建立超高效液相色谱-串联质谱法(ultra-performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)同时测定婴幼儿配方乳粉中2′-岩藻糖基乳糖(2′-fucosyllactose, 2′-FL)和乳糖-N-新四糖(lacto-N-neotetraose, LNnT)。样品经水提取,沉淀法除蛋白、针式水系滤膜净化,以乙腈和10 mmol/L甲酸铵水溶液(含0.1%甲酸,体积分数)为流动相,采用酰胺柱进行梯度洗脱,采用选择反应检测扫描模式(selective reaction monitoring, SRM)测定,外标法定量,稀释法评估溶剂效应、提取后添加法评估基质效应。结果表明,正离子模式下2′-FL和LNnT分别采用加合离子峰[M＋NH＋4]和[M＋H＋]较好,梯度条件、流速优化后使得2′-FL和LNnT均能在8 min内完成洗脱,溶剂效应考察发现2′-FL和LNnT存在严重的溶剂效应,但在消除试验中发现在稀释20倍的情形下,50%乙腈甲酸铵水溶液作为净化样液的稀释溶剂可消除影响,线性范围考察发现2′-FL和LNnT线性范围分别为5.0~150.0 ng/mL和2.5~75.0 ng/mL,基质效应考察发现2′-FL和LNnT均不存在基质效应。方法验证表明,2′-FL和LNnT各项精密度正确度参数均满足国标要求,该方法适用于婴幼儿配方乳粉中2′-FL和LNnT的同时测定。
A method was established to simultaneously determine 2′-fucosyllactose (2′-FL) and lactose-N-neotetraose (LNnT) in infant milk powder by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).The sample was extracted by water, precipitated protein, and purified through a needle-type water system filtration membrane.Then the sample solution was separated using an amide column by gradient elution, within acetonitrile and 10 mmol/L ammonium formate aqueous solution (containing 0.1% formic acid, V/V).The selective reaction monitoring mode was used to measure and quantify by external standards.In addition, it also evaluated the solvent effect and matrix effect by dilution method and extraction addition respectively.Results showed that the addition ion peaks [M+NH+4] and [M+H+] of 2′-FL and LNnT in the positive ion mode were better than others.After the gradient conditions and flow rate optimization, both 2′-FL and LNnT could be eluted within 8 min.However, it was found that 2′-FL and LNnT had serious solvent effects, while, in the elimination test, the effect could be eliminated using 50% acetonitrile ammonium formate aqueous solution as the diluted solvent, within 20 times dilution.The linear ranges of 2′-FL and LNnT were 5.0-150.0 ng/mL and 2.5-75.0 ng/mL, respectively.And there was no matrix effect in either 2′-FL or LNnT.The method verification showed that the precision and accuracy parameters of 2′-FL and LNnT met the requirements of the national standard, and this method was suitable for the simultaneous determination of 2′-FL and LNnT in infant formula.
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