Improving Lipidomic Coverage Using UPLC-ESI-Q-TOF-MS for Marine

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Improving Lipidomic Coverage Using UPLC-ESI-Q-TOF-MS for Marine Shellfish by Optimizing the Mobile Phase and Resuspension Solvents Yu-Ying Zhang, Yu-Xi Liu, Zheng Zhou, Da-Yong Zhou, Ming Du, Bei-Wei Zhu, and Lei Qin* National Engineering Research Center of Seafood, School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China Downloaded via BUFFALO STATE on July 28, 2019 at 13:41:34 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Reversed-phase ultrahigh-performance-liquid chromatography−mass spectrometry (UPLC−MS) is the typical method for the lipidomic analysis of most of biological samples, which was rarely used for the comprehensive lipidomic analysis of marine shellfish. Thus, a range of columns, modifiers, and resuspension solvents were evaluated using UPLC-electrospray ionization-quadrupole time-of-flight-MS to facilitate the ionization efficiency in both the positive and negative electrospray ionization (ESI(+)/(−)) modes for abalone lipids. Optimal lipidomic coverage was acquired with 10 mM ammonium formate in both ESI(+)/(−) modes. The selected resuspension solvents also influenced ionization efficiency through the matrix effect, and resuspension in methanol enhanced the signal intensities by reducing ion suppression. Because of the higher glycerophospholipid content in shellfish, bridged ethylene hybrid C8 columns showed clear advantages over charged surface hybrid C18 columns. A series of glycerophospholipids, lyso-glycerophospholipids, glycerolipids, and fatty acids in different shellfish can be annotated and semiquantified in one injection by the optimized method. KEYWORDS: Q-TOF, lipidomic, glycerophospholipids, glycerolipids, fatty acids, shellfish

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INTRODUCTION Various liquid chromatography (LC) platforms have been applied to describe the analysis of complex lipid extracts. The three most commonly used methods are reversed phase LC (RPLC), normal phase LC (NPLC), and hydrophilic interaction LC (HILIC).1 Among these methods, lipids are typically isolated on RP column,2 and this accounts for 70% of all published LC−mass spectrometry (LC−MS) articles approximately. Recent advances in LC−MS technology include column chemistries, smaller particle sizes, and novel identification methods.3 In terms of the columns employed, RPLC-based lipidomics are analyzed with shorter (50−150 mm; typically 100 mm) chromatographic columns (1−2.1 mm i.d.).1 Both C8 and C18 columns can provide good separation effects of tissue lipid mixtures. However, C18 columns are more appropriate to the analysis of lysophospholipids and fatty acids (FAs), while better separation and elution of lipid extracts are provided by C8 columns.4,5 With regard to lipid profiling of many shellfish,6−9 Ascentis Express HILIC column was used for evaluation. According to the hydrophilic functionalities of lipid species on HILIC columns, lipid classes are separated with their characteristic polar headgroup.10 However, RP separation methods provide good isolation of lipids.11 Because of the diversity of glyceride structures, such as a wide range of lengths of fatty acid chains, positions, and numbers of double bonds, polar head-groups, and other modifications, the separation methods are particularly important. Regarding lipidomics and lipid profiling appraisal, mass spectrometry is the most commonly reported tool due to its high resolution and sensitivity, whereas this technique is primarily coupled with liquid chromatography.12 Indeed, LC− © XXXX American Chemical Society

MS is considered as a promising technique on account of its increased specificity and structure characterization capabilities.13 Triple quadrupole linear ion trap mass spectrometry was used for identification of lipid species in many shellfish, such as dried clams,8 fresh edible clams,7 mussels,9 and edible whelk,6 with precursor-ion and neutral loss scanning modes. However, detection of the mass spectrum was required to be operated in terms of the characteristic fragmentation pattern. To date, on the basis of advances in instrumentation, the development of lipidomics has a certain leap such as the creation of high resolution mass spectrometers.14 One of the most successful techniques for the structural characterization of lipids is time-of-flight mass spectrometry, with fast acquisition speed, high resolution, excellent sensitivity, and precise mass accuracy.12,15 This technique promises accurate molecular mass and intact fragmentation information simultaneously without preselecting any characteristic ions. 16 However, electrospray-based ionization techniques tend to suffer from ion suppression and ion enhancement effects, and owing to the complexity of lipid extracts, the detection of analytes (or sample cleanup) is necessary before analysis to avoid matrix effects and ion suppression. In lipidomic acquisition modes, the precursor and product ions are obtained by primary and secondary spectra, respectively, yielding important information regarding the elemental compositions and structural elucidation.17 Received: Revised: Accepted: Published: A

February 27, 2019 July 3, 2019 July 11, 2019 July 11, 2019 DOI: 10.1021/acs.jafc.9b01343 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Experimental design of the study focused on selecting optimal mobile phase modifiers and resuspended solvents by UPLC-ESI-Q-TOFMS of abalone lipid extracts.

