Analysis of Mycotoxins in Cannabis Plant Material and Derivative Products by Immunoaffinity Enrichment LC-MS/MS

Importance of the Topic

Cannabis products, including raw plant material, concentrates, edibles, and topicals, can be susceptible to contamination by regulated mycotoxins such as aflatoxins (B1, B2, G1, G2) and ochratoxin A. Reliable detection of these toxins is essential for consumer safety, regulatory compliance, and quality assurance across medical, recreational, and industrial applications.

Objectives and Study Overview

The primary goal of this study was to develop and validate a comprehensive analytical workflow for quantifying key mycotoxins in diverse cannabis matrices. The method integrates immunoaffinity enrichment with liquid chromatography–tandem mass spectrometry (LC-MS/MS) to achieve high selectivity, sensitivity, and robustness. Performance characteristics such as linearity, limits of detection (LOD) and quantitation (LOQ), accuracy, precision, matrix effects, and system robustness were systematically evaluated.

Methodology and Instrumentation

• Sample Preparation: Homogenized cannabis flower, concentrates, tinctures, edibles, and topicals were spiked pre-extraction at multiple concentration levels (3–60 ng/g), equilibrated for 30–60 minutes, and extracted using isopropanol and methanol/water mixtures with zirconia beads and mechanical shaking.
• Clean-Up: Extracts were diluted and loaded onto AflaOchraTM immunoaffinity columns to selectively enrich aflatoxins and ochratoxin A, followed by elution and filtration.
• Quantification: A single-point standard addition calibration corrected for matrix effects, with paired extract aliquots spiked post-extraction.

Used Instrumentation

• Immunoaffinity Columns: AflaOchra® (VICAM®, Waters).
• Chromatography: Waters UPLC I-Class system with 2.1×100 mm C18 X-Bridge® column; mobile phases of 5 mM ammonium formate with 0.02% formic acid in water (A) and methanol (B); flow rate 0.5 mL/min; column temperature 30 °C; 10 µL injection.
• Mass Spectrometry: Xevo TQS-micro triple quadrupole detector with optimized gradient elution for baseline separation and detection of the targeted mycotoxins.

Main Results and Discussion

• Accuracy: Recoveries across five matrix classes ranged between 85% and 115% at three spike levels, demonstrating reliable quantitation in complex cannabis formulations.
• Precision: Repeatability studies (n=30) at mid-level concentration showed relative standard deviations below 15%, meeting regulatory precision requirements.
• Matrix Effects: Comparison of solvent-based and matrix-matched calibration slopes revealed minimal suppression or enhancement (±20%), effectively compensated by the standard addition approach.
• Robustness: System performance over 179 consecutive injections remained within control limits (UCL, CL, LCL) with no significant drift or trend violations, confirming method stability.

Benefits and Practical Applications

This validated protocol offers a streamlined and adaptable solution for routine mycotoxin surveillance in cannabis laboratories. Immunoaffinity enrichment ensures high selectivity, minimizing interference from complex botanical matrices. Coupled with LC-MS/MS, the method achieves regulatory limits of ≤5 ppb for aflatoxin B1, ≤20 ppb total aflatoxins, and ≤20 ppb ochratoxin A, supporting compliance and quality control in commercial and research settings.

Future Trends and Potential Uses

• Expansion to additional mycotoxins and emerging fungal toxins relevant to cannabis safety.
• Integration with high-throughput automation for large-scale testing and real-time monitoring.
• Development of portable LC-MS/MS platforms for on-site screening in cultivation and processing facilities.
• Application of advanced data analytics and machine learning to enhance detection limits and predict contamination trends.

Conclusion

The combination of immunoaffinity column clean-up and LC-MS/MS provides a robust, sensitive, and precise method for detecting regulated mycotoxins in diverse cannabis matrices. The validated workflow meets stringent regulatory requirements, demonstrates strong method performance, and offers versatility for routine quality control in analytical laboratories.

Reference

1. U.S. Food and Drug Administration Foods Program. Guidelines for the Validation of Chemical Methods in Food, Feed, Cosmetics, and Veterinary Products. 3rd ed.; October 2019.
2. USDA GIPSA. Grain, Fungal Diseases, and Mycotoxin Reference; Washington, D.C., September 2006.