Determination of mycotoxins – in cannabis and cannabis derived products with fluorescence detection

Significance of the Topic

The reliable detection of mycotoxins in cannabis and hemp products is essential for consumer safety and regulatory compliance. With expanding legal markets and recognized health benefits, controlling aflatoxins and ochratoxin A across diverse matrices prevents harmful exposure and ensures product quality.

Objectives and Study Overview

This work evaluates sample preparation strategies and a fluorescence detection method for quantifying the main mycotoxins defined by AOAC SMPR 2021.010 in cannabis biomass, derived products, and hemp oil. The goal is to compare four extraction workflows in terms of sensitivity, solvent usage, time, cost, and compliance with FDA and EU limits.

Methodology and Instrumentation

Four matrices were tested: hemp pellets, seeds, commercial flour, and oil. Sample preparation strategies included:

• P1: Solid–liquid extraction/liquid–liquid extraction (SLE/LLE)
• P2: QuEChERS with dispersive cleanup
• P3: Solvent extraction followed by CrossTOX immunoaffinity cleanup
• P4: Solid phase extraction using immunoaffinity columns (IAC SPE)

Quantification employed reversed-phase liquid chromatography with fluorescence detection. Calibration covered 0.5–30 ppb for aflatoxins and ochratoxin A and 50–1000 ppb for zearalenone. Method performance was assessed via recovery studies at two spiking levels and determination of LODs (S/N=3) and LOQs (S/N=10).

Instrumentation Used

• Pump: AZURA P 6.1L HPG (1000 bar)
• Autosampler: AZURA AS 6.1L with thermostat
• Column: Eurospher II C18, 150×2 mm
• Detector: Shimadzu RF-20A fluorescence
• Software: ClarityChrom 8.7

Main Results and Discussion

All methods achieved LODs and LOQs below 20 ppb, satisfying FDA and EU regulatory thresholds. Recoveries varied by matrix and workflow:

• P1 yielded rapid extraction but insufficient cleanup for solids.
• P2 improved peak shapes and cleanup yet required optimization for non-oil samples.
• P3 enhanced selectivity via CrossTOX columns with moderate recoveries.
• P4 (IAC SPE) provided consistent detection in all matrices, with recoveries ranging from 20% to 140%.

Complex matrices such as pellets, seeds, and flour demanded immunoaffinity cleanup. Hemp oil samples were amenable to simpler workflows.

Benefits and Practical Applications

• Fluorescence detection offers a cost-effective, user-friendly alternative to LC-MS/MS.
• Method meets regulatory requirements for food and feed safety.
• Flexible sample preparation allows adaptation to various cannabis products.

Future Trends and Opportunities

Emerging developments may include:

• Automation and miniaturization of immunoaffinity cleanup.
• Integration with advanced detectors or portable analyzers for on-site testing.
• Green chemistry approaches to reduce solvent use.
• Expansion of validated methods to additional mycotoxins and cannabinoids.

Conclusion

The study demonstrates that fluorescence-based LC methods, coupled with tailored sample workflows, reliably detect key mycotoxins in cannabis matrices. Immunoaffinity SPE provides the most robust performance for complex samples, while QuEChERS and solvent extraction can serve in simpler contexts following further optimization.

Reference

1. Guidance for Industry: Action Levels for Poisonous or Deleterious Substances in Human Food and Animal Feed, FDA, 2023.
2. Commission Regulation (EU) 2023/915 on maximum levels for certain contaminants in food.
3. AOAC SMPR 2021.010 Standard Method Performance Requirements for Mycotoxin Analysis in Cannabis.