abalone (Haliotis discus hannai Ino) was selected to discuss the lipidomic coverage due to higher glycerophospholipid (GPL) contents. Indeed, with regard to the delivery efficiency into different tissues and bioavailability of polyunsaturated fatty acids (PUFA), the form in GPL was higher than those in triacylglycerol (TAG). Thus, depending on the functional activities of the abalone products, the GPL composition of the lipids need to be identified.25 Over the past few decades, the lipid content of abalone has been examined in addition to the effect of diet on the abalone lipid content.26 The lipid profiles of different abalone tissues were investigated, and thirty-four lipid species consisting of lysophosphatidic acid (LPA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), triacylglycerol (TAG), steroid, terpenoid, and fatty acids (FAs) were detected.27 However, the number of identified lipid structures in abalone was significantly lower than those of other reported biological samples. To improve the lipid profile coverage of shellfish, different ionization efficiency, columns, modifiers, and resuspension solvents must be examined. The optimized methods are expected for the comprehensive lipidomic analysis of different shellfish samples. Thus, we herein report our evaluation of different combinations of the LC−MS modifiers and resuspension solvents most commonly covered in lipidomic studies with ultrahigh-performance-liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS) to ultimately facilitate the lipidomic coverage of lipid standards and shellfish lipids. The molecular species formed during ESI in the presence of different modifiers will also be characterized by MS and MS/MS with database comparison, and the signal intensities of the lipid species present in different resuspension solvents and mobile phase modifiers will be acquired using MS-DIAL software.

Water or aqueous of organic solvents (methanol, acetonitrile, isopropanol, and tetrahydrofuran) are commonly employed as a weak mobile phase, whereas tetrahydrofuran or isopropanol primarily dissolved in water, methanol, or acetonitrile in a strong mobile phase. To adjust the isolation selectivity or detection sensitivity, different modifiers can be mixed with the mobile phase to improve elution efficiency.18 Ammonium formate and ammonium acetate as the buffer salts are typically used in mobile phases at a concentration of 5−10 mM, whereas formic acid and acetic acid are employed with percentages of 0.05−0.2% (vol %).1 Indeed, different combinations of modifiers have been reported including ammonium formate or ammonium acetate,19,20 ammonium formate or ammonium acetate with formic acid,21,22 and ammonium acetate with acetic acid.23 Moreover, the various organic solvent-based protein removal methods are available for evaluation due to their protein precipitation efficiency, lipidomic coverage, and precision. Regarding serum specimens, acetone and methanol are efficient for protein removal, while methanol is the most efficient reagent for the separation of polar compounds.18 Although many recent studies exist into the use of HPLC− MS/MS methods to detect the lipid profiles of shellfish with precursor ion and neutral loss scanning,6−9 no systematic evaluation of the various mobile-phase modifiers and resuspension solvents has been conducted for RPLC−MSbased lipidomics to determine the optimal results for shellfish lipids. Currently, the various modifiers-based ionization efficiency are tested for the optimized detection of blood plasma lipids, where 10 mM ammonium formate and ammonium acetate can be used for comprehensive analysis of complex lipid mixtures in the ESI(+) and ESI(−) modes, respectively.24 It was also found that charged surface hybrid (CSH) C18 columns were advantageous in the detection of blood plasma lipids.24 In view of the various analytical approaches for blood plasma lipids, it was considered that the lipid profiles of marine shellfish could be studied following optimization. Among the wide range of available shellfish,

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MATERIALS AND METHODS

Materials. Abalone (Haliotis discus hannai Ino), scallop (Patinopecten yessoensis), mussel (Mytilus edulis), and clam (Ruditapes

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DOI: 10.1021/acs.jafc.9b01343 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article


Table 1. Effect of Mobile Phase Modifiers on Ionization Efficiency of Lipid Standards during UPLC-ESI-Q-TOF-MS Analysisa BEH-C8 column

CSH-C18 column

lipid standard

ESI mode

AmF

AmF/FA

AmAc

AmAc/FA

AmAc/AA

AmF

AmF/FA

AmAc

AmAc/FA

AmAc/AA

TAG(17:0/17:0/17:0) LPC(10:0) PC(17:0/17:0) PE(17:0/17:0) PG(17:0/17:0) PS(18:0/18:0) PA(17:0/17:0) PI(18:1/18:1) SM(d18:1/18:0) SM(d18:1/24:0) GalCer(d18:1/24:1) cholesterol LPC(10:0) LPA(18:0) PC(17:0/17:0) PE(17:0/17:0) PG(17:0/17:0) PS(18:0/18:0) PA(17:0/17:0) PI(18:1/18:1) SM(d18:1/18:0) SM(d18:1/24:0) GalCer(d18:1/24:1)

(+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (−) (−) (−) (−) (−) (−) (−) (−) (−) (−) (−)

79% 90% 99% 97% 98% 0% 96% 87% 99% 98% 97% 0% 98% 81% 71% 79% 87% 85% 61% 67% 92% 71% 89%

68% 97% 47% 52% 51% 0% 53% 33% 57% 44% 45% 0% 88% 95% 72% 74% 86% 82% 89% 73% 83% 60% 59%

21% 67% 36% 43% 37% 0% 40% 0% 31% 1% 20% 0% 75% 37% 0% 42% 79% 60% 28% 83% 41% 0% 0%

97% 96% 79% 75% 45% 0% 91% 0% 82% 80% 76% 0% 89% 61% 75% 79% 78% 66% 91% 0% 94% 69% 76%

