基于液相色谱-离子阱飞行时间质谱的大豆磷脂组分分析

Analysis of soybean phospholipids based on liquid chromatography-ion trap-time of flight-mass spectrometry

目的利用液相色谱-离子阱飞行时间质谱建立大豆磷脂组分分析方法,对大豆磷脂分子进行全面结构鉴定和定量分析,获得大豆磷脂分子组成及含量数据。方法以我国东北地区栽培的10个大豆品种为样品,利用亲水相互作用液相色谱-离子阱飞行时间质谱系统,通过亲水相互作用色谱柱(2.1 mm×150 mm,2.7μm)实现不同类型磷脂的分离,并通过离子阱-飞行时间质谱准确表征磷脂分子的头部基团类型、脂肪酰基结构和取代基位置,进行磷脂分子的定性,再采用精确质量数提取和内标法对鉴定出的磷脂分子进行定量。结果在10个大豆品种中共定性出101种磷脂分子,包括20种磷脂酰胆碱(phosphatidylcholine,PC)、15种磷脂酰乙醇胺(phosphatidylethanolamine,PE)、17种磷脂酰肌醇(phosphatidylinositol,PI)、12种磷脂酰甘油(phosphatidylglycerol,PG)、9种磷脂酸(phosphatidic acid,PA)、6种磷脂酰丝氨酸(phosphatidylserine,PS)、5种溶血磷脂酰胆碱(lysophosphatidylcholine,LPC)、5种溶血磷脂酰乙醇胺(lysophosphatidylethanolamine,LPE)、6种溶血磷脂酰肌醇(lysophosphatidylinositol,LPI)、4种溶血磷脂酰甘油(lysophosphatidylglycerol,LPG)和2种溶血磷脂酸(lysophosphatidicacid,LPA)。定量分析方法线性相关系数(R^(2))均>0.99,检出限≤60 ng/mL,精密度和准确度良好。受试大豆中的总磷脂含量为6873.1~12678.6μg/g,大部分大豆品种均能提供较为丰富的磷脂,尤其是东风-1、东农-690、吉育-301和绥农-61。受试大豆中的PC和PE含量较高,其后依次是PI、PG、PA和PS。在溶血型磷脂中,LPC含量最高,其后依次是LPE、LPI、LPG和LPA。结论本研究能够实现各类型磷脂的良好分离,并能准确表征磷脂分子的头部基团类型、脂肪酰基结构和取代基位置,获得了大豆磷脂分子组成及含量数据。

OBJECTIVE Using liquid chromatography-ion trap-time of flightmass spectrometry to establish a soybean phospholipids(PL)analysis method,carry out comprehensive structural identification and quantitative analysis of soybean PL molecule species,and obtain soybean PL molecular composition and content data.METHODS The PL profiles of 10 soybean varieties cultivated in northeast China were determined by a hydrophilic interaction liquid chromatography-ion trap-time of flight-mass spectrometry(HILIC-ESI-IT-TOF-MS)system.The hydrophilic interaction liquid chromatography realized class separation of PLs,and ion trap-time of flight-mass spectrometry realized the estimation of head group type,fatty acyl structure,and substituent position.The identified PL molecule species were quantified using accurate mass extraction and internal standard method.RESULTS A total of 101 PL molecular species from 11 classes were estimated in soybean seeds,including 20 phosphatidylcholines(PC),15 phosphatidylethanolamines(PE),17 phosphatidylinositols(PI),12 phosphatidylglycerols(PG),9 phosphatidic acids(PA),6 phosphatidylserines(PS),5 lysophosphatidylcholines(LPC),5 lysophosphatidylethanolamines(LPE),6 lysophosphatidylinositols(LPI),4 lysophosphatidylglycerols(LPG)and 2 lysophosphatidicacids(LPA).The limits of detection for the target object were≤60 ng/mL and R^(2) were all>0.99.The total concentration of PL ranged from 6873.1 to 12678.6μg/g in detected soybeans.Generally,most of the detected soybean cultivars were great resources of PL,especially SN-29,SN-61,HN-40 and DN-690.The concentrations of PC and PE in the tested soybean were higher,followed by PI,PG,PA and PS.Among the lysophospholipids,the concentration of LPC was the highest,followed by LPE,LPI,LPG and LPA.CONCLUSION The soybean PL component analysis method established based on the HILIC-IT-TOF-MS system can achieve good separation of various types of PL,and can accurately characterize the head group type,fatty acyl structure and substituent position of PL molecule species.The molecular composition and content data of soybean PL were obtained.