超高效液相色谱-串联质谱法检测绵羊组织中地昔尼尔残留的方法学研究

Methodology Study on Determination of Dicyclanil Residual in Sheep Tissue by Ultra-performance Liquid Chromatography-tandem Mass Spectrometry

【目的】建立一种绵羊组织中地昔尼尔及其代谢产物2,4,6-三氨基-5-氰基嘧啶质量浓度的超高效液相色谱-串联质谱(UPLC-MS/MS)检测方法。【方法】对采集的绵羊肌肉、脂肪、肝脏、肾脏组织进行前处理,以乙腈和七氟丁酸水溶液为流动相,利用Acquity UPLC Beh C18色谱柱进行梯度洗脱;流速为0.15 mL/min,柱温为30℃,进样量为5μL。在正离子扫描模式下通过多离子反应监测采集数据,采用外标法测定地昔尼尔及其代谢产物浓度,并考察该检测方法的线性范围、检测限、定量限、回收率、批内与批间精密度、准确度和稳定性。【结果】建立的检测方法灵敏度高,检测限为10μg/kg,定量限为20μg/kg,地昔尼尔和2,4,6-三氨基-5-氰基嘧啶的添加浓度为20~1000μg/kg,标准曲线的线性关系良好,相关系数均≥0.99。将地昔尼尔和2,4,6-三氨基-5-氰基嘧啶以高、中、低、定量限4个水平分别添加到各空白组织中,测得地昔尼尔和2,4,6-三氨基-5-氰基嘧啶的回收率为70%~110%,批内和批间变异系数均<15%。在考察的条件下,地昔尼尔和2,4,6-三氨基-5-氰基嘧在各组织中的稳定性良好。【结论】建立的UPLC-MS/MS检测方法专属性强、重复性好、灵敏度高,可适用于地昔尼尔乳剂在绵羊组织内的药物代谢动力学和残留研究。

【Objective】This study was constructed to establish a method for the determination of the mass concentration of dicyclanil and its metabolites 2,4,6-triamino-5-pyrimidinecarbonitrile in sheep tissue by ultra-high liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).【Method】Sheep tissues(muscle,fat,liver and kidney)were pretreated.Acetonitrile and heptafluorobutyric acid aqueous solution were used as mobile phases,Acquity UPLC Beh C18 column was used for gradient elution.The flow rate was 0.15 mL/min,the column temperature was 30℃and the injection volume was 5μL.The data were collected by multi-reaction monitoring in positive ion scanning mode,and the concentrations of dicyclanil and its metabolites were determined by external standard method.The linear range,detection limit,quantitative limit,recovery rate,intra-assay and inter-assay precision,accuracy and stability of the method were investigated.【Result】The established detection method had high sensitivity,the detection limit was 10μg/kg,and the limit of quantitation was 20μg/kg.The addition concentrations of dicyclanil and 2,4,6-triamino-5-pyrimidinecarbonitrile were in the range of 20 to 1000μg/kg,and the linear relationship between the standard curves was good,with correlation coefficients≥0.99.The recoveries of dicyclanil and 2,4,6-triamino-5-pyrimidinecarbonitrile were 70%to 110%when they were added to the blank tissues at high,medium,low and limited quantification levels,and the coefficient of variation within and between batches was less than 15%.The stability of dicyclanil and 2,4,6-triamino-5-pyrimidinecarbonitrile in different tissues was good under the conditions of investigation.【Conclusion】The established UPLC-MS/MS assay was specific,reproducible and sensitive,and could be used for pharmacokinetic and residual studies of dicyclanil emulsion in sheep tissues.