Rapid Evaporative Ionization Mass Spectrometry-Based Lipidomics

May 28, 2018 - Institute of Seafood, Zhejiang Gongshang University , Hangzhou ... Zhejiang Province Key Lab of Anesthesiology, The Second Affiliated ...
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Omics Technologies Applied to Agriculture and Food

Rapid evaporative ionization mass spectrometry based lipidomics tracking of grass carp (Ctenopharyngodon idellus) during in vitro multiple-stage digestion Yanan Lin, Haixing Wang, Wei Rao, Yiwei Cui, Xina Yu, Zhiyuan Dai, and Qing Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01644 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018

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Journal of Agricultural and Food Chemistry

Rapid evaporative ionization mass spectrometry based lipidomics tracking of grass carp (Ctenopharyngodon idellus) during in vitro multiple-stage digestion

Yanan Lin1, Haixing Wang2, Wei Rao3, Yiwei Cui1, Xina Yu1, Zhiyuan Dai1,4, Qing Shen1,4,*

1 Institute of Seafood, Zhejiang Gongshang University, Hangzhou, 310012, China 2 Zhejiang Province Key Lab of Anesthesiology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, 325035, China 3 Waters Corporation, Shanghai, 201206, China 4 The Joint Key Laboratory of Aquatic Products Processing of Zhejiang Province, Hangzhou, 310012, China

Conflict of Interest: All authors declare that they have no conflict of interest.

* Corresponding Author Prof. Qing Shen Tel: +86 0571 88071024. Fax: +86 15968148458. E-mail: [email protected]; [email protected]

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ABSTRACT

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A rapid evaporative ionization mass spectrometry (REIMS) method was

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developed for lipidomics tracking of Ctenopharyngodon idellus during in

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vitro multiple-stage digestion. The REIMS conditions were optimized to be

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the temperature of heating probe 500 ◦C, sample amount 30 mg, and flow rate

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of auxiliary solvent 100 µL—min–1. The results showed that the phospholipids

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were detected to be dominated with variety and quantity in the crude and

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multiple-stage digested samples. The enzymatic effect on the phospholipids is

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varied depending on the phospholipid classes, and the hydrolysis rate of

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phospholipids increased as the degree of unsaturation of the acyl chain

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increased. The principal component analysis (PCA) indicated that the ion at

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m/z 809.61, 811.63, and 857.52 were the most noticeable species digested

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during the process. This method exhibited great potential in fast lipidomics

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profiling for inspecting the characteristics of nutritional lipid absorption

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digestion in human gastrointestin.

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KEYWORDS:

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multiple-stage digestion; rapid evaporative ionization mass spectrometry;

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phospholipids.

lipidomics;

Ctenopharyngodon

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idellus;

in

vitro

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INTRODUCTION

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Phospholipids are a class of amphiprotic biomolecules with important

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physiological and nutritional functions for human body, which include a

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hydrophilic head and a hydrophobic tail comprising the hydrocarbon chains of

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two fatty acids.1 The biological membrane is constituted of lipid bilayer, and

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its permeability and fluidity attribute to the composition and distribution of

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phospholipids.2,3 Recently, researchers found that phospholipids can play a

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significant role in lipid absorption regulation, activating cells, enhancing the

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immunity and regeneration, and reducing hepatic lipid levels and

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inflammation by clinical and biological experiments.4 In addition, with the

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natural biocompatible, biodegradable and amphiprotic characteristics,

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phospholipids have been widely utilized as emulsifier, surfactantas, liposome

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membrane, and drug coat in food, cosmetics and pharmaceutical industries.5-8

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Therefore, tracking the phospholipids in vitro digestion can simulate and

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estimate their behavior in the human gastrointestinal process, providing a

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basis for designing phospholipid containing products.

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In vitro digestion simulating human gastrointestinal environment is

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generally considered to be a safe, rapid, inexpensive method without ethical

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restriction for investigating the gastrointestinal behavior of food and

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pharmaceutical, and establishing the physicochemical phenomena that

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governs their behavior.9,10 Currently, researchers used a static method that

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simulate the mouth, stomach, and small intestine phases, since it is postulated

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that passage of a sample through the mouth and stomach will appreciably

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alter its subsequent behavior in the small intestine.11,12 Meanwhile,

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gastrointestinal tract model can accurately mimic the sophisticated conditions

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in vivo, such as salt, bile acids, variable digestive enzyme and pH, etc.9

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Therefore, multiple-stage digestion is an effective tool simulating the transit of

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phospholipids and analyzing their behavior through the human digestive

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tract.