23% 57% 36% 39% 37% 0% 61% 0% 38% 2% 18% 0% 74% 99% 41% 42% 15% 53% 70% 0% 43% 0% 46%

77% 85% 36% 39% 23% 0% 0% 0% 37% 38% 33% 91% 78% 0% 64% 73% 55% 0% 0% 0% 44% 66% 62%

77% 79% 33% 35% 19% 37% 0% 0% 36% 32% 27% 0% 42% 0% 93% 96% 19% 0% 0% 0% 57% 89% 70%

12% 30% 26% 31% 17% 0% 0% 0% 20% 2% 14% 0% 28% 0% 35% 56% 17% 0% 0% 0% 33% 0% 33%

68% 90% 54% 46% 33% 0% 0% 0% 61% 48% 28% 89% 56% 30% 0% 35% 9% 0% 0% 0% 9% 0% 0%

14% 52% 27% 27% 18% 91% 0% 0% 25% 0% 12% 0% 56% 26% 0% 0% 46% 0% 0% 0% 0% 0% 0%

a

Legend: AmF, ammonium formate; AmF/FA, ammonium formate with formic acid; AmAc, ammonium acetate; AmAc/FA, ammonium acetate with formic acid; AmAc/AA, ammonium acetate with acetic acid. Each column with different modifier combinations has three replicate injections. The highest peak intensity is considered as 100%. philippinarum) were obtained from a local market in Dalian, China. After removal of the shells, the abalone foot muscles were cut into sections measuring 1 cm × 1 cm. Scallop muscle, mussel gonad, and clam viscera were separated for further study. All samples were stored at −80 °C immediately after collection for less than 2 weeks prior to use. Chemicals. HPLC-grade solvents and ionization modifiers were purchased from Spectrum Chemical (New Brunswick, Canada; chloroform, methanol, isopropanol and acetonitrile), Sigma-Aldrich (Madison, USA; ammonium formate, ammonium acetate, formic acid), and Aladdin (Shanghai, China; acetic acid). Lipid standards triacylglycerol (TAG; 17:0/17:0/17:0) and cholesterol were purchased from Sigma-Aldrich (Madison, WI, USA). Phosphatidylcholine (PC; 17:0/17:0), phosphatidylethanolamine (PE; 17:0/17:0), phosphatidylglycerol (PG; 17:0/17:0), and phosphatidic acid (PA; 17:0/17:0) were obtained from Aladdin (Shanghai, China). Lysophosphatidylcholine (LPC; 10:0), phosphatidylserine (PS; 18:0/18:0), phosphatidylinositol (PI; 18:1/18:1), sphingomyelin (SM; d18:1/18:0), sphingomyelin (SM; d18:1/24:0), and galactosyl(ß) ceramide (GalCer; d18:1/24:1) were acquired from Avanti Polar Lipids (Alabama, USA). Sample Pretreatment. Lipid extraction was employed with the Folch method.28 More specifically, the foot muscles of abalone (50 g) were homogenized with water (1:4, w/v) and extracted in triplicate using chloroform/methanol (2:1, v/v). The obtained extracts were then subjected to centrifuge at 9190g for 10 min, and the organic layers were shifted into two preweighed vials prior to evaporation with nitrogen. The resulting dried lipid extracts were resuspended using chloroform/methanol (2:1, v/v) or methanol containing PC (17:0/ 17:0) (internal standard for injection quality control). The final injection concentrations of the lipid extracts were 2 mg/mL for ESI(+) and 5 mg/mL for ESI(−). Samples were mixed with the support of a vortex for 10 s and subjected to centrifuge at 22 400g for 10 min to remove any impurities prior to LC−MS analysis. The lipids of scallop muscles, mussel gonads, and clam viscera were extracted by the same method as those from abalone. The dried lipid extracts were

redissolved using methanol with internal standard (PC (17:0/17:0), PE (17:0/17:0), PG (17:0/17:0), LPC (17:0), and TAG (17:0/17:0/ 17:0)) for semiquantitative analysis. Liquid Chromatography Condition. RPLC-based analysis were performed with UltiMate 3000 liquid chromatography system (Thermo Fisher, MA, USA). Analytes were separated on an Acquity UPLC BEH C8 column (2.1 × 100 mm2; 1.7 μm) (Waters, MA, USA) coupled with an Acquity BEH C8 VanGuard precolumn (2.1 × 5 mm2; 1.7 μm) (Waters). For comparison, an Acquity UPLC CSH C18 column (2.1 × 100 mm2; 1.7 μm) (Waters) coupled with an Acquity CSH C18 VanGuard precolumn (2.1 × 5 mm2; 1.7 μm) (Waters) was also used. Both columns were thermostated at 65 °C, and the flow rate was 0.6 mL/min. Mobile phase A consisted of acetonitrile/water (60:40 v/v) and mobile phase B was the mixture of isopropanol/acetonitrile (90:10 v/v), both containing different modifiers (Figure 1). The modifiers were dissolved in a small volume of water prior to mixing with the mobile phases to increase their solubility, and then degassed for 15 min.24,29 The following gradient elution was as follows: 0 min 15% B; 0−2 min 30% B; 2−2.5 min 48% B; 2.5−11 min 82% B; 11−11.5 min 99% B; 11.5−12 min 99% B; 12−12.1 min 15% B; 12.1−15 min 15% B. The injection volume was 5 μL, and the autosampler temperature was 10 °C. Ten millimolar sodium formate solution was used for internal calibration based on data processing software within 2 min under isocratic condition in the sample batch. Mass Spectrometric Analysis. Untargeted mass detection was performed on an impact II Q-TOF (Bruker, Karisruhe, Germany) in both the ESI(+)/(−) modes. The mass range was 100−1200 m/z (mass to charge ratio) with 5 spectra/s. The source parameters were as follows: the dry gas was 10 L/min at 250 °C, and the nebulizer pressure was 3 bar. In the positive mode, the collision energy was at 15−30 V, and the capillary voltages were 4500 V for ESI(+). In the negative mode, the collision energy was 15−40 V, and the capillary voltages were 3000 V for ESI(−). Auto MS/MS mode was selected as the data acquisition mode with the cycle time of 1.2−1.8 s. Tuning Mix solution (Santa Clara, CA, USA) was used for instrument C


