Analysis of phospholipids by NACE with on-line ESI-MS

Fei Gao1, 2

Zhengxiang Zhang1

Xiaofang Fu1

Wei Li1

Tao Wang2

Huwei Liu1

1Beijing National Laboratory forMolecular SciencesKey Laboratory of BioorganicChemistry and MolecularEngineering of Ministry ofEducation, Institute of AnalyticalChemistry,College of Chemistry andMolecular Engineering,Peking University,Beijing, P. R. China

2Division of Nephrology,Third Hospital,Peking University,Beijing, P. R. China

Received August 27, 2006Revised November 12, 2006Accepted November 29, 2006

Research Article

Analysis of phospholipids by NACEwith on-line ESI-MS

A hyphenated method of nonaqueous capillary electrophoresis coupled to electrosprayionization mass spectrometry (NACE-ESI-MS) is described for the simultaneous analysis ofphospholipids. The best results were obtained with a mixed solution of methanol/ACN(40:60 v/v) containing 20 mM ammonium acetate and 0.5% acetic acid, under the appliedvoltage of 30 kV and capillary temperature of 257C. ESI-MS measurements were performedin the negative mode with methanol/ACN (40:60 v/v) containing 50 mM ammonium ace-tate as sheath liquid at a flow rate of 2 mL/min. Different phospholipid classes have beensuccessfully separated within 16 min, and the molecular species of every single class havebeen identified by using MS2 or MS3, which generates characteristic fragments throughCID. The developed method has been applied to analyze the phospholipids extracted fromrat peritoneal surface and the molecular species of phospholipid classes are presented.

Keywords:

MS / NACE / Phospholipids DOI 10.1002/elps.200600533

1418 Electrophoresis 2007, 28, 1418–1425

1 Introduction

Phospholipids are essential cellular constituents possessingmultiple distinct yet critical roles in cellular function [1]. Themajority of cellular phospholipids form a membrane bilayerwhose integrity and physical characteristics are vital for lifeprocesses and provide an appropriate hydrophobic environ-ment for membrane protein function and interactions [2, 3].Moreover, phospholipids serve as reservoirs for energy stor-age, which can be rapidly accessed at times of demand.Finally, membrane phospholipids are the source of lipid sec-ond messengers generated by the actions of a variety ofintracellular enzymes [4–6]. According to the polar headgroup, phospholipids can be classified into multiple differentclasses (Fig. 1). Numerous molecular species, containing a

Figure 1. General structure and classes of phospholipids. Theclassification of phospholipids is based on the polar head moi-eties of X. R1 and R2 are used to designate undefined alkyl groupsat the sn-1 and sn-2 positions, respectively.

variety of long-chain fatty acid residues, are found in a singleclass of phospholipids.

To our knowledge, TLC has been the simplest techniquefor lipid analyses [7], but quantification by scanning of thestained chromatograms lacked precision because of problemsarising from nonuniformity of staining of both the phospho-lipids and the background. In recent years, normal-phase(NP)-HPLC has been used for the separation of phospholipidclasses with either evaporative light-scattering detection [8–

Correspondence: Professor Huwei Liu, College of Chemistry andMolecular Engineering, Peking University, Beijing 100871, P. R.ChinaE-mail: [email protected]: 186-10-6275-1708

Abbreviations: DMPC, 1,2-dimyristelaidoyl-sn-glycero-3-phos-phocholine; DMPE, 1,2-dimyristoyl-sn-glycero-3-phosphoetha-nolamine; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol;DMPS, 1,2-dimyristoyl-sn-glycero-3-phosphoserine; HOAc,acetic acid; MeOH, methanol; PE, glycerophosphoethanolamine;PtdCho, phosphatidylcholine; PtdGro, phosphatidylglycerol;PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; SM,sphingomyelin; TEA, triethylamine

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2007, 28, 1418–1425 CE and CEC 1419