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The rapid expanding research field, lipidomics, is built on the advances in

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multiple analytical technologies, such as liquid chromatography (LC), gas

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chromatography (GC), mass spectrometry (MS), and nuclear magnetic

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resonance (NMR) spectroscopy.13-15 One of the major analytical platforms in

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current lipidomics practice is a multidimensional mass spectrometry

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(MDMS)-based shotgun method.16,17 Hydrophilic interaction chromatography

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(HILIC) or reverse phase LC coupled to MS/MS is also commonly used for the

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separation and detection of phospholipids because of its resolution, sensitivity

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and reproducibility.18 However, the analytical procedure of these techniques is

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complex and labor-costing. Recently developed REIMS is a new and efficient

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technology which can realize real-time and accurate identification of tissues

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and allow in vivo, in situ tissue analysis.19,20 REIMS lipidomic profile displays

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many complex phospholipid molecular species originating from the cell

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membrane and make the analysis of lipids with minimal sample preparation 4

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and analysis time in a few seconds.21,22 By far, REIMS has been successfully

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applied to identify and classify the biological tissues,23 detect and resect tumor

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as the intelligent knife of medical surgery,21 characterize microorganisms,22

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and etc. Therefore, the application of REIMS in the research field of food

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science and technology has bright further, while related reports and

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publications are very rare.

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In this study, a REIMS method was developed and optimized for tracking

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the phospholipids in Ctenopharyngodon idellus during in vitro multiple-stage

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digestion. The results can contribute to revealing the characteristics of lipid

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nutrition absorption and the principle of release of phospholipid anchored

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compound in human gastrointestinal digestion.

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MATERIALS AND METHODS

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Materials and reagents.

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U·g–1), gastric pepsin (2000 U·mg–1) from porcine gastric mucosa, gastric

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lipase (3000 U—g–1), pancreatin (2500 U—mg–1 based on lipase) from porcine

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pancreas and bile salts from bovine bile were obtained from Sigma Aldrich (St.

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Louis, MO, USA). Chloroform, methanol, propan-2-ol, and acetone were

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chromatography grade from Merck (Darmstadt, Germany). The other

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chemicals and reagents are standard analytical grade and purchased from

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Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Salivary α-amylase from human saliva (1500

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Sample preparation and lipid extraction. The grass carp (C. Idellus)

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was selected as a representative sample because it is an important economic

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fish species in China containing medium content of fat. Fresh fish samples (C.

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Idellus, weight ca. 3 kg) were purchased from Wal-Mart Stores, Inc.

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(Hangzhou, China), placed in ice at a fish/ice ratio of 1:2 (w/w), and

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transported to the Institute of Seafood, Zhejiang Gongshang University,

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Hangzhou. Upon arrival, five fish samples without evident sign of parasite

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were collected and steamed. The white muscle from both sides of the fish

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sample was obtained by the mean of a mechanical deboner after removing the

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internal organs, heads, and tails of fish. Then, the muscle sample was ground

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to uniformity and stored at 4 ◦C. Five parallel samples from different fish

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bodies were prepared, and four technical replicates were applied.

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For extracting the crude lipid (including phospholipids, free fatty acids,

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glycerides, etc.), about 10 g of sample was weighed and homogenized with 30

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mL mixture of chloroform and methanol (2:1, v/v). Then, 10 mL of water was

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added, and the mixture was oscillated and centrifuged at 4 ◦C at 10000 r·min–1

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for 10 min. The organic phase (lower phase) was collected by pipette making

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sure to avoid the solid sample and upper phase. The residue was re-extracted

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with 20 mL of chloroform for another two times. Afterward, the collections of

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organic phase were combined and condensed by a rotary evaporator at 50 ◦C.

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After cooling down, 300 mL pre-cooled acetone (-20 ◦C) was poured into the

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mixture with vigorous shaking. The phospholipids were obtained by collecting 6

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the precipitation and nitrogen drying. The total amount of phospholipids at

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different digestion stage (g/100 g) was calculated using the equation, Quantity

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= Wp/Ws × 100, where Wp is the weight of phospholipids extracted from the

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digestion juice at different stage (g), while Ws is the weight of the initial fish

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sample (g).

