高效液相荧光色谱法检测婴幼儿配方食品中7种母乳低聚糖

Determination of 7 kinds of human milk oligosaccharides by high performance liquid chromatography with fluorescence detector

中文关键词:  HPLC-FLD  HMOs  酶解  衍生  定量检测

英文关键词:high performance liquid chromatography with fluorescence detector  human milk oligosaccharides  enzymatic digestion  derivatization  quantitative detection

中文摘要:

      目的 建立高效液相荧光色谱法(high performance liquid chromatography with fluorescence detector, HPLC-FLD)检测婴幼儿配方食品配方奶粉中7种母乳低聚糖(human milk oligosaccharides, HMOs)的分析方法。方法 婴幼儿配方食品复溶后, 首先通过酶解处理(β-半乳糖苷酶和淀粉葡萄糖苷酶)去除婴幼儿配方食品基质中存在的HMOs干扰物, 例如麦芽糖糊精和低聚半乳糖, 酶解后向样品溶液中添加内标物质, 后使用衍生化方法使得目标HMOs和内标物质均标记荧光基团(2-氨基苯甲酰胺), 即可运用HPLC-FLD对其进行检测。该方法使用HMOs外标法进行目标物的定量分析, 且标准物质信号均亦通过内标物矫正。结果 婴幼儿配方食品中7种HMOs可通过酶解、衍生及HPLC-FLD检测, 使用外标法进行定量分析。通过内标物质可矫正信号, 减少实验偏差。7种HMOs [2’-岩藻糖基乳糖(2’-fucosyllactose, 2’-FL)、3-岩藻糖基乳糖(3-fucosyllactose, 3-FL)、双岩藻糖基乳糖(difucosyllactoses, DFL)、乳糖-N-四糖(lacto-N-tetraose, LNT)、乳糖-N-新四糖(lacto-N-neotetraose, LNnT)、3’-唾液酸乳糖(3’-sialyllactose, 3’-SL)和6’-唾液酸乳糖(6’-sialyllactose, 6’-SL)]可检测范围分别为20.0~1247.9、18.3~1146.2、7.6~455.1、16.4~821.9、16.3~814.2、7.2~431.6、8.0~477.2 mg/100 g powder, 平均回收率为91%~110%, 相对标准偏差低于3%。结论 该方法可实现0~36月龄婴幼儿配方食品中7种HMOs的定量检测, 结果准确可靠。

英文摘要:

      Objective To establish analysis method for the determination of 7 kinds of human milk oligosaccharides (HMOs) in infant formula food by high performance liquid chromatography with fluorescence detector (HPLC-FLD). Methods After the infant formula food was redissolved, the HMO interferents present in the matrix of infant formula food, such as maltodextrin and oligosaccharides, were first removed through enzymatic hydrolysis (β-galactosidase and starch glucosidase). After enzymatic hydrolysis, internal standard substances were added to the sample solution, and derivatization methods were used to label both the target HMOs and internal standard substances with fluorescent groups (2-aminobenzamide), which could be detected by HPLC-FLD. This method used HMOs external standard method for quantitative analysis of the target substance, and the signal of the standard substance was also corrected by internal standard. Results The 7 kinds of HMOs in infant formula food could be quantitatively analyzed through enzymatic hydrolysis, derivatization, and HPLC-FLD detection by using external standard method. Signal correction coud be achieved through internal standard substances, and experimental bias was reduced. The detectable ranges for 7 kinds of HMOs [2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), difucosyllactoses (DFL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL)] were 20.0?1247.9, 18.3?1146.2, 7.6?455.1, 16.4?821.9, 16.3?814.2, 7.2?431.6, 8.0?477.2 mg/100 g powder, respectively, and the average recoveries were 91%-110% with relative standard deviations of less than 3%. Conclusion This method can achieve quantitative detection of 7 kinds of HMOs in formula foods for infants and young children aged 0?36 months, with accurate and reliable results.