动物营养学报 >

2022 , Vol. 34 >Issue 10: 6748 - 6757

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.10.067

研究论文 RESEARCH PAPER

动物肠道内容物中8种酸性低聚糖的液相色谱-质谱联用测定方法

  • 许丽卫 ,
  • 孙德慧 ,
  • 耿梅梅 ,
  • 陈闻 ,
  • 贺志雄 ,
  • 王久荣
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  • 中国科学院亚热带农业生态研究所, 亚热带农业生态过程重点实验室, 长沙 410125
许丽卫(1983-),女,河北定州人,高级工程师,博士,从事色谱分离与分析研究。E-mail:xlw818@isa.ac.cn

收稿日期: 2022-03-24

  网络出版日期: 2022-10-17

基金资助

中国科学院广州分院开放基金项目(2021000060)；湖南省重点研发计划项目(2020NK2049)

收起

Determination Method of Eight Acidic Oligosaccharides in Animals' Intestinal Contents by Liquid Chromatography-Mass Spectrometry

  • XU Liwei ,
  • SUN Dehui ,
  • GENG Meimei ,
  • CHEN Wen ,
  • HE Zhixiong ,
  • WANG Jiurong
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  • Key Laboratory for Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China

Received date: 2022-03-24

  Online published: 2022-10-17

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摘要

本试验旨在建立一种测定动物肠道内容物中3'-唾液乳糖(3'-SL)、6'-唾液乳糖(6'-SL)、3'-唾液基-N-乙酰乳糖胺(3'-SLN)、6'-唾液基-N-乙酰化乳糖胺(6'-SLN)、3'-唾液基-3-岩藻基乳糖(3'-S, 3-FL)、唾液基乳糖-N-四糖a(LSTa)、唾液基乳糖-N-四糖c(LSTc)和二唾液酰乳糖(DSL)8种酸性低聚糖的液相色谱-质谱联用方法。将肠道内容物冷冻干燥后粉碎，以水作为提取溶剂，超声提取、离心，重复提取2次，合并上层清液；上层清液用乙腈沉淀蛋白，离心并用固相萃取小柱净化，净化液过0.22 μm滤膜，进行液相色谱-质谱分析。以Acquity UPLC? BEH Amide色谱柱进行分离，8种酸性低聚糖在7 min内达到基线分离。结果表明：8种目标物的保留时间和峰面积的相对标准偏差均分别小于0.5%和11.0%。3'-SL、6'-SLN、LSTa、3'-S, 3-FL、LSTc、3'-SLN、DSL和6'-SL的线性范围分别为5.00~2 000.00 ng/mL、2.50~100.00 ng/mL、9.59~94.00 ng/mL、10.00~100.00 ng/mL、27.80~200.00 ng/mL、4.00~80.00 ng/mL、6.00~120.00 ng/mL、16.10~642.00 ng/mL。8种目标物质分别在线性范围内线性关系良好，相关系数均达到0.991以上，检测限在0.335~8.330 ng/mL，平均回收率为77.3%~122.9%。综上所述，该方法选择性强、样品用量少、操作简单，可用于定量测定不同肠段的内容物中8种酸性低聚糖，该方法的建立可为研究酸性低聚糖在动物健康中的作用机制提供技术支撑。

关键词： 肠道内容物; 酸性低聚糖; 液相色谱-质谱联用

本文引用格式

许丽卫 , 孙德慧 , 耿梅梅 , 陈闻 , 贺志雄 , 王久荣 . 动物肠道内容物中8种酸性低聚糖的液相色谱-质谱联用测定方法[J]. 动物营养学报, 2022 , 34(10) : 6748 -6757 . DOI: 10.3969/j.issn.1006-267x.2022.10.067