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In vitro multiple-stage digestion.

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idellus during in vitro digestion, artificial simulated oral, gastric and intestinal

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digestion was conducted following the harmonized protocol published by

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Minekus et al.9 and Martínez-Las et al.24

To track the phospholipids of C.

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At the oral digestion stage, salivary α-amylase was added to the simulated

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salivary fluid (SSF) to a final concentration of 75 U·mL–1, and the mixture was

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combing with the prepared sample by a ratio of 1:1 (w/v). The mixture was

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properly homogenized in a 50 mL falcon tube and incubated with agitation for

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2 min at 37 ◦C. For gastric phase simulation, pepsin and gastric lipase were

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added into the simulated gastric fluid (SGF) to a final concentration of 2000

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U·mL–1 and 120 U·mL–1 respectively, and mixed with the sample after oral

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digestion at a ratio of 1:1 (v/v). The pH was adjusted to 3 with HCl (1 M), and

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the mixture was incubated at 37 ◦C for 2 h with continuous shaking (50 rpm).

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Pancreatin is a mixture of enzymes extracted from porcine pancreas

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containing trypsin, pancreatic amylase and pancreatic lipase. Because lipid

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digestion was the focus of this study, the addition of pancreatin was on the

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basis of the lipase activity. Finally, the simulated intestinal fluid (SIF) with

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2000 U·mL–1 of pancreatin and 10 mM of bile salts were added into the

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gastric mixture at 1:1 (v/v) ratio, and the pH was adjusted to 7 with NaOH (1

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M). Afterward, the mixture was incubated at 37 ◦C and shook at 50 rpm for 2 h.

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At the end of each stage, the samples were taken to the boiling water bath

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immediately for 6 min to inactivate the enzymes. The phospholipids were

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extracted and weighed by the aforementioned methods. The experiment was

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performed in triplicate.

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The content of phospholipids of C.

Instruments and parameters.

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idellus during in vitro multiple-stage digestion was investigated by REIMS

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(Waters Co., Ltd., Shanghai, China) which mainly contained the electric

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heating probe (WSD151, Weller, Germany) and a quadrupole time-of-flight

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mass spectrometer (Xevo G2-XS, Waters Co., Ltd., Shanghai, China) coupled

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with a commercial available REIMS interface. Mass spectrometer was

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calibrated with sodium formate before analysis every day. For REIMS analysis,

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the aerosol formed via the electric heating probe was aspirated into the MS via

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a PTFE tube by a Venturi pump driven by 2 bar nitrogen and mounted on the

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REIMS interface. In REIMS interface, the auxiliary solvent of propan-2-ol was

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introduced into the interface via a stainless steel capillary (1/16ʹʹ O.D., 0.002ʹʹ

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I.D.) with leucine enkephalin added for lock mass correction. The aerosol and

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solvent were mixed and guided to a heated helix collision surface operated at

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around 750 ◦C to improve signal intensity and avoid contamination. The 8

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REIMS spectra were obtained at the rate of 1 scan/second between m/z 50 –

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1200 in negative mode and accumulated for each analysi. The MS data were

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analyzed by the statistical software package Masslynxv4.1. Only the peaks with

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signal-to-noise higher than 10 were selected and analyzed. For mass spectrum

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processing, background subtraction was applied to reduce the interring noise,

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automatic peak detection was used to center the spetrum, and then lock mass

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(Leu-enkephalin, m/z 554.2615) was run to calibrate the spectrum for exact

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mass.

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Statistical analysis.

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statistical analysis. The relative content of each peak was calculated using a

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peak area normalization method. Statistical analysis and the calculation of

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mean, standard deviation, and level of significance were performed by using

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SPSS 16.0 software (SPSS Inc.,Chicago, IL, USA). The ions showing significant

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difference were examined with one-way ANOVA (p < 0.05). 20 samples for

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each digestion stage was applied to principal component analysis (PCA) for

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the determination of the main sources of variability (phospholipid peaks and

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relative contents) presented in the data sets to establish the relation between

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crude and digested samples with phospholipids.

