Simultaneous Determination of Chlorothalonil and 4-Hydroxy-Chlorothalonil in Sulfur-Rich Vegetables by UHPLC-MS/MS with a Synergistic Enzyme Inhibition Strategy

Chlorothalonil and its toxic metabolite, 4-hydroxy-chlorothalonil, pose significant environmental and health risks. However, their simultaneous and accurate detection remains challenging due to their differing ionization efficiencies in mass spectrometry and the interference caused by enzymatic reactions in sulfur-rich vegetables. This study developed a UHPLC-MS/MS method for simultaneous detection of chlorothalonil and 4-hydroxy-chlorothalonil, using an atmospheric pressure chemical ionization (APCI) source, optimizing the probe temperature to 600 °C and a set of optimal chromatography parameters. A low-temperature and acidification synergistic enzyme inhibition strategy was developed, involving refrigerating samples and extraction reagents, acidifying with citric acid before sample homogenization, and extracting with formic acid/acetonitrile, significantly improving chlorothalonil recovery. Method validation demonstrated limits of detection (LOD) and quantification (LOQ) of 0.003 mg/kg and 0.01 mg/kg, respectively, with recoveries of 76.5-91.1% for chlorothalonil and 87.6-96.7% for 4-hydroxy-chlorothalonil. The method was successfully applied in monitoring the residue risks in sulfur-rich vegetables.