Identification of phospholipid structures in human blood by direct-injection quadrupole-linear ion-trap mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2080

Identification of phospholipid structures in human

blood by direct-injection quadrupole-linear ion-trap

mass spectrometry

Chang Wang, Jun Yang, Peng Gao, Xin Lu and Guowang Xu*National Chromatographic R&A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116023, P.R. China

Received 23 March 2005; Revised 28 June 2005; Accepted 7 July 2005

Direct-injection electrospray ionization mass spectrometry in combination with information-

dependent data acquisition (IDA), using a triple-quadrupole/linear ion trap combination, allows

high-throughput qualitative analysis of complex phospholipid species from child whole blood.

In the IDA experiments, scans to detect specific head groups (precursor ion or neutral loss scans)

were used as survey scans to detect phospholipid classes. An enhanced resolution scan was then

used to confirm the mass assignments, and the enhanced product ion scan was implemented as a

dependent scan to determine the composition of each phospholipid class. These survey and depen-

dent scans were performed sequentially and repeated for the entire duration of analysis, thus pro-

viding the maximum information from a single injection. In this way, 50 different phospholipids

belonging to the phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphati-

dylcholine and sphingomyelin classes were identified in child whole blood. Copyright# 2005 John

Wiley & Sons, Ltd.

Membrane phospholipids are a complex mixture of molecu-

lar species containing a variety of fatty acyl and head group

moieties. The head group is the functional group that defines

the specific class to which the phospholipid belongs, i.e.,

phosphatidylethanolamine (PE), phosphatidylserine (PS),

phosphatidylinositol (PI), phosphatidylcholine (PC) and

sphingomyelin (SM) classes, while the fatty acyl groups

distinguish the individual phospholipid molecular species

within each class (Scheme 1). In addition to being critical com-

ponents of cellular membranes, phospholipids interact with

all membrane proteins and many non-membrane proteins,

and mediate signal transduction.1 Phospholipids serve as a

reservoir for arachidonic acid (20:4, n–6) and other polyun-

saturated fatty acids that can be metabolized to biologically

active eicosanoids such as prostaglandins, thromboxanes,

leukotrienes and lipoxins.2,3 Due to their structural and func-

tional roles in mammalian cells, the understanding of the

composition, metabolism, and regulation of phospholipids

at the level of molecular species has become increasingly

important.

Analysis of these phospholipids has been performed using

chromatographic techniques such as thin-layer chromato-

graphy (TLC),4,5 and high-performance liquid chromatogra-

phy (HPLC).6–10 However, the identification of the different

phospholipids in most of these approaches is based only on

retention behavior in comparison to known standards.

Several fast and convenient phospholipid analytical methods

have been developed using thermospray or electrospray

mass spectrometry on-line with HPLC (LC/MS),11–15 and

have been employed for the phospholipid analysis of various

biological mixtures. Separation by LC before MS analysis can

clean up samples and distinguish different phospholipid

classes; however, the whole process usually requires more

than 30 min per sample. Short analysis times are critical for

high-throughput analysis.

For the combined purpose of analytical speed and analysis

of individual molecular species of the phospholipid classes,

MS-based methods hold the most promise. Brugger et al.16

took advantage of the fact that a common fragment is formed

from the head-group region of each member of a class of

complex polar lipids upon collisionally activated dissociation

(CAD), using electrospray ionization tandem mass spectro-

metry (ESI-MS/MS). When the head group is lost as a

charged fragment, precursor (Pre) ion scanning is used, and

when the head group is lost as a neutral fragment, neutral loss

(NL) scanning provides the specific detection.38 Speed and

selectivity are the main advantages of this method. Such

selective detection is particularly useful when negatively

charged lipids are being analyzed, since the negative ion

spectrum of lipid extracts is typically highly crowded due to

extensive overlap of species belonging to different head

Copyright # 2005 John Wiley & Sons, Ltd.

