Differences in the Triacylglycerol and Fatty Acid Compositions of

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Omics Technologies Applied to Agriculture and Food

Differences in the Triacylglycerol and Fatty Acid Compositions of Human Colostrum and Mature Milk Pu Zhao, Shuwen Zhang, Lu Liu, Xiaoyang Pang, Yang Yang, Jing Lu, and Jiaping Lv J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00868 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018

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Journal of Agricultural and Food Chemistry

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Differences in the Triacylglycerol and Fatty Acid

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Compositions of Human Colostrum and Mature

3

Milk

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Pu Zhao a, 1, Shuwen Zhang a, 1, Lu Liu a, Xiaoyang Pang a, Yang Yang a, Jing Lu a*,

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Jiaping Lv a*

7 8

a

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Institute of Food Science and Technology, Chinese Academy of

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Agricultural Science, Beijing 100193, People’s Republic of China

Key Laboratory of Agro-Food Processing and Quality Control,

11 12 13

1

P.Z and S.Z contributed equally to this work

14 15 16

*Corresponding Author: Jing Lu, Institute of Food Science and Technology CAAS,

17

Chinese Academy of Agricultural Sciences No. 2 Yuanmingyuan West Road, Haidian

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District, Beijing.

19

E-mail: [email protected]

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Jia-ping Lv, Institute of Food Science and Technology CAAS, Chinese Academy of

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Agricultural Sciences No. 2 Yuanmingyuan West Road, Haidian District, Beijing.

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E-mail: [email protected] 1

ACS Paragon Plus Environment


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Abstract

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Human colostrum is important for the immune system development and plays a

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protective role for infants. However, the comprehensive exploration of lipids, which

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account for 3-5% of milk, and their biological functions in human colostrum was

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limited. In present study, the triacylglycerol (TAG) and fatty acid (FA) compositions

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of human colostrum and mature milk were analyzed and compared. Variations were

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observed in both the TAG and FA compositions. The concentrations of 18:1/18:1/16:0

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TAG, high-molecular-weight and unsaturated TAGs were significantly higher in

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colostrum, whereas mature milk contained more low/medium-molecular-weight TAGs

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and medium-chain FAs. Furthermore, there were also specific TAGs in both

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colostrum and mature milk. Our data highlighted targets for further investigation to

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elucidate biological function of lipids in colostrum milk. In addition, the

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comprehensive analysis of TAGs in Chinese colostrum might help in designing infant

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formula for Chinese babies especially the preterm ones.

37 38

human

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Keywords:

40

UPLC-ESI-MS

colostrum,

mature

milk,

triacylglycerols,

41 42 43 44 2


fatty

acids,

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1. Introduction

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Human milk, which contains essential nutrients and other physiological substances, is

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regarded as the optimal food for neonates.1 Among these substances, a fat content of

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only 3-5% in milk can provide approximately 50% of the energy required by infants.2

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Ninety-eight percent of the milk fat consists of triacylglycerols (TAGs), which are

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molecules of glycerol esterified to three fatty acids (FAs), with the side-chains at the

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Sn-1, Sn-2 and Sn-3 positions. The different FAs at these three positions result in

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unique TAGs that play important roles in improving digestion and absorption,3 and

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determine the applications of TAGs. For example, TAG containing palmitic acid

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(C16:0), a FA with a higher melting point than the human body temperature (37°C),

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in the Sn-1/Sn-3 position can combine with Ca2+ or Mg2+ to form insoluble solids that

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may be excreted in hard stool.4 More than 20% of all fatty acids in human milk fat

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(HMF) are C16:0, with 60% of C16:0 in the Sn-2 position to reduce calcium loss and

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improve FA absorption.5-6 Only 35% of newborn to 6-month-old babies are currently

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breastfed globally. Therefore, developing infant formula (IF) with a composition

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similar to that of breast milk will help ensure suitable nutrition for all infants. Based

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on previous studies of TAGs in human milk,7-8 the addition of 18:1/16:0/18:1 TAG

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(OPO) is common when formulating IF. However, adding only OPO does not

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simulate the overall structure and composition of HMF. Therefore, investigating the

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changes in the TAGs over the lactation periods is important for accurately mimicking

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HMF for IF.

