quadrupole-linear ion trap mass spectrometry

Analytica Chimica Acta 525 (2004) 1–10

Structural identification of human blood phospholipids using liquid chromatography/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 China b Dalian Center Hospital, Dalian 116033, PR China

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

Abstract A normal-phase liquid chromatography/quadrupole-linear ion trap mass spectrometry method was developed for the separation and mass spectral characterization of the main phospholipid species in human blood. The instrument combines the capabilities of a triple quadrupole mass spectrometer and ion trap technology on a single platform. The optimal separation was achieved by using hexane/1-propanol as mobile phase and 0.6% formic acid, 0.06% ammonia as modifiers. The HPLC/MS technique was able to provide information about the molecular mass of individual homologues by positive and negative turbo ionspray. More complete characterization of fatty acid chains and of the polar head group was obtained by using a quadrupole collisionally activated dissociation (CAD) spectrum with ion trap sensitivity. The mass spectra and molecular species of phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC) and sphingomyelin (SM) are presented. © 2004 Elsevier B.V. All rights reserved. Keywords: Phospholipids; Linear ion trap; Ionspray; Liquid chromatography/mass spectrometry

1. Introduction Phospholipids are an important constituent in the biomembranes. Both the physical and chemical properties of the membrane bilayer can be affected by the variation of phospholipid compositions. Membrane phospholipids are a complex mixture of molecular species containing a variety of fatty acyl and head group compositions. In addition to their structural role, some phospholipids also participate in biological processes in various ways. Other phospholipids, such as polyphosphoinositides, are important in cellular signalling systems [1,2]. Phospholipids serve as a reservoir for arachidonic acid (20:4 n-6) and other polyunsaturated fatty acids that can be metabolized to biologically active eicosanoids such as prostaglandins, thromboxanes, ∗

Corresponding author. Tel.: +86 411 83693413; fax: +86 411 3693403. E-mail address: [email protected] (G. Xu).

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.07.065

leukotrienes and lipoxins [3,4]. Phospholipids have been given increased attention in many fields, for example as biomarkers in chemotaxonomical studies and in the making of liposomes for drug delivery or cosmetics/detergents. The commercial use of phospholipids is increasing in fields such as biomembranes, skin-care formulations and drug delivery. Analysis of these phospholipids has been carried out with chromatographic techniques such as thin-layer chromatography (TLC) [5,6], high-performance liquid chromatography (HPLC) [7–13]. Detection of phospholipids has been performed by different spectrophotometric techniques such as UV. However, with this technique, serious constraints are imposed on the mobile phase selection since underivatized phospholipids absorb near 200 nm with a low extinction coefficient [9,14,15]. A novel derivatization approach was proposed to increase the UV sensitivity of phospholipid analysis [15] using naproxen chloride. But this approach is labor intensive and is not suitable for routine and high throughput

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analysis. Alternatively, phospholipids can be analyzed by HPLC with evaporative light-scattering detection (ELSD) [16–18]. Although this technique is compatible with gradient elution and permits quantitation of phospholipids, the poor selectivity of this detector implies that the identification of the different phospholipids must be made by retention behavior in comparison to known standards. Mass spectrometry (MS) offers an attractive alternative for the analysis of phospholipid composition because of its high sensitivity, specificity, and (apparent) simplicity. However, MS has rarely been used for this purpose until very recently. The main reason for this is that the ionization methods available (fast-atom bombardment, etc.) cause extensive fragmentation of the lipid molecules, this along with the extreme complexity (hundreds of different molecular species) of most biological samples, has precluded compositional analysis. The introduction of “soft” ionization methods has completely opened new vista in this field. In particular, electrospray ionization-mass spectrometry (ESI-MS) has been shown to be a very promising technique [19]. However, it is important to have a chromatographic system separate the different phospholipid classes to avoid possible mass overlap. Thus, in the analysis of lipids taken from a complex biological matrix, there is a need for class separation by LC followed by species identification by mass spectrometry [20]. HPLC/MS is a very useful tool for lipid analysis [21–24]. In this work, we describe an improved chromatographic method for class separation of phospholipids in human blood. Product ion spectra following collisionally activated dissociation (CAD) of quasi-molecular ions in a tandem mass spectrometry was obtained to identify individual molecular species of each phospholipid class.