CSH-C18

BEH-C8

column

LPC PC PE MAG DAG TAG FA LPC LPE PC PE PI LPC PC PE MAG DAG TAG FA LPC LPE PC PE PI

lipid class

8 76 8 2 4 15 30 6 4 25 19 1 6 72 8 2 2 14 29 5 4 26 19 1

total no. lipids

(+) (+) (+) (+) (+) (+) (−) (−) (−) (−) (−) (−) (+) (+) (+) (+) (+) (+) (−) (−) (−) (−) (−) (−)

ESI mode 6% 38% 28% 12% 77% 70% 5% 10% 19% 7% 24% 0% 3% 17% 6% 29% 19% 60% 7% 9% 20% 18% 32% 14%

value 5 49 5 1 4 12 18 5 4 9 15 0 3 44 5 2 2 11 19 5 4 24 19 1

no. lipids

AmF

38% 27% 29% 18% 42% 92% 4% 12% 25% 23% 4% 7% 7% 19% 20% 0% 10% 45% 4% 4% 0% 22% 21% 0%

value 6 50 7 1 3 15 13 5 4 22 4 1 4 45 6 0 1 12 14 2 0 21 15 0

no. lipids

AmF/FA

35% 10% 7% 35% 6% 36% 35% 6% 28% 11% 29% 59% 4% 7% 10% 0% 0% 8% 34% 6% 0% 8% 18% 0%

value 3 26 4 2 1 8 28 2 3 14 17 1 1 27 4 0 0 5 25 2 0 9 11 0

no. lipids

AmAc

17% 27% 26% 35% 75% 82% 6% 0% 5% 1% 24% 6% 27% 28% 22% 0% 11% 50% 3% 0% 22% 0% 8% 0%

value 5 33 6 2 4 11 18 0 1 1 14 1 4 39 7 0 1 12 16 0 2 0 14 0

no. lipids

AmAc/FA

chloroform:methanol = 2:1

12% 10% 3% 39% 4% 23% 54% 7% 5% 16% 39% 84% 17% 6% 3% 0% 0% 20% 46% 4% 12% 3% 10% 0%

value 3 27 4 2 1 10 26 2 1 15 18 1 4 32 3 0 0 10 29 2 3 7 11 0

no. lipids

AmAc/AA

97% 100% 98% 35% 97% 71% 16% 82% 87% 24% 59% 0% 52% 50% 45% 97% 30% 58% 21% 96% 99% 99% 98% 94%

value 5 48 5 1 4 12 18 5 4 9 15 0 3 44 5 2 2 11 19 5 4 24 19 1

no. lipids

AmF

93% 61% 88% 44% 44% 76% 11% 81% 93% 75% 11% 9% 27% 40% 64% 0% 9% 35% 7% 25% 0% 58% 37% 0%

value 6 44 7 1 3 14 13 5 4 22 4 1 3 45 6 0 1 12 14 2 0 21 15 0

no. lipids

AmF/FA

15% 24% 23% 73% 8% 30% 89% 37% 41% 27% 60% 0% 0% 12% 17% 0% 0% 9% 53% 20% 0% 13% 19% 0%

value 2 26 4 2 1 8 28 2 2 14 17 0 0 27 4 0 0 5 25 2 0 9 11 0

no. lipids

AmAc

methanol

Table 2. Effect of Mobile Phase Modifiers on Intensity of Molecular Species of Abalone Lipid Extracts during UPLC-ESI-Q-TOF-MS Analysis

35% 57% 71% 91% 47% 50% 12% 0% 22% 2% 50% 9% 39% 55% 84% 0% 10% 38% 9% 0% 19% 0% 32% 0%

value 5 31 6 2 4 11 18 0 1 1 14 1 4 39 7 0 1 12 16 0 1 0 14 0

no. lipids

AmAc/FA

14% 20% 17% 71% 6% 13% 83% 41% 19% 30% 57% 0% 30% 15% 17% 0% 0% 18% 99% 33% 56% 10% 36% 0%

value

3 26 4 2 1 10 26 2 1 15 18 0 4 31 4 0 0 10 29 2 3 7 11 0

no. lipids

AmAc/AA

Journal of Agricultural and Food Chemistry Article

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Article


Figure 2. Fragmentation pathways of PC (14:0/16:0) in MS/MS. (A) MS/MS spectrum for the [M + H]+ at m/z 706.5342 of PC (14:0/16:0); (B) MS/MS spectrum for the [M+FA−H]− at m/z 750.5298 of PC (14:0/16:0); (C) fragmentation pathways of PC (14:0/16:0).