10] or ESI-MS [11–14]. The combination of HPLC with ESI-MS allows detecting even the least abundant lipid molecularspecies. By this approach, Taguchi and colleagues [13] identi-fied over 500 phospholipid molecular species from a singlepreparation of lipid extract from mouse B cell. We have suc-cessfully used this combined method to determine more than90 molecular species of phospholipids extracted from the ratperitoneal surface [14]. However, the obvious disadvantage ofmost HPLC methods in the analysis of phospholipids is thehigh cost for organic solvent consumption and time-con-suming, which would not meet the needs for high-through-put analysis. Compared with TLC and HPLC, CE offers notonly different selectivity and higher efficiency, but also fastermethod development and shorter analysis time, which makesit a powerful complement or an alternative to chromato-graphic separations. However, few reports have been pub-lished on the separation of phospholipids by CE [15–23].Considering the poor aqueous solubility of phospholipids,MEKC with UV detector [15, 16] or LIF detector [17, 18] wasfirst adopted in analysis of phospholipids. Although LIF cangive high sensitivity for phospholipids, the derivatizationprocess is complex and nonstoichiometric. Another com-monly used method is CZE with indirect UVdetection [19–21]due to the low UV absorbance of phospholipids. Raith et al.[22] measured the migration of four standard phospholipidsby nonaqueous CE on-line coupled to MS (NACE-MS). Intheir experiment, an extra pressure had to be applied at theinlet of capillary to guarantee a sufficient liquid flow in capil-lary and no any real samples have been tested. Guo et al. [23]separated and determined phospholipids in plant seeds usingNACE with UV detector, but the detection sensitivity was notsatisfactory for real samples.

Compared to aqueous solutions, several physical andchemical properties of organic solvents, such as relative per-mittivity, viscosity, auto-proteolysis constant, polarity andvolatility can be successfully exploited to adjust the selectivityupon modifying the electrophoretic mobilities. Conse-quently, NACE not only allows the separation of analytes thatare insoluble in water, but also might enable the separationof structurally very close related compounds [24–27]. Cou-pling of CE with ESI-MS has become a powerful combina-tion of high separation efficiency, high detection sensitivityand great capability for molecular characterization [28, 29].However, there are some constraints to be considered whenCE and MS are coupled on-line; the most important beingthe running solution compatibility with the ESI process [30].NACE may be considered an excellent solution to this issuebecause nonaqueous medium is virtually ideal in ESI pro-cess in providing stable spray and overall sensitivity. Lowsurface tension and high volatility favor stable formation ofthe spray and enhance sample ionization process that resultsin improved sensitivity compared with separation in aqueousbuffer system [31, 32].

In this study, we described the separation and character-ization of phospholipids by NACE coupled with ESI-IT-MSdetection. Good separation of different phospholipid classes

was achieved within 16 min and the molecular species ofevery single class have been determined by MSn techniques.The phospholipids from the rat peritoneal surface have beenanalyzed using the present method.

2 Materials and methods

2.1 Chemicals

Phospholipids, including glycerophosphoethanolamine (PE)from egg yolk, phosphatidylserine (PtdSer), phosphatidyli-nositol (PtdIns) and phosphatidylcholine (PtdCho) fromsoybean, 1-palmitoyl-2-oleoyl-phosphatidylglycerol (PtdGro),and ammonium acetate were obtained from Sigma (St.Louis, MO). Other internal standard phospholipids, such as1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), were pur-chased from Avanti Polar Lipids (Alabaster, AL). HPLC-grademethanol (MeOH) and ACN were purchased from DimaTechnology (Hong Kong, China). Analytical reagent-gradeacetic acid (HOAc), triethylamine (TEA) and ammoniumhydroxide were from Beijing Chemical Factory (Beijing,China).

2.2 Electrophoretic solutions and standard solution

preparation

The electrophoretic solutions were prepared by dissolvingappropriate amounts of ammonium acetate and HOAc in anACN/MeOH mixture. Stock standard phospholipid solutions(100 mg/mL), including PE, PtdCho, PtdSer, PtdIns andPtdGro, were dissolved in MeOH and diluted to appropriateconcentrations. Both of the electrophoretic solutions andstandard solutions were filtered through a 0.45-mm nylonmembrane and degassed by ultrasonication before use.

2.3 Extraction of phospholipids from Sprague

Dawley rat peritoneal surface

Phospholipids from rat peritoneal surface were extractedessentially as described earlier [14]. Briefly, Sprague Dawleyrats were anesthetized and sacrificed, and then 20 mL ofFolch solution (chloroform/methanol, 2:1 v/v) was infusedinto the peritoneal cavity for 30 s and drained completely asthe crude solution. The phospholipids in the crude solutionwere extracted by a modified Folch extraction technique [33,34]. Appropriate amounts of internal standards (e.g. DMPC(2 mg), DMPG (2 mg), DMPE (2 mg), DMPS (2 mg)) wereadded to 4.0 mL crude solution during lipid extraction. Thelipid extracts were dried by evaporation under nitrogen andthe samples were then stored dry at 2207C. Prior to analysis,the extracted samples were redissolved in 1 mL MeOH andfiltered through a 0.45-mm nylon membrane.


