Analytica Chimica Acta 525 (2004) 1–10

Structural identification of human blood phospholipids using liquidchromatography/quadrupole-linear ion trap mass spectrometry

Chang Wanga, Sigang Xieb, Jun Yanga, Qing Yanga, Guowang Xua,∗a National Chromatographic R&A Center, Dalian Institute of Chemical Physics,

The Chinese Academy of Sciences, Dalian 116011, PR Chinab Dalian Center Hospital, Dalian 116033, PR China

Received 6 June 2004; received in revised form 23 July 2004; accepted 23 July 2004Available online 3 September 2004

Abstract

A normal-phase liquid chromatography/quadrupole-linear ion trap mass spectrometry method was developed for the separation and massspectral characterization of the main phospholipid species in human blood. The instrument combines the capabilities of a triple quadrupolem ol as mobilep cular masso polar headg pectra andm e (lysoPC)a©

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ass spectrometer and ion trap technology on a single platform. The optimal separation was achieved by using hexane/1-propanhase and 0.6% formic acid, 0.06% ammonia as modifiers. The HPLC/MS technique was able to provide information about the molef individual homologues by positive and negative turbo ionspray. More complete characterization of fatty acid chains and of theroup was obtained by using a quadrupole collisionally activated dissociation (CAD) spectrum with ion trap sensitivity. The mass solecular species of phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholinnd sphingomyelin (SM) are presented.2004 Elsevier B.V. All rights reserved.

eywords:Phospholipids; Linear ion trap; Ionspray; Liquid chromatography/mass spectrometry

. Introduction

Phospholipids are an important constituent in the biomem-ranes. Both the physical and chemical properties of theembrane bilayer can be affected by the variation of phos-holipid compositions. Membrane phospholipids are a com-lex mixture of molecular species containing a variety of

atty acyl and head group compositions. In addition to theirtructural role, some phospholipids also participate in bi-logical processes in various ways. Other phospholipids,uch as polyphosphoinositides, are important in cellularignalling systems[1,2]. Phospholipids serve as a reser-oir for arachidonic acid (20:4n-6) and other polyunsat-rated fatty acids that can be metabolized to biologicallyctive eicosanoids such as prostaglandins, thromboxanes,

∗ Corresponding author. Tel.: +86 411 83693413; fax: +86 411 3693403.E-mail address:dicp402@mail.dlptt.ln.cn (G. Xu).

leukotrienes and lipoxins[3,4]. Phospholipids have begiven increased attention in many fields, for examplebiomarkers in chemotaxonomical studies and in the maof liposomes for drug delivery or cosmetics/detergents.commercial use of phospholipids is increasing in fields sas biomembranes, skin-care formulations and drug deli

Analysis of these phospholipids has been carried outchromatographic techniques such as thin-layer chromatphy (TLC) [5,6], high-performance liquid chromatograp(HPLC) [7–13]. Detection of phospholipids has been pformed by different spectrophotometric techniques sucUV. However, with this technique, serious constraintsimposed on the mobile phase selection since underivaphospholipids absorb near 200 nm with a low extinccoefficient [9,14,15]. A novel derivatization approach wproposed to increase the UV sensitivity of phospholipid aysis[15] using naproxen chloride. But this approach is laintensive and is not suitable for routine and high throug

003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2004.07.065

2 C. Wang et al. / Analytica Chimica Acta 525 (2004) 1–10

analysis. Alternatively, phospholipids can be analyzed byHPLC with evaporative light-scattering detection (ELSD)[16–18]. Although this technique is compatible with gradientelution and permits quantitation of phospholipids, the poorselectivity of this detector implies that the identification of thedifferent phospholipids must be made by retention behaviorin comparison to known standards.

Mass spectrometry (MS) offers an attractive alternativefor the analysis of phospholipid composition because of itshigh sensitivity, specificity, and (apparent) simplicity. How-ever, MS has rarely been used for this purpose until veryrecently. The main reason for this is that the ionization meth-ods available (fast-atom bombardment, etc.) cause extensivefragmentation of the lipid molecules, this along with the ex-treme complexity (hundreds of different molecular species)of most biological samples, has precluded compositionalanalysis. The introduction of “soft” ionization methods hascompletely opened new vista in this field. In particular, elec-trospray ionization-mass spectrometry (ESI-MS) has beenshown to be a very promising technique[19]. However, it isimportant to have a chromatographic system separate the dif-ferent phospholipid classes to avoid possible mass overlap.Thus, in the analysis of lipids taken from a complex biologicalmatrix, there is a need for class separation by LC followed byspecies identification by mass spectrometry[20]. HPLC/MSi

phicm lood.P so-c asss ulars

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Table 1Linear gradient composition

Time (min) A% B%

0 68 3220 20 8033 20 8038 68 3260 68 32

tracted essentially as described earlier[25]. Prior to analy-sis, the extracted samples were redissolved in 500�l chloro-form/methanol (2:1, v/v) and then was diluted by hexane/1-propanol (3:2, v/v).