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RESULTS AND DISCUSSION

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Optimization of REIMS conditions.

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was performed by online mass spectrometric analysis of the aerosol produced

MS spectral data in centroid mode was exported for

The REIMS study of phospholipids

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by electric heating evaporation.22 The temperature of sampling probe, sample

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volume, and flow rate of auxiliary solvent are critical and significant factors

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for the analysis of phospholipids. These factors were evaluated to optimize the

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REIMS conditions. The intensities of the peaks at m/z 783.52, 809.61, 835.54,

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857.52, and 883.54 were selected as inspection indicators.

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The temperature of sampling probe exerted significant influence on the

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intensity of phospholipid. In this study, the effect of temperature was

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evaluated in the range from 350 to 550 ◦C (Figure 1A). The results indicated

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that the sample can’t be sufficiently evaporated and ionized to produce aerosol

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at low temperature. With the increase of temperature, the signal intensities of

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the five selected peaks had a clear upward trend, and the maximum values

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were reached at 500 ◦C. When the temperature exceeded 500 ◦C, the intensity

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values decreased significantly, because high thermal energy can cause

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excessive sample evaporation and ionization and produce aerosol which

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contain many impurities. Therefore, the temperature of 500 ◦C was a superior

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condition which can be adopted for subsequent optimization tests.

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Sample volume is an important parameter for REIMS study, because a

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small amount of sample makes insufficient aerosol during evaporation, while

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excessive sample volume will dilute the thermal energy, leading to reduced

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peaks of phospholipids. The effect of sample volume on the ionization of

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phospholipids was depicted in Figure 1B, which indicated that there is a

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positive correlation between the signal intensity of phospholipid and the

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sample volume. The intensities of the selected peaks increased in the range

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from 15 to 30 mg of sample, and kept steady when the sample volume further

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increased to 35 mg. Therefore, 30 mg digestive sample is enough for REIMS

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analysis.

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In addition, a steady flow of isopropyl was used as auxiliary solvent and

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introduced directly into the atmospheric interface to enhance the signal

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intensity of phospholipid and wash the ion transfer pipeline. The effect of the

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loading flow rate of auxiliary solvent (10, 20, 50, 100 and 150 µL—min–1) on the

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signal intensity and stability of phospholipids were investigated. As shown in

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Figure 1C, the signals of phospholipid became more and more intensive when

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the flow rate was increased gradually from 10 to 100 µL—min–1, and the highest

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values were obtained at 100 µL—min–1. When the flow rate of auxiliary solvent

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was continuously increased, a light drop of the signals was occurred, and the

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signals of phospholipid became unstable and fluctuated dramatically.

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Therefore, the flow rate of auxiliary solvent was set at 100 µL—min–1 for

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promising a series of intensive and high resolution signals.

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Total volume of phospholipids at different digestion stage.

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prerequisite to compare the total quantity of phospholipids at different

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digestion stages for tracking the changes of phospholipids. The digestion

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efficiency and quantity of phospholipids in vitro oral, gastric and intestinal

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digestion were evaluated and compared with that of non-digested sample and

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the results were presented in Figure 2. The phospholipid digestion efficiency

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of non-digested sample was set as the start point (0%). There is a slight

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increase of digestion efficiency in the oral digestion stage (5.89%), and this

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upward trend became significant during the gastric digestion stage (25.22%).

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The highest digestion efficiency was obtained at the intestinal digestion stage

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(61.75%). The absolute phospholipid content was also depicted in the Figure.

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This value decreased in sequence during the oral, gastric and intestinal

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digestion, and the trend of drop became more and more fierce along with the

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digestion process. In particular, the absolute phospholipid content fell from

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1.37 to 0.26 (%) in the intestinal digestion stage. This phenomenon could be

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explained by the diversity and plenty of phospholipase in intestinal tract. After

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the ingestion of food, phospholipids were mainly physically digested in the

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mouth and stomach. The function of the oral cavity was mainly the

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mechanical mastication and homogenization, and the stomach is subjected to

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primary emulsification of lipid by gastric acid and reduce the surface tension

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of lipids. In intestinal digestion, the SIF was composed of bile salts and

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pancreatin from porcine pancreas containing lipase, phospholipase and

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cholesterol esterase. Pancreatic lipase can catalyze the hydrolysis of fatty acids

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of phospholipids and release fatty acids and acyl lysophospholipids. In

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addition, bile salts and enzymes have a synergistic effect on the hydrolysis of

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phospholipids.25

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The phospholipids in fish tissue can be

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Identification of phospholipids.

