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March 15, 2018 - Health Ranger - Mike Adams
14:17
CBD lab science mass spec analysis walkthrough at CWC Labs
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All right, welcome everyone.
This is Mike Adams, the Health Ranger here at CWC Labs, editor of naturalnews.com.
I'm showing you cannabidiol, the molecule, in 3D form.
And you'll notice that it has 21 carbons, 30 hydrogens, and 2 oxygens.
The red elements here are, of course, the oxygens and...
If you want to look at this in just sort of a standard format, it looks more like this.
Here.
So this is what we're looking for.
I'm going to show you how we analyze this using mass spec instrumentation.
And I'm going to show you using a method that we developed here at CWC Labs.
It's been printed up in some science posters and exhibited at some science expos already.
But I want to show you how this is done, because a lot of people are curious.
So we run the sample through a chromatography and also a mass spec instrumentation.
In this case, we're using an Agilent LC-MS time-of-flight instrument.
And we get, at first, let me turn these off.
First, we get the chromatogram.
Now this is showing how the sample elutes chemicals over time.
So the x-axis here is time starting at zero and going up to about 15 minutes.
This is a 15 minute run, and you can see then that certain chemicals come out at certain times through the chromatography.
You'll notice the response up here is 10 to the sixth, and our highest response here is a little under 4 times 10 to the sixth, which is good.
That's a good range.
That's where we want it to be.
What you don't know, though, from looking at this is what masses these are.
If you go back to cannabidiol, you'll notice that the mass here is 314.462 Daltons.
Once you add ionization to that, you get M plus H, and you actually end up with a mass of 315.2319.
So if we're looking for 315.2319, you might grab a peak, extract the spectrum, the MS spectrum, and see.
Look in this range.
Do you see 315?
The answer is no.
So we know that is not cannabidiol.
That's not CBD. That's something else.
So how do we find cannabidiol in all of these different masses that exist?
The answer is you do a mass extraction.
It's called an EIC. This is extracting ion chromatogram.
And I'm going to put in 315.2319 and give it a range of 20 ppm.
And boom!
There's our peak of CBD. Let me integrate that chromatogram.
So now we have an area of the peak, a response area from the instrument 2917516.93, as well as the retention time 9.731.
Now if we take a look at this and extract the MS spectrum, you'll see that we have 315.2317, and we have a carbon isotope variant of it, it looks like at 316.2347.
So this is good.
We found our CBD. Actually, I got to tell you, this isn't a hemp extract.
This is a standard.
So if we were to open a hemp extract, let me show you what that looks like.
This is an actual hemp sample, hemp extract chromatogram from a high CBD, low THC hemp strain.
And you'll immediately notice there's a very large peak here.
You might wonder, what's in that?
Well, as you can see, that is not...
Anything related to CBD. In fact, it's this stuff, whatever that is, at 191.1642.
But that's not CBD. That's not what we're looking for.
So what are we going to do with this?
We're going to extract the chromatogram that we want at the mass of 315.2319, which, as you remember, is cannabidiol plus ionization mass, M plus H. So now, look at that.
We get the chromatogram that we want.
Oops.
I want to integrate that.
Okay, now, this is a real live sample of hemp.
I'm going to actually modify this and show you.
So, you're looking at several peaks here.
What is this big peak on the left?
It turns out that is cannabidiol.
But what are these peaks?
These peaks, it turns out, are trace amounts of THC. Very low concentrations of THC, but they're in there.
Why does THC have the same mass as CBD? Well, to answer that question, you can go, well, note back on ChemSpider that the average mass is 314.462, and that the molecular formula is C21H30O2. So you can go back here and you can search for THC, and you'll find, let's use, yeah, let's use this one.
Look, it's exactly the same mass, 314.462, same number of carbons, hydrogens, and oxygens.
What does that tell you?
It tells you that cannabidiol is exactly the same mass as THC. So how do we tell them apart?
By retention time.
Notice that the THC is coming out 11 minutes, 11.5, and so on, 12.3, whereas CBD is coming out at maybe 9.7, well, yeah, 9.712.
That's what it's showing.
So that's how we can tell them apart.
if you don't have a mass spec instrument you can't differentiate these based on masses and you don't really know exactly what you're looking at that's why mass spec instrumentation is vastly superior for hemp extract analysis versus just chromatography and uv or diode array uv analysis which doesn't have mass discrimination so this is again an actual hemp sample we can zoom out look at the entire thing And you can see that CBD is one of the largest or most prominent chemical
constituents of this hemp sample, but not the largest.
Whatever this is, and I'm not sure what it is, is much larger.
And this peak is notable as well, but it's not CBD or THC. We can look at the spectrum, and what do we find?
Huh.
Look at this mass, 359.2214, with a carbon isotope at 316.2246.
That's because about 1% of the carbons are, well, they exist at carbon-13 instead of carbon-12, so you're going to see an isotopic spread, and that's a good verification.
But you might wonder, what is at 359.2214?
Well, as it turns out, that is CBD-A. So if we do extract a chromatogram at 359.2214, Correct number is 2, 2, 1, 9.