2.3. High performance liquid chromatography

An HP 1100 series HPLC system (Agilent Technologies,Palo Alto, CA, USA) was used. The LC separation was per-formed on a diol column (Nucleosil, 100-5 OH, Germany)(250 mm× 3.9 mm, i.d.× 5.0�m, particle size). The totalflow rate was 0.7 ml min−1. The flow from the LC was splitusing a Micro-Splitter Valve such that the flow to the electro-spray was approximately 0.28 ml min−1. The column tem-perature was at 35◦C. The linear solvent gradient is shownin Table 1. Solvent mixture A: hexane/1-propanol/formicacid/ammonia (79/20/0.6/0.07, v/v); solvent mixture B: 1-propanol/water/formic acid/ammonia (88/10/0.6/0.07, v/v).

2.4. Mass spectrometry

The mass spectrometric detection was performed on aQTRAP LC/MS/MS system from Applied Biosystems/MDSSciex (USA) equipped with a turbo ionspray source. Thisinstrument is based on a triple-quadrupole ion path usingt eter.T ion-a witht asss leq rod-u rapidi oods thec MS”( mu-l tureo ducti thirdq

teelE ) or4 s sett op-t r re-l yingg t was

s a very useful tool for lipid analysis[21–24].In this work, we describe an improved chromatogra

ethod for class separation of phospholipids in human broduct ion spectra following collisionally activated disiation (CAD) of quasi-molecular ions in a tandem mpectrometry was obtained to identify individual molecpecies of each phospholipid class.

. Experimental

.1. Chemicals

1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,almitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine,leoyl-2-hydroxy-sn-glycero-3-phosphocholine and sphomyelin (SM) from chiken egg were from Avanti Poipids (Alabaster, AL, USA).l-�-Phosphatidyl-l-serine, dialmitoyl sodium salt was from Sigma (St. Louis, MO, US,6-Di-tert-butyl-4-methylphenol was from Aldrich-ChemSteinheim, Germany). Formic acid and all the solvents wPLC grade (TEDIA, USA), ammonia (25%) is analytirade from Lian-Bang (Shenyang, China).

.2. Sample preparation

The phospholipid standards were dissolved (appately 1 mg/ml) in chloroform/methanol (2:1, v/v), and f

her diluted with hexane/1-propanol (3:2, v/v).Heparinised human whole blood was pooled from

nteers. The lipids in the 500�l blood samples were e

he final quadrupole as a linear ion trap mass spectromhus, the QTRAP instrument combines all of the functlity of a classical triple quadrupole mass spectrometer

he capabilities of a very high sensitivity linear ion trap mpectrometer[26]. The combination of highly selective tripuadrupole MS/MS scans and high sensitivity ion trap pct scans on the same instrument platform provided

dentification of phospholipids of extracted human blample. The survey scan of phospholipids eluted fromhromatographic column is performed in the “enhancedEMS), single quadrupole mode where ions were accuated then filtered in the Q3-linear ion-trap. The strucf phospholipid are elucidated by the “enhanced” pro

on scan mode (EPI) where ions were trapped in theuadrupole before filtration.

The split HPLC effluent entered the MS through a sS ionization needle set at 5500 V (in positive ion mode500 V (in negative ion mode) and a heated capillary wa

o 250◦C. The ion source and ion optic parameters wereimized with respect to the positive or negative moleculaated ions of the phospholipid standards. The nitrogen dras and turbo gas were both at 40 psi, the curtain gas tha

C. Wang et al. / Analytica Chimica Acta 525 (2004) 1–10 3

to prevent the contamination of the ion optics was set at 30 psi.The declustering potential (DP) was set at 80 psi. The otherparameters as follows: EMS as survey scan (mass rangem/z450–950, scan speed 1000 Da s−1, trap time 20 ms) and EPIas dependent scan (scan speed 1000 Da s−1, trap time 150 ms,collision energy set at +35 eV in the positive-ion mode andfrom −50 to−35 eV in the negative-ion mode).

