In Situ Method for Real-Time Discriminating Salmon and Rainbow

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New Analytical Methods

An in-situ method for real-time discriminating salmons and rainbow trout without sample preparation using iKnife and rapid evaporative ionization mass spectrometry based lipidomics Gongshuai Song, Mengna Zhang, Yiqi Zhang, Haixing Wang, Shiyan Li, Zhiyuan Dai, and Qing Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00751 • Publication Date (Web): 05 Apr 2019 Downloaded from http://pubs.acs.org on April 7, 2019

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Journal of Agricultural and Food Chemistry

An in-situ method for real-time discriminating salmons and rainbow trout without sample preparation using iKnife and rapid evaporative ionization mass spectrometry based lipidomics

Gongshuai Song1, Mengna Zhang1, Yiqi Zhang1, Haixing Wang2, Shiyan Li3, Zhiyuan Dai1, Qing Shen1,* 1 Zhejiang Province Joint Key Laboratory of Aquatic Products Processing, Institute of Seafood, Zhejiang Gongshang University, Hangzhou, 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 Aquatic Products Quality Inspection Center of Zhejiang Province, Hangzhou 310012, China

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

The authors declare no competing financial interest.

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ACS Paragon Plus Environment


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ABSTRACT

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The domestic rainbow trout producers issued a standard with an aquatic association

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that classified rainbow trout as salmon, which raised the concern of consumers on the

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fish parasites infection. Herein, an in situ method was developed using “iKnife” and

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rapid evaporative ionization mass spectrometry based lipidomics for real-time

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discrimination of salmons and rainbow trout without sample preparation. A total of 12

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fatty acids and 37 phospholipid species was identified and imported into statistical

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analysis for building a in-situ and real-time recognition model. The ions with |p(corr)|

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> 0.5 and |p| > 0.03 were shown to be responsible for allocating samples, and the ions

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with high correlation values, such as of m/z 747.50, 771.49 and 863.55, indicated

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large weights in identification of the salmons and rainbow trout. The results indicated

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that this technology could be employed as a front-line test method to ensure the

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authenticity of salmon products.

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Keywords: Salmon; rainbow trout; lipidomics; rapid evaporative ionization mass

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spectrometry; real-time identification.

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INTRODUCTION

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Salmon, is an economically important fishery products in contemporary daily life,

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representing an increasingly popular food for human consumption.1 It is thought to be

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nutritious healthy due to the high protein and omega-3 fatty acids content in the fish.

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The red fleshed fillet is eaten in a variety of ways, such that the Japanese enjoy the

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raw fillet on sushi or sashimi, while it appears as smoked salmon or cured lox on

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northwest of America. There are many salmon species, such as Atlantic salmon

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(Salmo salar), king salmon (Oncorhynchus tshawytscha), etc. Recently in China,

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twelve companies, most of which are rainbow trout producers, issued a controversial

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standard with an aquatic association that classified rainbow trout (Oncorhynchus

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myhiss) as salmon. This case has aroused wide attention because it raised the concern

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of consumers on the fish parasites infection. Paragonimus westermani and Clonorchis

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sinensis are the two major parasites in rainbow trout, which can induce human hepatic

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disease and cholangiocarcinoma.2 Some media reports claimed that a lot of what is

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sold as salmon in China is in fact rainbow trout because of a close resemblance and

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commercial profits. To facilitate the regulation of such violation, a high-throughput

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and efficient analytical method for real-time discrimination of salmons and rainbow

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trout is urgently needed.

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Phospholipids are the essential structural constituents of biological membranes and

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considered as nutrients because of positive nutritional properties, primary

3



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physiological and biological functions.3 The phospholipids containing n-3 fatty acyl

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chains are thought to be superior to the conventional ethyl ester and glyceride types in

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promoting the development of infant brain and eye function, lowering blood pressure,

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reducing cardiovascular disease, etc.4 In addition to the nutritional functions, some

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phospholipid species are reported to be potential indicators to differentiate biological

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species. For instance, Shen et al. developed the shotgun lipidomics strategy for

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species differentiation based on four classes of phospholipids.5 However, the variation

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of the position of double bonds and the length of fatty acyl chains make the chemical

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structure of phospholipid molecules much complicated, which raised a challenge to

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determine and characterize the specific phospholipid molecular species.6

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Owing to the rapid progress on lipidomics, multiple analytical technologies have

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been developed for comprehensive analysis of lipid profile in various biological

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samples, such as

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chromatography (GC),8 liquid chromatography (LC),9 mass spectrometry (MS), etc.10

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The multidimensional mass spectrometry (MDMS)-based shotgun method is one of

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the main analytical platforms in current lipidomics.11 In addition, reverse phase LC

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coupled to MS/MS or hydrophilic interaction chromatography (HILIC) is also

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frequently used for the separation and detection of phospholipids by reason of its

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reproducibility, sensitivity, and resolution.12 However, the experimental operation of

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these techniques is complicated, laborious and time-consuming. The alternative

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approach is on the basis of rapid evaporative ionization mass spectrometry (REIMS),

nuclear magnetic resonance (NMR) spectroscopy,7 gas

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which allows in situ identification of target samples without preparation by real-time

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analysis of released aerosol during electrosurgical dissection with an intrinsic coupled

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intelligent knife (iKnife).13 REIMS profile displays many complex phospholipid

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molecular species mainly originating from the cell membrane, whose analysis

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commonly takes a few seconds and can provide the histological tissue identification

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with > 90% correct classification performance.14,15 There are various studies reported

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its successful application in identification of biological tissues,16 characterization of

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microorganisms,13 tumors detection and resection tumor,14 etc. Based on these

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applications, REIMS technology is supposed to find the application niche in the

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research field of food security with particular emphasis on salmon and rainbow trout

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authenticity.