*Correspondence to: G. Xu, National Chromatographic R&ACenter, Dalian Institute of Chemical Physics, The ChineseAcademy of Sciences, Dalian 116023, P.R. China.E-mail: [email protected]/grant sponsor: State Ministry of Science and Technologyof China; contract/grant number: High-Tech R & D Plan2003AA223061.Contract/grant sponsor: Foundation for Distinguished YoungScholars from National Natural Science Foundation of China;contract/grant number: 20425516.Contract/grant sponsor: Knowledge Innovation Program of theChinese Academy of Sciences; contract/grant number:K2002A12, K2003A16.

group classes.17 This approach has been used for analysis of

crude lipid extracts;16,18–20 however, the major drawback of

this approach is that the molecular species at the level

of individual fatty acid residues can not be identified.17

Information-dependent data acquisition (IDA) proce-

dures22 are optimized to generate large amounts of useful

MS/MS data, e.g., through the combination of a specific

survey scan, a high-resolution scan and a product ion scan. In

a typical IDA experiment an MS1 survey scan is performed to

generate a peak list (m/z values) of all ions present. The peak

list is subjected to a set of user-defined criteria to filter out

unwanted precursor ions, and the remaining ions are then

subjected to MS/MS analysis. This cycle is repeated through

the duration of the acquisition to generate large amounts of

informative data. In general, an IDA procedure combines two

or more different scan modes in a sequential way within the

same analytical run. In the case of phospholipid analysis, this

approach enables a head-group-specific scan and a product

ion scan to be combined.

In this work we investigated the possibility of analyzing

phospholipid species in human whole blood by combining

direct-injection electrospray ionization mass spectrometry

(DI-ESI-MS) and IDA. The total analysis time was only

2.0 min. Product ion spectra were obtained using CAD of

intact ionized molecular species in a tandem mass spectro-

meter to identify individual molecular species of each

phospholipid class.

EXPERIMENTAL

ChemicalsPhospholipid standards were from Avanti Polar Lipids

(Alabaster, AL, USA) or Sigma (St. Louis, MO, USA). 2,6-

Di-tert-butyl-4-methylphenol was from Aldrich-Chemie

(Steinheim, Germany). Formic acid and all the solvents

were HPLC grade from TEDIA (Fairfield, USA), and ammo-

nia (25%) was analytical grade from Lian-Bang (Shenyang,

China).

Sample preparationHeparinized whole child blood was from Dalian Children’s

Hospital. The lipids in 500mL of blood samples were

extracted essentially as described earlier.21 Briefly, 0.28 mL

of water was added to 500 mL of the blood sample; then

2 mL of methanol with 0.01% (w/v) 2,6-di-tert-butyl-4-

methylphenol and 4 mL of chloroform were added, and the

solution was sonicated for 60 s both before and after adding

the chloroform. The solution was then vortex-mixed for 30 s

and incubated for approximately 1 h at room temperature.

Finally, 2 mL of water were added, the solution was mixed

again, and centrifuged at 2600 g for 10 min. The lower chloro-

form phase was sampled and dried under vacuum. Prior to

analysis, the extracted samples were redissolved in 500 mL

of chloroform/methanol (2:1, v/v) and then diluted 10 times

with methanol.