3. Results and discussion

3.1. Separation of phospholipid classes

Initially, the modifiers in mobile phase were 0.6% (v/v)formic acid and 0.06% (v/v) triethylamine (TEA). This didnot, however, give an optimal separation of the different phos-pholipid classes, because PC, PS and PI elute very closelyand make class determination more complicated. Further-more, there are so many molecular species in each class ofphospholipids, it is necessary to increase separation resolu-tion as much as possible. Most importantly, TEA has beenreported to be strongly absorbed on the surfaces of the vac-uum manifold and parts therein[27]. This absorption maysuppress the ionization of less basic compounds in positivemode for low molecular mass substances due to the TEA sig-n ntom vedr ni thep tiono ationmo theb em-p dp oro-

pholipi

form as mobile phase can produce danger to public health. Incontrast, hexane or 1-propanol is less toxic.

The addition of 0.6% (v/v) formic acid and 0.07% (v/v)ammonia in mobile phase improved separation of the mainphospholipid classes. Under this condition, PE was firstlyeluted, followed by PS(PI), PC, SM and lysoPC in a suc-cessive manner.Fig. 1 shows the total-ion current (TIC) ofphospholipids in human blood in the negative-ion HPLC-ESI/MS. Because different molecular species within a classhave a same polar head, their retention time in HPLC is verysimilar. The retention time difference of compounds withinone same class is less than that between two different classes.

3.2. Species characterization of the phospholipid classes

The molecular mass peaks from the different phospholipidclasses were detected using positive or negative ion full-scanESI-MS analysis. Many classes of phospholipids possess netnegative charge at neutral pH. Accordingly, negative-ion ESI-mass spectra of these phospholipids can be effectively ob-tained with [M − H]− as the molecular ion peak (such asPS, PI). However, PC, PE, lysoPC and SM are zwitterionicmolecules, and therefore either positive or negative-ion massspectra of these phospholipid classes are accessible throughESI-MS. In this work, the PE, PS, PC, lysoPC and PI speciesw en-t

3

thee nsi os-p de,s ctive

al atm/z102. However, the addition of 0.06% ammonia iobile phase as modifier instead of TEA highly impro

esolution. It was reported[28] that inclusion of ammonia infusion solvent eliminated sodium adduct formation inositive ion mode, thus greatly simplifying the interpretaf the spectra. Comparing this chromatographic separethodology to earlier solvent/modifier systems[20], notnly the resolution has been highly improved, but alsoad effect on the column life due to the higher column terature (55◦C) has been avoided. Uran et al.[25] separatehospholipids using chloroform and methanol. But chl

Fig. 1. Total ion chromatogram of LC/ESI-MS analysis of phos

ds of extracted human blood sample. Conditions are given inSection 2.

ere mainly analyzed in negative-ion mode, SM were idified in positive-ion mode.

.3. Phosphatidylethanolamine (PE) species

Negative-ion HPLC-ESI/MS analysis of PE species inxtracted human blood is shown inFig. 2a. In the negative-iopectra, molecular species of PE mainly give the [M − H]−

ons. The fatty acid composition of negative ions of phholipids can be determined by MS/MS in the EPI moince formation of fatty acid anions represents an effe

4 C. Wang et al. / Analytica Chimica Acta 525 (2004) 1–10

Fig. 2. (a) Negative-ion EMS of PE species from human blood sample. (b) EPI spectrum ofm/z738.7 as shown in (a) representing the [M − H]− ion ofdiacyl-PE. (c) EPI spectrum ofm/z750.8 as shown in (a) representing the [M − H]− ion of pPE.

fragmentation pathway of negatively charged ions of phos-pholipids containing ester-bound fatty acids[29]. The identi-fication of diacyl and plasmalogen species was based on themolecular ion andsn-1 andsn-2 carboxylate anions observed.For diacyl species, both carboxylate anions corresponding tosn-1 andsn-2 substituents were present in the negative ionmode. For plasmalogen species, only one carboxylate an-ion corresponding tosn-2 acyl substituents was observed,because thesn-1 group did not cleave to form an anion[35].