2. Experimental 2.1. Chemicals 1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1oleoyl-2-hydroxy-sn-glycero-3-phosphocholine and sphingomyelin (SM) from chiken egg were from Avanti Polar Lipids (Alabaster, AL, USA). l-␣-Phosphatidyl-l-serine, dipalmitoyl sodium salt was from 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 (TEDIA, USA), ammonia (25%) is analytical grade from Lian-Bang (Shenyang, China). 2.2. Sample preparation The phospholipid standards were dissolved (approximately 1 mg/ml) in chloroform/methanol (2:1, v/v), and further diluted with hexane/1-propanol (3:2, v/v). Heparinised human whole blood was pooled from volunteers. The lipids in the 500 ␮l blood samples were ex-

Table 1 Linear gradient composition Time (min)

A%

B%

0 20 33 38 60

68 20 20 68 68

32 80 80 32 32

tracted essentially as described earlier [25]. Prior to analysis, the extracted samples were redissolved in 500 ␮l chloroform/methanol (2:1, v/v) and then was diluted by hexane/1propanol (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 performed on a diol column (Nucleosil, 100-5 OH, Germany) (250 mm × 3.9 mm, i.d. × 5.0 ␮m, particle size). The total flow rate was 0.7 ml min−1 . The flow from the LC was split using a Micro-Splitter Valve such that the flow to the electrospray was approximately 0.28 ml min−1 . The column temperature was at 35 ◦ C. The linear solvent gradient is shown in Table 1. Solvent mixture A: hexane/1-propanol/formic acid/ammonia (79/20/0.6/0.07, v/v); solvent mixture B: 1propanol/water/formic acid/ammonia (88/10/0.6/0.07, v/v). 2.4. Mass spectrometry The mass spectrometric detection was performed on a QTRAP LC/MS/MS system from Applied Biosystems/MDS Sciex (USA) equipped with a turbo ionspray source. This instrument is based on a triple-quadrupole ion path using the final quadrupole as a linear ion trap mass spectrometer. Thus, the QTRAP instrument combines all of the functionality of a classical triple quadrupole mass spectrometer with the capabilities of a very high sensitivity linear ion trap mass spectrometer [26]. The combination of highly selective triple quadrupole MS/MS scans and high sensitivity ion trap product scans on the same instrument platform provided rapid identification of phospholipids of extracted human blood sample. The survey scan of phospholipids eluted from the chromatographic column is performed in the “enhanced MS” (EMS), single quadrupole mode where ions were accumulated then filtered in the Q3-linear ion-trap. The structure of phospholipid are elucidated by the “enhanced” product ion scan mode (EPI) where ions were trapped in the third quadrupole before filtration. The split HPLC effluent entered the MS through a steel ES ionization needle set at 5500 V (in positive ion mode) or 4500 V (in negative ion mode) and a heated capillary was set to 250 ◦ C. The ion source and ion optic parameters were optimized with respect to the positive or negative molecular related ions of the phospholipid standards. The nitrogen drying gas and turbo gas were both at 40 psi, the curtain gas that was

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to prevent the contamination of the ion optics was set at 30 psi. The declustering potential (DP) was set at 80 psi. The other parameters as follows: EMS as survey scan (mass range m/z 450–950, scan speed 1000 Da s−1 , trap time 20 ms) and EPI as dependent scan (scan speed 1000 Da s−1 , trap time 150 ms, collision energy set at +35 eV in the positive-ion mode and from −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 did not, however, give an optimal separation of the different phospholipid classes, because PC, PS and PI elute very closely and make class determination more complicated. Furthermore, there are so many molecular species in each class of phospholipids, it is necessary to increase separation resolution as much as possible. Most importantly, TEA has been reported to be strongly absorbed on the surfaces of the vacuum manifold and parts therein [27]. This absorption may suppress the ionization of less basic compounds in positive mode for low molecular mass substances due to the TEA signal at m/z 102. However, the addition of 0.06% ammonia into mobile phase as modifier instead of TEA highly improved resolution. It was reported [28] that inclusion of ammonia in infusion solvent eliminated sodium adduct formation in the positive ion mode, thus greatly simplifying the interpretation of the spectra. Comparing this chromatographic separation methodology to earlier solvent/modifier systems [20], not only the resolution has been highly improved, but also the bad effect on the column life due to the higher column temperature (55 ◦ C) has been avoided. Uran et al. [25] separated phospholipids using chloroform and methanol. But chloro-