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The aim of this study was to develop and optimize a REIMS method for high

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throughput and effective discriminating the salmons (S. salar and O. tshawytscha)

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from freshwater rainbow trout (O. myhiss) on the basis of in situ lipidomics profiling.

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The results can contribute to revealing the characteristics of fish species and

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providing reference for assessing the adulteration in the market.

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MATERIALS AND METHODS

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Materials and reagents. The Atlantic salmon (S. salar) and king salmon (O.

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tshawytscha) are the most popularly consumed salmon species, but frequently

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adulterated by rainbow trout (O. myhiss) because of their close resemblance. The 5



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Atlantic salmon and king salmon were bought from Zhejiang Ocean Family Co. Ltd.,

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which were cultured in Norway and New Zealand, respectively. The rainbow trout

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was bought from Longyangxia Aquafarms Co. Ltd., which was cultured in Qinghai,

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China. After capture, the salmons and rainbow trout were frozen and preserved at -60

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◦C.

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Gongshang University by cold-chain transportation. There were different parts of the

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fish body including belly, back and tail collected and tested (Figure 1). A total of 18

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samples (6 samples for each species) from different parts of fish body including belly,

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back and tail was collected and tested in three technical replicates. Leucine

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enkephalin (purity 97%) was bought from Sigma-Aldrich (St. Louis, MO, USA).

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2-propanol, methanol, and acetone were chromatographic grade and purchased from

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Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). High purity water with a

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resistivity of 18.2 MΩ·cm was obtained using a Milli-Q system (Millipore, Bedford,

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USA).

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REIMS analysis. The fish fillets were analyzed directly without further processing.

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The detection of lipids in salmons and rainbow trout was carried out on a REIMS

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system (Waters Co., Ltd., Shanghai, China). Fish samples were analyzed by

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electrosurgical evaporation using the electrosurgical knife (WSD151, Weller,

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Germany), and online mass spectrometric analysis was performed on a quadrupole

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time-of-flight mass spectrometer (Xevo G2-XS, Waters Co., Ltd., Milford, MA). The

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electrosurgical knife consists of a cutting electrode containing cutting blade and a

The fish was immediately transported to the Institute of Seafood, Zhejiang

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handpiece equipped with a 4 m × 4.11 mm o.d., 2.53 mm i.d. PTFE tube on the

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atmospheric interface of the MS. A puff of aerosol containing gaseous ions was

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generated once the knife touched the tissue and aspirated into the MS via the PTFE

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tube using a Venturi pump driven by 2 bar nitrogen. For lock mass correction, leucine

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enkephalin (m/z 554.2615) dissolved in the auxiliary solvent of 2-propanol (2 ng/μL)

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was introduced into a heated helix collision surface processed at roughly 750 ◦C using

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a stainless steel capillary (1/16ʹʹ O.D., 0.002ʹʹ I.D.). The mass spectra were acquired in

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the range of m/z 50 – 1200 in both positive and negative ion modes with a scan time

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of 0.5 s. Besides, the condition of MS/MS experiment was optimized by tuning the

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collision energy (CE) from 20 to 80 eV. Each fillet sample was cut in 20 mm length

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strip by the electrosurgical knife with repeated operation for 6-8 times. There were

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five scans near peak center in total ion current graph being summed up to calculate

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the cps of each peak. The data were processed by the Masslynxv4.1 software with

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background subtraction, automatic peak detection, and peaks centroid, and the peak

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list with absolute counts could be exported in to text file. Real-time recognition of

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sample identity was performed using the software of LiveID (Waters Co., Ltd.,

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Milford, MA).

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Data analysis. The peaks with signal-to-noise are higher than 10 were selected and

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analyzed. The mean value, standard deviation, and level of significance were

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calculated using Kingsoft WPS software and SPSS 16.0 software (SPSS Inc., Chicago,

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IL, USA). The data showing significant difference were determined by the analysis of 7



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variance (ANOVA, p < 0.05). The data matrix was directly imported into SIMCA

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14.1 (Umetrics, Sweden) for broader processing options. The chemometrics tools,

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principal component analysis (PCA), partial least-squares to the latent structures

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(PLS), and orthogonal PLS (OPLS) were applied for modeling information-rich data.