As an example,Fig. 2b and c show the negative EPI spectraof deprotonated diacyl and plasmalogen PE species, respec-tively. The fragment ions detected atm/z255, 279 and 303corresponded to C16:0, C18:2, and C20:4 fatty acid residues(carboxylate anion fragments) of [M − H]−, respectively(Fig. 2b). The fragment ions atm/z452 andm/z476 cor-respond to the C16:0 lysoPE and C18:2 lysoPE ions (PE haslost one of the two fatty acid residues), respectively. In ad-dition, the C20:4 lysoPE fragment ion is in low abundance.The position of the acyl chains in the glycerol backbone ofthe phospholipid molecule is obviously important for their de-gree of dissociation. This phenomenon has previously beenreported by several publications[29–33]. There has been dis-

crepancy on which carboxylate anion yields the most intensepeak in the product ion spectrum in these reports. One re-port stated that the intensity of the fatty acid fragment in thesn-2 position was approximately twice that of the fatty acidin thesn-1 position[29] and another report stated that therewas no preferential loss of the fatty acid moiety from eithersn-1 or sn-2 position[30]. Hvattum et al.[33] reported thatthe abundance ratio of the carboxylate anions relates to manyfactors, such as collision energy, the phospholipid class, andthe fatty acids attached to thesn-2 position. In this paperwe adopt that the phospholipids isolated from animals mostoften contain a saturated fatty acid insn-1 position and anunsaturated fatty acid insn-2 position[34]. Them/z738.7 istherefore identified as C16:0/C20:4 or C18:2/C18:2 diacyl PEspecies.

In the negative-ion EPI spectrum of deprotonated plas-malogen PE atm/z750.8 (Fig. 2c), the presence of a rela-tively more intense lysoplasmenylethanolaminelike ion peak(m/z464, C18:0 lysopPE;m/z436, C16:0 lysopPE) relative toits phosphatidylethanolamine counterpart from the examinedplasmenylethanolamine molecular species that contain C20:4and C22:4 acyl chains atsn-2 position indicates the presenceof pC18:0/C20:4 or pC16:0/C22:4 plasmalogen PE species.

C. Wang et al. / Analytica Chimica Acta 525 (2004) 1–10 5

Table 2Molecular species identification of phosphatidylethanolamines (PE)

PL class Negative ion [M − H]− (m/z) Combinations of molecular species

Diacyl-PE and pPE 714.7 C16:0/C18:2716.8 C16:0/C18:1720.7 pC16:0/C20:5722.7 pC16:0/C20:4726.7 pC18:0/C18:2 pC18:1/C18:1728.7 pC18:0/C18:1736.7 C18:2/C18:3 C16:1/C20:4

C16:0/C20:5738.8 C16:0/C20:4 C18:2/C18:2740.7 C16:0/C20:3 C18:1/C18:2742.8 C16:0/C20:2 C18:1/C18:1

C18:0/C18:2744.7 C18:0/C18:1 C16:0/C20:1750.7 pC18:0/C20:4 pC16:0/C22:4

C16:1/C22:6 C18:1/C20:6760.6 C18:2/C20:5

C18:3/C20:4762.7 C16:0/C22:6 C18:2/C20:4764.7 C16:0/C22:5 C18:1/C20:4766.7 C18:0/C20:4 C18:2/C20:2

C16:0/C22:4778.8 pC18:0/C22:4 pC20:0/C20:4788.8 C18:1/C22:6 C18:2/C22:5

C18:0/C22:6 C18:1/C22:5790.7 C18:2/C22:4

C20:2/C20:4

This product ion ratio likely results from the relatively higherstability and formation rate of thesn-1 vinyl ether-likedproduct ion from plasmenylethnolamine anion in compari-son to those that result from the loss of thesn-1 carboxylicacid (or derivative) from deprotonated diacy PE species[29].Using the LC-ESI/MS technique, more than 30 PE specieswere identified from extracted human blood sample, and arelisted inTable 2.

Under the positive-ion mode, the EMS spectra of PE give[M + H]+, [M + Na]+, and [M + H-141]+ ions (the figurenot shown). However, in the EPI spectrum of [M + H]+ ions,except the abundant [M + H-141]+ ions, the other fragmentions (such as [lyso-PE + H]+ ion) is in a low abundance (thefigure not shown). As a result, the identification of PE specieswas mainly performed in negative ion mode in spite of thehigher sensitivity in positive-ion mode.