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form as mobile phase can produce danger to public health. In contrast, 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 main phospholipid classes. Under this condition, PE was firstly eluted, followed by PS(PI), PC, SM and lysoPC in a successive manner. Fig. 1 shows the total-ion current (TIC) of phospholipids in human blood in the negative-ion HPLCESI/MS. Because different molecular species within a class have a same polar head, their retention time in HPLC is very similar. The retention time difference of compounds within one 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 phospholipid classes were detected using positive or negative ion full-scan ESI-MS analysis. Many classes of phospholipids possess net negative charge at neutral pH. Accordingly, negative-ion ESImass spectra of these phospholipids can be effectively obtained with [M − H]− as the molecular ion peak (such as PS, PI). However, PC, PE, lysoPC and SM are zwitterionic molecules, and therefore either positive or negative-ion mass spectra of these phospholipid classes are accessible through ESI-MS. In this work, the PE, PS, PC, lysoPC and PI species were mainly analyzed in negative-ion mode, SM were identified in positive-ion mode. 3.3. Phosphatidylethanolamine (PE) species Negative-ion HPLC-ESI/MS analysis of PE species in the extracted human blood is shown in Fig. 2a. In the negative-ion spectra, molecular species of PE mainly give the [M − H]− ions. The fatty acid composition of negative ions of phospholipids can be determined by MS/MS in the EPI mode, since formation of fatty acid anions represents an effective

Fig. 1. Total ion chromatogram of LC/ESI-MS analysis of phospholipids of extracted human blood sample. Conditions are given in Section 2.

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Fig. 2. (a) Negative-ion EMS of PE species from human blood sample. (b) EPI spectrum of m/z 738.7 as shown in (a) representing the [M − H]− ion of diacyl-PE. (c) EPI spectrum of m/z 750.8 as shown in (a) representing the [M − H]− ion of pPE.

fragmentation pathway of negatively charged ions of phospholipids containing ester-bound fatty acids [29]. The identification of diacyl and plasmalogen species was based on the molecular ion and sn-1 and sn-2 carboxylate anions observed. For diacyl species, both carboxylate anions corresponding to sn-1 and sn-2 substituents were present in the negative ion mode. For plasmalogen species, only one carboxylate anion corresponding to sn-2 acyl substituents was observed, because the sn-1 group did not cleave to form an anion [35]. As an example, Fig. 2b and c show the negative EPI spectra of deprotonated diacyl and plasmalogen PE species, respectively. The fragment ions detected at m/z 255, 279 and 303 corresponded to C16:0, C18:2, and C20:4 fatty acid residues (carboxylate anion fragments) of [M − H]− , respectively (Fig. 2b). The fragment ions at m/z 452 and m/z 476 correspond to the C16:0 lysoPE and C18:2 lysoPE ions (PE has lost one of the two fatty acid residues), respectively. In addition, the C20:4 lysoPE fragment ion is in low abundance. The position of the acyl chains in the glycerol backbone of the phospholipid molecule is obviously important for their degree of dissociation. This phenomenon has previously been reported by several publications [29–33]. There has been dis-

crepancy on which carboxylate anion yields the most intense peak in the product ion spectrum in these reports. One report stated that the intensity of the fatty acid fragment in the sn-2 position was approximately twice that of the fatty acid in the sn-1 position [29] and another report stated that there was no preferential loss of the fatty acid moiety from either sn-1 or sn-2 position [30]. Hvattum et al. [33] reported that the abundance ratio of the carboxylate anions relates to many factors, such as collision energy, the phospholipid class, and the fatty acids attached to the sn-2 position. In this paper we adopt that the phospholipids isolated from animals most often contain a saturated fatty acid in sn-1 position and an unsaturated fatty acid in sn-2 position [34]. The m/z 738.7 is therefore identified as C16:0/C20:4 or C18:2/C18:2 diacyl PE species. In the negative-ion EPI spectrum of deprotonated plasmalogen PE at m/z 750.8 (Fig. 2c), the presence of a relatively more intense lysoplasmenylethanolaminelike ion peak (m/z 464, C18:0 lysopPE; m/z 436, C16:0 lysopPE) relative to its phosphatidylethanolamine counterpart from the examined plasmenylethanolamine molecular species that contain C20:4 and C22:4 acyl chains at sn-2 position indicates the presence of pC18:0/C20:4 or pC16:0/C22:4 plasmalogen PE species.