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evaporated and ionized in both positive-ion and negative-ion modes. However,

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the signals of phospholipids in positive-ion mode were suppressed by

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impurities, such as glycerides, because of the protonating competition during

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the rapid evaporative ionization. The phospholipids in negative-ion mode are

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obviously more abundant and informative, due to the substantial negative

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charge of the headgroup.26 Therefore, negative-ion mode was used in the

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subsequent experiments, and the extract of phospholipids from non-digested

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C. idellus was used as a control group. The mass spectrum was recorded in

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real-time as the electric heating probe touched the sample. The identities of

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phospholipids were interpreted by investigating the characteristics of ions in

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the database LIPID MAPS and comparing them with those in related

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literature, and the results of phospholipids analysis at different digestion

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stages were summarized in Table 1. Some ions with intensive signals were

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confirmed by MS/MS analysis. For example, the ion of 885.5 could be

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tentative identified as [PI(38:4)-H]- by exact mass value, which could be

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confirmed by the characteristic fragments of m/z 283.1 (18:0) and m/z 303.1

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(20:4), indicating the detailed structure [PI 18:0/20:4-H]-. The phospholipids

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identified in non-digested sample, oral, gastric, and intestinal digested sample

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majorly dominated in the m/z range of 700 to 950. A total of 35 phospholipid

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molecular species were tentatively identified, which could be divided into

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three phospholipid classes, including PC, PE, and PI. However, PS, PA and PG

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were not observed in this experiment, and it could be attributed to the low

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abundance of PS and PA in fish tissue and the rare existence of PG in animal

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muscle tissue.27 As shown in the spectrum, the m/z of the most phospholipid

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peaks came out as an odd number (such as 809.61), indicating that the

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nitrogen-containing moiety of PC or PE has lost during ionization. It is usually

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a good rule to speculate the molecular formula of phospholipids. For example,

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the m/z 739.57 could be tentatively explained as [PC O-38:2-N(CH3)3]– or [PE

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O-38:2-NH3]–, and the m/z of PC O-38:2 and PE O-38:2 became identical

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after losing the nitrogen-containing moiety. This phenomenon is not

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frequently observed in modern mass spectrometers which are usually

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equipped with soft ionization techniques, such as electrospray ionization,

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atmospheric pressure chemical ionization, and matrix assisted laser

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desorption ionization. The intensive energy of electric heating probe make the

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rapid evaporative ionization a kind of hard ionization, leading to the loss of

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nitrogen-containing moiety possible. It is worth to mention that some peaks

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were composed of more than one phospholipid species. As described, the

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overlap of [PI 34:4 - H]–, [PE 44:6 - NH3]– and [PC 44:6 - N(CH3)3]– peaks

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produced the peak at m/z 829.53. This phenomenon could be attributed to the

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similarity of structure between PC with PE and the equivalent molecular mass

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after losing some groups. Some phospholipid molecular species has positive

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nutritional properties, such as m/z 795.51 (PC 42:9 & PE 42:9), m/z 929.51

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(PI 42:10), etc., whose sn-1 and sn-2 moieties involved eicosapentaenoic acid

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or docosahexaenoic acid regarded as important protective agents for health

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promotion and disease prevention.28

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Influence of digestion on the quantity of phospholipids.

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spectra of phospholipids extracted from non-digested crude sample and oral,

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gastric and intestinal digested samples were depicted in Figure 3. It was

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obvious that in vitro simulated digestion had a great influence on

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phospholipids, not only on the mass spectrometric profile, but also on the

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specific intensity of phospholipids. Before digestion, the peaks of phospholipid

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in crude sample had the best signal-to-noise (S/N) ratio and signal diversity

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involving 35 phospholipids species. The ion at m/z 809.61 was the most

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abundant, which could be tentatively interpreted as the overlap of [PC 42:2 -

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N(CH3)3H]– and [PE 42:2 - NH3]–, followed by the ions at m/z 857.52 ([PI