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The m/z and content of each phospholipid ion were subjected to PCA, PLS, and

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OPLS for evaluating the relationship between salmons and rainbow trout based

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lipidomics. The peak area normalization method was used to calculate the relative

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content of each peak. Characterization of each lipid molecular species was performed

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by MS/MS analysis or comparing accurate mass information with the LIPID MAPS

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prediction tool (http://www.lipidmaps.org/tools/index. html).

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RESULTS AND DISCUSSION

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REIMS Parameters Optimization. REIMS analysis of fish fillet samples was

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carried out on the basis of online mass spectrometric analysis of the aerosol produced

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by electrosurgical evaporation. The MS profile of aerosol depends upon the factors of

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REIMS procedure, including the operation power of electrosurgical knife, sensitivity

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and accuracy of MS analysis, and sample volume. Therefore, the optimization of

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those parameters was performed by serial selection of cutting power conditions, flow

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rate of auxiliary solvent, and fish meat quantity, using the top 20 peaks as

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representative indicators.

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The cutting power setting of the electrosurgical knife is the most significant

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physical factor for REIMS analysis of biological tissues.16 In view of the overall

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signal intensity of sample, the cutting power was optimized in the range from 15 to 35

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W. The results were depicted in Figure 2, and it could be found that the signal

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intensity of each peak enlarged gradually along with the increase of power from 15 to

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25 W, and reached the highest value at 25 W. Afterwards, the signal-to-noise ratio of

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all peaks underwent a sustained decline. Finally, a cutting power of 25 W was chosen,

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and the maximal signal intensity was acquired.

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During REIMS process, the spectral characteristics highly depends on the condition

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of atmospheric interface of MS. To enhance the overall signal intensity with well

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reduced noise, the leucine enkephalin contained 2-propanol was introduced directly

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into the atmospheric interface for lock-mass correction and interferent washing, and

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its flow rate was tuned from 60 to 140 μL·min–1. There was a well-defined experience

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being adopted to investigate the effect of the flow rate of auxiliary solvent on the

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signal-to-noise ratio.12 It can be seen from Figure S1A that the signals of the target

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analytes increased gradually when the flow rate of the solvent was increased from 60

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to 120 μL·min–1. But when the flow rate was further raised to 140 μL·min–1, a decline

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of intensity was occurred, and the signals of spectra became dramatically fluctuated.

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Therefore, 120 μL·min–1 was chosen as the flow rate of auxiliary solvent.

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After the cutting power of electrosurgical knife and the flow rate of auxiliary

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solvent were settled down, the minimum sample volume was taken into consideration

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to avoid deficient aerosol generated and provide continuous output signal.17 The

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effect of fish meat quantity ranged from 4 to 12 g on the intensity was studied. The

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results in Figure S1B showed that there was a growth in the signal intensity of five

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characteristic peaks when the sample volume raised from 4 to 10 g. Thus, a volume of

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10 g fish fillet was necessary for the REIMS process.

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Characterization of MS profile.

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Low m/z region. The fish tissue samples were analyzed in both positive-ion and

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negative-ion modes. It was found that the fatty acids were not informative in

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positive-ion mode (Figure S2), but easily deprotonated and well acquired in

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negative-ion mode. As shown in Figure 3, the REIMS spectra for salmons and

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rainbow trout were obtained in the range of m/z 200-500. There was a difference

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observed on the signal intensities in the spectra referred to the Atlantic salmon

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(2.72e7, Figure 3A), king salmon (2.60e7, Figure 3B) and rainbow trout (2.16e7,

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Figure 3C), respectively. The ions of m/z 255.23 and m/z 327.23 were the base peaks,

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and the major signals in relation to the primary isotopologues of the detected isotopic

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clusters were suggested as fatty acids. In comparison with the specific ions in the

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LIPIDMAPS database, the fatty acids were identified to match to the m/z values with

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a reasonable degree of tolerance, and the results were summarized in Table 1. There

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were 12 molecular species of fatty acids being tentatively identified with mass

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accuracy in the range of 1.53 to 8.83 ppm, while the rest signals were unable to be

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elucidated due to the oxidation of fatty acids, such as carbonylation, epoxidation and

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hydroxylation. C16:0, C16:1, C18:1, C18:2, C18:3, C20:5, and C22:6 were the major

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fatty acids in salmons and rainbow trout, which was in line with the results of

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previously reported studies.18 In particular, the ions of m/z 301.21 and m/z 327.27 in

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the spectra were corresponding to n-3 polyunsaturated fatty acids of C20:5 (EPA) and

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C22:6 (DHA) respectively, which are reported to be the primary protective agents for

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disease prevention, health maintenance and promotion.19,20 The relative content of

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EPA and DHA was the highest in king salmon (55.63%), followed by Atlantic salmon

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(52.52%) and rainbow trout (46.36%). In addition, the highest proportion of saturated

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fatty acids (C16:0, C18:0 and C20:0, 24.58%) in rainbow trout was found.