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calibration. The reference ions m/z 622.0290, 922.0098 in the ESI(+) mode and m/z 601.9790, 1033.9881 in the ESI(−) mode were chosen to check the mass accuracy during the acquisition. Raw Data Processing. Untargeted data acquired on the Q-TOF were analyzed by MS-DIAL (http://prime.psc.riken.jp/ Metabolomics_Software/).30 On the basis of the calibration curves of sodium formate, lipid molecular mass accuracy was corrected prior to the data analysis. The MS and MS/MS spectra were extracted by MS-DIAL through mass spectral deconvolution. Three of the most important indicators, such as retention time, mass accuracy, and isotope ratio, were used to annotate the lipids molecular species, while the structural elucidation was performed to match the MassBank and LipidBlast databases through MSMS spectra similarity.31,32 The details on data processing in Tables 1 and 2 were shown in the Supporting Information. Displayed as relations between sets by Venn Diagram (http://bioinfogp.cnb.csic.es/tools/venny/index.html), 75, 51, 63, and 71 lipid species repesented BEH-C8 column in ESI(+)/ (−) mode and CSH-C18 column in ESI(+)/(−) mode with 10 mM ammonium formate modifier. Analysis of variance (ANOVA) and principal component analysis (PCA) were performed by SPSS v9.0 (SPSS Inc., Chicago, IL) and MetaboAnalyst 4.0 (https://www. metaboanalyst.ca/MetaboAnalyst/faces/home.xhtml).

RESULTS AND DISCUSSION

Analysis of Lipid Profile by UPLC-ESI-Q-TOF-MS. Selection of the ionization mode employed in LC−MS analysis is vital for the determination of a lipidomic profile. Indeed, a single methodology is unable to consist of all analytes. However, the ionization efficiency can be facilitated by the use of mobile-phase modifiers since this leads to the formation of different types of adducts. The positive mode is the most typical mode for LC−MS analysis on account of ionizing various lipid classes effectively.1 However, negative ionization mode also displays excellent ionization efficiency for some lipid classes such as PG, PS, PA, and PI.33 Thus, we herein detected LPC, PC, PE, PS, SM, and GalCer as [M + H]+ ions, and PG, PA, and PI as [M+NH4]+ ions. On the basis of the sodium in the extraction process or the system pipeline, monoacylglycerol (MAG), diacylglycerol (DAG), and TAG were formed with [M + Na]+ adducts. In addition, LPA, LPE, PE, PG, PS, PA, PI, and FA were detected as [M−H]− adducts, whereas LPC, PC, SM, and GalCer formed various major ions (i.e., formate, E


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Journal of Agricultural and Food Chemistry [M+HCOO]− or acetate, [M+CH3COO]−), depending on the mobile phase modifier employed. Precursor ion and neutral loss scans were utilized to examine the lipid compositions. In the positive mode, the glycerophospholipid (GPL) groups were assigned, while in the negative mode, the derived fatty acid fragments were determined. The GPLs exhibited a regular fragmentation pattern according to the loss of the polar groups.9 Thus, on the basis of the measured [M + H]+ molecular ion at m/z 706.5342, this unknown compound was analyzed (Figure 2A). In addition, the characteristic fragment, such as m/z 184.0738 in the ESI(+) mode, was employed to selectively determine LPC, PC, or SM. Following collision-induced dissociation (CID), product ions with m/z values of 750.5298, 690.5045, 480.3142, 255.2324, 241.2195, and 227.2006 were observed in the MS/MS spectrum (Figure 2B). Among these fragments, product ions such as m/z 255.2324 and 227.2006 were confirmed as the fatty acid of 14:0 and 16:0, respectively. These fragments are typical of the fatty acids, thereby confirming the sn-1 and sn-2 structures of the GPLs.34 Several other product ions were visible in the MS/MS spectrum, including fragments at m/z 750.5298, 690.5045, 480.3142, and 241.2195, which were identified as [M+FA−H]−, [M−CH3]−, [LPC 16:0−CH3]−, and the [RCOO−O+2H]− of 16:0 (Figure 2C). Thus, the unknown compound was identified as PC (14:0/16:0). PE, PG, PS, PI, LPC, and SM exhibited extremely similar fragmentation patterns to PC and were also determined according to a similar strategy. Effect of Mobile-Phase Modifiers on Lipid Standard Signal Intensity. Modifiers are extensively recommended to mix in the mobile phase, which can enhance lipid isolation and detection. As such, various combinations of salts (ammonium formate and ammonium acetate) and acids (formic acid and acetic acid) were employed to form [M + H]+, [M+NH4]+, [M−H]−, [M+HCOO]−, and [M+CH3COO]− ions instead of the formation of [M+K]+ and [M + Na]+ ions during the electrospray process, as these modifiers can significantly improve signal intensities.22,29,35−37 Moreover, sodium adducts tend to exhibit poor reproducibility and stability, with reduced signal intensities and complicated analytes. Thus, it was postulated that buffers such as ammonium salts can lower the surface tension of the solvent droplets during the ESI process, thereby enhancing the ionization effects.29 The experimental design employed in our study is shown in Figure 1, where 10 mM ammonium formate or ammonium acetate and 0.1% formic acid or acetic acid were used to modify the lipid elution and ionization. The lipid standards and the abalone lipid extracts were separated using high-pressure tolerance columns, and an Acquity UPLC BEH C8 column was filled with ethylene-bridged hybrid (BEH) particles to increase the separation efficiency and the retention stability of neutral compounds. In addition, an Acquity UPLC CSH C18 column was also examined as this column incorporated reproducible low concentration charges to the particle surfaces. It was found that the CSH column produced an excellent peak shape and exhibited a suitable column efficiency in the typical buffer salt mobile phase system or in the low ionic strength mobile phase system. The above columns were selected due to their common report in metabolomics and lipidomics studies.1 Furthermore, mobile phases A consisting of acetonitrile/water (60:40 v/v) and mobile phases B consisting of isopropanol/ acetonitrile (90:10 v/v) were employed, which are frequently used in lipidomics. Moreover, the mixture of reagents provided

