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.

BACKGROUND: Cannabis sativa flowers are a complex plant matrix that poses significant analytical challenges due to their chemical composition and the low regulatory limits established for consumer safety. OBJECTIVE: To optimize and validate a QuEChERS-UHPLC-MS/MS method for the simultaneous determination of aflatoxins B1, B2, G1, G2, and ochratoxin A in Cannabis sativa flowers. METHODS: Critical parameters influencing extraction efficiency were systematically evaluated through a 3³ factorial design, including solvent acidification, extraction time, and evaporation temperature. Optimal conditions were achieved using acetonitrile acidified with 0.1% v/v formic acid, 15 min vortex extraction, and evaporation below 50 °C. RESULTS: Under these conditions, recoveries ranged between 90-109%, with precision (RSD 2.5-8.3%) and trueness demonstrating satisfactory method performance. Linearity was confirmed with R² > 0.99, and LOQs of 1.0 µg/kg for aflatoxins and 5.0 µg/kg for ochratoxin A, consistent with European Pharmacopoeia requirements. Application to 30 cannabis flower samples revealed that all commercial products complied with international maximum residue limits (MRLs), whereas some non-regulated samples exceeded the total aflatoxin limit (4 µg/kg), mainly due to elevated aflatoxin B2. CONCLUSION: These findings provide the first data on mycotoxin occurrence in cannabis flowers from Uruguay and highlight the importance of monitoring non-regulated products, emphasizing the need for appropriate quality-control strategies. HIGHLIGHTS: The proposed method provides a simple, robust, and high-throughput approach for routine quality control of cannabis flowers.