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High m/z region. Although the phospholipid can be evaporated and ionized in both

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positive and negative-ion modes, the signals in positive-ion mode are likely to be

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suppressed by impurities due to the protonating competition during the process of

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rapid evaporative ionization (Figure S2), while those in negative-ion mode are

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obviously more informative because of the substantial negative charge of the

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headgroup.21 As shown in Figure 4, the mass spectra were mainly filled by intact

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phospholipid molecular species in the range of m/z 600-1000, which were tentatively

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identified

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phosphatidylinositol (PI), and phosphatidic acid (PA).22 The PE molecular species

as

phosphatidylcholine

(PC),

phosphatidylethanolamine

11


(PE),


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could be ionized as [M-NH3]–, forming a structurally PA-like fragment, while the PC

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and PI molecular species were deprotonated as [M-H]–.23 The peaks displayed in the

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figure were corresponding to distinct phospholipid molecular species, resulting from

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different fatty acyl substitutions at the glycerol backbone. There was a significant

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difference on the mass spectrometric profiles and specific signal intensity of

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phospholipids between the salmons and rainbow trout. The spectra of Atlantic salmon

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(Figure 4A) and king salmon (Figure 4B) showed a close resemblance, which had

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better intensity and signal-to-noise ratio than that of rainbow trout (Figure 4C). The

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ion of m/z 790.53 was calculated to be the most abundant one, which could be

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tentatively identified as [PE 40:6-H]– and [PC O-38:6-H]–, followed by the ions of

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m/z 745.48 and m/z 762.50 characterized as [PE 38:6-NH3]– and [PE 38:6-H]–,

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respectively. After structural analysis, a total of 37 phospholipid molecular species

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was successfully identified with mass error less than 12.31 ppm. Their relative

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contents were calculated by a normalization method on the basis of the ionization

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efficiency of each phospholipid molecular species highly depends on the polar

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headgroup while the dipole moment of hydrophobic tails are nearly negligible.12 As

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summarized in Table 2, PE and PC were the key glycerophospholipids accounted for

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the highest percentage in the fish tissue cells, such as the ions of m/z 790.53 (the

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overlap of [PE 40:6-H]– and [PC O-38:6-H]–), m/z 816.55 (the overlap of [PE

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42:7-H]– and [PC 38:0-H]–), and m/z 836.52 (the overlap of [PE 44:11-H]– and [PC

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40:4-H]–), and the relative contents of PE and PC molecular species showed the fairly

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close resemblance between Atlantic salmon and king salmon, while that in the

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rainbow trout was nearly half less. For example, the contents of the ion of m/z 836.52

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(the overlap of [PE 44:11-H]– and [PC 40:4-H]–) in Atlantic salmon and king salmon

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were 3.40% and 3.72% respectively, whereas that in the rainbow trout was only

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1.73%. PC can organize into lipid bilayers due to the overall cylindrical molecular

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shape.24 PE is another important class of phospholipid, which can be synthesized by

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adding cytidine diphosphate ethanolamine to diglyceride and releasing cytidine

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monophosphate.4 In additon, PI is a negatively charged phospholipid molecular that is

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the minor component in the cytosolic side of eukaryotic cell membranes. The contents

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of polyunsaturated phospholipid molecular species, 907.53 ([PI 40:7-H]–), 909.54 ([PI

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40:6-H]–), and etc., were even lower. The difference of lipidomics profiles among the

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salmons and rainbow trout was studied in terms of lipid class, putative identification

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and intensity of glycerophospholipids. The quality of rainbow trout was much poorer

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than Atlantic salmon and king salmon.

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MS/MS analysis. To confirm the results of structural identification, MS/MS analysis

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was further applied to the major components of the lipidomics profiles. The fragments

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of phospholipid became smaller along with the increased CE value, and the MS/MS

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spectra were acquired with optimized CE. The detailed composition of fatty acyl

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chains (sn-1/sn-2) of phospholipids, including the number of double bonds and length

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of carbon chain, could be well interpreted by specific fragments. Figure 5A illustrated

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a typical mass spectrum of salmon, which exhibits a large number of prominent ions. 13



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The two labeled ions were selected as representative examples, and identified as

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719.47 (PE 16:0/20:5) and 909.54 (PI 18:0/22:6) by the corresponding spectra shown

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in Figure 5B and Figure 5C. In detail, the identification of m/z 719.47 could be

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interpreted on the basis of the characteristic fragment ions. The base peak fragment

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ion of m/z 409.24 indicated the loss of a fatty acid in ketene form as

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[M-H-R'1CH=C=O]–. The ion of m/z 391.22 was produced by cutting off a fatty acid

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in form of carboxylic acid as [M-H-R1COOH]–, along with two free fatty acids as the

247

carboxylate anions form as [R16:0COO]– for m/z 255.23 and [R20:5COO]– for m/z

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301.21. According to the spectrum of the ion of m/z 909.54, the base peak fragment

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ion of m/z 581.36 was originated from the loss of a fatty acyl chain in form of ketene

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as [M-H-R'2CH=C=O]–. The ion of m/z 419.26 represents the loss of a fatty acid

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(sn-1/sn-2) in the form of carboxylic acid as [M-H-R2COOH]–. In addition, the ions of

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m/z 283.25 and 327.23 represents the free acid as the carboxylate anions form

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reflecting [R18:0COO]– and [R22:6COO]–, respectively.25,26 As a result, the composition

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of fatty acyl chains in the significant phospholipid molecules was identified and the

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results were shown in Table 2. The ions of m/z 764.51 ([M-H]–, the overlap of (PE

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18:0/20:5) and (PC O-16:0/20:5)), 808.49 ([M-H]–, the overlap of (PE 20:5/22:6) and

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(PC 18:1/20:3)), 903.50 (PI 18:3/22:6), etc., the fatty acyl chain composition of whose

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ions contained EPA or DHA with high nutritional values.

