Determination of Pesticides and Mycotoxins in Cannabis Flower as Defined by Legalized U.S. State Recreational Cannabis Regulations

Importance of the Topic

The rapid expansion of legal recreational cannabis markets in the United States has heightened the need for reliable residue testing to protect consumers. State regulators have defined stringent maximum levels of pesticides and mycotoxins in dried flower products. A robust analytical method is critical to verify compliance across diverse state regulations and ensure product safety.

Objectives and Overview

This work presents an integrated LC/MS/MS workflow for simultaneous quantitation of over 90 pesticides and key mycotoxins in cannabis flower. The method is tailored to meet action levels set by California, Michigan, Nevada, Oregon, and Washington for recreational cannabis compliance. It covers sample homogenization, SPE cleanup, chromatographic separation, mass spectrometric detection, and data reporting.

Methodology

• Sample Preparation
  1. Weigh 1 g of dried cannabis flower and homogenize with acetonitrile using ceramic beads.
  2. Centrifuge to separate solids, then perform gravity-fed SPE cleanup with C18 cartridges.
  3. Collect eluate, bring to a fixed volume for a 1/250 dilution, and prepare injection using an aqueous–organic sandwich technique.
• Chromatography and Detection
  1. LC System: Agilent 1260 Infinity II binary pump, multisampler (100 µL loop), multicolumn thermostat.
  2. Column: 3 × 100 mm, 2.7 µm phenylhexyl, 55 °C, flow rate 0.5 mL/min.
  3. Gradient: 30% to 100% methanol over 9 min, total cycle time 13–14 min.
  4. Mass Spectrometer: Agilent 6470 or Ultivo QQQ with Jet Stream ESI, operated in positive/negative modes.
  5. MRM transitions optimized via MassHunter Optimizer for each analyte.

Injector pretreatment uses aqueous sandwiching to prevent peak distortion from high organic content, preserving symmetry across early-eluting compounds.

Instrumentation Used

• Agilent 1260 Infinity II HPLC (pump G7112B, multisampler G7167A, thermostat G7116A)
• Agilent 6470 or Ultivo (6465BA) QQQ MS with Jet Stream ESI source
• SampliQ C18 SPE cartridges
• Agilent InfinityLab ultrapure methanol, deionized water, formic acid, ammonium formate
• MassHunter software for acquisition, quantitation, and reporting

Main Results and Discussion

• Calibration and Linearity: Matrix-matched standards down to 100 ppt achieved R² ≥ 0.990 for all targets.
• Lower Limits of Quantitation: LLOQs on flower basis ranged from 2.5 ppb to 100 ppb, meeting or exceeding regulatory action levels.
• Recovery and Precision: Mean recoveries between 88% and 131% for pesticides; 100%–105% for mycotoxins; precision within regulatory criteria.
• Chromatographic Performance: Overlapped injections and pretreatment ensured consistent peak shapes and high throughput.

Practical Benefits

• Unified analysis of pesticides and mycotoxins reduces instrument time and sample handling.
• SPE cleanup combined with injector dilution yields robust recoveries without extensive reoptimization.
• Automated batch review and custom reports accelerate compliance testing.

Future Trends and Potential Applications

Emerging needs include screening for novel cannabis contaminants (e.g., residual solvents, heavy metals, transformation products). Integration of high-resolution MS, automation, and LIMS will further improve throughput, data integrity, and traceability in cannabis testing laboratories.

Conclusion

An efficient LC/MS/MS method has been validated for simultaneous determination of state-regulated pesticides and mycotoxins in cannabis flower. The protocol combines streamlined SPE cleanup, optimized chromatography, and sensitive MS detection to satisfy the strictest action levels. Paired with automated data processing, it delivers a turnkey solution for routine quality control laboratories.

References

1. Agilent Technologies. A Fast Analysis of the GC/MS/MS Amenable Pesticides Regulated by the California Bureau of Cannabis Control, Application Note 5994-1019EN, 2019.
2. Agilent Technologies. Analysis of Challenging Pesticides Regulated in the Cannabis and Hemp Industry with the Agilent Intuvo 9000 GC/MS/MS System, Application Note 5994-1604EN, 2019.