the effective elution and isolation of complex lipid extracts. Selection of the resuspension solvents to optimize the chromatographic conditions using the same analytical column was also important, and so the dried lipids were resuspended using the mixture of chloroform/methanol (2:1, v/v)9 or methanol38 for MS analysis. The ionization efficiency of the lipid standards under the various modifier combinations was evaluated using both the BEH-C8 and CSH-C18 columns by measuring the signal intensity of each peak, as shown in Table 1. Thirteen lipid standards were employed to represent 12 lipid classes. The polarity ranged from highly polar lipids to extremely nonpolar lipids. Information regarding the retention times, m/z values, and adduct ions of the lipid standards are given in Table S1(1)(3). Among various tested modifiers, although many lipid species could be detected under both the ESI(+)/(−) modes, the identified compound profiles were different. In the positive ionization mode, some lipids indicated particularly extreme differences between various modifier combinations such as cholesterol. It was only visible upon injection with ammonium formate as well as ammonium acetate with formic acid. Hence, it was apparent that the buffer modifiers significantly influenced the lipidomics coverage in LC−MS studies. In general, it is accepted that neutral polar lipids (TAGs, DAGs, cholesterol) and electrically neutral lipids (PCs, SMs) can be effectively ionized in positive ion mode.33,39 Furthermore, the majority of nonpolar lipids were observed to exhibit relatively weak ionization efficiency using acid additives. This was particularly evident during the evaluation of the glycerides due to the formation of sodium or ammonium adducts, while in comparison, the most intense intensities of the GPL and lyso-GPL originated from their [M + H]+ adducts.29 Moreover, one single modifier, namely ammonium formate, provided an excellent ionization efficiency for all 12 lipid classes in positive mode on the BEH-C8 column including LPC, PC, PE, PG, PA, SM, TAG, and GalCer. In general, the most intense peak can be acquired under acidic condition (i.e., pH 3.5−4.0), owing to the acid facilitates protonation as the analytes are more basic than the reagents.29,40 However, a number of compounds did not demonstrate conventional behavior. Perhaps, analytes were either more easily identified under the basic conditions41 or were not affected by the pH. Interestingly, some studies have reported that protonated adducts form under basic conditions, while deprotonated adducts form under acidic conditions, and this was termed the “wrong-way-round ionization”.29 Thus, the use of acidifiers gave somewhat lower signal intensities for some lipid standards. Indeed, we obtained superior results using the BEH column, on account of all detected lipids in the ESI(+) mode showed excellent peak intensities with ammonium formate in the absence of formic acid. In contrast, ammonium acetate yielded poor results for the vast majority of lipids, although the addition of formic acid improved its performance, especially in the case of TAG, LPC, and PA. In general, the positive ionization mode is employed for nontargeted analysis, as it tends to ionize much more numbers of compounds. In contrast, lipid species bearing anionic groups are ionized more effectively in the ESI(−) mode. Ammonium formate gave optimal results in the ESI(−) mode using the BEH column, and in particular in the cases of LPC (10:0) and SM (d18:1/18:0). As higher mobile phase pH values increased the deprotonation of some lipids, ammonium formate (pH F


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Figure 3. Venn diagram representing the lipid species overlap between BEH-C8 column and CSH-C18 column with 10 mM ammonium formate modifier in ESI(+) and ESI(−) mode.