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Multivariate analysis of REIMS data. The REIMS data obtained from 18 salmons

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and rainbow trout tissue samples (6 samples for each species) were directly processed 14


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by the LiveID software. A lipidochemometric model was built using embedded

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statistical approaches. The identity of each fillets sample was real-time displayed

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during REIMS test with accuracy > 96.58%. A representative software interface was

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demonstrated in Figure 6, where the upper right circle indicated the identity of the

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sample, the upper left recorded the historic results, and the bottom demonstrated the

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spectrum of total ion current. To explain the intrinsic principle and find out the

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potential marker, the principle component analysis (PCA) and orthogonal partial

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least-squares discriminant analysis (OPLS-DA) were applied separately.27

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The difference of phospholipid molecular species among Atlantic salmon (AS),

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king salmon (KS), and rainbow trout (RT) were statistically evaluated by the

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SIMCA-P software. Firstly, a total of 37 peaks of each sample was subjected to the

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unsupervised PCA was applied to reduce the multi-dimensionality of the preprocessed

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data to typical two-dimensional analysis system characterized by principal

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components with the minimum loss of original information. The score plot of PCA

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(Figure 7A) showed that both salmon and rainbow trout samples were clustered

276

individually into three statistically characteristic groups (AS, KS, and RT),

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distributing at different places in the figure without any outliers. The first principal

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component (t1) explains 43.60% of the variation and the second (t2) 30.60%, which

279

indicates the good group separation of the three samples.28 Figure 7B plots the weight

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of each variable. The majority of the putative variables were clustered together around

281

the central zero line, while the most influential ones were scattered from the center. 15



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The ion of m/z 790.53 (PE & PC 18:0/22:6; O-18:1/20:5) is the most predominant ion,

283

followed by the ions of m/z 762.50 (PE 18:1/20:5), 788.52 (PE & PC 18:1/22:6;

284

16:0/20:0), and 764.51 (PE & PC 18:0/20:5; O-16:0/20:5).

285

The OPLS-DA was performed to detect discrimination between salmons and

286

rainbow trout. The species-responsive ions were evaluated on the S-plot of OPLS-DA

287

for the predictive and orthogonal variation.29 The cut-off value of |p(corr)| > 0.5 and

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|p| > 0.03; statistical significance of p < 0.05 in t test; and intensity change > 2-fold

289

between the each group were set to define the relevant ions responsible for the

290

variance of the observations as advised in previous publications.30,31 In the S-plot

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(Figure 7C), there were 80 samples (including replicates) imported for building the

292

model (R2(Y) = 0.921, Q2(Y) = 0.892), among which the ions with |p(corr)| > 0.5 and

293

|p| > 0.03 were shown to be responsible for allocating samples. The ions of m/z

294

747.50, 771.49 and 863.55 owned high correlation values, indicating their large

295

weights in distinguishing the fish species. In addition, the similar results were

296

obtained from the VIP-plot on the basis of OPLS-DA model (Figure 7D). The

297

VIP-plot summarizes the importance if the variables both to explain X-matrix (mass

298

spectrometric fingerprint) and to correlate to Y-variable (classification of samples in

299

groups). The larger VIP value indicates more important (VIP > 1).32 The ions of m/z

300

747.50, 771.49 and 863.55 with the VIP values of 1.47, 1.46 and 1.47 respectively

301

have significant effect on the dataset and separation of salmons and rainbow trout.

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Therefore, the ions of m/z 747.50 (PE 18:0/20:5), 771.49 (PE 18:1/22:6) and 863.55 16


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(PI 16:0/20:1) could be used as candidate markers for the discrimination of salmons

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and rainbow trout.

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Method validation The robustness of REIMS based lipidomics method for

306

discriminating the salmons from freshwater rainbow trout was validated in terms of

307

accuracy and reproducibility. The fillet of king salmon was used as a representative

308

sample and the peaks of fatty acids (m/z 255.23, 301.21, and 327.23) and

309

phospholipids (m/z 762.50, 790.53, and 909.54) were selected. The accuracy and

310

reproducibility were evaluated based on the intra-day and inter-day precision,

311

respectively. Inter-day variability test was carried out by king salmon tissue samples

312

in triplicate for three consecutive days, whereas for intra-day precision was calculated

313

by means of error propagation of duplicate electrosurgical cutting ofsamples within

314

the same batch. As shown in Table 3, the values for intra-day accuracy (RSD) were

315

less than 7.06%, fell within the range of 4.37 - 6.35%, and the values for inter-day

316

reproducibility (RSD) fell within the range of 5.86 - 7.49% for all lipid molecular

317

species. The results indicated that the proposed method is accurate and reproducible.

