Direct Mass Spectrometric Analysis of Ozonides: Application to

Kathleen A. Harrison and Robert C. Murphy*. Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson ...
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Anal. Chem. 1996, 68, 3224-3230

Direct Mass Spectrometric Analysis of Ozonides: Application to Unsaturated Glycerophosphocholine Lipids Kathleen A. Harrison and Robert C. Murphy*

Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson Street, Denver, Colorado 80206

The reaction of ozone with double bonds present in glycerophosphocholine lipids results in formation of ozonides that can be directly analyzed by mass spectrometry as either positive or negative molecular ion species generated by electrospray ionization. Polyunsaturated fatty acyl groups esterified to the phospholipid yielded a mixture of ozonide species with the maximum number of ozone molecules added equal to the total number of double bonds. Ozonide decomposition resulted in ω-aldehyde and ω-carboxylic acid products as revealed by ESIMS. Collisional activation of the ozone adducts for monoand polyunsaturated phospholipids gave rise to fragment ions indicative of the position of the double bonds in these molecules. The major decomposition pathway for either positive or negative ozonide ion species involved charge remote fragmentation of the ozonide initiated by homolytic cleavage of the peroxide bridge followed by rearrangement to form the ω-aldehyde and ω-carboxylate acyl species. The reaction of ozone with phospholipids containing polyunsaturated fatty acyl groups is a useful method to probe the position of double bonds by electrospray ionization mass spectrometry. Development of fast atom bombardment and electrospray ionization (ESI) has permitted the direct mass spectrometric analysis of complex lipid substances including glycerophospholipids.1,2 Both positive and negative ions are readily derived from such compounds including glycerophosphocholine lipids (GPC) indicative of the molecular weight of the molecule. Collisioninduced decomposition (CID) of these molecular ion species has been shown to yield an abundant array of product ions which reveal relevant structural features that now make it possible to characterize endogenous molecular species of glycerophospholipids isolated from cells.3,4 A more challenging analytical task has been to structurally characterize positions of double bonds within polyunsaturated fatty acyl groups as well as structurally characterize oxidized fatty acyl groups such as ozonides present in glycerophospholipids. This is particularly true when only small quantities of material are available. High-energy collisional activation of carboxylate anions and subsequent charge site remote fragmentation has been used to determine positions of double bonds in fatty acyl groups of (1) Jensen, N.; Gross, M. L. Mass Spectrom. Rev. 1988, 7, 41. (2) Kim, H. Y.; Wang, T. L.; Ma, Y. C. Anal. Chem. 1994, 66, 3977. (3) Murphy, R. C.; Harrison, K. A. Mass Spectrom. Rev. 1994, 13, 57-75. (4) Kerwin, J. L.; Tuininga, A. R.; Ericsson, L. H. J. Lipid Res. 1994, 35, 10021114.

3224 Analytical Chemistry, Vol. 68, No. 18, September 15, 1996

Scheme 1

glycerophospholipids.5,6 Additional studies have suggested that tandem mass spectrometry does provide structural information for epoxides,7 hydroperoxides,8 and hydroxy9,10 substituents in fatty acids and phospholipids. More diverse structures such as products of the reaction of ozone with unsaturated glycerophospholipids have not been widely studied. Ozone adds rapidly almost quantitatively to carbon-carbon double bonds to form an initial 1,2,3-trioxolane (molozonide) which undergoes rearrangement (Scheme 1) to the more stable 1,2,4-trioxolane (Criegee ozonide) via a carbonyl oxide and aldehyde intermediates.11 Ozone is known to react with phospholipids containing polyunsaturated fatty acids esterified to the glycerol backbone. These lipid ozonides can propagate further lipid peroxidation, activate lipases, and lead to the release of lipid mediators.12,13 Furthermore, the technique of ozonolysis has been used for many years to aid in the structural elucidation of unsaturated natural products14 for determination of double bond position. The direct analysis of lipid ozonides by mass spectrometry has been limited because of their chemical reactivity. Oxidation or reduction of a Criegee ozonide leads to carboxylic acids or aldehydes, respectively, marking the original unsaturated carbon atoms. This feature of ozonolysis has been used as a means to structurally characterize the unsaturated precursor through analysis of degradation products by gas chromatography/mass (5) Jensen, N.; Tomer, K. B.; Gross, M. L. Lipids 1986, 21, 580. (6) Bryant, D. K.; Orlando, R. C.; Fenselau, C.; Sowder, R.; Henderson, L. E. Anal. Chem. 1991, 63, 1110. (7) Bernstrom, K.; Kayganich, K.; Murphy, R. C. Anal. Biochem. 1991, 198, 203-211. (8) MacMillan, D. K.; Murphy, R. C. J. Am. Soc. Mass Spectrom. 1995, 6, 11901201. (9) Zhang, J. Y.; Nobes, B. J.; Wang, J.; Blair, I. A. Biol. Mass Spectrom. 1994, 23, 399-405. (10) Wheelan, P.; Zirrolli, J. A.; Murphy, R. C. Biol. Mass Spectrom. 1993, 22, 465-473. (11) Criegee, R. Angew. Chem., Int. Ed. Engl. 1975, 14, 745-752. (12) Pryor, W. A. Free Radical Biol. Med. 1994, 17, 451-465. (13) Wright, D. T.; Adler, K. B.; Akley, N. J.; Dailey, L. A.; Friedman, M. Toxicol. Appl. Pharmacol. 1994, 127, 27-36. (14) Hamberg, M. Anal. Biochem. 1971, 43, 515. S0003-2700(96)00302-2 CCC: $12.00

