Validation of a QuEChERS-UHPLC-MS/MS method for the determination of aflatoxins and ochratoxin A in cannabis flowers: Application to regulated and non-regulated products.

Pereira M.; Tissot F.; Machado I. · Journal of AOAC International · 2026

**Background & Methods** This analytical validation study developed and optimized a QuEChERS-UHPLC-MS/MS method for simultaneous detection of aflatoxins (B1, B2, G1, G2) and ochratoxin A in Cannabis sativa flowers. A 3³ factorial design systematically evaluated critical extraction parameters including solvent acidification, extraction time, and evaporation temperature, with validation testing performed on 30 commercial and non-regulated cannabis flower samples. **Key Findings** • **Method Performance**: Optimal extraction conditions (acetonitrile acidified with 0.1% formic acid, 15-minute vortex extraction, evaporation <50°C) achieved recoveries of 90-109% with acceptable precision (RSD 2.5-8.3%) and linearity (R² >0.99). Limits of quantification were 1.0 μg/kg for aflatoxins and 5.0 μg/kg for ochratoxin A, meeting European Pharmacopoeia standards. • **Compliance and Safety Findings**: All 30 commercial cannabis products complied with international maximum residue limits (MRLs). However, non-regulated samples showed concerning contamination, with some exceeding the total aflatoxin limit of 4 μg/kg, primarily due to elevated aflatoxin B2 levels. • **Epidemiological Significance**: This study provides the first mycotoxin occurrence data for cannabis flowers from Uruguay and establishes baseline contamination patterns, highlighting disparities between regulated and unregulated products. **Dosage & Administration** Not reported. **Safety & Adverse Effects** Not reported. **Evidence Quality** This is a laboratory analytical validation study rather than a clinical trial, limiting direct assessment of clinical significance. Strengths include rigorous factorial design optimization, comprehensive method validation meeting international standards, and application to real-world samples. Limitations include the relatively small sample size (n=30), geographic specificity to Uruguay limiting generalizability, and lack of data on actual health impacts from detected mycotoxin levels. The study's primary value is methodological—establishing a validated analytical tool—rather than providing epidemiological or clinical evidence. The findings warrant regulatory attention but require larger, multi-regional prospective studies to establish the true health burden of mycotoxin contamination in cannabis products. The distinction between regulated and non-regulated products suggests quality control standards significantly impact consumer safety.

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