Direct-injection ESI-MSDI-ESI-MS was performed using a QTRAP LC/MS/MS sys-

tem from Applied Biosystems/MDS Sciex (Concord, ON,

Canada) with a turbo-ionspray source (operated without

hot turbo-gas). Spectra were acquired in the positive or nega-

tive ion mode. This instrument is based on a triple-quadru-

pole ion path in which the final quadrupole can be used as

a linear ion trap mass spectrometer. Thus the QTRAP instru-

ment combines all of the functionality of a classic triple-quad-

rupole mass spectrometer with the capabilities of a very high

sensitivity linear ion trap.22 The combination of highly selec-

tive triple-quadrupole MS/MS scans and high sensitivity ion

H2C

CH

H2C O P O

OH

OR2 C OO

O C

O

R1

CH2CH2NH3+

H2C

CH

H2C O P O

OH

OR2 C OO

O CH2 CH2 R

CH2CH2NH3+

H2C

CH

H2C O P O

OH

OR2 C OO

CH2CHCOOH

NH2

O C

O

R1

H2C

CH

H2C O P O

OH

OR2 C OO

O C

O

R1

HO

HO

OH

OH

OH

H2C

CH

H2C O P O

OH

OR2 C OO

O C

O

R1

CH2CH2N(CH3)3+

CH

H2C O P O

OH

OC

O

CH2CH2N(CH3)3+

R2

CHHO R1

HN

Phosphatidylinositol (PI)Phosphatidylserine (PS)

Phosphatidylethanolamine (PE)

Phosphatidylcholine (PC) Sphingomyelin (SM)

Plasmalogen PE (pPE)

Scheme 1. Structures and abbreviations of common phospholipids. R1 and R2 represent different fatty acyl chains.

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453

2444 C. Wang et al.

trap product scans on the same instrumental platform turned

out to be ideal to provide rapid identification of phospholi-

pids in child blood extracts.

In this work the survey scan of the phospholipids was

performed using the ‘enhanced MS’ mode (EMS, in which the

first two quadrupoles are operated in funnel (RF-only) mode

and ions are accumulated and then mass-analyzed in the (Q3)

linear ion trap), or using a precursor ion scan (Pre ion scan, in

which Q1 sweeps a given mass range with Q3 fixed to

transmit the diagnostic fragment ion), or using a neutral loss

scan (NL scan, in which both Q1 and Q3 scan a given mass

range together but with a constant mass difference between

the ranges scanned). The structures of the phospholipds were

elucidated using the ‘enhanced’ product ion (EPI) scan mode

in which the product ions are trapped in Q3 (in trap mode)

before mass analysis. With the QTRAP system this approach

can be realized using a single instrument platform with IDA.

The phospholipid extracts from child whole blood samples

were introduced into the mass spectrometer using the

autosampler of an HP 1100 series HPLC system (Agilent

Technologies, Palo Alto, CA, USA). A mobile phase of 100%

methanol was continuously injected into the mass spectro-

meter at a flow rate of 100 mL/min, and aliquots (10mL) of

sample were directly introduced into the carrier flow and

thus on into the ionization source without sample pre-

separation. In order to increase the available IDA cycles per

injection, a homemade union replaced the LC column with

no precautions to avoid dead volume, so the duration of

sample flow into the ionization source was extended to

�1 min. The chloroform in the injection solution may have

increased the peak width to some extent. The different

phospholipid classes were identified using a Pre ion scan or

NL scan as the survey scan (range m/z 650–950, scan time

0.5 s, scan speed 4000 Da s�1, collision energy 40–50 eV); an

enhanced resolution (ER) scan was used to confirm phos-

pholipid mass assignments. An EPI scan was used as the

dependent scan (range m/z 100–950, scan speed 4000 Da s�1,

trap time 100 ms, collision energy 40–50 eV). The survey and

dependent scans were performed sequentially, and repeated

for the entire duration of analysis, thus providing maximum

information from a single injection. The instrument para-

meters were optimized to provide maximum signals from the

(positive or negative) precursor ions of the phospholipid

standards. The nitrogen drying gas and turbo gas were at 45

and 40 psi back-pressure, respectively. The curtain gas that

prevents contamination of the ion optics was set at 30 psi. The

declustering potential (DP) was set at 80 V.

RESULTS AND DISCUSSION

ESI MS1 spectraESI mass spectra (MS1) of the phospholipids in extracts of

child whole blood were obtained in both positive and nega-

tive ion EMS mode (Fig. 1). Many classes of phospholipids

Figure 1. DI-ESI-MS analysis of a child whole blood phospholipid extract: (a) positive ion and

(b) negative ion ESI mass spectra of the same extract.