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Colostrum is the milk produced during the first several days after parturition. 3



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Colostrum is important for the development of the immune system and protects

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infants against allergies and chronic diseases. Infants could gain long-term metabolic

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benefits by consuming colostrum during the first several days of life.9 To the best of

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our knowledge, information about the TAGs in human colostrum is scarce. Martin

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and coworkers analyzed the TAGs in human colostrum and mature milk by using

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enzymatic degradation,10 but information on individual molecular species was absent.

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Benefitting from the development of analytical techniques in recent years, 24 TAGs

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were determined in Danish human colostrum, transitional, and mature milk.11 Thirty

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TAGs were compared in Spanish human colostrum and mature milk.12 Most recently,

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Chinese human transitional and mature milk were analyzed by using supercritical

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fluid chromatography (SFC) coupled with quadruple time-of-flight mass spectrometry

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(Q-TOF-MS).13 The lipid composition of breast milk is influenced by diet, age, parity

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and stage of lactation.14 Large variations exist between the Western and Asian diets

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and the TAG data for Western colostrum is thus not representative of Chinese data.

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The TAGs with specific FA chain composition in Chinese colostrum could aid in the

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determination of the nutritional value and health benefits of colostrum and the

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exploration of the effect of diet on TAGs.

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For TAG analysis, most studies focus on developing suitable analytical methods,

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including traditional methods, such as enzymatic degradation using thin layer

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chromatography (TLC) and gas chromatography (GC),15 as well as new methods,

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such as high-performance liquid chromatography-tandem mass spectrometry

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(HPLC-MS/MS) with atmospheric pressure chemical ionization (APCI)16 for plant oil 4


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and electrospray ionization (ESI) for milk fat.17 The methods used for sample

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preparation and TAG separation for human milk were developed by Ten-Doménech

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and co-workers. In their study, 42 TAGs were characterized and quantified in human

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milk by HPLC with evaporative light scattering detection and tandem mass

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spectrometry (HPLC-ELSD MS/MS).18 TAGs in milk of different species have also

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been analyzed by HPLC-APCI-MS.11 To the best of our knowledge, in most of the

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works related to TAG analysis in milk, the detailed TAGs in Chinese human milk at

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different lactation stages have not been well characterized. Thus, in the present study,

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we analyzed the TAGs in colostrum and mature Chinese human milk by the advanced

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ultra-performance liquid chromatography-tandem mass spectrometry equipped with

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an electrospray ionization source (UPLC-ESI-MS) method reported by M Guan.19 In

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such a method, one sample can be analyzed in 30 mins, and a small sample amount

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can yield highly accurate and precise results. More than 66 different TAGs were

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characterized and quantified. And there were also significant differences between

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human colostrum and mature milk, such as colostrum milk had more UUU (three

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unsaturated fatty acids in Sn-1, 2, 3 positions) and LLL (three long chain fatty acids in

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Sn-1, 2, 3 positions) types of TAGs, whereas mature milk had more low- and

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medium-molecular-weight TAGs, etc. The variations and patterns of the TAGs

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observed in the present study can form the basis for IF production.

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2. Materials and methods

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2.1. Chemicals

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Internal

standard

TAG,

1,

3(d5)-diheptadecanoyl-2-heptadecenoyl-glycerol 5



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(d5-(17:0/17:1/17:0) TAG), was purchased from Avanti Polar Lipids (Birmingham,

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AL, USA). Standard FAs (Supelco 37-component FAME mix) were obtained from

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Sigma-Aldrich Co. LLC. HPLC-grade acetonitrile, methanol (MeOH), isopropanol

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(IPA), chloroform (CHCl3), formic acid, ammonium formate and toluene (PhMe)

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were purchased from Fisher Scientific (Pittsburgh, PA, USA). Analytical-grade

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anhydrous sodium carbonate, anhydrous sodium sulfate and acetyl chloride were

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purchased from Sinopharm Chemical Reagent Co., Ltd.

