Establishment And Application Of Quantitative Detection Method For Oligosaccharides In Human Milk

Background: Human milk is recognized as the best source of nutrition for newborns.It is rich in human milk oligosaccharides(HMOs),which are the third largest component of human milk after lactose and lipids.HMOs have multiple physiological functions that are beneficial to the growth and development of infants and children.Defining the content and the influencing factors of HMOs in human milk are of great significance for developing infant formulas that similar to human milk.HMOs are absence with chromophore,which results in its low sensitivity on the detector,and derivatization using reductive amination can greatly improve the sensitivity.Mass spectrometry has excellent accuracy and reproducibility in targeted analysis.Based on reductive amination reaction,this research uses ultra-high performance liquid chromatography tandem mass spectrometry platform to establish a simultaneous quantitative detection method for 6 HMOs,and applies the method to human milk sample to analyze the effects of lactation and geographic differences on the target HMOs content.Methods: Based on the reductive amination reaction,2’-fucosyllactose(2’-FL),3’-fucosyllactose(3’-FL),3’-sialyllactose(3’-SL),6’-sialyllactose(6’-SL),lacto-N-fucopentaose-I(LNFP-I)and lacto-N-fucopentaose-V(LNFP-V)were derivatized with 2-aminobenzoic acid(2-AA),2-aminobenzamide(2-AB),and 2-aminoacridone(AMAC),respectively.Combining the scanning results of high-resolution mass spectrometry and triple quadrupole mass spectrometry,a mass spectrometry multiple reaction monitoring(MRM)method was established and the cone voltage and collision energy were optimized,and then the optimal derivatizing agent was determined.After the optimization of the liquid phase conditions,the ultra-high performance liquid-tandem mass spectrometry(UPLC-MS)platform was used to establish a synchronous and fast MRM mass spectrometry method for these 6 HMOs.And the method was validated from four aspects: linear regression,detection limit,recovery rate and precision.Then the established method was used to quantitatively analyze these 6 HMOs in human milk samples.The data were analyzed by independent sample T test and one-way analysis of variance(ANOVA).Results: 1.Establishment of simultaneous quantitative methods of 6 HMOs in breast milkHMOs were derivatized with 2-AA,2-AB and AMAC,respectively.After comparison,2-AA was selected as the optimal derivatizing agent.After the optimization of the liquid phase conditions,a 25 m M ammonium formate aqueous solution was selected as the aqueous phase,and pure acetonitrile was used as the organic phase.A quantitative method for the determination of human milk oligosaccharides was established using an ultra-high performance liquid chromatography-tandem mass spectrometry platform,and the corresponding methodological verification was passed.The established method has simple pre-processing steps,short analysis time,and can complete the separation and quantification of 6 target HMOs in 14 minutes.The linearity and stability of each HMO are excellent,the precision range is 1.0-7.9 %,and the recovery rate is 83.2-119.9 %,2’-FL,3’-FL have a limit of quantification(LOQ)of 1.5 mg/L,3’-SL,6’-SL,LNFP-I have a LOQ of 5.0 mg/L,and LNFP-V has a LOQ of 4.5 mg/L.The precision and accuracy of the method are good,which can meet the accurate quantification of 6 HMOs in human milk.2.Application of quantitative methods for HMOs in breast milkWe applied the previously established method to quantitative analysis of these 6 HMOs on a total of 103 breast milk samples from Qingdao,Wuhan and Hohhot.The results showed that the method can be well applied to human milk matrix,the contents of 2’-FL,3’-FL,3’-SL and 6’-SL in all samples were both higher than LOQ,but LNFP-I was not detected in 9 samples and LNFPV was not detected in 5 samples.The content of 2’-FL,3’-FL,3’-SL,6’-SL,LNFP-I and LNFP-V in the sample was 7.4-4900.3 mg/L,28.2-3192.1 mg/L,113.1-613.1 mg/L,20.6-1281.6 mg/L,6.4-5354.6 mg/L,and 4.7-136.4 mg/L,respectively.By comparing different lactation periods,it was found that except for 3’-FL and LNFP-V,the content of the other 4 HMOs in mature milk decreased significantly,while 3’-FL and LNFP-V showed opposite trends.The results of regional comparison showed that there were significant differences in 2’-FL and LNFP-V content in mature milk between Qingdao,Wuhan and Hohhot.The 2’-FL content decreased in order from Wuhan,Qingdao and Hohhot.The median(inter-quartile range)of corresponding content was 1966.1(2441.7)mg/L,1266.7(1164.9)mg/L,and 24.9(1497.9)mg/L.The LNFP-V,on the contrary,increased in order.Conclusions: The methodological verification shows that the simultaneous quantitative detection method of HMOs in human milk by mass spectrometry can be used for the quantitative analysis of 2’-FL、3’-FL、3’-SL、6’-SL、LNFP-I and LNFP-V in human milk.Lactation stages and geographical differences have different effects on the content of each HMO.More detailed information on pregnant women and subsequent large sample studies are needed for further exploration.