Phospholipid structures in human blood by DI-ESI-QqLIT-MS 2445


possess a net negative charge at neutral pH; accordingly,

negative ion ESI mass spectra of such phospholipids (PS, PI

and PE) can be effectively obtained with [M–H]� as the pre-

cursor ions. However, PC, PE and SM are zwitterionic mole-

cules, and therefore either positive or negative ion ESI mass

spectra of these phospholipid classes are readily accessible.

Thus, the positive ion spectrum in Fig. 1(a) contains the

[MþH]þ and [MþNa]þ ions of PE, PS, PC and SM, while

the negative ion spectrum in Fig. 1(b) gives intense signals

for the [M–H]� ions for PS, PI and PE, as well as [M–15]�

(demethylated) ions for PC and SM.

IDA procedureFragmentation of polar head groups of phospholipids allows

the specific detection of phospholipid classes by Pre ion or NL

scanning. However, the presence of alkali cations in the sam-

ple or matrix gives rise to some disadvantageous effects on

the analysis of positive ion spectra of phospholipids. First,

the cationization produces multiple ion signals for any parti-

cular molecular species, thus reducing the intensity of signals

for [MþH]þ ions and producing some degree of ion suppres-

sion.24,25 Second, the alkali-cationized species of some phos-

pholipid classes (e.g., PS and PE) also undergo fragmentation

of the polar head,26 which complicates the head-group-speci-

fic spectra. Furthermore, removal of salts from small amounts

of phospholipid samples can be very difficult. Accordingly,

most of the phospholipid classes (except PC) were analyzed

in the negative ion mode. The head-group-specific scans were

based on the fact that a common fragment is formed from the

head-group region of each member of a given class of polar

lipids upon CAD, when using ESI-MS/MS.16

Child whole blood was analyzed using the IDA sequence:

Pre or NL scan! enhanced resolution scan (ER)! enhanced

product ion scan (EPI).

In such an IDA experiment, the Pre ion scan or the NL scan

is considered as the survey scan, and the EPI scan is the

dependent scan. Phospholipid structural information is

obtained by this technique on two levels. The first level,

referred to as the class mass profile, is provided from the Pre

ion or NL survey scan; the second level, referred to as

providing fatty acyl formula data, is derived from the EPI

scan. As an example, the application of this IDA experiment

to identification of PS species is elucidated in detail in the

following.

Figures 1 and 2 were obtained for the same sample. It can

be observed in Figs. 2(a) and 2(b) that many peaks that

were completely buried in the chemical background of the

conventional mass spectrum (MS1) in Fig. 1(b) have been

revealed using neutral loss scanning for 87 Da16 in the

negative ion mode; this neutral loss is diagnostic for PS.

These overlapping and partially buried peaks can be resolved

by the Pre ion spectra. The resolution of the classical NL or Pre

ion scan modes is not as high as that using the enhanced mode

exploiting the trap functionality; the role of the classical

triple-quadrupole scans is solely the selection of the

precursor ion for the EPI experiment. Furthermore, it is

important to obtain unit mass resolution when the phospho-

lipids differ in mass by 2 Da or less. In the IDA procedure

used here, the resolution is reduced because of fast scan

speed (4000 Da s�1), as exemplified by Fig. 2(a), in which the

peak width is more than 10 Da. To solve this problem, an

enhanced resolution (ER) scan was included between the

initial survey scan (Pre ion or NL) and the EPI experiment. It

was found that the resolution and general performance of the

method were greatly improved by adding an ER scan to the

IDA procedure (Fig. 2(b)).