3.4. Phosphatidylinositol (PI) and phosphatidylserine(PS) molecular species

As shown inFig. 3a, the negative-ion HPLC-ESI/MS anal-ysis gives the main [M− H]− ions for either PS or PI species.The molecular species of PS and PI are discriminated accord-ing to nitrogen rule. In the negative ion mode, PS (with onenitrogen atom) shows signals at even-numberedm/zvalues,w odd-n

ainedb sly

[35]. The EPI spectrum of the [M − H]− ion of PI atm/z885.8 is shown inFig. 3b. The carboxylate anion fragmentions atm/z283.4 (C18:0), 303.4 (C20:4) are detected. ThelysoPI ions atm/z581.4 (C18:0) and atm/z601.5 (C20:4)were probably in the cyclic form, those atm/z599.5 (C18:0)andm/z619.3 (C20:4) fragment ions may be probably in theopen form. In addition, two specific PI related negative ions atm/z259.4 (inositol phosphate) and 241.2 [inositol phosphate– H2O]− are produced. Therefore,m/z885.8 was detectedas C18:0/C20:4 PI. The PI species identified in human bloodsample are listed in.

Similarly to PE and PI classes, the negative-ion EPI spec-tra of [M − H]− of PS provides the information on the fattyacid substituents. As shown inFig. 3c, C18:0 (m/z283.4)and C22:6 (m/z327.3) as the carboxylate anions confirmedthe fatty acyl substituents atsn-1 andsn-2 positions. More-over, fragment ions atm/z437.3, 481.2 correspond to C18:0and C22:6 lysophosphate in the open form, two abundantions atm/z419.5, 463.4 corresponding to C18:0 and C22:6lysophosphate in the cycle form are also observed. The diag-nostic ion [M− 87− H]− of PS species is obviously detectedatm/z747.6. Then, them/z834.8 is identified as C18:0/C22:6PS.Table 3shows the PS species in the extracted humanblood sample. Like PE, the positive-ion EMS spectra of PSprovided [M + H]+, [M + Na]+, and [M + H-185]+ ions( +i ntifyfi

hereas PI (without nitrogen atom) shows signals atumbered values.

The negative ion EPI mass spectra of PI species obty ESI-MS/MS were similar to those reported previou

the figure not shown). And the EPI spectra of [M + H]ons showed no abundant ions that can be used to ideatty acid substituents but a single distinct [M + H-185]+ons.

6 C. Wang et al. / Analytica Chimica Acta 525 (2004) 1–10

Fig. 3. (a) EMS scan negative-ion spectrum of PI and PS species in human blood sample. (b) EPI ofm/z885.8 as shown in (a) representing the [M − H]− ionof PI. (c) EPI spectrum ofm/z834.8 as shown in (a) representing the [M − H]− ion of PS.

3.5. Phosphatidylcholine (PC) andlysophosphatidylcholine (lysoPC) molecular species

EMS spectra of PC species in human blood sample bothin positive and negative ion LC-ESI/MS are shown inFig. 4aand b. The positive-ion spectrum gave the main [M + H]+and [M + Na]+ ions of intact PC, whereas the negative-ionspectrum predominantly gave the [M − 15]− and [M + 45]−ions. In positive-ion ESI/MS mode, EPI spectrum of [M +H]+ atm/z760.7 (Fig. 4c) showed intense fragment ion atm/z 184.2 which readily gave the head group informationof PC class. The fragment ion atm/z577.6 resulting fromloss of polar head was also observed. The deacylated ions atm/z522.3 and 496.5 corresponding to the loss of C16:0 andC18:1 acyl chain, respectively were present in lower abun-

dance. In contrast, the EPI spectrum in negative [M − 15]−at m/z 744.8 (representing demethylated PC) provided in-tense abundant fatty acid fragments atm/z255.4 (C16:0) and281.4 (C18:1)(Fig. 4d). Additionally, C16:0 lysoPC atm/z480.5 and C18:1 lysoPC atm/z506.3 ions also give the sim-ilar structural information of PC species. Accordingly, them/z744.8 is detected as C16:0/C18:1 PC. The ions clustersatm/z802.8, 830.7 and 854.8 are the HCOO− adduct ionsof PC. These ions can also be used to identify PC molecularspecies in the negative-ion mode[23] (the figure not shown).Although PC species in the positive-ion mode showed moresensitivity than in the negative-ion mode, however, fatty acidanalysis of PC species required negative-ion EPI scan mode,since CAD of [M + H]+ ions of PC results in the formationof a single intense fragment ion atm/z184 representing the

C. Wang et al. / Analytica Chimica Acta 525 (2004) 1–10 7

[H2O3PO–CH2–N(CH3)3]+ ion. The detected PC species arelisted inTable 4.