© 1996 American Chemical Society

spectrometry techniques with nanomolar quantities of material. The intact ozonide of methyl oleate has been analyzed by direct probe introduction and chemical ionization mass spectrometry,15 and several fragment ions were observed which were informative as to the location of the 1,2,4-trioxolane group in the molecule. Santrock et al.16 used fast atom bombardment mass spectrometry to analyze the products of the ozonolysis of 1-palmitoyl-2oleoylglycerophosphocholine liposomes but observed only degradation products from the formed ozone adduct. We report for the first time the mass spectrometry by electrospray ionization of ozone addition products of glycerophosphocholine lipids containing polyunsaturated fatty acyl groups. Unique fragmentations of the ozonides were observed after collision-induced decomposition of the molecular ion species from both positive and negative ions. Ozone adduct formation and direct analysis by ESI-MS/MS was carried out with picomole quantities of phospholipid, and this technique represents a significant improvement over a conventional ozonolysis followed by GC/MS analysis of products to determine double bond location in unsaturated fatty acyl groups esterified to glycerophosphocholine lipids. EXPERIMENTAL SECTION Materials. All solvents were HPLC grade (Fisher, Fair Lawn, NJ) and reagents of the highest grade commercially available. All phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL) or Sigma Chemical Co. Oxygen used to generate ozone was 99.9% pure, and argon used as collision gas in the tandem mass spectrometer was 99.9% pure. 1-Palmitoyl-2-(5-hydroxyeicosatetraenoyl)glycerophosphocholine was generated by free-radical oxidation of 1-palmitoyl2-arachidonoyl-GPC and purified by reverse phase HPLC as previously described.17 1-Palmitoyl-2-oleoyl-2[H]9-glycerophosphocholine was made from 1-palmitoyl-2-oleoylglycerophosphoethanolamine and deuteriomethyl iodide previously described.18 The reaction with ozone was carried out on a dry, thin film of lipid as previously described19 with minor modifications. Ozone was generated in a commercial apparatus (Supelco, Belefonte, PA) by passing a stream of oxygen (20 mL/min) through a tesla coil fitted with a glass sleeve around the high-voltage electrode. The lipid was prepared by vacuum evaporating a solution of 100-200 ng in a glass capillary (5 cm × 2 mm). The ozone/oxygen gas mixture was passed for 2-8 min through the glass apparatus in which the lipid-containing capillary had been placed. The ozone leaving the apparatus was vented through a beaker of water. The lipid was dissolved by adding 50 µL of the electrospray mobile phase (methanol/20 mM ammonium acetate, 85:15) to the capillary and analyzed within 1 h. Mass spectrometry was carried out using a Sciex API-III+ tandem quadrupole mass spectrometry (Thornhill, Ontario, Canada). An orifice potential of 35-45 V was used to minimize collisioninduced dissociation of ions in the region prior to the first quadrupole lens. Flow injection into the mass spectrometer was employed using 5 µL of the solution containing 10-20 ng of (15) Wu, M.; Church, D. F.; Mahier, T. J.; Barker, S. A.; Pryor, W. A. Lipids 1992, 27, 129-135. (16) Santrock, J.; Gorski, R. A.; O’Gara, J. F. Chem. Res. Toxicol. 1992, 5, 134141. (17) Harrison, K. A.; Murphy, R. C. J. Biol. Chem. 1995, 270, 17273-17278. (18) Kayganich-Harrison, K.; Murphy, R. C. Anal. Biochem. 1994, 221, 16-24. (19) Wheelan, P.; Zirrolli, J. A.; Morelli, J. G.; Murphy, R. C. J. Biol. Chem. 1993, 268, 25439-25448.