When a single-step MS/MS method such as Pre ion or NL

scanning is used to characterize phospholipid mixtures, even

though a precursor ion may be observed as a single mass peak

it is possible that several different unresolved (isobaric)

molecules can be dissociated simultaneously and detected.34

Thus, when a single Pre ion or NL scan is used for a head-

group survey (i.e., to survey a selected class), only the

summed numbers of carbon atoms and of double bonds

within individual phospholipid molecules can be obtained;

precise information on the pairs of fatty acyl groups on the sn-

1 and sn-2 positions cannot be elucidated.37 However, in a

typical IDA experiment as used in this work, a Pre ion or NL

survey scan is performed and processed to locate mass peaks

of interest (candidates), and product ion (dependent) scans

are acquired for these precursor ions. This cycle is repeated

through the duration of the acquisition to generate large

amounts of informative data. Thus, head-group-specific

scans, and product ion scans for the species identified,

were acquired sequentially in the same run by the IDA

procedure. Then, even if two (or more) isobaric precursors

are present, the several fatty acyl anions observed in the

overlaid product ion spectrum must be combined pairwise in

order to give the fixed total mass of the precursor, so that

some resolution of isobaric precursors can be obtained. This

approach does not distinguish between positional (sn-1/sn-2)

isomers.

While this IDA procedure is very powerful, improper

selection of the candidate precursor ions may result in the

generation of large amounts of poor or worthless data.

The IDA option in the Analyst software used to control the

QTRAP system includes a group of ion-filtering tools, such as

a time-based windowed exclusion list that allows the user to

specify an exclusion retention time (RT) window width to

indicate ions that should be used as candidates only for

certain time periods during an IDA run. If the precursor ion is

present in the survey scan within its specified time window

and if its intensity is above a specified threshold value, the

system will then acquire the PRI spectrum. The present IDA

procedure provides very good MS/MS spectra when the

precursor ion signal is strong. We have found that the key to

elucidating the structures of phospholipid species of low

abundance is to define an inclusion list indicating precursor

ions that should be preferentially used as candidate pre-

cursor ions during an IDA run. Because of the advantages of

this IDA procedure, enabling head-group-specific scans and

product ion scans to be combined together with this selective

filtering function, the candidate ion selectivity is greatly

improved.

In general, in order to obtain the maximum information,

3–4 injections were needed in view of the limited ‘chromato-

graphic’ peak width available; about 4–5 EPI spectra of

phospholipid species could be obtained per injection in the

current experimental conditions. The entire information on

molecular species within each phospholipid class, together

2446 C. Wang et al.


with some EPI spectra of precursors with high abundance,

were obtained from the first injection. The other molecular

species can be obtained from the succeeding injections by

properly using inclusion and exclusion lists. It should be

noted that the number of EPI spectra per injection depends on

the ‘chromatographic’ peak width and carrier flow rate; thus

a moderate dead volume can be beneficial in the present

context.

Figure 2. DI-ESI-MS analysis of phospholipids using a negative ion IDA experiment.

(a) Detection of PS by conventional neutral loss (87Da) scanning. (b) Enhanced resolution

(ER) neutral loss scan of the same extract over a limited range. (c) Using IDA to identify a

precursor ion of interest (m/z 810.6, upper spectrum), immediately followed by an

enhanced product ion (EPI) scan to display fragment ions that provide information on

the fatty acyl moieties in the molecule. The chromatograms on the left-hand side of

(c) represent TIC profiles for the two scan modes.



Figure 2(c) displays the IDA results. The top left box shows

the TIC for the NL (87 Da) scan for PS species in the child

blood sample, the top right box is the ER mass spectrum of the

region around m/z 810.6 (a target PS based on Fig. 2(b)) taken

at 0.667 min, the bottom left box shows the extracted ion

chromatogram (XIC) for the [M–H]� ion (m/z 810.6), and the

bottom right box is the EPI spectrum of m/z 810.6 recorded

immediately after the ER spectrum shown in the top right

box. In this EPI spectrum, the fragment ions detected at 283.4

and 303.3 correspond to C18:0 and C20:4 fatty acid residues

(carboxylate anion fragments) of [M–H]�, respectively.

Additionally, the fragment ion at m/z 437.2 corresponds to

C18:0 lysophosphate in the open form, and the abundant ion

at m/z 419.3 corresponds to C18:0 lysophosphate in the cyclic

form. The diagnostic ion [M–87–H]� for PS species is clearly

detected at m/z 723.5.

