Multitoxin analysis of trichothecenes, including NX toxins, in rice and wheat

Fusarium species biosynthesize trichothecenes that contaminate food and poses health risks. Among these, NX toxins produced by F. graminearum and F. culmorum , are structurally similar to deoxynivalenol (DON). Accurate measurement of these mycotoxins in food is essential for food safety. We developed UPLC-QDA and UHPLC/PDA methods to simultaneously quantify nine trichothecenes, in particular NX2-M1, NX2, and NX3. In this study, two complementary analytical workflows, UPLC-QDA (single-quadrupole mass spectrometry) and UHPLC/PDA—were developed and validated for the simultaneous determination of nine trichothecenes in rice and wheat matrices. Sample cleanup was comparatively performed using two solid-phase extraction (SPE) procedures, OASIS® HLB and Mycosep® 227, to assess their performance in reducing matrix effects and improving recovery. The UPLC-QDA method enabled the reliable quantification of all target analytes, including the NX-series, with recovery values between 70 and 103 % and intra-day precision (RSD) below 13 %. Conversely, UHPLC/PDA was unable to detect NX2-M1 and NX3 due to their poor UV absorption. Mycosep® 227 exhibited superior cleanup efficiency and lower signal suppression compared to OASIS® HLB, particularly in wheat extracts. Detection limits ranged from 1–34 µg/kg for UPLC-QDA and 4–26 µg/kg for UHPLC/PDA. Overall, the study provides a validated and practical framework for the simultaneous quantification of both regulated and emerging trichothecenes, highlighting the necessity of MS-based detection for accurate determination of NX toxins in complex cereal matrices.