With so many different types of samples being sent to cannabis testing labs, is it realistic to expect a single sample extraction technique to work for all of them? Join Justin Steimling, Restek’s LC Applications Laboratory Manager, as he discusses some considerations that go into choosing a sample extraction method based on the nature of the sample being analyzed.
And check out the analysis of different sample matrices for the presence of cannabinoids, with sample preparation information provided in the notes for each chromatogram:
- Cannabis Concentrate (HPLC-UV)
- Cannabis Concentrate (UHPLC-UV)
- Cannabis Flower (UHPLC-UV)
- Cannabis Flower (UHPLC-UV)
- Cannabis Chocolate (UHPLC-UV)
- Cannabis Hard Candy (UHPLC-UV)
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Hello, my name is Justin Steimling. I’m the LC applications manager at Restek.
Question: What's one key to a successful cannabis potency analysis and why?
I think one of the most important aspects of doing cannabis testing, particularly potency determination, is the sample preparation itself. And the reason that I say sample preparation is so important is just the fact that there are so many cannabis matrices out there that it really makes it difficult to just use one method for everything.
Question: How do you match sample prep to the sample?
I think that matching an extraction procedure to a sample is probably the most important thing that you consider when you’re doing method development because you want to consider the composition of the sample, and you want to be able to use that to drive purposeful method development. And what I mean by that… When you have samples like oils which are high in fats, you have edibles which can be high in carbohydrates.
And the way that you actually do the preparation for those two different things can be completely different because you have to consider solubility, if there’s anything that is added to the sample as a formulation in something like a balm or a lotion. And there’s also things to consider when you’re doing certain types of edibles like chocolate for example. Chocolate contains a lot of carbohydrates, but it also contains a lot of lipids. So, just being aware of the sample composition that you’re trying to analyze is incredibly important because that’s really what drives purposeful method development.
Question: Can you give us some examples?
So, the variety of the samples itself contributes to the extraction process because you have to consider how you’re actually going to get the most efficient extraction of cannabinoids from your sample. Take for instance you have something like gummy bears or a hard candy composite, that’s something that’s mainly compromised of sugars. Well, I could homogenize that sample and extract with an organic solvent, and I could see cannabinoids. But I might not get the best extraction efficiency that way. So, a strategy to handle that particular sample would be to dissolve the actual matrices into something like water, so I fully solubilize the sample. And then I extract it with an organic solvent. There are other sample types such as chocolates and lotions where you can take a different type of approach. For example in chocolates, it’s actually really effective to take the sample and heat it up slightly. So, you just melt it down so the sample is essentially homogenized. And once you put that into an organic solvent, you can be sure that you’re doing a very efficient extraction.
Question: Let’s talk some more about homogenization. It’s a pretty important step.
Ensuring that your sample is homogenous is super important because you want to make sure that what you are going to report as your result is actually representative of the entire sample. Depending on where you sample, you might find that in a cannabis flower, you might have a high concentration in one area, particularly in the trichome’s, and a low concentration closer to the leaves and the stem.
As far as mechanical homogenization is concerned, when you’re working with the cannabis flower itself, a good technique is just to use a blender. But if you’re going to use a blender, I have found that it’s really effective if you pair that with the use of dry ice because one of the things that’s nice about using a blender and dry ice is that you’ll produce a sample that is not only homogenous, but the particle size is nice and small. The sample is free flowing, and it’s very easy to work with.
You can also use something such as a ball mill if you’re looking at homogenizing your flower. One of the things that is difficult when you’re working with a ball mill is that some of the trichome’s can actually stick to the ball. So, you want to make sure that you rinse that off before you proceed with your extraction.
Question: Okay, once we have our extract, how about cleanup?
Once you’ve extracted your sample, now you have to consider different types of clean ups that you’re going to have to do post extraction. And it really depends on your sample type. If you are extracting something like a gummy bear, odds are you don’t really have to do a whole lot of cleanup because that sample is going to be pretty clean. But if you do an extraction…again, referencing the chocolate bar. Now you’re going to have a lot of dissolved fats in that sample, and you want to tailor your post extraction clean up to the particular matrix. And the example of the chocolate bar, something that is actually particularly effective is if you take the sample, put it in the freezer for 30 minutes, and let all the lipids actually crash out. And in the industry, that’s known as winterizing, which is effectively a temperature induced precipitation. But if you do these sorts of strategies, you’ll find that the sample cleanup that you do now will actually be beneficial when you move the sample analysis to the instruments itself.
Question: How do we know if our extraction was efficient?
Determining how effective your sample extraction and clean up was is really the million dollar question in this industry. There are a number of ways that you can actually go about determining the efficiency of your extraction. One of the easy ways to do it is if you have a sample that doesn’t actually solubilize into your extraction solvent. And this is something more like a flower. You can actually go back and re-extract. And if you’re finding that in your re-extraction, you’re pulling more cannabinoids into your extraction solvent, odds are you need to rework your extraction solvent and your extraction itself in order to make sure that you’re doing a more efficient extraction.
Question: What if your sample dissolves in the extraction solvent?
There are still strategies available if your sample does dissolve in the extraction solvent. One of the most popular methods is to consider doing standard addition. There is a problem with doing standard addition in the fact that the reference standards that you can get that are DEA exempt are actually pretty low in concentration compared to the actual cannabis products or cannabis itself that you’re going to be testing.
One of the best ways to handle the low concentration of standard compared to the cannabis product itself is to actually scale down the size of your extraction. A lot of times, you’re going to be working with a standard solution that is around one mg per milliliter. So, you know that for every milliliter you have, you have one milligram. So, you can only work with so much additional matrix on top of that in order to make the actual concentrations relevant. So, if you scale down your actual extraction, you will find that you’re able to create a more potent sample that is actually…that shows a great disparity between the original sample and the post spike sample. So, you’ll really be able to evaluate the efficiency of your extraction via standard addition.
if you originally start out with one gram of sample, there’s no reason that you can’t scale it down to 0.1 grams as long as you’re also matching and scaling down the extraction solvent, going from 10 mLs down to 1 mL. It will show that your extraction efficiency is still performing the same. It’s just performed on a lot smaller scale.
Question: Thanks, Justin! Any final thoughts?
I really feel for all these cannabis testing labs that have to deal with these wide variety of matrices. I understand that you get a lot of samples and you don't really have a whole lot of time, but the good news is that it doesn't actually take a whole lot of time to do extraction efficiency experiments, and what it costs in time it certainly adds more in value. I hope you found this Q&A session to be helpful. Please feel free to reach out to us at Restek if you have any questions.