The positions of the acyl chains on the glycerol backbone of

the phospholipid molecule are believed to be important for

their relative propensity for dissociation. Product ion scan-

ning in negative ion mode can be used to identify the fatty

acyl chains,26 but there is disagreement in the literature on

which carboxylate anion (sn-1 or sn-2) yields the most intense

peak in the product ion spectrum.26,30–33 Hvattum et al.33

reported that the abundance ratio of the carboxylate anions

depends on many factors, such as collision energy, the

phospholipid class, and the nature of the fatty acid attached

to the sn-2 position. Houjou et al.34 and Hsu et al.35 determined

the acyl positions based on relative intensities of lyso-

phospholipid related fragment ions of [MþH]þ or [MþLi]þ,

respectively, in the positive ion mode. In the present work the

acyl positions are tentatively assigned on the basis of the

finding that phospholipids isolated from animals most often

contain a saturated fatty acid in the sn-1 position and an

unsaturated fatty acid in the sn-2 position.36 Therefore, m/z

810.6 is identified as C18:0/C20:4 PS, although the acyl

positions are only inferred. The other PS species identified by

this method are listed in Table 1 and the corresponding EPI

spectra are shown in Fig. 3.

Similarly, Fig. 4 shows the results of ER scans in negative

and positive ESI-MS modes for the detection of other

phospholipid classes. The Pre ion scan form/z 24116 provided

information on the PI molecular species in the child blood

sample (Fig. 4(a)). It was found that multiple molecular

species of phosphatidylinositols containing polyunsaturated

fatty acids (e.g., m/z 833.6, 857.5 and 885.6, corresponding to

C16:0/C18:2 PI, C16:0/C20:4 PI and C18:0/C20:4 PI, respec-

tively) were present (Fig. 4(a)). In addition, PIs containing a

monounsaturated fatty acid, e.g., C18:0/C18:1 PI (m/z 863.6),

was also detected (Fig. 4(a)). The corresponding EPI spectra

are shown in Fig. 5.

By using Pre ion scanning for m/z 196,16 more than ten PE

molecular species were detected (Figs. 4(b) and 6). In addition

to the plasmalogen PE molecular species (e.g., m/z 748.5 and

750.5, corresponding to pC16:0/C22:5 or pC18:1/C20:4 and

to pC18:0/C20:4 or pC16:0/C22:4, respectively), ethanola-

mine glycerophospholipids were predominantly composed

of diacyl molecular species (e.g., m/z 738.5, 766.5 and 790.6,

corresponding to C16:0/C20:4 or C18:2/C18:2 PE, to C18:0/

C20:4 PE or C16:0/C22:4 PE, and to C18:0/C22:6 PE,

respectively).

The EPI spectra of PI and PE were similar to those of the PS

in negative ion mode, revealing the fatty acid residues, since

formation of fatty acid anions represents an effective

fragmentation pathway of negatively charged ions of

phospholipids containing ester-bound fatty acids.26 How-

ever, in the EPI spectra of pPE, only carboxylate anions in the

sn-2 position can be observed because the sn-1 vinyl ether has

relatively higher stability.23,26

SM molecular species were identified by Pre ion scanning

for m/z 168 (dimethylethanolamine phosphate) in the

negative ion mode, as demonstrated in Fig. 4(c). Because

SM molecular species do not contain ester-bound fatty acids,

the ion atm/z 168 is the main fragment observed.27 As PC and

SM both contain a choline phosphate head group, Pre ion

scanning form/z 184,16,18,29 representing the [H2O3PO–CH2–

CH2–N(CH3)3]þ ion, identified [MþH]þ ions for PC and SM

in the positive ion mode. Discrimination between the PC and

SM molecular species was achieved by exploiting the

Table 1. Identification of phospholipid species in child whole

blood using DI-ESI-MS IDA experiments

Class Ion m/zCombinations of molecular

species

PE [M–H]� 714.6 C16:0/C18:2[M–H]� 738.5 C16:0/C20:4 C18:2/C18:2[M–H]� 740.5 C18:1/C18:2 C16:0/C20:3[M–H]� 748.5 pC16:0/C22:5 pC18:1/C20:4[M–H]� 750.5 p C18:0/C20:4 p C16:0/C22:4[M–H]� 762.5 C16:0/C22:6[M–H]� 764.5 C16:0/C22:5 C18:1/C20:4[M–H]� 766.5 C18:0/C20:4 C16:0/C22:4[M–H]� 790.6 C18:0/C22:6