318

Blind sample application. The proposed REIMS based lipidomics method was

319

applied to twenty randomly picked blind samples from local supermarket. The iKnife

320

cut the samples in a 20 mm length strip with power 25 W, and a stream of aerosol was

321

immediately produced and transferred into the MS analyzer. The identification results

322

were given by the software real-time. Eight samples were tentatively classified as KS

323

because of the highest cut-off value of m/z 863.55 (|p(corr)| = 1.31 and |p| = 0.24) and 17



324

lower cut-off values of m/z 747.50 (|p(corr)| = 1.02 and |p| = 0.17) and 771.49

325

(|p(corr)| = 0.99 and |p| = 0.15) respectively. Ten blind samples were identified as AS

326

due to the lowest cut-off value of m/z 747.50 (|p(corr)| = 0.93 and |p| = 0.11).

327

According to the cut-off values of three characteristic ions of m/z 747.50 (|p(corr)| =

328

1.28 and |p| = 0.19), m/z 863.55 (|p(corr)| = 0.80 and |p| = 0.07), and m/z 771.49

329

(|p(corr)| = 0.85 and |p| = 0.09), the rest was classed as RT. The identity of each blind

330

sample during REIMS test with accuracy > 96.31%. Moreover, for verification and

331

confirmation of the identification results, the REIMS data were imputed into the

332

SIMCA-P software for building the PCA model and it indicated that three KS, four

333

AS and three RT samples were well clustered. Therefore, the result revealed ensure

334

the authenticity of salmon products and demonstrated that the real-time REIMS

335

technique based lipidomics method performed great advantage in fast discrimination

336

of the three salmon strains with the significant economic value.

337

In summary, a REIMS technique with high throughput, efficiency, and accuracy

338

was developed for in situ discrimination of salmons and rainbow trout. Based on the

339

well-established model, the identity of blind fish fillets could be real-time given

340

without any sample preparation. The principle behind was inspected by the

341

characterization of lipid peaks in the spectra and multivariate analysis. A total of 12

342

fatty acids and 37 phospholipid molecular species were identified in the samples. The

343

results of S-plot and VIP-plot indicated the obvious difference among salmons and

344

rainbow trout. The ions of m/z 747.50, 771.49 and 863.55 showed significant effect 18


Page 18 of 41

Page 19 of 41


345

on the dataset, which could be used as candidate markers for the discrimination of

346

salmons and rainbow trout. The successful application of this method indicated its

347

advantages in adulteration real-time detection and violation regulation. Furthermore,

348

the method has high relevance to the field of food quality and safety, which makes it a

349

significant technique in safety test including oxidation characterization differentiation

350

of krill oil, butterfish and yellow croaker identification, and etc in our future study.

351

Acknowledgment.

352

This

353

(2018YFD0901103), the the Natural Science Fund for Young Scholars of China

354

(31601542), the Zhejiang Provincial Public Welfare Technology Research Project

355

(LGN18C200001), and the Quality & Safety of Aquatic Products Technical Support

356

Team (QS2018001). The authors thank Dr. Wei Rao (Waters Corporation, Shanghai,

357

China) for REIMS system operation.

358

Conflict of Interest.

359

The authors declare that there is no conflict of interest.

360

Ethical approval.

361

This article does not contain studies with human participants. All applicable

362

international, national, and/or institutional guidelines for the care and use of animals

363

were followed.

work

was

supported

by

National

Key

R&D

19


Program

of

China


364

Supporting Information

365

The supporting information includes a figure illustrating the optimization of REIMS

366

parameters and a representative spectrum of king salmon acquired in positive ion

367

mode.

368

Figure S1. The effect of the (A) flow rate of auxiliary solvent and (B) salmon tissue

369

quantity on the signal intensity of phospholipid ions.

370

Figure S2. A representative REIMS positive ion spectrum of king salmon.

371

20


Page 20 of 41

Page 21 of 41


372

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H.

Correction

to

nontargeted

metabolite

481 482

26


profiling

in

compatible

Page 27 of 41


Figure captions Figure 1. The fillets of (A) king salmon (O. tshawytscha), (B) Atlantic salmon (S. salar), and (C) rainbow trout (O. myhiss) in row.

Figure 2. Effect of the cutting power of the electrosurgical knife.

Figure 3. Representative REIMS negative ion spectra of Atlantic salmon (A), king salmon (B) and rainbow trout (C). The features in the spectra mainly suggested fatty acids in the range of m/z 200-500.

Figure 4. Representative REIMS obtained in negative-ion mode of Atlantic salmon (A), king salmon (B) and rainbow trout (C). The spectra mainly feature glycerophospholipids in the range of m/z 600-1000.

Figure 5. Representative MS spectra of the two negative ions, m/z 719.47 and 909.54, identified as PE 16:0/20:5 and PI 18:0/22:6 respectively.

