同位素内标-气相色谱-质谱法测定婴幼儿配方乳粉中缩水甘油脂肪酸酯的含量

Determination of Glycidic Fatty Esters in Infant Formula Milk Powder by Isotopic Internal Standard-Gas Chromatography-Mass Spectrometry

将奶粉样品(4.000 0g)的氨性乙醇溶液用乙醚(25,15,15 mL)和石油醚(25,15,15mL)先后3次提取其脂肪;合并上层液相,蒸发除去溶剂后于(100±2)℃烘干,于4℃保存。称取上述脂肪(0.100 0g)2份,分别置于塑料管A和B中[A管用于测定3-氯-1,2-丙二醇(3-MCPD)酯和由缩水甘油酯转化的3-MCPD的含量,B管用于测定3-MCPD酯的含量],于两管中各加叔丁基甲基醚-乙酸乙酯(8+2)混合液500μL及2.00mg·L-1d5-3-MCPD棕榈酸二酯标准溶液100μL,再加0.1mol·L-1甲醇钠-甲醇溶液1.0mL进行水解,5min后立即向A管中加入中和剂C1 200μL,并控制酸度在pH 1.0左右;同时向B管中加入中和剂C2 100μL,控制其酸度在pH 4.0左右,使水解反应终止。在A管及B管中各加正己烷3mL提取,以除去油脂类杂质。取下层水相经硅藻土小柱净化,用乙酸乙酯(3,3,15mL)洗脱3次,合并洗脱液并蒸干,加异辛烷2mL溶解残渣。于此溶液中迅速加入七氟丁酰基咪唑100μL使衍生化,30min后加入饱和氯化钠溶液2mL使反应终止。取上清液进样进行同位素内标法气相色谱-质谱分析。按公式以差量法计算缩水甘油脂肪酸酯(GEs)的含量。GEs的线性范围为13.4~402μg·L-1,检出限(3S/N)为0.015mg·kg-1。测得回收率为95.2%~103%,测定值的相对标准偏差(n=6)小于4.0%。

Sample of milk powder （4. 000 0 g） was extracted thrice with ether （25, 15, 15 mL） and petroluem ether （25, 15, 15 mL） in succession to have the fat separated into the ether upper layers, which were combined and evaporated to dryness and dried at （100±2）℃. The dried fat was kept at 4℃. Two portions of the fat were weighed （0. 100 0 g） and placed separately into 2 plastic tubes, marked A and B. To both of the tubes, 500 gL of a mixture of tert-butyl methyl ether and ethyl acetate （8±2） and 100μL of 2. 00 mg·L^-1 ds-3-MCPD dipalmitate standard solution were added and both of the solutions in Tube A and Tube B were hydrolyzed for exactly 5 min by adding 1.0 mL of 0. 1 mol·L^-1 sodium methylate-methanol solution. At the end of 5 min, 200μL of neutralizing solution C1 and 100 μL of neutralizing solution C2 were added immediately to the solutions in Tube A （pH 1.0） and Tube B （pH 4. 0） respectively, to stop the hydrolytic reaction. The solutions in the 2 tubes were extracted separately with 3 mL of n-hexane to remove fatty impurities. The lower aqueous phases were taken and treated separately by passing through a micro-column packed with diatomite and eluting thrice with 3, 3, 15 mL of ethyl acetate successively. The eluates were combined and evaporated to dryness, and the residue was taken up with 2 mL of isooctane. 100μL of HFBI were added to each of the 2 solutions from Tube A and B separately and derivatization was carried out for 30 min and stopped exactly at the end of 30 min by adding 2 mL of saturated NaC1 solution. Supernatant from Tube A was used for determination of ester of 3-MCPM and 3-MCPD transformed from glycidic ester in total and supernatant from Tube B was used for determination of ester of 3-MCPD. Both of the determinations were carried out by GC-MS with isotopic internal standard. Amount of glycidic aftty ester （GE＇ s） in the sample was found by substraetion as shown by the formula given. The linearity range for GEs was found between 13. 4 to 402μg·L^-1 , with detection limit （3S/N） of 0. 015 mg·kg^-1. Values of recovery and RSD （n= 6） found were ranged from 95.2% to 103% and less than 4. 0% respectively.