Table 1. Positive Ions Obtained by Electrospray Ionization of Ozone Adducts of Glycerophosphocholine Lipids Having Unsaturated Fatty Acyl Groups at the sn-2 Position glycerophosphocholine lipid molecular speciesa ozone adduct (product) tetraozonide triozonideb diozonideb monoozonideb unreacted starting material ω-carboxylate (first double bond)c ω-aldehyde (first double bond)c ω-aldehyde triozonide loss of H2O ω-aldehyde diozonide loss of H2O ω-aldehyde monoozonide loss of H2O other ions ω-carboxylate-K+ adduct ω-carboxylate-Na+ adduct

16:0/ 18:1

808 (90) 760 (5) 666 (35)

[2H9]16:0/ 18:1

817 (92) 769 (27) 675 (33)

16:0/ 18:2

18:0/ 20:4

1002 (23) 954 (5) 854 (100) 906 (7) 806 (8) 858 (6.5) 758 (16) 810 (56) 666 (18) 638 (17)

650 (100) 659 (100) 650 (51)

622 (100)

738 (27) 720 (9.8)

886 (13) 868 (5.1) 798 (26) 780 (13) 710 (45) 692 (17)

704 (20) 688 (12) 636 (11)

713 (23) 697 (13) 645 (31)

704 (9.3) 688 (5.6) 636 (5.7)

650 (17) 522 (14) 608 (12)

a The observed mass-to-charge ratio corresponding to [M + H]+ is reported (except as noted) with relative abundance in parentheses. b Mixture of isomers. c The carboxylic acid or aldehyde moiety at the sp2 carbon atom closest to the glycerol ester of the original fatty acyl group.

oxidized lipid. All of the sample was sprayed into the mass spectrometer at a flow rate of 50 µL/min. Tandem mass spectrometry of ozone adducts was performed with collision energy at 18-20 eV and a collision gas thickness of 220 × 1013 argon atoms/cm2. The resultant ions are reported as truncated, nominal masses, but the observed masses of the molecular ion species were typically 0.5-0.6 u above the nominal mass because of the total number of hydrogen atoms in these glycerophosphocholine species. RESULTS AND DISCUSSION 1-Palmitoyl-2-oleoylglycerophosphocholine (16:0/18:1GPC).20 Reaction of ozone with 16:0/18:1-GPC led to the rapid formation of the 16:0/18:1-ozonide-GPC, which could be observed as the [M + H]+ at m/z 808 in the electrospray mass spectrum. This ion at m/z 808 was 48 u greater than the protonated molecular ion for unreacted 16:0/18:1-GPC (m/z 760) (Table 1). Additional alkali metal adduct ions were observed at m/z 830 and 846 corresponding to the sodium and potassium adduct ions, respectively. The abundant ions observed at m/z 650 and 666 were consistent with the protonated molecular ions expected for 1-palmitoyl-2-(9-carboxynonanoyl)-GPC and 1-palmitoyl-2(9-oxononanoyl)-GPC, the known decomposition products of (20) Abbreviations for individual glycerophosphocholine molecular species used are as follows: n:j/s:t-GPC (e.g. 16:0/20:4-GPC) where n is the number of carbon atoms in the sn-1 substituent and j is the number of double bonds in the sn-1 hydrocarbon chain; s is the number of carbons and t is the number of double bonds in the sn-2 fatty acyl substituent. GPC stand for glycerophosphocholine as the sn-3 polar head group bonded as a phosphate ester to glycerol. The recommended nomenclature for phospholipids results in complex names and this nonstandard abbreviation system is useful to fully describe the glycerophosphocholine molecular species in this study.

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Scheme 2

Figure 1. (A) Product ions obtained from collision-induced decomposition in a tandem quadrupole mass spectrometer of m/z 808 [M + H]+ ion obtained from 1-palmitoyl-2-oleoyl-ozonide-GPC. (B) Mass spectrum following collision-induced decomposition of the acetate adduct ion designated as A- or [M + OAc]- from 1-palmitoyl-2-oleoylozonide-GPC observed at m/z 866. The fragment ions at m/z 634 and 664 arise following the suggested loss of neutral species from the acetate adduct ion A-. Collision energy for both MS/MS experiments was 20 eV (laboratory frame of reference) using argon as collision gas.