PS [M–H]� 790.7 C18:0/C18:0[M–H]� 808.6 C18:1/C20:4 C18:0/C20:5[M–H]� 810.6 C18:0/C20:4[M–H]� 812.6 C18:0/C20:3[M–H]� 832.6 C18:2/C22:5[M–H]� 834.7 C18:0/C22:6[M–H]� 836.6 C18:0/C22:5

PI [M–H]� 833.5 C16:0/C18:2[M–H]� 857.5 C16:0/C20:4[M–H]� 859.6 C18:1/C18:2[M–H]� 861.6 C18:0/C18:2[M–H]� 863.6 C18:0/C18:1[M–H]� 885.6 C18:0/C20:4[M–H]� 913.7 C18:0/C22:4

SM [M–CH3]� 685.6 34:2[M–CH3]� 687.6 34:1[M–CH3]� 713.5 36:2[M–CH3]� 711.5 36:3[M–CH3]� 745.5 38:0[M–CH3]� 749.5 39:5[M–CH3]� 769.5 40:2[M–CH3]� 771.6 40:1[M–CH3]� 797.7 42:2[M–CH3]� 799.7 42:1[M–CH3]� 829.6 44:0

PC [MþH]þ 758.5 34:2[MþH]þ 760.6 34:1[MþH]þ 762.6 34:0[MþH]þ 780.6 36:5[MþH]þ 782.6 36:4[MþH]þ 784.6 36:3[MþH]þ 786.6 36:2[MþH] 808.6 38:5[MþH] 814.7 38:2

2448 C. Wang et al.


Figure 3. Enhanced product ion (EPI) spectra of [M–H]� ions of PS species (except m/z 810.6, see Fig. 2(c)) from child blood

sample obtained by DI-ESI-MS with IDA. (a–f) EPI spectra of m/z 790.7, 808.6, 812.6, 832.6, 834.7 and 836.6, respectively.

Fatty acid carboxylate ions are observed as follows: m/z 283, 18:0; m/z 281, 18:1; m/z 301, 20:5; m/z 303, 20:4; m/z 305, 20:3;

m/z 325, 22:7; m/z 327, 22:6; m/z 329, 22:5.



Figure 4. Specific detection of phospholipid classes in an unprocessed lipid extract from child

whole blood by DI-EMS-MS with IDA. (a) Detection of PI by Pre ion scanning form/z 241 (collision

energy 50 eV) in the negative ion mode. (b) Detection of PE by Pre ion scanning for m/z 196

(collision energy 40 eV) in the negative ion mode. (c) Detection of SM by Pre ion scanning for m/z

168 (collision energy 35 eV) in the negative ion mode. (d) Detection of [MþH]þ ions of SM and PC

by Pre ion scanning for m/z 184 (collision energy 40 eV) in the positive ion mode.

2450 C. Wang et al.


Figure 5. Enhanced product ion (EPI) spectra of [M–H]� ions of PI species from child blood sample obtained by DI-ESI-MS with

IDA. (a–g) EPI spectra of m/z 833.6, 857.5, 859.6, 861.6, 863.6, 885.6 and 913.7, respectively. Fatty acid carboxylate ions are

observed as follows: m/z 255, 16:0; m/z 283, 18:0; m/z 281, 18:1; m/z 279, 18:2; m/z 303, 20:4; m/z 331, 22:4.



