Pesticide remediation of Cannabis oil- Myclobutanil removal by flash chromatography

Myclobutanil is a fungicide of considerable concern to the cannabis industry.  Its removal from extracted cannabinoid oil is the hot topic among processors and extractors.  In today’s post we will present how flash chromatography can be used for the remediation of the pesticide, thereby significantly reducing it from the final product in a single step.

Flash column chromatography has been applied to cannabis oils.  We have previously posted an orthogonal approach to isolating cannabinoids from winterized extract.  Give it read if you have not seen it.

 

Myclobutanil, aka Myclo and commercially known as Eagle 20®, is a commonly used fungicide applied in agriculture to reduce and often prevent powdery mildew and leaf spots, Figure 1.

Without going into detail about the pesticide’s toxicity, it’s needless to say that the presence of Myclo in consumer products is highly regulated by state public health agencies.  Depending on your geographical location, acceptable limits of this pesticide can range in the low parts-per-million (ppm) or even part-per-billion (ppb) concentrations, Table 1.

Table 1. State action limits for myclobutanil

StateCAORNV
Action level (ppm)0.020.29

What can be done to remove Myclo from cannabis oils?  Some work we recently did shows a successful attempt to remove a significant amount of Myclobutanil from THC distillate by way of flash chromatography.  Flash column chromatography has already been shown useful to isolate cannabinoids.

Figure 1. Myclobutanil chemical structure

To perform the study we started with about 50 grams of THC distillate and then added a calculated amount of Eagle 20 to formulate a Myclo concentration of a whopping 111.5 ppm.  Yep, that’s a lot!  Typical Myclo concentrations are in the range of 1-10 ppm if its present at all.  So this experiment will show how even a highly contaminated extract can be successfully cleaned up.

For the flash chromatography portion of the experiment we used a Biotage Isolera™ Spektra One flash system, a 30 gram Biotage® SNAP Ultra C18 column and a 25 gram Biotage SNAP KP-Sil column.

To assure that the purified THC was free of Myclo, the collected material was analyzed using a Shimadzu GCMS-QP2020.

First, we ran a normal-phase purification experiment utilizing the 25g SNAP KP-Sil column loaded with 2.5 grams of Myclo-spiked sample.   That is a mass load ratio (sample/column) of 10%.  An optimized step gradient with common organic solvents provided a single peak comprised mostly of THC cannabinoid, Figure 2.  Fractions 2, 3 were pooled and evaporated to remove any trace amounts of organic solvent.  The dried sample was solvated in acetonitrile (ACN) and injected into the gas chromatography-mass spectrometer (GC-MS) system.

Figure 2. Normal-phase flash chromatography of a THC extract containing 111.5 ppm of Myclobutanil. The peak seen here contains purified cannabinoids and minimal amount of other compounds.

A flash purification investigation wouldn’t be complete without including reversed-phase (RP) chromatography.  For this experiment, we utilized a 30g SNAP Ultra C18 column loaded with Myclo-spiked sample to a mass/mass ratio of 3%.

Sometimes chromatographic separation is enhanced with a pH modifier.  To explore this potential, we decided to split the RP purification experiments into two runs and evaluate both neutral and acidic gradients.   For the acidic gradient, formic acid was added to the solvent at a concentration of 1%.  Optimized linear gradients of ethanol+water and ethanol+water+formic acid provided single peaks of mostly THC cannabinoid, Figure 3.  Fractions 6-11 were pooled in both runs and evaporated to dryness.  The resulting dried sample was then solvated in ACN and injected into the GC-MS system.

Figure 3. Reversed-phase purification of Myclobutanil-spiked THC. Top – water/ethanol gradient, bottom – water/ethanol/formic acid gradient. The chromatograms indicate no impact from the formic acid.