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2.2. Sample collection

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Twenty-six colostrum samples (collected on the 4th day of lactation) and 40 mature

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milk samples (collected on the 30th day of lactation) were obtained from healthy

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volunteer mothers in the morning in the hospital in Beijing and were transported to

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the laboratory on ice. All the participating mothers were informed of the project in

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advance. The samples were stored at -80°C before analysis.

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2.3. Extraction of human milk lipids

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According to the Bligh and Dyer method,20 one human milk sample was mixed with

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CHCl3, MeOH and Milli-Q water (1:1:2:0.8, v/v/v/v), and the mixture was oscillated

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for 30 min. CHCl3 and Na2SO4 (2%, w/v) were added for a final volume ratio of

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2:2:1.8 (MeOH: CHCl3: water, v/v/v). The mixture was oscillated again for 15 min

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and centrifuged at 2250 g for 30 min. The organic fraction was filtered through filter

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paper containing 2 g of Na2SO4, dried under a nitrogen stream and stored at -80°C

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before analysis.

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2.4. TAG composition of HMF detected by UPLC-MS/MS 6


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Referring to the method of M Guan,19 the extracted lipids were dissolved in CHCl3/

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MeOH (1:2, v/v) at 5 mg/mL and then underwent a 50-fold dilution using MeOH with

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an internal standard (d5-(17:0/17:1/17:0) TAG). Samples were loaded through a

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UPLC system (I-class Acquity UPLC, Waters) with an Autosampler. The lipids were

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separated on a BEH C18 reversed-phase column (1.7µm, 2.1 mm ID × 100 mm,

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Waters Corporation). The column was maintained at 60°C during the whole process.

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Mobile phase A was acetonitrile/water (1:1, v/v), and mobile phase B was IPA/

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acetonitrile (9:1, v/v), both of which contained 10 mM ammonium formate and 0.1%

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formic acid. The flow rate was 0.3 mL/min, with a linear gradient of solvent B from

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70% to 85% over 28 min, and then to 70% at 28.1 min, followed by maintenance at

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70% for 1.9 min, and 5µL samples were injected for analysis by MS (API 4500

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Q-Trap, AB SCIEX) which was equipped with an ESI source. Samples were detected

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in the positive ionization mode and both the nebulizer and desolvation gases were

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nitrogen. The typical operating parameters were set as follows: curtain gas (CUR) 25;

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collision gas (CAD) medium; ion source gas 1 (GS1) 45; ion source gas 2 (GS2) 50;

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electrospray voltage 5500V; and a temperature of 550°C.

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The detected method was the same with M Guan. In brief, firstly, the parent ions were

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obtained using the scan mode, with the declustering potential (DP) set to 100V, the

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mass range set to 400-1200m/z, and the scan rate set to 1000Da/s. Parent ion of TAGs

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in colostrum and mature milk can be obtained using the scan mode; then an

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instrument

154

(IDA)-enhanced product ion (EPI) mode was established for the qualification of

method

in

MRM

(Q1=Q3)-information

7


dependent

acquisition


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TAGs with specific FA chain compositions; finally, for the quantification of TAGs

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with specific FA chain compositions, MRM (Q1≠Q3) mode was applied. Specific ion

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pairs of each TAG isomers were selected and used in MRM survey channels, which

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was shown in Table 1. The isomeric species of TAGs were determined by referring to

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the mass-to- charge ratio (m/z) and abundance. The peak areas of TAGs isomers were

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collected and compared with the peak areas of d5-17:0/17:1/17:0 TAG to calculate the

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concentrations of TAGs in colostrum and mature samples.

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2.5. FAs detected by GC

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The sample pretreatment method followed the Chinese national standard

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541327-2010. 0.5g sample was weighed and then it was placed in a screw-cap test

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tube. Then, 5 mL of PhMe and 6 mL of acetyl chloride-MeOH (1:9, v/v) were added

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to the tube. After the tube was shaken, it was filled with a nitrogen stream for 60 s.

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The mixture was placed in an 80°C water bath, and shaken six time every 20 min

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intervals. After the mixture cooled to room temperature, it was transferred to a 50 mL

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centrifuge tube and washed 3 times with 3 mL of 6% Na2CO3. The mixture was

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centrifuged at 5000 rpm for 5 min, and then the upper phase was transferred to a gas

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vial with a 0.2 µm organic filter.