columns). For all other lipid standards, the retention time shifts were >20 s for both columns. The ionization efficiency was also affected by the shifts of retention times owing to the different contents of the volatile organic solvent.40 Signal Intensities of Lipid Extracts Using Different Ionization Modifiers. Because of high economic value of cultured marine products, and higher GPL contents, the Japanese abalone (Haliotis discus hannai Ino) was selected to discuss the lipidomic coverage.46 The ionization efficiency and the structure identification of all lipid classes was performed simultaneously using five modifier combinations in the ESI(+)/(−) modes. The obtained results were evaluated according to the total peak numbers and peak intensities, which were determined using MS-DIAL software. Detection of the various lipid species is summarized in Table 2. In total, 125 species from six lipid classes including LPC, PC, PE, MAG, DAG, and TAG were identified in positive ion mode, and 88 were identified from six lipid classes including FA, LPC, LPE, PC, PE, and PI in negative ion mode. Further details regarding all abalone lipids detected in the ESI(+)/(−) modes can be found in Table S1(2)(4). Table 2 lists the different peak heights recorded under positive and negative ionization modes using ammonium formate alone for both columns, and also in the presence of formic acid for the BEH column. More specifically, LPC, PC, PE, and DAG exhibited slightly higher peak intensities on BEH column with ammonium formate; however, the peak intensities for MAG in ESI(+) mode using the CSH column were slightly greater than that for the BEH column. Following tests on the lipid standards, it was found that all lipid classes showed a more than two-fold decrease in the signal intensity with ammonium acetate instead of ammonium formate. In addition, in negative ion mode, ammonium formate was the most suitable buffer modifier for the CSH column (see Table 2). As no PA or PS was present in the lipid extracts, the CSH column gave superior results to the BEH column. In terms of the lipidomic coverage provided by the BEH and CSH columns

6.6) was mixed into the mobile phase to improve the ESI(−) ionization efficiency for many lipid species. However, this buffer pH was only slightly lower than that of ammonium acetate. The significant decrease in the signals corresponding to PC, PE, SM, and GalCer suggested an additional suppression effect by acetate ions in the mobile phase. In terms of [M−H]− ion yielded in the acid solutions, the “wrong-way-round ionization” mechanism was generated in the ESI(−) mode. It was assumed that the addition of acidic additives improved the reduction process of analytes because the electrospray droplets were negatively charged more efficiently. Because of the evaporation of the spray droplets in the ESI source, lower pH value-based vacuum environment was more appropriate for the reduction reaction.42,43 However, the conjugate anion of the acid was also involved in the ionization process, for which the negative charge was involved with gas phase proton affinity.42,44 In the ESI(−) mode, the use of acids as modifiers also resulted in some analytes exhibiting higher peak intensities, for example, PA and LPA. For all lipid standards, superior results were obtained using the BEH column, in particular for PA (17:0/17:0) and PS (18:0/ 18:0), which gave poor peak shapes when the CSH column was employed. Indeed, PA and PS are typically isolated on RP column as extensively broad peaks, while NPLC and HILIC as well as direct infusion are commonly employed for these classes.45 Overall, for samples containing PA and PS, the BEH column should be used for separation in negative ion mode. Different mobile phase pH values were also found to influence the chromatographic retention times, which could in turn lead to improvement in the signal intensities on both columns, although it should be noted that the different stationary phase materials of the two columns also influence the retention mechanisms.29 When ammonium formate was employed as the mobile phase, the highest retention time shifts were found for TAG (17:0/17:0/17:0) (3.79 min for BEH and CSH columns), SM (d18:1/24:0) (2.63 min for BEH and CSH), and GalCer (d18:1/24:1) (2.47 min for BEH and CSH G


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Figure 4. Detection of selected free fatty acids in abalone lipid extracts by ESI(−) mode with different modifiers. Lipids were separated on a BEHC8 column with (A) 10 mM ammonium formate modifiers and (B) 10 mM ammonium formate and 0.1% formic acid modifier.

with 10 mM ammonium formate in the ESI(+)/(−) modes, a Venn diagram (Figure 3) shows that the set of 142 identified lipid species consisted of 47 overlapping compounds, 28 from the BEH column, and 19 from the CSH column in the ESI(+) mode. Furthermore, two lipid species were identified from the BEH column and 19 from the CSH column in the ESI(−) mode, in addition to 49 mutual lipid species. These results suggest that the greatest number of lipid species can be determined using the BEH column in the ESI(+) mode and the CSH column in the ESI(−) mode. It was also found that formic acid (0.1%) was a poor modifier in the ESI(−) mode for the majority of anionic lipids, as the conjugate anion of the acid participated in the ionization process, carrying the negative charge, and was involved in gas phase transfer. Importantly, the intensities of fatty acids were significantly suppressed for both columns where formic acid was employed in negative ion mode. Indeed, the addition of ammonium formate was essential for free fatty acids, allowing shorter retention times to be observed compared to the use of other modifiers with lower mobile phase pH values (Figure 4). However, a number of reaction mechanisms exist into the analytes during the ESI source spray process including acid or basic reactions in the droplets, gas phase proton transfer, the formation of ions in the solvent, and electrolytic oxidation or reduction.29 In addition, due to matrix effects resulting in ion suppression and influencing adduct formation, the ionization effects of the lipid standards were not consistent with the lipid extracts present in the shellfish. The obtained results suggested that many abalone lipid species were detected with lower or no signal intensities if inappropriate modifiers were employed. As mentioned previously, the optimized condition was obtained with 10 mM ammonium formate in the ESI(+) mode on BEH column and in the ESI(−) mode on CSH column (see Figure 5), thereby indicating the significant impact of the mobile phase modifier on the lipidomic coverage. However, Table 2 and Figure 3 show acceptable coverage results on a BEH column