Fractions from all purification runs were analyzed for content and purity by GC-MS, Table 2.  This data shows that flash chromatography using neutral pH reversed-phase flash chromatography removed 99.7% of the myclobutanil from the sample and normal-phase flash removed 99.6%. The data show adding formic acid did not improve myclobutanil remediation efficiency.

Table 2. Myclobutanil remediation efficiency results

 Spiked THCNormal-phaseReversed-phaseReversed-phase with acid
Myclobutanil content (ppm)111.540.440.311.09

The 99 plus percent clearance in both applications is quite an accomplishment provided the very high starting Myclo concentration in our distillate sample.  It’s common chromatographic knowledge that sample load ratios can impact separation efficiency.  What if our sample had only 10 ppm concentration of Myclo instead of 111 ppm?  What if we utilized the orthogonal approach and ran both normal- and reversed-phase separations in step sequence to remove Myclo from a single sample?  Can scavengers resins be utilized to reduce/remove pesticides?  I encourage you to stay tuned to our Cannabis Blog as we’ve got much more planned in the very near future.

 

Biotage would like to thank NorCal Analytical for their contributions in these experiments.

So, how have you addressed removing pesticides from plant extracts?  Please share so that our readers can learn.

Click here to access a survey regarding this blog; I’d like to see your thoughts.

9 thoughts on “Pesticide remediation of Cannabis oil- Myclobutanil removal by flash chromatography”

  1. Hi Brian,

    Thank you for the generous post. I am already specing the F400L (through Wayne Daneker) for contaminated THC oil remediation but am returning to this post to further refine my manufacturing proforma estimate calculations.

    Wayne has conveyed to me a potentially scalable method for PHIC remediation of hemp (CBD-dominant) extracts with reversed-phase media and 4 column volumes (CV) EtOH:H2O to elute CBD followed by 4 CV EtOH for THC + other hydrophobes + column wash.

    Examining this post it appears that on reversed-phase media at 3% loading ~6 CV of the EtOH:H2O gradient (that you don’t specify) are needed to elute. On the F400L that is a very large volume (~336 L). Whereas, on the normal-phase media at a much higher loading of 10% has an elution volume of perhaps 3 CV. Here though you only mention a step gradient of unspecified identity.

    1-Would you be willing to specify the aforementioned gradient?
    2-Did you ever do that follow-up with the expanded pesticide panel?
    3-In your orthogonal approach post on reverse-phase using MeOH:H2O gradient 80%-90%, after 5 CV, 3 CV elutes THC and ~2 more CV the THC-A. Have you explored the MeOH:H2O gradient for remediation?

    1. Hi Pratham,
      Thank you for commenting. Your question is answered in the blog itself. See the paragraph immediately following the action limits table- “In this experiment, we attempt to remove a significant amount of Myclobutanil from THC distillate by way of flash chromatography.”
      Flash removal of pesticides is likely more effective with a purer form of Cannabis oil. My preferred product form is distillate. “Cleaner “in” column, cleaner “out” of column
      Thanks again,

      Brian

  2. Would like all the info I can get. Need to put me on your email list. Working with Stafford Shealy. It was nice speaking with you and look forward learning more moving forward.

  3. This is amazing research. I am a newbie and will be paying attention to your work! Its genius and desperately needed. I wonder about the ACN you used- is there no chemical affect on the oil from the acetonitrile or contamination? Does the Myclo attach to the silica and you simply drain the oil from the bottom? Do you have any experience with the industrial flash system by biotage? Thank you for your work and all that you are doing for the purity of medicine.

    1. Katherine, Thank you for the positive feedback.
      A few responses to your questions:
      The ACN was only used to solvate a very small sample of purified thc isolate prior to GCMS analysis.
      Yes, Myclo and THC were bound to silica and then eluted or retained on column with an organic solvent step gradient.
      Absolutely, check the Biotage website under Industrial Products for the Flash 150 and Flash 400 systems.

    1. Thank you Chris. I have another experiment planned in the coming weeks with an expanded pesticide panel. Look for a follow up on this posting in late December or early January.

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