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Table 2 Molecular species identification of phosphatidylethanolamines (PE) PL class

Negative ion [M − H]− (m/z)

Combinations of molecular species

Diacyl-PE and pPE

714.7 716.8 720.7 722.7 726.7 728.7 736.7

C16:0/C18:2 C16:0/C18:1 pC16:0/C20:5 pC16:0/C20:4 pC18:0/C18:2 pC18:0/C18:1 C18:2/C18:3 C16:0/C20:5 C16:0/C20:4 C16:0/C20:3 C16:0/C20:2 C18:0/C18:2 C18:0/C18:1 pC18:0/C20:4 C16:1/C22:6 C18:2/C20:5 C18:3/C20:4 C16:0/C22:6 C16:0/C22:5 C18:0/C20:4 C16:0/C22:4 pC18:0/C22:4 C18:1/C22:6 C18:0/C22:6 C18:2/C22:4 C20:2/C20:4

738.8 740.7 742.8 744.7 750.7 760.6 762.7 764.7 766.7 778.8 788.8 790.7

This product ion ratio likely results from the relatively higher stability and formation rate of the sn-1 vinyl ether-liked product ion from plasmenylethnolamine anion in comparison to those that result from the loss of the sn-1 carboxylic acid (or derivative) from deprotonated diacy PE species [29]. Using the LC-ESI/MS technique, more than 30 PE species were identified from extracted human blood sample, and are listed in Table 2. Under the positive-ion mode, the EMS spectra of PE give [M + H]+ , [M + Na]+ , and [M + H-141]+ ions (the figure not shown). However, in the EPI spectrum of [M + H]+ ions, except the abundant [M + H-141]+ ions, the other fragment ions (such as [lyso-PE + H]+ ion) is in a low abundance (the figure not shown). As a result, the identification of PE species was mainly performed in negative ion mode in spite of the higher sensitivity in positive-ion mode. 3.4. Phosphatidylinositol (PI) and phosphatidylserine (PS) molecular species As shown in Fig. 3a, the negative-ion HPLC-ESI/MS analysis gives the main [M − H]− ions for either PS or PI species. The molecular species of PS and PI are discriminated according to nitrogen rule. In the negative ion mode, PS (with one nitrogen atom) shows signals at even-numbered m/z values, whereas PI (without nitrogen atom) shows signals at oddnumbered values. The negative ion EPI mass spectra of PI species obtained by ESI-MS/MS were similar to those reported previously

pC18:1/C18:1 C16:1/C20:4 C18:2/C18:2 C18:1/C18:2 C18:1/C18:1 C16:0/C20:1 pC16:0/C22:4 C18:1/C20:6

C18:2/C20:4 C18:1/C20:4 C18:2/C20:2 pC20:0/C20:4 C18:2/C22:5 C18:1/C22:5

[35]. The EPI spectrum of the [M − H]− ion of PI at m/z 885.8 is shown in Fig. 3b. The carboxylate anion fragment ions at m/z 283.4 (C18:0), 303.4 (C20:4) are detected. The lysoPI ions at m/z 581.4 (C18:0) and at m/z 601.5 (C20:4) were probably in the cyclic form, those at m/z 599.5 (C18:0) and m/z 619.3 (C20:4) fragment ions may be probably in the open form. In addition, two specific PI related negative ions at m/z 259.4 (inositol phosphate) and 241.2 [inositol phosphate – H2 O]− are produced. Therefore, m/z 885.8 was detected as C18:0/C20:4 PI. The PI species identified in human blood sample are listed in. Similarly to PE and PI classes, the negative-ion EPI spectra of [M − H]− of PS provides the information on the fatty acid substituents. As shown in Fig. 3c, C18:0 (m/z 283.4) and C22:6 (m/z 327.3) as the carboxylate anions confirmed the fatty acyl substituents at sn-1 and sn-2 positions. Moreover, fragment ions at m/z 437.3, 481.2 correspond to C18:0 and C22:6 lysophosphate in the open form, two abundant ions at m/z 419.5, 463.4 corresponding to C18:0 and C22:6 lysophosphate in the cycle form are also observed. The diagnostic ion [M − 87 − H]− of PS species is obviously detected at m/z 747.6. Then, the m/z 834.8 is identified as C18:0/C22:6 PS. Table 3 shows the PS species in the extracted human blood sample. Like PE, the positive-ion EMS spectra of PS provided [M + H]+ , [M + Na]+ , and [M + H-185]+ ions (the figure not shown). And the EPI spectra of [M + H]+ ions showed no abundant ions that can be used to identify fatty acid substituents but a single distinct [M + H-185]+ ions.

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Fig. 3. (a) EMS scan negative-ion spectrum of PI and PS species in human blood sample. (b) EPI of m/z 885.8 as shown in (a) representing the [M − H]− ion of PI. (c) EPI spectrum of m/z 834.8 as shown in (a) representing the [M − H]− ion of PS.