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36:4 - H]–) and m/z 811.63 ([PC 42:1 - N(CH3)3H]– & [PE 42:1 - NH3]–). After

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oral digestion, although there is a reduction of the signal intensities from

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1.35e7 to 2.57e6, the diversity of phospholipid molecular species didn’t

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changed much, because of the absence of lipase in the oral environment. The

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lingual lipase secreted by lingual serous glands in mouth wasn’t considered

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here, because it works on medium- and long-chain triglycerides only but not

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phospholipids. Along with the progress of multistage digestion, the

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background noise of the mass spectra became more and more serious, and the

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signal intensity of phospholipid decreased to 9.72e5 and 4.22e5 for gastric and

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intestinal digestion stages, respectively. The specific dynamic changes of 15

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intensity were listed in detail in Table 1. After comprehensive analysis, it can

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be seen that the absolute intensity (peak area, cps) were reduced for all the

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phospholipids during the digestion process. The enzymatic effect on the

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phospholipids is varied generally depending on the phospholipid classes. The

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PI decreased more significantly than the PC and PE. For example, the most

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intensive peak, m/z 857.52 ([PI 36:4 - H]–), decreased dramatically from

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9.19% to 3.71% after gastrointestinal digestion, and some PI peaks were

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digested completely, such as the m/z 879.50 ([PI 38:7 - H]–), 903.50 ([PI 40:9

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- H]–), 931.53 ([PI 42:9 - H]–), etc. On the contrary, the relative percentage of

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PC and PE increased gradually, attributing to the marked drop of PI. For

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example, the relative percentage of m/z 811.63 at different stages were 6.03%

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(oral phase), 10.21% (gastric phase), and 13.57% (intestinal phase). In

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addition, it was observed that the phospholipids with high degree of

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unsaturation are more easily digested. The polyunsaturated phospholipid

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molecular species, 903.50 ([PI 40:9 - H]–), 931.53 ([PI 42:9 - H]–), and etc,

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were digested completely. It has been reported that the hydrolysis rate of

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phospholipids increased as the degree of unsaturated of the acyl chain

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increased.25 The hydrolyzing selectivity of porcine pancreatic lipase is also

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related to location of double bonds in the fatty acid, which appears to be

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depending on the distance of the first double bond from the ester linkage

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being hydrolyzed.29

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It is well known that PCA is an effective method

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PCA statistical analysis.

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used for reducing the number of dimensions present in the whole data matrix

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with only a minimum loss of original information. To determine the main

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variability and establish the relation between phospholipids and digestion,

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PCA was applied to analyze the data produced by REIMS. It normalized the

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relative amounts of the phospholipids in crude and digested samples in a

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reduced dimension plot for revealing the difference between each digestion

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stage. As shown in Figure 4A, there are generally three major clusters

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observed in the score plot. The lipidomic profile data from crude and orally

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digested samples were mixed into a single cluster. It can be explained by the

330

limited digestive effect in the oral digestion stage due to the absent of

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phospholipase. The other two samples of gastric and intestinal digestion

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stages were distinctly separated by using only the first two PCA components.

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The separation effect of the second principal component (PC2) was not as

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good as that of PC1. In detail, the first principal component (PC1) separated

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the three clusters by explaining 65.8 cumulative percent (cum %) of the

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variance in the REIMS data set, and PC2 explained 11.7 cum %. The first two

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PCA components with 77.5 cum % of the total variance could be considered to

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be significant enough to demonstrate the variation of the digestion stages

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among these samples. Figure 4B revealed the loadings for the variables in the

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first two PCA components clearly, in which the most influential phospholipid

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molecular species could found out. It showed that most of the phospholipid

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molecular species were clustered at the central zero line, whereas the rest were

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dispersed far away from the center. The species with higher loading values

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and maximum variance in the data were mostly influenced by the digestion

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treatment. For instance, the ion at m/z 797.54 ([PG 38:4]–), m/z 809.61 ([PE

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42:2 - NH3]– & [PC 42:2 - N(CH3)3]–), 811.63 ([PE 42:1 - NH3]– & [PC 42:1 -

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N(CH3)3]–), 857.52 ([PI 36:4 - H]–), etc. were the most noticeable species, and

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their content varied dramatically along with the digestion process. All of these

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ions showing significant difference were examined with one-way ANOVA (p