1420 F. Gao et al. Electrophoresis 2007, 28, 1418–1425

2.4 CE

All experiments were performed with an HP3D CE system(Agilent Technologies, Palo Alto, CA). Fused-silica capillaries(50 mm id6362 mm od) were obtained from PolymicroTechnologies (Phoenix, AZ) with a total length of 60 cm.Other conditions are described as follows: injection at50 mbar for 5 s, applied voltage of 30 kV, temperature 257C.Before use, the new capillaries were washed sequentiallywith 0.1 M sodium hydroxide for 10 min, water for 10 min,MeOH for 10 min, and running solution for 5 min. Betweenruns, the capillary was rinsed with running solution for3 min.

2.5 ESI-MS

The CE system was coupled on-line to a Trap XCT IT massspectrometer (Agilent Technologies) equipped with an ESIsource and operated in the negative ion mode. The exit tip ofthe capillary was orthogonally inserted into the electrosprayinterface. To maintain stable electrospray, the capillary tipwas positioned ,0.1 mm outside the stainless-steel needleand a ca. 10-mm portion of the polyimide coating wasremoved. The coaxial sheath liquid consisted of 50 mMammonium acetate in MeOH/ACN (40:60 v/v), at 0.2 mL/min delivered by an isocratic pump (Agilent Technologies)through a splitter set at 1:100, resulting in a flow rate of 2 mL/min into the nebulizer. The electrospray chamber and meta-lized entrance of the glass capillary were operated at 4.0 kV inthe negative-ion mode. The nitrogen drying gas flow-rate wasapprox. 4.0 L/min at 2007C with a nebulizer pressure of69 kPa. The ion source and ion optic parameters were opti-mized with respect to the negative molecular ions of thephospholipids standards. The MS data were collected underfull-scan mode in the range of 500–1000 m/z at a rate of fiveMS spectra per time point. Identification of ion peaks wasachieved by MSn experiments, which were carried out withAutoMSn operation function. The scan range for AutoMSn

was 100–1000 m/z with isolation and fragmentation width4.00 m/z. The low mass cutoff was set at slightly less thanone-third the precursor m/z value.

3 Results and discussion

3.1 Separation of phospholipids by NACE

MeOH, ACN, and their mixtures are the most frequentlyemployed organic solvents in NACE separations [31, 35, 36].As reported previously, varying the percentage of ACN inMeOH can lead to changes of migration times due to thechanges of viscosity (Z) and relative permittivity (e) of theelectrophoretic medium. Ammonium acetate is the mostfrequently used electrolyte in NACE systems and TEA orHOAc are often added to adjust the acid-base properties ofthe medium. In our case, 20 mM ammonium acetate has

been utilized and the effects of the percentage of ACN, and ofelectrolyte under basic (addition of TEA) and acidic (additionof HOAc) conditions have been investigated for the separa-tion of phospholipids.

The low viscosity combined with moderately low polaritymakes ACN an especially useful solvent for adjustment ofseparation selectivity in NACE systems. The alteration ofACN/MeOH composition results in changes of separationselectivity for some long chain surfactants [37, 38]. To moni-tor the influence of the solvent composition on the selectivityof phospholipids, the apparent mobility of phospholipids,varying with ACN/MeOH ratio is illustrated in Fig. 2. Theresults demonstrated that apparent mobilities of phospholi-pids increased as the content of ACN was increased exceptfor PtdSer (Fig. 2). The patterns of apparent mobility forphospholipids resulted from the combination of the changesof EOF and the variance of ion mobilities of phospholipids inthe MeOH/ACN mixtures. As described by Salimi-Moosavi etal. [37], the EOF initially increased upon addition of ACN,but decreased beyond 70% ACN. The decrease in EOFbeyond 70% ACN may be attributed to an increasing pKa

value of the silanol groups, which has a major influence onthe EOF at high ACN concentrations. Meanwhile, changes ina higher relative permittivity and a lower viscosity of theseparation media predict steadily increasing ion mobilities,as described by the von Smoluchowski equation [39] withoutconsidering the solvation interactions. However, withincreasing ACN content beyond 70%, the ion-ion interac-tions will take place for anionic phospholipids because ACNis dipolar-aprotic solvent lacking hydrogen bonding andhaving lower electrophilic properties [40]. The solvationinteractions slow the electrophoretic mobilities of anionicphospholipids (from cathode to anode) such as PtdGro and

Figure 2. Influence of ACN percentage on the apparent mobilityof phospholipids. NACE conditions: fused-silica capillary60 cm650 mm ID; running solution, MeOH/ACN (varying compo-sition) containing 20 mM ammonium acetate and 0.2% aceticacid; applied voltage, 30 kV; temperature, 257C; injection,50 mbar for 5 s; MS conditions: negative ion mode; capillaryvoltage 4 kV; nebulizer gas, N2 69 kPa; dry gas, 4 L/min at 2007C;sheath liquid, MeOH/ACN (40:60,v/v) containing 50 mM ammo-nium acetate, at 2 mL/min.