Figure 6. Real-time analysis of king salmon tissue using the prototype recognition software.

Figure 7. (A) The score plot of PCA the identified phospholipid in three salmon tissue samples (AS, Atlantic salmon; KS, king salmon; RT, rainbow trout). Six parallel samples for each species were tested with three technical replicates; (B) The corresponding loading plot of PCA; (C) The loading S-plot constructed on the basis of

27



the OPLS-DA of the contribution ions comparing salmons vs. rainbow trout; (D) The VIP graph of all relevant ions analyzed in the multivariate dataset.

28


Page 28 of 41

Page 29 of 41


Table 1. The Probable Chain Composition And Relative Content Of Fatty Acids From The m/z Range Of 200 To 400 In Salmons And Rainbow Trout Using REIMS Method.

Measured ion (m/z)

Extract value (m/z)

Error

Chain

(ppm)

composition

Atlantic salmon

King salmon

Rainbow trout

Intensity

Content

Intensity

Content

Intensity

Content

(cps, 105)

(%)

(cps, 105)

(%)

(cps, 105)

(%)

253.2164

253.2173

3.55

16:1

6.80

0.84

8.12

1.49

5.10

1.02

255.2322

255.2330

3.13

16:0

97.90

12.07

83.1

15.23

88.10

17.62

277.2166

277.2173

2.53

18:3

29.80

3.68

6.31

1.16

7.61

1.52

279.2324

279.2330

2.15

18:2

38.10

4.70

22.80

4.17

40.80

8.17

281.2480

281.2486

2.13

18:1

142.00

17.45

68.00

12.45

61.70

12.35

283.2568

283.2543

8.83

18:0

38.40

4.73

30.10

5.52

32.30

6.46

301.2168

301.2173

1.66

20:5

92.60

11.42

43.90

8.05

45.90

9.18

303.2312

303.2330

5.94

20:4

18.00

2.22

17.50

3.20

15.50

3.10

305.2505

305.2486

6.22

20:3

7.02

0.87

4.78

0.88

8.40

1.68

307.2658

307.2643

4.88

20:2

5.46

0.67

1.01

0.16

6.13

1.23

311.2105

311.2116

3.53

20:0

2.14

0.26

1.59

0.29

2.47

0.50

327.2325

327.2330

1.53

22:6

333.00

41.10

546.00

47.58

216.00

37.18

29



Page 30 of 41

Table 2. The Probable Attribution And Relative Abundance Of Detected Spectrometric Peaks From The m/z Range Of 600 To 1000 In Salmons And Rainbow Trout Using REIMS Method. Measured

Probable

Putative

Extract

Error

Atlantic salmon

King salmon

ion (m/z)

ionization state

identification

value (m/z)

(ppm)

Intensity

Intensity

(cps,

Abundance (%)

(cps,

60.40

0.66

5.60

36.00

699.4970

1.29

PA O-16:1/22:6

705.4865

[M-NH3]–

PE 16:0/20:5

721.4789 *

[M-NH3]–

745.4814 *

Intensity

2.92

0.05

106.89

2.14

0.39

4.12

0.07

100.34

2.03

196.00

1.14

64.10

1.07

111.00

2.29

5.39

87.00

0.95

40.50

0.68

56.7

1.17

719.4657

2.22

391.00

4.26

263.00

4.40

234.00

4.83

PE 16:1/20:3

721.4814

3.47

146.00

1.60

81.80

1.37

76.90

1.59

[M-NH3]–

PE 18:1/20:5

745.4814

0.00

603.00

6.49

499.00

8.34

414.00

6.55

747.5062 *

[M-NH3]–

PE 18:0/20:5

747.4970

12.31

344.00

3.28

325.00

5.43

518.00

7.63

749.5150 *

[M-NH3]–

PE 18:1/20:3

749.5127

3.07

162.00

1.77

120.00

2.00

140.00

2.89

760.4956

[M-H]–

PE 16:1/22:6

760.4923

4.34

57.00

0.62

n.d.

n.d.

n.d.

n.d.

762.5076 *

[M-H]–

PE 18:1/20:5

762.5079

0.39

850.00

8.27

530.00

8.86

405.00

6.43

764.5167 *

[M-H]–

PE 18:0/20:5;

764.5236

9.03

642.00

7.00

471.00

7.87

395.00

6.10

766.5378

[M-H]–

766.5392

1.83

139.00

1.52

87.10

1.46

68.00

1.40

PA 16:1/20:4

693.4501

3.89

695.4618

[M-H]–

PA 16:1/20:3

695.4657

699.4979 *

[M-H]–

PA 16:0/20:2

705.4827

[M-H]–

719.4673 *

104)

Abundance

(cps,

693.4474

104)

Abundance (%)

[M-H]–

104)

Rainbow trout

(%)

PC O-16:0/20:5 PE 18:3/20:1; PC O-18:1/18:3 ]–

771.4906

[M-NH3

PE 18:1/22:6

771.4915

1.17

92.80

1.01

43.19

0.68

17.23

0.43

784.4885

[M-H]–

PE 18:3/22:6;

784.4923

4.84

83.50

0.91

3.97

0.07

n.d.

n.d.