We can build a chromatogram of just the CBDA, and there it is.
So now we have CBDA. Let me actually turn these off.
Okay, in red, this is CBD. In green, this is CBDA, cannabidiolic acid.
Now what's interesting about this is that a lot of people try to analyze hemp extracts using gas chromatography.
Well, gas generates a lot of heat Obviously, and it destroys the CBDA because it gets deteriorated at high heat.
So your CBDA often reverts to CBD and you get an inaccurate quantitation of these constituents of a hemp extract.
But using what we're doing here at CWC Labs, which is a liquid form, we can not only discriminate CBD from THC based on retention time, but we can also use mass discrimination to pull out the CBDA Separate from CBD without destroying the CBDA. Isn't that interesting?
Now you might wonder, what is this little peak down here?
I'm pretty sure that's THCA, actually.
So THC has also an acid form as well.
So anyway, this shows you that.
Now, let's go to the next step and look at how we plot the concentrations on a quantitation curve.
All right, now I'm going to show you how to quant CBD using the Agilent instrumentation.
So first, all right, we are creating a new batch and we're going to bring in, let's see, seven samples of CBD standards to create the batch.
Now we're going to change these to calibration samples and do a fill down and put in the levels.
Oops.
We are skipping number seven because we had a robot pipetting error.
We're going to do a new method and in the method we're going to set up our compound CBD let's see target mass over charge ratio 2319 retention time let's see let's go back to our screen here the CBD came out at 9.712 right so we'll go back to doing 9.71 that should be plenty there's our chromatography we're looking good retention times delta right We can do like 0.6.
We don't want it to run into the THC, so we'll set that up that way.
Now for our concentrations, nanograms per milliliters is not actually what we want, but we'll ignore that for the moment.
Our high concentration will be 100 ppb, roughly.
It's actually micrograms per milliliter, but we'll just use that for now, just for the demonstration purposes.
Let's see, 2, 5, 10, 20, 40, 60, 80, 100.
We're not using number seven.
Validate the method.
No warnings.
We'll exit.
We'll apply it to the data.
And now we will do a quadratic fit on our curve.
So looking in this section, we now have our quantitation curve from 2 ppb up to 100 ppb.
And you notice our coefficient, our curve fit is 0.9993, which is considered very, very, very good.
And so this is our quantitation curve, our standards curve.
that we compare our extracts to.
So, look at our response is about 5.5 times 10 to the 5th response on our instrument, which shows you that this instrument is capable of detecting even two parts per billion of cannabidiol with a response of 10 to the 4th.
1.7 times 10 to the 4th.
That's how sensitive this is.
It's incredibly sensitive.
We're going to bring in a 2000 dilution here.
Analyze that batch.
Now, Put this back to a quadratic curve because that's the correct fit.
Now you see this purple arrow here?
That's showing that this sample is showing a concentration of 42.7.
And it knows that because it sees all these other standards with their concentration.
So it turns out that this is actually 42.7...
And the units are micrograms per milliliter, and if you multiply that by 2,000, you get the concentration in the original product.
So I guess if you want to do the math on that, what is that?
47.77 times 2,000.
And divide that by 1,000.
So this is 95.54 milligrams per milliliter.
If you want to get fluid ounces on that, There you go.
It's about 2,818 milligrams per fluid ounce of CBD in this product.
That is how we find this in a product.
So that is how we find CBD, CBDA, THC, or other molecules in the chromatogram and the ion-extracted chromatograms of hemp samples using Agilent instrumentation and using our quantitation curve using good chromatography and by the way what's really cool actually about all this is we could run this we could easily run these i mean look at this standard this is a hundred parts well it's actually micrograms
per milliliter don't don't pay attention to ppm but we could run this easily uh an order of magnitude higher you see how it's times 10 to the fifth we could go we could go 10 to the sixth on this so we could multiply everything by 10 on this and still be in great shape and get even a little bit smoother chromatography although this is not bad chromatography at all and remember you can also look at any section of this let's say you want to look at this section and you want to look at the spectrum and here you go you can actually you can actually see some
of the spectra here these are all compounds that are in hemp these are all other terpenes and other nutrients and chemicals don't pay attention to the 922 that's actually one of the reference masses that we're using for the instrument We also have another one of those down here, 121.
Right in here.
Yeah, that's another reference mass, so don't pay attention to that.
But all these other masses in here are very, very interesting things.
And we can actually use fine compounds over here on the left, the fine compounds by molecular feature.
It will give us a really good guess on what those compounds could be.
It can actually tell us that it thinks that's, you know, CBD or cannabidiol, or you can compare it to...
You can identify compounds.
You can search a database.
You can find by formula.
You can do all kinds of things in the formula.
Anyway, thanks for watching.
I hope you found this to be an interesting tutorial in the analytical quantitation of cannabinoids in hemp extracts.
My name is Mike Adams.
I'm known as the Health Ranger.
I'm the founder of cwclabs.com.
And we're currently not offering CBD analysis to other customers because we're kind of busy with our in-house stuff.
But in the future, we might pick that up.
So contact us if you're interested in our methods or using us to quantitate your CBD products.
Thanks for listening.
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