在线固相萃取-高效液相色谱-高分辨质谱法测定植物中11种水溶性有机酸

Determination of 11 Water-Soluble Organic Acids in Plants by Online Solid-Phase Extraction Coupled with High Performance Liquid Chromatography-High Resolution Mass Spectrometry

采用在线固相萃取-高效液相色谱-四极杆-静电场轨道阱高分辨质谱建立植物中11种水溶性有机酸(乳酸、富马酸、琥珀酸、苹果酸、酒石酸、香豆酸、没食子酸、柠檬酸、香草酸、阿魏酸、新绿原酸)的分析方法。样品经匀浆、超声、过滤、稀释后,经在线Oasis MAX柱(20 mm×2.1 mm,30μm)富集净化,采用BEH C18AX色谱柱(100 mm×2.1 mm,1.7μm)分离,以含10 mmol/L甲酸铵的0.9%甲酸和0.9%甲酸-乙腈为流动相,梯度洗脱。在负离子扫描模式下,以Full MS/dd-MS2模式进行分析,内标法定量。结果表明,11种有机酸可在9 min内完成分离分析,各组分的精确质量数偏差小于5.00×10^(-6),在1~100μg/L质量浓度范围内线性关系良好,相关系数(R^(2))大于0.999,方法检出限为0.1~3.2μg/kg,定量限为0.3~9.6μg/kg。以芡实和小白菜为基质样品,在2个不同加标水平下,11种有机酸的平均添加回收率分别为92.2%~111%和94.2%~110%,日内相对标准偏差分别为1.4%~4.9%和0.8%~5.6%。所建方法简便、快速、准确、可靠,已应用于8种植物(白萝卜、胡萝卜、土豆、小白菜、香蕉、鸭梨、葡萄、芡实)中有机酸含量的分析,可以作为快速分析植物中水溶性有机酸的新方法。

An analytical method for the determination of 11 water-soluble organic acids(lactic acid,fumaric acid,malic acid,tartaric acid,coumaric acid,gallic acid,citric acid,vanillic acid,ferulic acid,and neochlorogenic acid)in plants by online solid-phase extraction(SPE)coupled with high performance liquid chromatography-quadrupole orbitrap high resolution mass spectrometry(HPLC-Q-Orbitrap-HRMS)was developed.Samples were homogenized,ultrasonically extracted,?ltrated,diluted,and puri?ed on an Oasis MAX column(20 mm×2.1 mm,30μm).The analytes were separated on separated on a BEH C18AX column(100 mm×2.1 mm,1.7μm)by gradient elution using a mobile phase consisting of 0.9%formic acid solution containing 10 mmol/L ammonium formate and 0.9%formic acid in acetonitrile.The MS analysis was performed by full MS/dd-MS~2 scanning in the negative ion mode.Quantitation was performed using the internal standard method.Under the optimum conditions,the 11 organic acids were well separated within 9 minutes,and the accurate mass deviation for each compound was less than 5.00×10^(-6).The developed method showed good linearity in the range of 1–100μg/L with correlation coef?cients(R^(2))greater than 0.999.The limits of detection(LOD)and quanti?cation(LOQ)ranged from 0.1 to 3.2μg/kg and from 0.3 to 9.6μg/kg,respectively.The average recoveries of the 11 organic acids in Euryale ferox seeds and pakchoi spiked at two concentration levels were 92.2%–111%and 94.2%–110%with intraday relative standard deviations(RSDs)of 1.4%–4.9%and 0.8%–5.6%,respectively.The method was successfully applied to the analysis of organic acids in white radish,carrot,potato,pakchoi,banana,pear,grape,and E.ferox seeds.This method is simple,fast,accurate,and reliable,and can be used as a new approach for the rapid analysis of water-soluble organic acids in plants.