3.5. Phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) molecular species EMS spectra of PC species in human blood sample both in positive and negative ion LC-ESI/MS are shown in Fig. 4a and b. The positive-ion spectrum gave the main [M + H]+ and [M + Na]+ ions of intact PC, whereas the negative-ion spectrum predominantly gave the [M − 15]− and [M + 45]− ions. In positive-ion ESI/MS mode, EPI spectrum of [M + H]+ at m/z 760.7 (Fig. 4c) showed intense fragment ion at m/z 184.2 which readily gave the head group information of PC class. The fragment ion at m/z 577.6 resulting from loss of polar head was also observed. The deacylated ions at m/z 522.3 and 496.5 corresponding to the loss of C16:0 and C18:1 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 intense abundant fatty acid fragments at m/z 255.4 (C16:0) and 281.4 (C18:1)(Fig. 4d). Additionally, C16:0 lysoPC at m/z 480.5 and C18:1 lysoPC at m/z 506.3 ions also give the similar structural information of PC species. Accordingly, the m/z 744.8 is detected as C16:0/C18:1 PC. The ions clusters at m/z 802.8, 830.7 and 854.8 are the HCOO− adduct ions of PC. These ions can also be used to identify PC molecular species in the negative-ion mode [23] (the figure not shown). Although PC species in the positive-ion mode showed more sensitivity than in the negative-ion mode, however, fatty acid analysis of PC species required negative-ion EPI scan mode, since CAD of [M + H]+ ions of PC results in the formation of a single intense fragment ion at m/z 184 representing the

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[H2 O3 PO–CH2 –N(CH3 )3 ]+ ion. The detected PC species are listed in Table 4. The lysoPC species detected of human blood sample in negative-ion ESI/MS is shown in Fig. 5a. Because lysoPC corresponding to PC has lost one of the two fatty acid residues, the lysoPC species have less simple struc-

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

Fig. 4. EMS spectrum of PC species in human blood sample. (a) Positive-ion. (b) Negative-ion. (c) EPI spectrum of m/z 760.7 as shown in (a) representing the [M + H]+ ion of PC. (d) EPI spectrum of m/z 744.8 as shown in (a) representing the [M − 15]− ion of PC.

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Table 3 Molecular species identification phosphatidylinositols (PI) and phosphatidylserines (PS) in human blood sample PL Class

Negative ion [M − H]− (m/z)

Combinations of molecular species

PI

833.6 857.8 859.7 861.8 863.7 883.7 885.8 887.7 909.7 911.8 913.7

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

734.8 748.7 760.8 762.8 782.8 786.7 788.8 790.8 808.7 810.7 832.7 834.8 836.8 838.8

C16:0/C16:0 C15:0/C18:0 C16:0/C18:1 C16:0/C18:0 C16:0/C20:4 C18:0/C18:2 C18:0/C18:1 C18:0/C18:0 C18:1/C20:4 C18:0/C20:4 C18:0/C22:7 C18:0/C22:6 C18:0/C22:5 C18:0/C22:4

PS

C18:0/C18:4 C18:1/C18:2 C18:1/C18:1

C18:1/C18:1

C18:0/C20:5 C18:3/C22:4

3.6. Sphingomyelin (SM) species SM consists of phosphorycholine ester-bound to a ceramide (Fig. 6c). As seen from Fig. 6a, the SM species in human blood gave the main peaks of [M + H]+ and [M + Na]+ ions. Spectrum of the EPI scan of [M + H]+ ion at m/z 731.7

Fig. 5. (a) Negative-ion EMS spectrum of lysoPC species in human blood sample. (b) EPI spectrum of m/z 508.5 as shown in (a) representing the [M − 15]− ion of lysoPC.

in the positive ion mode (Fig. 6a) only provided abundant 184 fragment ion representing the choline ion(the figure not shown). In contrast, selection and collisional activation of [M + Na]+ at m/z 753.7 yields several informative product ions (Fig. 6b). The most abundant product ion is m/z 694.7, which corresponds to the loss of trimethylamine([M + Na-59]+ ). Other product ions at m/z 570.5, 548.5 and 530.5 correspond to three ceramidelike cations (i.e., [M + Na-183]+ , [M + Na205]+ , and [M + Na-205–H2 O], respectively). The product ion at m/z 147 corresponds to the sodiated five-member cyclophosphane. The ion at m/z 264.4 is the LCB ion with 18 carbon atoms [CH3 (CH2 )12 CH CH–CH C(NH2 )–CH2 ]+ . In addition to LCB ions, the ion at m/z 308.5 seems to be due