The lysoPC species detected of human blood samplein negative-ion ESI/MS is shown inFig. 5a. BecauselysoPC corresponding to PC has lost one of the two fattyacid residues, the lysoPC species have less simple struc-

F[

ture than that of PC species. In EPI spectrum of [M− CH3]− ion at m/z 508.5(Fig. 5b), the carboxylate an-ions at m/z 283.5 indicated the C18:0 fatty acid com-position. And them/z 508.5 is demonstrated as C18:0lysoPC.Table 4provides the lysoPC species in human bloodsample.

ig. 4. EMS spectrum of PC species in human blood sample. (a) Positive-ionM + H]+ ion of PC. (d) EPI spectrum ofm/z744.8 as shown in (a) representing

. (b) Negative-ion. (c) EPI spectrum ofm/z760.7 as shown in (a) representing thethe [M − 15]− ion of PC.

8 C. Wang et al. / Analytica Chimica Acta 525 (2004) 1–10

Table 3Molecular species identification phosphatidylinositols (PI) and phos-phatidylserines (PS) in human blood sample

PL Class Negative ion[M − H]− (m/z)

Combinations ofmolecular species

PI 833.6 C16:0/C18:2857.8 C16:0/C20:4 C18:0/C18:4859.7 C18:0/C18:3 C18:1/C18:2861.8 C18:0/C18:2 C18:1/C18:1863.7 C18:0/C18:1883.7 C18:1/C20:4885.8 C18:0/C20:4887.7 C18:0/C20:3909.7 C18:0/C22:6911.8 C18:0/C22:5913.7 C18:0/C22:4

PS 734.8 C16:0/C16:0748.7 C15:0/C18:0760.8 C16:0/C18:1762.8 C16:0/C18:0782.8 C16:0/C20:4786.7 C18:0/C18:2 C18:1/C18:1788.8 C18:0/C18:1790.8 C18:0/C18:0808.7 C18:1/C20:4 C18:0/C20:5810.7 C18:0/C20:4832.7 C18:0/C22:7 C18:3/C22:4834.8 C18:0/C22:6836.8 C18:0/C22:5838.8 C18:0/C22:4

3.6. Sphingomyelin (SM) species

SM consists of phosphorycholine ester-bound to a ce-ramide (Fig. 6c). As seen fromFig. 6a, the SM species inhuman blood gave the main peaks of [M+ H]+ and [M+ Na]+ions. Spectrum of the EPI scan of [M + H]+ ion atm/z731.7

Table 4Identification of phosphatidylcholine (PC) and lysophosphatidylcholines (lysoPC) molecular species in human blood sample

PL Class Negative ion [M − 15]− (m/z) Combinations of molecular species

PC 716.7 C16:0/C16:1718.7 C16:0/C16:0742.8 C16:0/C18:2744.8 C16:0/C18:1746.8 C16:0/C18:0766.8 C16:0/C20:4 C18:2/C18:2768.8 C16:0/C20:3 C18:1/C18:2770.8 C18:0/C18:2790.8 C16:0/C22:6 C18:1/C20:5

C18:2/C20:4794.8 C18:0/C20:4 :3

C18:2/C20:2796.7 C18:0/C20:3 :2

C16:0/C22:3

lysoPC 478.5 C16:1480.5 C16:0494.5 C17:0504.5 C18:2

Fig. 5. (a) Negative-ion EMS spectrum of lysoPC species in human bloodsample. (b) EPI spectrum ofm/z508.5 as shown in (a) representing the [M− 15]− ion of lysoPC.

in the positive ion mode (Fig. 6a) only provided abundant184 fragment ion representing the choline ion(the figure notshown). In contrast, selection and collisional activation of [M+ Na]+ atm/z753.7 yields several informative product ions(Fig. 6b). The most abundant product ion ism/z694.7, whichcorresponds to the loss of trimethylamine([M + Na-59]+).Other product ions atm/z570.5, 548.5 and 530.5 correspondto three ceramidelike cations (i.e., [M + Na-183]+, [M + Na-205]+, and [M + Na-205–H2O], respectively). The production atm/z147 corresponds to the sodiated five-member cy-clophosphane. The ion atm/z264.4 is the LCB ion with 18carbon atoms [CH3(CH2)12CH CH–CH C(NH2)–CH2]+.In addition to LCB ions, the ion atm/z308.5 seems to be due