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The detection method was performed according to Chavarri et al.21 with a slight

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modification of the capillary column and temperature program. Samples were

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analyzed by GC (7890B Agilent) with a capillary column (Agilent HP-88 100 m×0.25

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mm×0.2 µm). The carrier gas (nitrogen) flow rate was 1 mL/min, and the oven

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temperature was increased from 80°C (1min) to 200°C at 2°C /min, held for 2 min, 8


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increased to 230°C at 5°C /min and held at 230°C for 15 min. The temperature of the

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flame ionization detector was 300°C. The qualitative and quantitative methods used

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an external standard (Supelco 37 component FAME mix) and area normalization.

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2.6.Statistical analysis

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All measurements were made in duplicate. IBM SPSS 22.0 was used to determine

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significant differences of the data.

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3. Results

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3.1. The characterization of TAGs based on UPLC-ESI-MRM (Q1=Q3)-IDA-EPI

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mode

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The UPLC-ESI-MRM (Q1=Q3)-IDA-EPI mode19 was applied for the determining

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TAGs. In this mode, Q1 was set as the same as Q3 to lock the parent ions. The locked

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parent ions will be fragmented in EPI experiment if the intensity of parent ions

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exceeds the threshold of IDA. From the real-time fragments of certain parent ions, the

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compositions of TAGs can be deduced.

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The total ion chromatograms (TICs) of human colostrum and mature milk were

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showed in Fig. 1(A) and Fig. 1(B) respectively. Fig. 1(a-f) has showed the extracted

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ion chromatograms (XICs) of 40-2～40-0 TAGs in colostrum and their corresponding

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MS/MS spectra of ammonium adducts. The TAGs were separated according to the

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combined effects of the number of carbon atoms in FA moieties and the number of

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double bonds. To be specific, the retention times of 40-2, 40-1 and 40-0 TAGs were

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5.16 min, 6.16 min and 7.43 min, respectively, after eliminating the isobaric

198

interferences.22 9



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Fig. 1(d-f) has showed the MS/MS spectra of the ammonium adducts of 40-2, 40-1

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and 40-0 TAGs detected in MRM (Q1=Q3)-IDA-EPI mode. For example, in Fig. 1(d),

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product ions at m/z 411.4, 519.5 and 491.3 were corresponding to the neutral loss (NL)

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of NH3 and 18:2, 10:0 and 12:0 FAs of ammonium adduct of 40-2 TAG, respectively,

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40-2 TAG can thus be deduced as 18:2/12:0/10:0 TAG. Similarly, 40-1 TAG eluted at

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6.16 min was deduced as 18:1/12:0/10:0 TAG shown in Figure 1(e). However, for

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some complex TAGs, such as 40-0 TAG (RT: 7.43 min), was deduced as a mixture of

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16:0/12:0/12:0 TAG, 18:0/12:0/10:0 TAG and 16:0/14:0/10:0 TAG (Table 1).

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In total, 68 and 66 TAGs were characterized in colostrum and mature human milk

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respectively.

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3.2.The quantitative analysis of TAGs in colostrum and mature milk

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After the characterization of each TAG isomer, the ion pairs, which were showed in

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Table 1, were carefully chosen and UPLC-ESI-MRM (Q1≠Q3) mode was applied for

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the quantification of TAGs with specific FA chain (Table 1). The TAGs with contents

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exceeding 5mol% were O/O/P, 18:2/18:1/16:0 TAG (L/O/P), 18:1/18:1/18:1 TAG

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(O/O/O),

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18:2/18:2/16:0 TAG (L/L/P) (Table 1). Among these TAGs, the L/L/P content was

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less than 5mol% in colostrum milk. Considering the major TAGs that accounted for

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more than 1mol% of the total content, colostrum milk contained more O/O/P, O/O/O,

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O/L/O, O/P/P, 18:2/16:0/16:0 TAG (L/P/P), 18:1/18:0/18:1 TAG (O/S/O) and

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16:0/16:0/16:0 TAG (P/P/P) than mature milk (p

Differences in the Triacylglycerol and Fatty Acid Compositions of

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