where ammonium formate was employed in negative ion mode. Therefore, for the use of the BEH column alone, ammonium formate was recommended as the modifier of choice for detection of the shellfish lipid extracts. Figure 5 shows the retention times of the lipids on the BEH and CSH columns in ESI(+)/(−) modes where 10 mM ammonium formate was employed as the modifier. Several peaks in Figure 5 are marked with numbers in Table S1(2)(4). The labels in Figure 5A and C are the same substances, and the labels in Figure 5B and D are the same substance. Obviously, retention times of the same compound on different chromatographic columns were quite different. The retention times for the lipid species were also found to vary in the presence of different additives, as these additives can alter the lipid charge states and influence their interactions with the column material. The differences in retention times also had a significant effect on the ionization efficiency owing to the altered contents of the mobile phase,40 and differences in the peak intensities for the different analytes were observed for the BEH and CSH columns. Compared with the lipid standard results, the ionization efficiency of lipid extracts was slightly different on both columns with different additives (Tables 1 and 2). Except for ammonium acetate with formic acid or acetic acid modifiers, the percentages of intensities were similar to those of lipid standards on BEH column in the positive mode with methanol as resuspension solvent. In the negative mode, the lipidomic coverage of lipid extracts was quite different with the lipid standards on both columns. Especially, PI(18:1/18:1) had better response on BEH column, while the PI class showed higher intensities on CSH column with ammonium formate modifier. However, lipidomic coverage was affected by resuspension solvent. Because of the matrix effect of lipid extracts, ion suppression may affect the intensities of different lipids classes. Effect of Resuspension Solvent on Lipid Extract Signal Intensities. In general, the resuspension solvent H


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Figure 5. MS deconvolution spectrum separation of abalone lipids in (A) ESI(+) and (B) ESI(−) on a BEH-C8 column with 10 mM ammonium formate modifier and (C) ESI(+) and (D) ESI(−) on a CSH-C18 column with 10 mM ammonium formate modifier. Numbers in A and C correspond to the compounds listed in Table S1(2); numbers in B and D correspond to the compounds listed in Table S1(4). The orders of numbers represented the retention times of compounds.

Method Validation Across Different Shellfish. The optimized UPLC-ESI-Q-TOF-MS method was applied to analyze the different lipid profile across three shellfish (scallop muscles, mussel gonads, and clam viscera). BEH-C8 column and ammonium formate modifier were selected to separate the lipids. The lipid species and contents of different shellfish were shown in Table S2. Totally 113 lipid molecular species containing LPC, LPE, PC, PE, PS, PI, PG, TAG, and FAs were identified in the ESI(+)/(−) modes (mass shift was 3-fold higher signal intensity than chloroform/methanol (2:1, v/v) when ammonium formate was used as the modifier on a BEH column. The matrix effect can partly explain these findings, as the higher signal intensity may indicate a reduction in ion suppression in methanol, and so this solvent was selected for analysis of the shellfish lipid extracts. I


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Figure 6. PCA results for lipid species in three different shellfish. (A) Score scatter plot. (B) Loading scatter plot. Numbers of lipid species in loading scatter plot are correspond to the compounds listed in Table S2.

In conclusion, the use of the same mobile phase modifier for both the ESI(+)/(−) modes not only enhanced the lipidomic coverage but also reduced the total operation time. The use of 10 mM ammonium formate as the modifier resulted in high signal intensity for the majority of lipids using the ESI(+) mode for a bridged ethylene hybrid (BEH)-C8 column or the ESI(−) mode for a charged surface hybrid (CSH)-C18 column, both without further acidification. Ammonium acetate was not recommended for use as the modifier due to the significant reduction in signal intensities. In general, due to the higher GPL content, the use of a BEH column gave better results for lipid detection from shellfish extracts; for other animal or plant samples with higher TAG content, a CSH column may be more suitable. Moreover, the selection of resuspension solvent also had a significant impact on the ionization efficiency. Resuspension in methanol improved the lipidomic coverage likely due to reduced ion suppression. The optimized method is expected to lead a more comprehensive lipidomic analysis of different marine shellfish samples.

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Ming Du: 0000-0001-5872-8529 Lei Qin: 0000-0002-3902-3192 Funding

This work was supported by the National Natural Science Foundation of China (31601432) and the National Key Research and Development Program of China (2016YFD0400404). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED UPLC-ESI-Q-TOF-MS, ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry; ESI(+)/(−), positive/negative electrospray mode; LC, liquid chromatography; RPLC, reversed-phase LC; NPLC, normal-phase LC; HILIC, hydrophilic interaction LC; GPL, glycerophospholipid; CSH, charged surface hybrid; BEH, bridged ethylene hybrid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol; PA, phosphatidic acid; LPA, lysophosphatidic acid; TAG, triacylglycerol; FAs, fatty acids; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; SM, sphingomyelin; GalCer, galactosyl ceramide; MAG, monoacylglycerol; DAG, diacylglycerol

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01343.

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Lipid species used for evaluation of mobile-phase modifiers, lipid composition and content in different shellfish (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86 411 86332275. Fax: +86 411 86323262. ORCID

Da-Yong Zhou: 0000-0001-5010-6418 J


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Improving Lipidomic Coverage Using UPLC-ESI-Q-TOF-MS for Marine

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