Jeff Tries Cannabis Part 6: HPLC Method Development

Jeff's headGuest Author: Jeff Layne

Well – it seems that we have a little more work to do with cannabis potency testing. I had originally planned to have the last article be the last of this HPLC method development series, however, we had a request to develop a similar separation using methanol rather than acetonitrile. The rationale behind this was that the customer would be analyzing biological samples, and they were afraid that the high amount of acetonitrile would lead to protein precipitation problems on-column. There most likely are other valid reasons for wanting to use methanol as well, so it’s worth a shot.

Now, I don’t think we need to go through our entire method development process step-by-step, so I am just going to state that we followed the same basic workflow as we did previously, but using methanol rather than acetonitrile as the organic component of our mobile phase. The biggest challenge here is that methanol is much more viscous than acetonitrile, so it generates a lot more backpressure. If you have a UHPLC system, that is not a problem, but a lot of our customers are from smaller labs and haven’t been able to make the jump to UHPLC yet. So, we wanted to see if we could achieve our goals while keeping the pressure under 250 or 300 Bar.

I will admit, we greatly struggled to come up with a decent method, and came up with no success. We started the process using the same 150 x 4.6 mm Kinetex® 2.6 µm Polar C18 column that we had used for the acetonitrile version, but the pressure generated when using the methanol was simply too great. But then we had a breakthrough when a scientist working in a cannabis testing lab recommended to us that we try a shorter column – a 50 x 2.1 mm in fact! I didn’t have high hopes for this to work either, because if we couldn’t get 12 peaks to resolve on a 150 mm long column, how can we possibly expect it to work on a column with 1/3 the efficiency? However, I ended up being proven wrong. It worked very well, arguably better than our version using acetonitrile (Fig. 1). We also found that our original Kinetex 2.6 µm C18 chemistry provided a better selectivity under these conditions than the Kinetex 2.6 µm Polar C18 that we used for the acetonitrile version from our previous article (Fig. 2).

Figure 1. Separation of 12 cannabinoids using methanol and Kinetex 2.6 µm C18 50 x 2.1 mm

Figure 2. Separation of the 12 cannabinoids using acetonitrile and Kinetex 2.6 µm Polar-C18 150 x 4.6 mm

In comparing the two methods, we can see that the method using methanol is actually a bit longer, with the last peak eluting at about 9.5 min, compared to about 5.5 min when using acetonitrile. However, I prefer the methanol method due to the improved resolution between the peaks, with the critical pair being CBN and CBGA, which have a resolution value to 1.55 under these conditions and on this system (an Agilent 1100 Binary system).

It is interesting to note that the selectivity, in terms of analyte elution orders, is much different when using methanol rather than acetonitrile (compared in Table 1). For instance, the first obvious difference is the reversal of elution order for the first two peaks – CBDVA and CBDV. The acid analogue (CBDVA) elutes first when using acetonitrile, but elutes after CBDV when using methanol. Likewise, note than the acid analogues (CBDVA, CBDA, CBGA) all elute AFTER the parent molecule when using methanol, whereas they elute BEFORE the non-acid form when using acetonitrile.

Table 1. Comparison of elution order and retention times when using the acetonitrile (ACN) method and the methanol (MeOH) method.
6.1 table

Mechanistically, it’s hard to understand the chemistry that underlies this elution reversal. Using acetonitrile, the analytes appear to follow a more classical reversed phase mechanism, with the more polar acid analogues eluting first. But when you switch to methanol, the more polar analogue is being more strongly retained. Neat stuff – if anyone has a plausible mechanistic explanation, I would love to hear your thought/hypotheses. From a practical perspective, though, we can see that selecting between acetonitrile and methanol will be a very valuable tool in our final method performance.

“Final” method using methanol:

Column: Kinetex 2.6 µm C18 50 x 2.1 mm
MPA: Water with 0.1% Formic acid (**other acids would work as well)
MPB: Methanol with 0.1% Formic acid (**other acids would work as well)
**For the cannabinoids – we do not know how the different acidic modifiers might affect the retention behavior of matrix peaks
Gradient: 60 – 85%B over 10 minutes (2.5% per min)
Flow Rate: 0.5 mL/min; initial pressure about 240 Bar
Temperature: 50°C

So, thank you very much for your suggestion—it looks great! Now we have two viable solutions for the analysis of these cannabinoids. I would not call these “final” methods because these are just standards and there is probably work to be done in optimizing depending on matrix interferences, but I hope that these at least provide you with a good starting point for your further method development work. From the data we have shown so far in this blog series, you should be able to use LC columns, combined with the different acidic modifiers, and the choice of methanol or acetonitrile to optimize a method to work for your particular sample matrix.

Make sure to check back for a future article series that shifts gears and moves from potency testing to looking at the use of LC-MS to analyze cannabis-industry related pesticides.

2 Replies to “Jeff Tries Cannabis Part 6: HPLC Method Development”

  1. Perhaps the reversal of elution order can be explained by formation of hydrogen bonds of polar cannabinoids with MeOH?
    MeOH would surround the polar groups like COOH and OH providing more methyl groups, making the associated complexes less polar… just speculation.

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