Screening for Bifenazate (Floramite) in Medical Marijuana Using QuEChERS and GC-FID – Is it Possible?

Bifenazate (CAS# 149877-41-8) is an acaricide made by Uniroyal Chemical and sold under the trade name Floramite.  Floramite is registered in the US for control of mites on a wide variety of plants, and is widely employed in greenhouses and other indoor growing environments.  Its effective control of spider mites leads to its application by medical marijuana growers too, even though bifenazate is not registered for use on cannabis.

Although the Pesticide Action Network (PAN) Pesticide Database indicates that bifenazate has only slight acute toxicity and is an unlikely carcinogen, its intake by medical marijuana users who already have health issues is not preferred.  Therefore, analytical screening methods for bifenazate in marijuana are necessary to assure that patients are receiving a high quality medicine.

Given that many medical marijuana patients are often dosed with “edibles”, foods that contain marijuana with pre-determined cannabinoid content, it makes sense to consider the maximum residue limits (MRLs) for bifenazate on plant product commodities in lieu of the absent MRL for cannabis (for analytical method development purposes NOT health reasons, as the tolerance level for an unregistered pesticide is ZERO in the US).  The USDA Foreign Agricultural Services Pesticide MRL Database indicates that MRLs for bifenazate on plant product commodities range from 0.1 to 7 ppm, depending on the commodity.

We recently showed a QuEChERS sample preparation approach with cartridge SPE cleanup and GCxGC-TOFMS for determining pesticides in marijuana.  The complexity of the sample extracts and the desire to work at the ppb range for over 80 pesticides is what dictated the sophisticated analysis approach, but is that approach always necessary?  Maybe not for screening purposes for bifenazate, but let’s see.

I snagged a marijuana QuEChERS extract and my bifenazate standard and went to work.  Previously...  Finely ground marijuana, approximately 2 g, was wet by shaking with 10 mL acetonitrile and 10 mL organic-free water in a 50 mL centrifuge tube and then allowed to soak for one hour.  After vortexing vigorously for 30 min, QuEChERS EN salts were added and the sample was shaken by hand for 1 min, followed by phase separation with a Q-Sep 3000 centrifuge for 5 min.  The supernatant was removed and stored in a refrigerator.  Back to the present...  To make it as easy as possible, no further cleanup was done on the QuEChERS marijuana extract prior to its division into different sample vials that were spiked with bifenazate at various concentration levels to be analyzed using simple GC with flame ionization detection (FID), the equipment available to most medical marijuana laboratories.  The goal here was to determine when the response for bifenazate became non-linear due to matrix interference from the complex extract.

Prior to analysis of the bifenazate-spiked cannabis extracts, bifenazate in acetonitrile standards were analyzed on an Agilent 6890 GC with a 15m x 0.25mm x 0.25µm Rxi-5Sil MS GC column, by 1µL split injection into a Restek Premium 4mm Precision liner with wool.  The inlet was at 275°C and the split ratio was 10:1.  Helium carrier was used at 1 mL/min constant flow and the GC oven was programmed as follows: 80°C (1.5 min), 12.5°C/min to 330°C (0.50 min).  The FID used nitrogen makeup gas and was at 350°C.

The calibration curve for bifenazate in acetonitrile standards showed an excellent correlation coefficient of 0.9997 from 0.5 to 200 ppm (in solution).  But things got more difficult when moving to the spiked extract and you can see why upon inspection of the 50 ppm spike chromatogram.  Bifenazate elutes in a chromatographically-cluttered cannabinoid region, relatively near to the large THC peak to its right.  The bifenazate calibration curve turned out surprisingly good in spite of this sample complexity, but closer inspection reveals issues with the lower points in the curve as noted by the response factors creeping up (due to interference contribution to peak area) and the Calculated Concentrations (from the Calibration Curve) showing larger deviations from the Certified Concentrations.

The final thing that needs to be done is relate all of this back to ppm values in the original marijuana sample based on the amount extracted, the QuEChERS solvent volume, and the spike amount.  And, I’m basing the numbers on “100% extraction recovery” (actual recovery needs to be verified on "incurred" residues in cannabis).  When that is done, we’ve been looking at 1000, 500, 250, 100, 50, 25, 10, 5, and 2.5 ppm, bifenazate in marijuana.  The workable range though is likely from 1000 to 10 ppm, and that is ONLY for this particular sample, as another cannabis might contain compounds that interfere with bifenazate under these GC conditions.

In conclusion, the method presented here for bifenazate in marijuana will likely allow only gross-level screening work, which may still be of value to medical marijuana laboratories.  GC-MS with selected ion monitoring should allow a better determination, certainly one that is better for confirmation over FID, when using the same QuEChERS extraction technique.