Abstract

The purpose of this experiment was to establish a method based on liquid chromatography-mass spectrometry to determine the eight acidic oligosaccharides of 3'-salivary lactose (3'-SL), 6'-salivary lactose (6'-SL), 3'-sialyl-N-acetylated lactose (3'-SLN), 6'-sialyl-N-acetylated lactose (6'-SLN), 3'-sialyl-3-fucosyl lactose (3'-S, 3-FL), sialyl lactose-N-tetrasaccharide a (LSTa), sialyl lactose-N-tetrasaccharide c (LSTc) and disasialyl lactose (DSL) in animals' intestinal contents. First, the intestinal contents were freeze-dried and pulverized into powder, then ultrasonic extraction with water, centrifuged, and the supernatant was taken. The procedures repeated once and supernatant was merged. Supernatant was treated with acetonitrile, centrifuged. The supernatant was purified by solid phase extraction column, and the purified liquid was filtered by 0.22 μm membrane for liquid chromatography-mass spectrometry analysis. The eight acidic oligosaccharides were separated by Acquity UPLC? BEH Amide column within 7 minutes of eight acidic oligosaccharides. The results showed that the relative standard deviations of retention time and peak area of eight target analytes were less than 0.5% and 11.0%. The linear range of 3'-SL, 6'-SLN, LSTa, 3'-S, 3-FL, LSTc, 3'-SLN, DSL and 6'-SL were 5.00 to 2 000.00 ng/mL, 2.50 to 100.00 ng/mL, 9.59 to 94.00 ng/mL, 10.00 to 100.00 ng/mL, 27.80 to 200.00 ng/mL, 4.00 to 80.00 ng/mL, 6.00 to 120.00 ng/mL, 16.10 to 642.00 ng/mL, respectively. The linearity of the eight target analytes was good in the linear range, the correlation coefficients were all above 0.991, the detection limits were ranged from 0.335 to 8.330 ng/mL, and the average recoveries were 77.3% to 122.9%. In conclusion, the method has high selectivity, low sample consumption and simple operation, which can be used for the determination of eight acidic oligosaccharides in contents of different intestinal segments. The established method can provide technical reference for the healthy mechanism study of acidic oligosaccharides in animals.

Key words： intestinal contents; acidic oligosaccharides; liquid chromatography-mass spectrometry

参考文献

[1] YANG G Q,YIN Y,LIU H Y.Effects of dietary oligosaccharide supplementation on growth performance, concentrations of the major odor-causing compounds in excreta, and the cecal microflora of broilers[J].Poultry Science,2016,(95):2342-2351.
[2] CINDY L B,STEPHANIE F B,LAURENCE L N.Maternal short-chain fructooligosaccharide supplementation influences intestinal immune system maturation in piglets[J].PloS One,2014,(9):153-156.
[3] 刁其玉,屠艳,张乃锋.羔羊配方代乳品的研制与应用[C]//第三届中国羊业发展大会.兰州:中国畜牧业协会,2006:250-255. DIAO Q Y,TU Y,ZHANG N F.Development and application of lamb formula milk substitute[C]//The Third China Sheep Industry Development Conference.Lanzhou:China Animal Agriculture Association,2006:250-255.(in Chinese)
[4] 和玉丹,万培伟,王庚.益生元促进动物肠道免疫的作用机理[J].江西畜牧兽医杂志,2020(1):5-9. HE Y D,WAN P W,WANG G.Effect of prebiotics on intestinal immunity in animals[J].Jiangxi Journal of Animal Husbandry&Veterinary Medicine,2020(1):5-9.(in Chinese)
[5] DANIELA G,ADRIANA DI T,SALVATORE C.Oligosaccharides, polyamines and sphingolipids in ruminant milk[J].Small Ruminant Research,2018,160:23-30.
[6] JOLANTA L K,MAGDALENA O P.Sialylated oligosaccharides and glycoconjugates of human milk. the impact on infant and newborn protection, development and well-being[J].Nutrients,2019,11:306-329.
[7] YAN J Y,DING J J,LIANG X M.Chromatographic methods for the analysis of oligosaccharides in human milk[J].Analytical Methods,2017,9(7):1071-1077.  
[8] KARINA M T,ANTONIO M,ANA CRISTINA F V A,et al.Validation and application of a method for the simultaneous absolute quantification of 16 neutral and acidic human milk oligosaccharides by graphitized carbon liquid chromatography-electrospray ionization-mass spectrometry[J].Food Chemistry,2019,274:691-697.
[9] AUSTIN S,BENET T.Quantitative determination of non-lactose milk oligosaccharides[J].Analytica Chimica Acta,2018,1010:86-96.
[10] ZHANG W Y,WANG T,CHEN X X,et al.Absolute quantification of twelve oligosaccharides in human milk using a targeted mass spectrometry-based approach[J].Carbohydrate Polymers,2019,219:328-333.
[11] AMANDA J. FISCHER,NILUSHA M,et al.The effect of heat treatment of bovinecolostrum on the concentration of oligosaccharides in colostrum and in the intestine of neonatal male Holstein calves[J].Journal of Dairy Science,2018,101:401-407.
[12] MA L,MCJARROW P,JAN MOHAMED H JAN B,et al.Lactational changes in the human milk oligosaccharide concentration in Chinese and Malaysian mothers'milk[J].International Dairy Journal,2018,87:1-10.
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