PtdIns, and lead to a higher apparent mobilities even inhigher ACN concentration. However, PtdSer bears twonegative charges compared to PtdIns and PtdGro, the de-crease of ion mobility might not be enough to compensatethe decrease of EOF at higher ACN content, and as a result,the apparent mobility would be decreased (Fig. 2). On theother hand, the solvation interactions might have little effecton the zwitterionic phospholipids such as PC and PE, whoseelectrophoretic mobilities (from anode to cathode) areincreased with ACN content as described above. The increaseof ion mobilities might counteract the decrease of EOF athigher ACN concentration. These results suggested that theuse of mixed organic solvents might be a useful approach forthe optimization of separation of compounds that differ onlyslightly in electrophoretic mobility. In this study, 60% ACNrepresented the condition for the best selectivity for allphospholipids.

Since acid-base properties are one of the most importantfactors influencing the selectivity of NACE separation [24,40], the effect of basic and acidic additives on the separationof phospholipids was explored and is shown in Fig. 3. Theaddition of TEA leads to further deprotonation of phosphategroup, which in turn increases ion mobilities of phospholi-pids. Even though the EOF increased with addition of smallamount (e.g. 0.2%) of TEA, it was smaller in magnitudecompared to the increased electrophoretic forces of phos-pholipids. Therefore, the apparent mobilities of phospholi-pids were decreased. With the increased TEA content, PtdInsand PtdGro were unable to be separated. Under acidic con-ditions with the addition of HOAc, the EOF was slowed downdue to the suppression of deprotonation of the silanol groupson the capillary inner surface and the change in the chargestate of phospholipids. In our experiments, 0.5% HOAcallowed sufficient separation and better peak shapes prob-ably because of lower analyte interactions with the silica wall.Collectively, a separation medium composed of MeOH/ACNat a ratio of 40: 60 v/v containing 20 mM ammonium acetateand 0.5% HOAc was finally chosen for the separation of

Figure 3. Influence of basic and acidic additives on the apparentmobility of phospholipids in mixture of MeOH/ACN (40:60 v/v).Other conditions as in Fig. 1.

phospholipids (Fig. 4). It can be seen that different anionicand zwitterionic phospholipid classes have been well sepa-rated within 16 min.

3.2 NACE-ESI-MS of phospholipids

NACE-ESI-MS analysis of phospholipids was performedusing 20 mM ammonium acetate and 0.5% HOAc in a mix-ture of ACN/MeOH (60:40 v/v) as a background electrolyte(BGE) in negative ionization mode. The instrument param-eters such as capillary positioning, sheath liquid flow rateand composition, nebulizing gas were all optimized to pro-duce the highest signal intensity of each phospholipid class.The capillary tip was set 0.1–0.2 mm protruding from theESI needle to assure a stable spray and strong signal. Thecomposition and the flow rate of sheath liquid demonstratedgreat effects on the ionization efficiency of analytes in theMS source. A sheath-liquid consisting of 50 mM ammoniumacetate in the mixture of ACN/MeOH (60:40 v/v) at 2 mL/minprovided a stable spray and highest response in MS due tohigher volatility, low surface tension and low viscosity of

Figure 4. Total ion electropherogram (TIE) of 0.2 mg/mL phospholipid standard samples by NACE-ESI-MS. NACE conditions: fused-silicacapillary, 60 cm650 mm, ID; running solution, MeOH/ACN (40/60 v/v) containing 20 mM ammonium acetate and 0.2% HOAc; applied volt-age, 30 kV; temperature, 257C; injection, 50 mbar for 5 s; MS conditions: negative ion mode; capillary voltage 4 kV; nebulizer gas, N2 69 kPa;dry gas, 4 L/min at 2007C; sheath liquid, MeOH/ACN ( 40:60 v/v) containing 50 mM ammonium acetate, 2 mL/min.



