Abstract:

This study employed ultrahigh-performance liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry with optimized chromatographic and mass spectrometric conditions （UPLC-Q-Orbitrap HRMS）， to investigate the contamination status of mycotoxins in silage alfalfa from Gansu Province. By optimizing chromatographic and mass spectrometric conditions and adopting a dual purification mode of gel permeation chromatography （GPC） and solid-phase extraction （SPE） to reduce matrix effects， mycotoxin levels in silage alfalfa samples from 84 sampling sites in 14 regions of Gansu Province were analysed. The results indicated that the optimized UPLC-Q-Orbitrap HRMS developed in this study could accurately and simultaneously detect 18 mycotoxins in silage alfalfa， namely： aflatoxins B1 and B2， G1 and G2 and M1 and M2 （AFB1， AFB2， AFG1， AFG2， AFM1 and AFM2， respectively）， ochratoxins A， B and C （OTA， OTB and OTC， respectively）， T-2 toxin， HT-2 toxin， deoxynivalenol （DON）， fuminosin B1， B2 and B3 （FB1， FB2 and FB3， respectively）， zearalenone （ZEN）， deoxynivalenol-3-glycoside （D3G） and 15-acetyldeoxynivalenol （15-ACDON）. The most frequently detected toxins included AFB1， AFB2， DON， FB1， FB2， FB3， and ZEN， along with masked toxins such as D3G and 15-ACDON. Among the 84 sampling sites， all 18 mycotoxins were detected in samples from Long County and Wen County， with AFB1， OTA， T-2， DON， FB1， FB2， and ZEN significantly exceeding reference tolerance limits （P<0.05）. In Pingliang， Qingyang， Tianshui， Dingxi， Baiyin and Lanzhou， 17 mycotoxins were detected， primarily AFB1， AFB2， AFG1， AFM1， DON， FB1， FB2， FB3， ZEN， D3G and 15-ACDON， with 3 mycotoxins （AFB1， DON， FB1） exceeding reference tolerance limits. In Wuwei， Jinchang， Zhangye， and Jiuquan， 9 toxins were detected， primarily AFB1， DON， FB1， and ZEN， all within reference tolerance limits. No mycotoxins were detected in samples from Diebu County， Xiahe County， Jishishan County in the Gannan and Linxia regions. This study demonstrates that the developed UPLC-Q-Orbitrap HEMS method enables high-throughput， highly accurate detection of 18 mycotoxins in silage alfalfa， including masked mycotoxins that are prone to being overlooked. Mycotoxin concentrations and types in Gansu ensiled alfalfa exhibited distinct regional characteristics. Pearson correlation and redundancy analysis revealed that average temperature during the ensiling period and altitude were the significant factors influencing mycotoxins content and types， with explanatory powers of 59.70% and 31.20%， respectively.

Key words: Gansu, alfalfa, silage, mycotoxins, association analysis