30


Page 31 of 41


PC 16:1/20:1 788.5222 *

[M-H]–

790.5379 *

[M-H]–

808.4911 *

[M-H]–

PE 18:1/22:6;

788.5236

1.78

594.00

5.47

355.00

5.94

144.00

2.96

790.5392

1.64

1370.00

20.91

1300.00

21.81

1060.00

16.86

808.4923

1.48

145.00

1.58

43.40

0.73

n.d.

n.d.

810.5079

5.68

152.00

1.65

67.60

1.13

n.d.

n.d.

812.5236

1.23

188.00

2.05

56.20

0.94

37.30

0.77

814.5392

1.10

155.00

1.69

67.50

1.13

64.50

1.33

816.5549

2.08

308.00

3.36

182.00

3.04

78.40

1.62

818.5705

7.82

106.00

1.16

73.00

1.22

76.50

1.58

834.5079

0

197.00

2.15

87.10

1.46

64.40

1.33

836.5236

2.63

311.00

3.40

222.00

3.72

84.10

1.73

838.5392

5.25

94.10

1.03

70.90

1.19

56.1

1.11

857.5186

1.98

16.30

0.18

7.20

0.12

n.d.

n.d.

PC 16:0/20:0 PE 18:0/22:6; PC O-18:1/20:5 PE 20:5/22:6； PC 18:1/20:3 810.5033 *

[M-H]–

PE 20:4/22:6; PC 18:1/20:2

812.5226 *

[M-H]–

814.5383 *

[M-H]–

816.5532

[M-H]–

818.5641 *

[M-H]–

834.5079 *

[M-H]–

836.5214 *

[M-H]–

PE 20:3/22:6; PC 18:1/20:1 PE 20:2/22:6; PC 16:1/22:0 PE 20:1/22:6; PC 18:0/20:0 PE 20:5/22:1; PC O-18:0/22:6 PE 22:6/22:6; PC 20:0/20:5 PE 22:5/22:6; PC 18:3/22:1

838.5348

[M-H]–

PE 22:4/22:6; PC 18:3/22:0

857.5203

[M-H]–

PI 16:1/20:3

31



Page 32 of 41

859.5367

[M-H]–

PI 16:1/20:2

859.5342

2.91

38.60

0.42

3.29

0.06

10.30

0.21

863.5505

[M-H]–

PI 16:0/20:1

863.5525

2.32

59.90

0.65

53.80

0.90

15.00

0.31

881.5181

[M-H]–

PI 18:3/20:3

881.5186

0.57

61.30

0.67

59.80

1.00

15.60

0.32

883.5337 *

[M-H]–

PI 18:3/20:2

883.5342

0.57

252.00

2.74

139.20

2.66

74.70

1.54

885.5486 *

[M-H]–

PI 18:3/20:1

885.5499

1.47

221.00

2.41

147.00

2.45

55.50

1.15

887.5610

[M-H]–

PI 18:3/20:0

887.5655

5.07

42.60

0.46

30.70

0.51

131.00

2.70

903.5000

[M-H]–

PI 18:3/22:6

903.5029

3.21

1.38

0.01

1.35

0.02

37.40

0.77

907.5325

[M-H]–

PI 18:1/22:6

907.5342

1.87

34.90

0.38

28.70

0.48

231.00

4.80

909.5498 *

[M-H]–

PI 18:0/22:6

909.5499

0.11

332.00

6.62

249.00

4.17

985.00

10.89

911.5602

[M-H]–

PI 20:0/20:5

911.5655

5.81

98.10

1.07

65.30

1.09

63.80

1.32

Note: PI, phosphatidylinositol; PA, phosphatidic acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine.

n.d., not detected * the ions were structurally confirmed by MS/MS analysis. The rest ions were tentatively assigned by consulting with other results published previously.5, 11, 12

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Table 3. The Accuracy And Reproducibility Of The Proposed REIMS Method Using Selected Ion Peaks Of King Salmon.

Selected ion peaks

Probable

Intra-day accuracy

Inter-day reproducibility

m/z

attribution

Relative content (%)

RSD (%)

RSD (%)

255.23

16:0

14.31 ± 0.71

6.14

6.73

301.21

20:5

7.72 ± 0.48

5.51

5.86

327.23

22:6

46.78 ± 2.62

5.23

6.75

762.50

PE 38:6

8.46 ± 0.50

6.35

7.04

790.53

PE 40:6;

21.62 ± 0.96

4.37

6.28

4.45 ± 0.32

7.06

7.49

PC O-38:6 909.54

PI 40:6

33



(A)

(B)

(C) Figure 1

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m/z Figure 2

35



Figure 3

36


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Figure 4

37



[PE 16:0/20:5-H]– 719.47

[PI 18:0/22:6-H]– 909.54

Figure 5

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Figure 6

39



(B)

(A)

(C)

(D)

Figure 7

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TOC

41


In Situ Method for Real-Time Discriminating Salmon and Rainbow

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