organic solvents. It should be noted that a neutral salt such asammonium acetate provides a more stable ionization pro-cess and results in a lower baseline noise compared withbasic additives such as ammonium hydroxide. In addition,phospholipids containing a choline group, such as PtdChoand sphingomyelin (SM), are more easily detected as the ac-etate adduct when ammonium acetate is present in thesheath liquid. A typical total ion electropherogram under theoptimal conditions is illustrated in Fig. 4. The molecular ionpeaks from the different phospholipid classes were detectedby full-scan ESI-MS analysis in negative mode. Most ofphospholipids possess net negative charge under ourexperiment conditions. Accordingly, negative-ion ESI massspectra of these phospholipids can be effectively obtained

with [M-H]– as the molecular ions, such as PtdIns, PtdSerand PtdGro. Even though PtdCho and PE are zwitterionicmolecules, they can be also analyzed by negative mode. Inour study, the species of PE were detected as the [M-H]– ions,and PtdCho, which has a choline group at the polar head,was detected as the acetate adduct, i.e. [M159]–.

In order to determine the phospholipid species, MSn

experiments were conducted with the IT mass spectrometer.Negative ion fragments on MS2 and MS3 spectra were pro-duced after CID and used to determine the structure of mo-lecular species. The main fragments were lysophospholipid-like and carboxylate anions [41–43]. Both types of productfragments might be used for species determination (Table 1and Fig. 5). The IT MS was set in the full-scan MS3 mode for

Table 1. Molecular ions and product ions of phospholipids used in species characterization by NACE-ESI-MSn

Phospholipids Molecular ions Main fragments detectedby MS2

Main fragmentsdetected by MS3

PtdCho [M1CH3COO]– [M–CH3]– [lyso-PtdCho–CH3]

–,[FA-H]–

PE [M–H]– [FA–H]–, [lyso-PE–H]–

PtdSer [M–H]– [FA–H]–, [M–Ser]–,[lyso-PtdSer–Ser–H]–,[lyso-PtdSer–Ser–H–H2O]–

PtdIns [M–H]– [FA–H]–, [lyso-PtdIns–H]–,[lyso-PtdIns–H–H2O]–

PtdGro [M-H]– [FA–H]–, [lyso-PtdGro–H]–,[lyso-PtdGro–H–H2O]–

Figure 5. Product ion spectra atone m/z value from each of thephospholipid classes obtainedby NACE-ESI-MSn in negativemode. (A) MS3 of PtdCho (m/z816.5) showing the 16:0/18:2fragments; (B) MS2 of PtdSer (m/z 782.5) showing 18:2/18:2 frag-ments; (C) MS2 of PtdIns (m/z833.5) showing 16:0/18:2 frag-ments; (D) MS2 of PtdGro (m/z747.5) showing the 16:0/18:1fragments; (E) MS2 of PE (m/z716.5) showing the 16:0/18:1fragments. The expressions ofmolecular species are the sameas in Table 3.



PtdCho and full-scan MS2 mode for other classes since MS2

spectra of PtdCho gave only one dominant product ion[M-15]– because of loss of CH3 from the choline group. It isnecessary to further fragment the product ion in order toprovide the information on the fatty acid substituents forsuccessful structure identification.

3.3 Method evaluation

Under optimized conditions, the generated CE electric cur-rent was low (15 mA), which considerably reduces Jouleheating and facilitates successful CE-ESI-MS on-line cou-pling. To assess method repeatability, consecutive injections(n = 5) of the phospholipid mixture were carried out. Therepeatability of migration time and peak area for each phos-pholipid were acceptable, as summarized in Table 2, wherethe RSDs of migration time and peak area was �2.0% and�6.0 %, respectively. The LOD for all detected phospholipidsvaried from 1.0 mg/mL for PtdGro to 8.0 mg/mL for PtdSer,which are nearly ten times lower than the values obtained byCE-UV [23]. The sensitivity will be further improved usingsheathless interface [44], or by preconcentration techniques,such as on-line sample-staking step [45], on-line RP-HPLCpacking [46] or an impregnated membrane [47].

The accuracy quantitation of phospholipids as a complexmixture of closely related compounds by ESI-MS has notbeen performed in our study because the efficiency in gen-erating ions not only depends on the polar head group of theindividual phospholipid classes, but also relies on the chainlength and unsaturation of fatty-acyl substituent [48]. Thus,the standards for each species need to be run to obtain abso-lute quantitation, but there was not each standard available.