Table 4 Identification 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

716.7 718.7 742.8 744.8 746.8 766.8 768.8 770.8 790.8

C16:0/C16:1 C16:0/C16:0 C16:0/C18:2 C16:0/C18:1 C16:0/C18:0 C16:0/C20:4 C16:0/C20:3 C18:0/C18:2 C16:0/C22:6 C18:2/C20:4 C18:0/C20:4 C18:2/C20:2 C18:0/C20:3 C16:0/C22:3

794.8 796.7 lysoPC

478.5 480.5 494.5 504.5 506.5 508.5 528.5

C16:1 C16:0 C17:0 C18:2 C18:1 C18:0 C20:4

C18:2/C18:2 C18:1/C18:2 C18:1/C20:5 C18:1/C20:3 C18:1/C20:2

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Fig. 6. (a) Positive-ion EMS spectrum of SM species in human blood sample. (b) EPI spectrum of m/z 753.7 as shown in (a) representing the [M + Na]+ ion of SM. (c) Composition of SM.

to C18:0 [(CH2 CH N)COC17 H35 ]+ which reflect the fatty acid composition. Therefore, the [M + Na]+ ion at m/z 753.7 is composed of dC18:1/C18:0 (the designation of SM is in the form of d-LCB/FA, with d referring to the stereochemistry of the long chain base (LCB) and FA referring to fatty acid, shown in Fig. 6c). However, the abundance of LCB ion and FA ion in the positive-ion EPI spectrum is relatively low, sometimes they can’t be detected. Table 5 lists the identified SM species in human blood. From the characterization of phospholipids described above, it could be known that in the negative ion mode, CAD using QTRAP mass spectrometry gave primarily the sn-1 and sn-2 carboxylate anions together with lyso-phosphatidic acid with neutral loss water, and these structurally informative fragment ions are almost same as those obtained with a triple quadrupole mass spectrometer [33,36]. However, collision-induced dissociation (CID) using an ion-tap mass spectrometer [25], the product ions were mainly sn-1 and sn-2 lyso-phospholipids with neutral loss of ketene in combination with neutral loss of the polar head group. The difference may be from the different fragmentation mechanisms between triple quadrupole mass spectrometer and ion trap mass spectrometer [37]. The QTRAP instrument is based on

a triple-quadrupole ion path using the final quadrupole as a linear ion trap mass spectrometer. According the fragment ions obtained with our method, the fragmentation mechanism in the EPI scan mode under QTRAP mass spectrometer is more similar to triple quadrupole mass spectrometer.

Table 5 Molecular species identification of sphingomyelins (SM) in human blood sample PL Class

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

Combinations of molecular species

SM

711.7 723.7 725.7 739.7 741.7 751.7 753.7 755.7 779.7 783.7 823.6 853.7

33:1 34:2 d18:1/C16:0 35:1 35:0 dC18:2/C18:0 dC18:1/C18:0 36:0 38:2 38:0 dC18:1/C23:0 43:0

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4. Conclusions The normal-phase HPLC-ESI/MS method described is proved to be valuable for the optimal separation and identification of different phospholipid species in human blood. In the present study, we chose hexane/1-propanol as mobile phase instead of chloroform/methanol. Furthermore, the use of ammonia as a mobile phase modifier in place of TEA not only improved the resolution but also avoided the bad effect of TEA on the ionization of basic compounds in the positive mode. The combination of highly selective triple quadrupole MS/MS scans and high sensitivity ion trap product scans on the QTRAP instrument provides rapid identification of phospholipids of extracted human blood sample. Using the present method, information regarding the molecular mass, the polar head group and the fatty acyl substituents can be obtained from the positive or negative ion EMS and EPI scan mode. Using the present HPLC/MS system, more than 100 phospholipid species (including isomers) were identified. Among them, C18:1/C20:6 PE and C18:0/C22:7 PS have not been reported to be present in blood, they may be new finding in the lipid field The fragmentation patterns of phospholipids obtained under positive-ion or negative-ion ionization conditions are helpful in characterizing different classes of phospholipid species in complex samples. We believe that this method will be valuable in lipid research such as in studying phospholipid profiles in different biological samples.

Acknowledgements This study has been supported by the high-technology development plan “863 project” (2003AA223061) of State Ministry of Science and Technology of China and the Knowledge Innovation Program of the Chinese Academy of Sciences (K2003A16).

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