高效液相色谱-串联质谱法测定水产中6种吲哚类代谢物

Determination of 6 Indole Metabolites in Aquatic Products by High Performance Liquid Chromatography Tandem Mass Spectrometry

关键词:  吲哚  代谢物  高效液相色谱-串联质谱法  水产品

Key words:  indole  metabolites  HPLC-MS/MS  aquatic product

摘要:

目的 建立高效液相色谱串联质谱技术同时检测水产品中6种吲哚类代谢物的方法,考察水产品储藏过程中吲哚含量的变化情况。方法 样品均质后,使用80%乙腈水溶液(含0.1%甲酸)提取,使用HLB固相萃取柱净化。采用Agilent EclipsePlus C₁₈ RRHD(2.1×100 mm,1.8 μm)色谱柱进行分离,以0.1%甲酸水溶液和0.1%甲酸乙腈溶液为流动相按比例进行梯度洗脱。使用APCI离子源,多重反应监测模式,吲哚-3-甲醛采用内标法定量,其余代谢物采用外标法定量。结果 6种吲哚类代谢物在10~200 μg.L_^–1 内呈现良好的线性关系相关系数均>0.99,其中吲哚、3-吲哚乙酸、3-甲基吲哚检出限为2 μg.kg^_-1,定量限5 μg.kg^_-1 ,吲哚-3-甲醛、吲哚-4-甲醛、吲哚-5-甲醛的检出限为1 μg.kg^_-1,定量限为2 μg.kg_-1^。6种吲哚类代谢物的平均回收率在63.18 %～102.58 %之间,相对标准偏差(RSD)在1.6 %～10.2 %之间。随着水产品储藏时间的延长,吲哚代谢物的种类和含量随时间的增长而增加。结论 该方法操作流程简便,结果准确可靠,可用于水产品中吲哚类代谢物的检测。

Abstract:

ABSTRACT: OBJECTIVE Establish a method for simultaneous determination of 6 indole metabolites in aquatic products using high-performance liquid chromatography tandem mass spectrometry, and investigate the content of indole during the storage process of aquatic products. METHODS After homogenization, the samples were extracted by 80% acetonitrile aqueous solution (containing 0.1% formic acid) and purified by HLB solid-phase extraction column. The chromatographic separation was performed on an Agilent EclipsePlus C18 RRHD（2.1×100 mm，1.8 μm），using 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution as mobile phases with a programmed gradient. APCI ion source was used with multiple reaction monitoring mode. Indole-3-formaldehyde was quantified using internal standard method, while other metabolites were quantified using external standard method. RESULTS The calibration curves of the 6 indole metabolites showed good linear correlation coefficient（>0.99） in the range of 10-200 μg·L-1. The limits detection were 1 μg·kg-1 for indole-3-formaldehyde, indole-4-formaldehyde, indole-5-formaldehyde and 2 μg·kg-1 for other metabolites. The limits quantification were 2 μg·kg-1 for indole-3-formaldehyde, indole-4-formaldehyde, indole-5-formaldehyde and 5 μg·kg-1 for other metabolites.The average recovery rates of 6 indole metabolites are between 63.18% and 102.58% with RSDs between 1.6% and 10.2%. As the storage time of aquatic products increased, the types and contents of indole metabolites were increased. CONCLUSION This method was simplicity, accurately, and provides a new way for the detection of 6 indole metabolites in aquatic products.