3.4 Analysis of phospholipids extracted from

Sprague Dawley rat peritoneal surface

A surface layer mainly consisting of phospholipids has beenfound on the peritoneal mesothelial cell surface [49]. Severalstudies have demonstrated that phospholipids, especiallyPtdCho, could decrease the peritoneal fluid absorption rateand increase peritoneal fluid removal [50]. In this study,phospholipids on the surface were extracted by modified

Table 2. LOD and RSDs of migration time and peak area for eachphospholipid class

Phospholipids RSD (%) (n = 5) LOD(mg/mL)Migration time Area

PtdCho 1.7 6.0 2.0PE 1.9 5.4 3.2PtdSer 1.2 4.8 8.0PtdIns 1.2 5.2 2.0PtdGro 1.5 5.3 1.0

Folch method and have been separated and identified by thedeveloped NACE-ESI-MS method. Figure 6 demonstratesthe separation of phospholipids from this surface, showingthe main phospholipids such as PtdCho, PE and PtdIns. Itshould be noted that PtdSer was not detected in this samplebecause of its poor response in ESI-MS. By comparing themigration times and the mass spectra, we found no PtdGromolecule species except the internal standard, in agreementwith the results obtained by HPLC-ESI-MS [14]. The molec-ular species for individual phospholipid class were deter-mined by MSn techniques as described above (Table 3). Theresults showed that SM, one kind of sphingolipids alsopresent in the rat peritoneal surface. Although the PthChoand SM migrated together, they could be easily distinguishedby MSn technique. We also observed that there were twosubclasses, phosphatidylethanolamine and plasmalogen,existing in PE class. Compared with HPLC-ESI-MS, theNACE-ESI-MS technique detected less molecular speciespeaks due to its lower concentration sensitivity, mainlycaused by the limited sample injection volume. A solution tothis problem requires the concentration of the PL fractions tobe improved or the use of some on-line preconcentrationtechniques [45, 46].

4 Concluding remarks

The presented results clearly demonstrated the potential ofhyphenated NACE-ESI-MS method for the separation andcharacterization of phospholipids in biological samples. The

Figure 6. NACE-ESI-MS electro-pherogram of the phospholipidsextracted from the peritonealsurface of rats. Experimentalconditions as in Fig. 4.



Table 3. Identification of the main molecular species of phos-pholipids in a rat peritoneal surfacea)

Phospholipid class Peak (m/z) Molecular speciesb)

PtdCho 764.6 14:0/16:0792.6 16:0/16:0818.6 16:0/18:1816.6 16:0/18:2846.7 16:0/20:1; 18:0/18:1844.6 18:0/18:2840.6 16:0/20:4; 18:2/18:2868.6 16:0/22:4; 18:0/20:4

SM 761.6 16:0789.7 18:0

PE 714.5 16:0/18:2742.5 16:0/20:2; 18:0/18:2738.5 16:0/20:4766.5 16:0/22:4, 18:0/20:4762.5 16:0/22:6722.5 p16:0/20:4750.6 p16:0/22:4; p18:0/20:4774.5 p18:0/22:6

PtdIns 861.6 16:0/20:2; 18:0/18:2857.5 16:0/20:4; 18:2/18:2883.6 16:0/22:5; 18:1/20:4887.6 18:0/20:3885.5 18:0/20:4909.6 18:0/22:6

a) p (Plasmenyl) indicates plasmalogen molecular species.b) The expressions of molecular species of phospholipids are

designated two fatty acyl residues attached to the sn-1 and sn-2 positions of glycerol backbone, where the carbon number ofthe fatty acid is indicated first, followed after a colon by thenumber of double bonds.

organic solvent composition and electrolyte nature played animportant role in the separation of phospholipids due totheir integrated effects of viscosity, relative permittivity, sol-vation interactions and acid-base properties. The bestseparation was achieved with MeOH/ACN (40:60 v/v) con-taining 20 mM ammonium acetate and 0.5% HOAc. Inaddition, because of its low surface tension and highvolatility, the nonaqueous medium was suitably coupled on-line with ESI-MS. Compared with CE-UV method, thedeveloped NACE-ESI-MS method demonstrated higher sen-sitivity and additional information on molecular structure.Finally, this method was successfully applied to the analysisof main phospholipids extracted from the rat peritoneal sur-face. We believe that NACE-ESI-MS is an alternative toHPLC-ESI-MS for the analysis of phospholipids in differentbiological samples.

The authors are grateful to Dr. X. Tian and the First Hospitalof Peking University for the assistance in animal experiments.This work was financially supported by NSFC (Grant No.20335010 and 20675004).

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Analysis of phospholipids by NACE with on-line ESI-MS

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