UPLC-HR-MS/MS法对吡唑醚菌酯原药中主要杂质的定性分析

采用超高压液相色谱-质谱（UPLC-MS）以及高能诱导裂解（HCD）技术分析吡唑醚菌酯原药中的杂质，以C18反相色谱柱梯度洗脱，ESI正离子模式测定。通过解析原药主成分与杂质的裂解规律发现，在二级质谱图中，同时存在偶电子离子和奇电子离子的碎片。结合原药的合成路线，推测3种杂质可能的结构及来源：杂质A产生于硝基羟胺化还原步骤中；杂质B由羟胺中间体与二氯乙烷溶剂反应产生；杂质C是苄溴化反应步骤中过量的溴导致的。经过比对杂质对照品的色谱、质谱数据，进一步确证了杂质的定性分析结果。该方法对吡唑醚菌酯原药的纯化、生产工艺改进、质量监控具有指导意义。

Qualitative Analysis of the Main Impurities in Pyraclostrobin by UPLC-HR-MS/MS

In the prescreening stage of full analysis for pesticide, direct analysis methods (GC-MS, LC-MS, LC-NMR etc.) and synthetic verification method were often combined to obtain better qualitative results. Three main impurities in pyraclostrobin technical concentrate (TC) were isolated and analyzed using liquid chromatography-mass spectrometry (UPLC-MS) and higher energy collision induced disassociation (HCD). The liquid chromatograms were determined on UPLC equipped with C18 reversed phase column. The corresponding mass data was collected in positive ion mode of ESI on a high-resolution accurate-mass Orbitrap. The HCD MS/MS spectra of pyroclostrobin and impurity B and C showed that both the even-electron fragment cations and the odd-electron cations were observed. The structures of three impurities were estimated from the MS spectra, MS/MS spectra and the synthetic route. The structure of impurity A was estimated by comparing its MS/MS spectra with pyraclostrobin, it may be generated from the nitro-derivative in the hydrogenation step. In the MS/MS data of impurity B, m/z 63.000 1 was observed and attributed to fragment of ClCH₂CH₂⁺, thus impurity B may be generated by the reaction of hydroxylamine intermediate with the solvent dichloroethane. Impurity C may be due to the excess amount of Br₂ in the bromination process, it was difficult to determine position of extra Br atom. From the perspective of systhesis process there were two possibilities: 1) Br atom was attached to the benzyl group; 2) Br atom was attached to the pyrazole ring. For impurity C, m/z 194.080 8 was observed instead of m/z 271.991 7, which indicated that the Br atom was more likely to be attached to the pyrazole ring part. In order to confirm the above results, standards of impurity A, B and C were synthesized and analyzed using LC-MS. The results showed that the retention time, mass data of the standards were in accordance with the impurities in TC. It could be concluded that the main impurities of pyraclostrobin came from the side-reaction of the intermediates, and all of them showed good response in the ESI mass spectra. This study would benefit not only the preparation and quality control, but also the registration of pyraclostrobin.