LC-MS/MS determination of mycotoxins in cannabis and cannabis derived products using different sample preparations

Significance of the Topic

The rapid expansion of hemp and cannabis products for medicinal and recreational use has heightened the need for rigorous safety testing. Mycotoxins such as aflatoxins B1/B2, G1/G2, ochratoxin A and zearalenone can contaminate plant biomass and derived products, posing health risks. Regulatory bodies including the FDA and the European Commission have set stringent limits for these toxins in food and feed. Developing robust analytical workflows to detect mycotoxins at parts-per-billion levels is therefore essential for consumer safety and regulatory compliance.

Objectives and Study Overview

This study aimed to evaluate different sample preparation strategies in combination with LC–MS/MS detection to quantify key mycotoxins in various cannabis matrices. Four preparation protocols—solid–liquid extraction (SLE)/liquid–liquid extraction, QuEChERS with dispersive cleanup, CrossTOX column cleanup and immunoaffinity solid-phase extraction (IAC SPE)—were assessed across hemp pellets, seeds, flour and oil. Performance metrics included recovery rates, limits of detection (LOD), limits of quantification (LOQ), processing time, cost and solvent usage.

Methodology

The analytes were separated on a reversed-phase C18 column under a gradient of water and methanol (both containing 0.1% formic acid) at 0.5 mL/min and 60 °C. Detection employed a triple-quadrupole MS in positive electrospray ionization using multiple reaction monitoring (MRM). Calibration curves covered six levels (0.25–100 ppb depending on the toxin). LOD was defined at S/N = 3 and LOQ at S/N = 10. Recovery experiments used samples spiked at the fifth calibration level.

Instrumentation Used

• AZURA P 6.1L HPG pump
• AZURA AS 6.1L cooled/heated autosampler
• AZURA CT 2.1 column thermostat
• Sciex Triple Quad 5500+ with QTRAP capability
• Eurospher II C18 column (150 × 2 mm, 2 µm)

Main Results and Discussion

All four cleanup strategies achieved regulatory LOD/LOQ requirements (<20 ppb for aflatoxin sums, 0.5–80 ppb for ochratoxin A, 20–400 ppb for zearalenone). Recovery rates depended on matrix complexity and cleanup strength. The simple SLE/LLE and standard QuEChERS workflows yielded incomplete recoveries in solid matrices (pellets, seeds). CrossTOX improved cleanup marginally but still missed low-level analytes. The IAC SPE approach provided consistent >80% recoveries across all toxins and matrices. Trade-offs emerged between cleanup efficiency, processing time (30–45 min/sample) and consumable cost (€6.80–39.50).

Benefits and Practical Applications

Combining selective sample preparation with LC–MS/MS enables reliable quantification of multiple mycotoxins in complex cannabis products. The immunoaffinity SPE method delivers the highest sensitivity and reproducibility, making it suitable for laboratories enforcing strict regulatory limits. Simpler methods may still be employed for less challenging matrices or preliminary screening.

Future Trends and Potential Applications

Future work may explore hybrid approaches, such as pairing QuEChERS with immunoaffinity cleanup to balance throughput and selectivity. Automation of sample preparation and micro-extraction techniques could reduce solvent use and labor costs. Expanding validated methods to additional mycotoxins and emerging cannabis derivatives will support comprehensive safety monitoring across the rapidly evolving market.

Conclusion

This comparison highlights that matrix complexity dictates the choice of cleanup procedure. Immunoaffinity SPE offers superior recovery and meets stringent regulatory requirements for mycotoxin analysis in hemp and cannabis products. Tailored workflows can optimize laboratory efficiency while ensuring consumer safety.

References

• [1] FDA Guidance for Industry: Action Levels for Poisonous or Deleterious Substances in Human Food and Animal Feed, U.S. Food & Drug Administration, 2024
• [2] Commission Regulation (EU) 2023/915 on maximum levels for certain contaminants in food, European Commission, April 2023
• [3] AOAC SMPR® 2021.010: Standard Method Performance Requirements for Quantitative Analysis of Mycotoxins in Cannabis Biomass and Cannabis-Derived Products, AOAC International, 2021