| Oligosaccharides are one of the most abundant solid compounds in mammalian milk.It is found that milk oligosaccharides have important biological significance for the growth and development of young children.In this study,two analytical methods were established and evaluated to determine the oligosaccharides in bovine and human milk.And the changes of oligosaccharides under different lactation conditions were studied.1.Determination of milk oligosaccharides by high-performance liquid ion trap tandem time of flight-mass spectrometry method:Solid phase extraction column(Sep-Pak C18 column)and porous graphitized carbon were used to separate and purify the milk oligosaccharides.Then,the oligosaccharides were derivated by 2-Aminoacridone(AMAC).Finally,AMAC derivated milk oligosaccharides were analyzed and identified by using high-performance liquid ion trap tandem time of flight-mass spectrometry(HPLC IT-TOF-MS)with optimized HPLC conditions.The mobile phase consisted of acetonitrile(mobile phase A)and mobile phase B:10 mmol/L ammonium formate(pH 4.5)solution.The flow rate was 0.5 mL/min.The column temperature was 35 ? and the wavelength was 254 nm.The elution conditions were as follows:0?27 min,95%?85%A;27 min?29 min,85%A;29 min?31 min,85%?84%31 min?33 min,84%A;33 min?37 min,84%-83%A;37 min?48 min,83%?79%A;48 min?64 min,79%?73%64 min?66 min,73%A;66 min?74 min,73%?69%A;74 min?78 min,69%?68%A;78 min?85 min,68%?65%2.The milk oligosaccharides' results showed that(1)The number and content of oligosaccharides in colostrums decreased with the increase of the number of days,and the number of acid oligosaccharides in bovine colostrums changed greatly,while the number of neutral oligosaccharides was not significant.The content of acid oligosaccharides in mature milk is lower than that of colostrum.The highest abundance of oligosaccharides was Hex2Neu5Ac1 3 days before lactation,and the highest abundance of oligosaccharides in mature milk was Hex3.(2)The number of oligosaccharides increased from 1 month to 7 months after lactation,and the number of oligosaccharides reached the maximum at the 7th month of lactation,about 23 oligosaccharides were identified.Since the 7 months after lactation,the number of oligosaccharides began to decrease.The change of acid oligosaccharides and neutral oligosaccharides was consistent with the number of milk oligosaccharides.The major oligosaccharides through the whole lactation stage were Hex2HexNAc1 and Hex3.(3)The number of oligosaccharides did not change much in milk from cows from first to fifth tires,the content of oligosaccharides gradually decreased slightly as the increase of tires.No significant differenceswere observed in the content of oligosaccharides in milk from cows with first,second,fourth and fifth tires.The number and content of oligosaccharides began to decrease rapidly,after fifth tires.3.The qualitative and quantitative methods for the determination of 12 oligosaccharides in human milk were established.The conditions of' the gradient elution were as follows:the mobile phase A:10 mmol/L ammonium formate solution,mobile phase B:acetonitrile;flow rate of 0.3 mL/min;column temperature 50 ?.0?10min,95%?75%B;10?15min,75%B;15?20min,75%?65%B;20?21min,65%?10%B;21?24min,10%?25min,10?95%B;25?35min,95%B.The standard curves of 12 oligosaccharides were linear in the given concentration range,R2 was above 0.990.the recoveries ranged from 80%to 120%,and the RSD values were less than 10%.4.Large variations were observed in the content of oligosaccharides among individual human milk.Furthermore,the content of oligosacchareidse changed significantly between colostrum and mature human milk.5.The results showed that the ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry could only quantitatively analyze 12 kinds of oligosaccharides,and there were significant limitations,and high performance liquid chromatography Ion trap time-of-flight mass spectrometry is more universal. |
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