Figure 6. Enhanced product ion (EPI) spectra of [M–H]� ions of PE species from

child blood sample obtained by DI-ESI-MS with IDA. (a–i) EPI spectra of m/z 714.6,

738.5, 740.5, 748.5, 750.5, 762.5, 764.5, 766.5 and 790.6, respectively. Fatty acid

carboxylate ions are observed as follows:m/z 255, 16:0;m/z 281, 18:1;m/z 279, 18:2;

m/z 303, 20:4; m/z 305, 20:3; m/z 327, 22:6; m/z 329, 22:5; m/z 331, 22:4.

2452 C. Wang et al.


nitrogen rule; PC (with one nitrogen atom) showed [MþH]þ

signals at even-numbered m/z values, whereas SM (with two

nitrogen atoms) showed [MþH]þ signals at odd-numbered

values. The possible reason why some SM molecular species

detected by Pre ion scanning form/z 168 in negative ion mode

(Fig. 4(c)) could not be detected by Pre ion scanning for m/z

184 in positive ion mode (Fig. 4(d)) is that PC molecular

species in blood samples are much more abundant than SM,

and ionization of the latter could have been suppressed. The

fatty acid residues of PC can be effectively determined by

negative ion EPI scans;23 alternatively, Hsu et al.28 identified

PC species in phospholipid mixtures using Liþ adducts as

precursors in positive mode. Recently, by selecting the [M–

CH3]� ion as the precursor, Houjou et al.34 identified the

fatty acyl chains of PC species, sphingosine or sphinganine

derivatives, and N-acyl species of SM species. These appro-

aches may provide some help to more fully characterize the

PC and SM species.

Table 1 lists all the phospholipid species detected in the

child blood sample using the DI-ESI-MS method with IDA.

Comparing the present approach with HPLC/MS meth-

ods, head-group-specific scans can identify some phospho-

lipid classes as shown here; however, some low abundance

but functionally important phospholipids, such as phospha-

tidic acid, lysobisphosphatidic acid, and cardiolipin, cannot

be analyzed in this way as they produce no diagnostic

fragments (charged or neutral) in MS/MS, so no specific scan

mode is available.17 On the other hand, the advantage of the

present method is obvious; the run time is greatly reduced

(only several minutes) due to no HPLC pre-separation, which

makes the method suitable for high-throughput screening

analysis and rapid identification of particular phospholipid

targets. Some further steps may be implemented to improve

this method, such as use of a short pre-column, slower

flow rate, or combination of a short microcolumn with a

nanospray source.

CONCLUSIONS

DI-ESI-MS in combination with IDA experiments without

prior analyte pre-separation was investigated as a high-

throughput method for the characterization of phospholipids

from child whole blood. Pre ion and NL scan modes are much

less sensitive than the enhanced product ion mode, but they

are used here only to discover the most interesting analytes.

The IDA procedure used here combines two or more different

scan modes in a sequential way within the same run, which

enables head-group-specific scans and product ion scans to

be combined. The use of inclusion and exclusion lists became

mandatory for biological samples to benefit fully from this

approach. In this way phospholipid information, including

mass profiles and fatty acyl formulae, can be obtained in a

single run. Because many biological matrices such as urine

or blood are complex, many peaks of interest that might

have been hidden previously in the chemical background

of the conventional mass spectrum are clearly identified

using head-group-specific scans within the IDA procedure.

This approach is advantageous for targeted analyses because

of the short analysis time (only several minutes). We believe

that this approach has potential application for high-

throughput determinations of some phospholipids in biolo-

gical samples, since it requires little sample preparation, is

rapid, and can be readily automated.

AcknowledgementsThis study was supported by the High-Tech R & D Plan

(2003AA223061) of the State Ministry of Science and Techno-

logy of China, the Foundation (No. 20425516) for Distin-

guished Young Scholars from the National Natural Science

Foundation of China and the Knowledge Innovation Pro-

gram (K2002A12, K2003A16) of the Chinese Academy of

Sciences.

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Identification of phospholipid structures in human blood by direct-injection quadrupole-linear ion-trap mass spectrometry