506.5 C18:1508.5 C18:0528.5 C20:4

C18:1/C20

C18:1/C20

C. Wang et al. / Analytica Chimica Acta 525 (2004) 1–10 9

Fig. 6. (a) Positive-ion EMS spectrum of SM species in human blood sample. (b) EPI spectrum ofm/z753.7 as shown in (a) representing the [M + Na]+ion ofSM. (c) Composition of SM.

to C18:0 [(CH2CH N)COC17H35]+ which reflect the fattyacid composition. Therefore, the [M + Na]+ ion atm/z753.7is composed of dC18:1/C18:0 (the designation of SM is inthe form of d-LCB/FA, with d referring to the stereochem-istry of the long chain base (LCB) and FA referring to fattyacid, shown inFig. 6c). However, the abundance of LCB ionand FA ion in the positive-ion EPI spectrum is relatively low,sometimes they can’t be detected.Table 5lists the identifiedSM species in human blood.

From the characterization of phospholipids describedabove, it could be known that in the negative ion mode, CADusing QTRAP mass spectrometry gave primarily thesn-1andsn-2 carboxylate anions together with lyso-phosphatidicacid with neutral loss water, and these structurally informa-tive fragment ions are almost same as those obtained witha triple quadrupole mass spectrometer[33,36]. However,collision-induced dissociation (CID) using an ion-tap massspectrometer[25], the product ions were mainlysn-1 andsn-2 lyso-phospholipids with neutral loss of ketene in com-bination with neutral loss of the polar head group. The differ-ence may be from the different fragmentation mechanismsbetween triple quadrupole mass spectrometer and ion trapmass spectrometer[37]. The QTRAP instrument is based on

a triple-quadrupole ion path using the final quadrupole as alinear ion trap mass spectrometer. According the fragmentions obtained with our method, the fragmentation mecha-nism in the EPI scan mode under QTRAP mass spectrometeris more similar to triple quadrupole mass spectrometer.

Table 5Molecular species identification of sphingomyelins (SM) in human bloodsample

PL Class Positive ion[M + Na]+ (m/z)

Combinations ofmolecular species

SM 711.7 33:1723.7 34:2725.7 d18:1/C16:0739.7 35:1741.7 35:0751.7 dC18:2/C18:0753.7 dC18:1/C18:0755.7 36:0779.7 38:2783.7 38:0823.6 dC18:1/C23:0853.7 43:0

10 C. Wang et al. / Analytica Chimica Acta 525 (2004) 1–10

4. Conclusions

The normal-phase HPLC-ESI/MS method described isproved to be valuable for the optimal separation and iden-tification of different phospholipid species in human blood.In the present study, we chose hexane/1-propanol as mobilephase instead of chloroform/methanol. Furthermore, the useof ammonia as a mobile phase modifier in place of TEA notonly improved the resolution but also avoided the bad effectof TEA on the ionization of basic compounds in the positivemode. The combination of highly selective triple quadrupoleMS/MS scans and high sensitivity ion trap product scans onthe QTRAP instrument provides rapid identification of phos-pholipids of extracted human blood sample. Using the presentmethod, information regarding the molecular mass, the polarhead group and the fatty acyl substituents can be obtainedfrom the positive or negative ion EMS and EPI scan mode.Using the present HPLC/MS system, more than 100 phos-pholipid species (including isomers) were identified. Amongthem, C18:1/C20:6 PE and C18:0/C22:7 PS have not beenreported to be present in blood, they may be new finding inthe lipid field The fragmentation patterns of phospholipidsobtained under positive-ion or negative-ion ionization condi-tions are helpful in characterizing different classes of phos-pholipid species in complex samples. We believe that thism yingp

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ethod will be valuable in lipid research such as in studhospholipid profiles in different biological samples.

cknowledgements

This study has been supported by the high-technoevelopment plan “863 project” (2003AA223061) of Sinistry of Science and Technology of China and the Knodge Innovation Program of the Chinese Academy ofnces (K2003A16).

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