn State University with the help of Professor Frank Dorman and a Police Officer Specialist. Because derivatization is a chemical reaction, the derivatization reagent gets used up during the derivatization reaction. Because plant matrix contains many other derivatizable compounds like sugars and sterols, these other compounds may compete for the derivatizing reagent, possibly resulting in the reagent getting used up before all of our analytes of interest can be derivatized.

So how can we be sure our derivatization is going to completion in the presence of matrix? There are a couple things we can do, the first of which is really simple. We can see in our procedure that we use a hefty 50µL of derivatizing reagent per 100µL of cannabis extract. We know that our extract contains a lot less than 50mg of plant matrix, not all of which is derivatizable. This means that by adding 50mg of BSTFA per 100µL of sample, we can be confident that we have a significant excess of derivatizing reagent as compared to derivatizable groups in our sample. Excess derivatizing reagent means that it will never be completely used up, ensuring the reaction will go to completion no matter what.

A more quantitative way to test derivatization efficiency in a matrix where you can’t get blanks is to evaluate analyte linearity with differing amounts of matrix. For example, if you derivatize four THCA-containing samples prepared using 10, 20, 50, and 100µL of cannabis extract and plot the area of THCA versus sample amount, you should end up with a straight line if your derivatization is going to completion. If it’s not, then you’ll likely see THCA area fall off for the samples containing more matrix since the derivatization reagent is being used up before all the analyte in the higher matrix level sample is derivatized. To test our procedure, we did just that. We can see that our linearity looks beautiful for all of the cannabinoids, indicating the derivatization does indeed go to completion.

Deriv Linearity2

CBDA Cal Curve
In addition to verifying that the derivatization reaction goes to completion in the presence of plant matrix, we also verified the procedure using several different samples which were generated at the same time as the sample shown in the figure above. Our preliminary work is still looking good, which is exciting, but what about all of the other matrices cannabis chemists have to work with? Well, we’re planning on moving the work forward into edible matrices next, so stay tuned for an update!

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