Simultaneous Determination of NX Toxins and Six Trichothecenes in Rice and Wheat Using UPLC with Mass Spectrometric and Photo Diode Array Detection

Trichothecenes are a major class of mycotoxins produced by Fusarium species, commonly contaminating cereal crops and posing health risks to humans and animals. Among them, NX-type A trichothecenes, including NX2-M1, NX2, and NX3, represent a subclass of type A trichothecenes structurally related to deoxynivalenol (DON), with their toxicological profiles are under investigation. Accurate quantification in food is essential for risk assessment and regulatory monitoring. This study presents two validated analytical methods for multi-mycotoxin detection: ultra-performance liquid chromatography coupled with a single quadrupole detector (UPLC/QDA), and UPLC with photodiode array detection (UPLC/PDA). Nine mycotoxins were targeted, including major type B trichothecenes and the emerging NX analogs. Sample preparation was optimized using two solid-phase extraction (SPE) methods OASIS® HLB and Mycosep® 227 on spiked rice and wheat samples. Chromatographic separation was performed using reversed-phase columns with gradient elution. Linearity was assesed in both solvent and matrices by spiking blank samples. The compounds were analysed in matrices by spiking sample extracts of a blank (mycotoxin free) rice and wheat samples. Good linearity (lack of fit test with p<0.05) and good determination coefficient R2 ≥0.990 was recorded in the range from 5 to 1000 µg/kg for all mycotoxins. Both methods demonstrated high analytical performance, with recoveries ranging from 70% to 103% and relative standard deviations (RSDs) below 13%. While the UPLC/PDA method proved effective for most trichothecenes but failed to detect NX2-M1 and NX3 due to their weak ultraviolet absorption characteristics. Conversely, UPLC/QDA enabled sensitive and selective detection of all analytes, including NX toxins, benefiting from mass spectrometric selectivity. Limits of detection (LODs) and quantification (LOQs) ranged from 1–34 µg/kg. Overall, both methods are suitable for routine mycotoxin screening, with UPLC/QDA offering broader applicability, especially for UV-inactive compounds like NX2-M1 and NX3.