关键词: 高效液相色谱-二极管阵列检测, 福美双, 小麦粉, 面粉改良剂

Abstract:

Thiram is an important dithiocarbamate (DTC) fungicide. In the United States and the European Union, the limit range of thiram is 0.1-15 mg/kg in fruits and vegetables, but there is no specific limit for grains. The maximum residue limit (MRL) for wheat is 1 mg/kg (calculated as carbon disulfide, CS₂) in the National Food Safety Standard (GB 2763-2019). At present, the relevant regulation methods in China are targeted at the detection of dithiocarbamates and are incapable of detecting thiram specifically. CS₂ is produced by the reaction of dithiocarbamate and acid, and it is then determined by spectrophotometry or GC; this renders the quantification of dithiocarbamate indirect. HPLC and HPLC-MS/MS methods are also reported for the detection of thiram. Most of the literature focuses on the determination of thiram in vegetables, fruits, soil, etc. In these methods, thiram is converted into dimethyldithiocarbamate (DMD) anions in an alkaline buffer solution, and DMD can be determined by HPLC-UV or LC-MS. However, ziram can also be converted into the DMD anion under alkaline conditions. Therefore, thiram cannot be distinguished from ziram, and this may produce false-positive results. Research has shown that in the presence of sulfite, thiram is converted into a DMD-sulfite adduct, which can be a marker for the selective determination of thiram. Furthermore, thiram can be directly detected by HPLC and HPLC-MS/MS after extraction with dichloromethane, chloroform, hexane, cyclohexane, ethyl acetate, or methanol and clean-up by solid phase extraction in vegetables and fruits. However, until now, few studies have reported the determination of thiram in wheat flour and flour improvers. Therefore, it is of great importance to develop a method for thiram in wheat flour. In this study, an analytical method based on HPLC-DAD was developed for the determination of thiram in wheat flour and flour improvers. The wheat flour and flour improver samples were extracted using acetonitrile. After shaking for 15 min, the samples were ultrasonicated for 10 min in an ice-water bath. The supernatant was filtered before separation on a ZORBAX plus-C18 column (150 mm×4.6 mm, 5 μm). The samples were eluted with a water-acetonitrile solvent system and detected at 280 nm. In this research, the extraction solvent, extraction solvent volume, ultrasonic conditions, chromatographic column, determination wavelength, and mobile phase were optimized. The retention times and UV spectra were used for qualitative analysis, and the external standard method was used to quantify thiram. Stability tests of standard stock solutions, a series of standard solutions, and extraction solutions were also performed. The standard stock solutions could be stored for at least 21 d, and the series of standard solutions could be stored for 14 d under refrigeration at 4 ℃. The standard solution was either exposed to light at room temperature for 4 h or kept in dark at room temperature for 48 h, and no obvious degradation was observed. This revealed that thiram was stable in acetonitrile solution during our investigation. It was suggested that the extraction solution should be analyzed as soon as possible. The linear range was 0.30-30.0 μg/mL. The peak area of the analyte showed a good linear relationship with its corresponding concentration, and the correlation coefficient (r²) was 0.99999. When the spiked levels were 1.5, 3.0, and 15 mg/kg, the spiked recoveries of thiram were 89.6%-98.3%, with relative standard deviations of 1.6%-3.9% (n=6). The limits of determination and quantification for thiram were 0.5 mg/kg and 1.5 mg/kg, respectively. The results revealed that this method is simple, rapid, and specific, in addition to having high precision, good repeatability, and a low limit of detection. The method is thus suitable for the daily routine analysis of thiram in wheat flour and flour improvers.

Key words: high performance liquid chromatography-diode array detection (HPLC-DAD), thiram, wheat flour, flour improvers