16:0/18:1-ozonide-GPC.15,16 Alkali metal adduct ions were also observed for these species at m/z 672, 688, and 704. At higher orifice potentials used for the electrospray, the abundance of the ozonide ion at m/z 808 was somewhat reduced relative to the decomposition products observed at m/z 650 and 666. It is not clear whether the ions observed at m/z 666 and 650 in the electrospray mass spectrum reported in Table 1 are the result of collision-induced dissociation in the ionization/desolvation region of the mass spectrometer or exist as chemically discreet products of ozonide decomposition prior to ESI-MS analysis. Tandem mass spectrometry was used to determine the collision-induced dissociation behavior of the 16:0/18:1-ozonide-GPC. The ion at m/z 808 (Figure 1A) decomposed to yield major product ions at m/z 666 and 650 as well as other ions characteristic of all GPC molecular species, viz. m/z 184 and 222.3,4 A possible mechanism leading to the formation of these two ions proceeds by initial homolytic cleavage of the peroxide bridge of the trioxolane followed by rearrangement to yield either the aldehyde functionality at carbon-9 (m/z 650) or the carboxylic acid functionality at carbon-9 (m/z 666) (Scheme 2). Negative ions generated by electrospray ionization of 16:0/ 18:1-ozonide-GPC were similar in many respects to those observed as positive ions. The most abundant ions appeared at m/z 866, 708, and 664 (data not shown), at low orifice voltages. The ion at m/z 866 corresponded to the acetate adduct of 16:0/18:1-ozonideGPC. This adduct has been previously observed for other glycerophosphocholine lipids.21 The ion at m/z 708 corresponded to the acetate adduct of the 1-palmitoyl-2-(9-oxononanoyl)-GPC, 3226

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and the ion at m/z 664 would correspond to the [M - H]- ion formed for 1-hexadecanoyl-2-(9-carboxynonanoyl)-GPC. The electrospray mass spectrum of GPC molecular species containing dicarboxylic acids (esterified at the sn-2 position) typically form [M - H]- anions rather than [M - CH3 ]- 19 or [M + acetate]anions. (sn-1 and sn-2 refer to a stereospecific numbering (sn) system used to assign the positions of substituents as sn-1, sn-2, and sn-3 for the three carbon atoms in glycerol using the IUPACIUB recommended nomenclature of lipids.) It was previously observed that these acetate adduct ions for glycerophosphocholine lipids decompose as the orifice potential is increased.21 At an orifice potential of -100 V, ions at m/z 634, 666 (corresponding to the hemiacetal of m/z 634 formed with methanol in the mobile phase), and 664 were observed as abundant anionic species whereas the ion at m/z 866 was no longer present. Collisioninduced decomposition of acetate adduct ions of glycerophosphocholine lipids typically yields abundant carboxylate anions corresponding to each group esterified at the sn-1 and sn-2 positions as well as loss of a methyl group from the choline residue [M CH3]- as methyl acetate. However, collision-induced decomposition of m/z 866 [M + OAc]- yielded abundant product ions at m/z 634 and 664 corresponding to the 9-oxononanoyl ester at sn-2 as an [M - CH3]- anion and the 9-carboxynonanoyl ester at sn-2 [M - H]- anion (Figure 1B). The mechanism of formation of these ions would also be consistent with Scheme 2 for a charge site remote rearrangement of the trioxolane ring with a possible participation of the acetate anion in removing the proton from carbon-9 facilitating formation of m/z 664. Three separate carboxylate anions were observed at lower mass at m/z 171, 201, and 255 corresponding to the groups esterified to both the sn-2 and sn-1 positions of GPC ozonide decomposition products. m/z 255 corresponded to a carboxylate anion for the sn-1 hexadecanoic acid group while m/z 171 and 201 would be the expected carboxylate anions for 9-oxo and 9-carboxymethyl acyl groups, the latter being formed by N-methyl transfer from the choline residue to the 9-dicarboxylic acid.18 Since the collision-induced dissociation of m/z 866 in the second quadrupole did not yield an ion at m/z 708, this ion observed in the initial electrospray mass spectrum likely corresponded to the 9-oxo compound present as a discreet product in the initial ozonolysis mixture. Collision-induced decomposition of (21) Harrison, K. A.; Murphy, R. C. J. Mass Spectrom. 1995, 30, 1772-1773.

Scheme 3

16:0/18:1-ozonide-2[H]9-GPC confirmed the identification of the product ions at m/z 664, 634, and 201 as [M - H]- , [M - CH3]-, and methyl nonanedioate from the mass shift of these ions to m/z 673, 640, and 204, respectively (data not shown). Homolytic cleavage of the peroxide bridge of the 1,2,4trioxolane depicted in Scheme 2 could lead to a series of other fragment ions observed in the collision-induced decomposition of the positive ions generated by electrospray ionization. The positive ion at m/z 621 could be formed by radical cleavage of the bond between carbon atoms 8 and 9 initiated by the oxygencentered radical at carbon-9 (Scheme 3). In a more complicated rearrangement, this initial oxygen-centered radical at carbon-9 could lead to cleavage of the carbon 2, 3 bond by way of a transition state involving two six-membered rings (Scheme 3), resulting in the ion at m/z 537. A less abundant ion at m/z 550 could result from cleavage between carbons 3 and 4 with double bond formation between carbons 2 and 3 by a similar mechanism driven by the same oxygen-centered radical. 1-Palmitoyl-2-linoleoylglycerophosphocholine (16:0/18: 2-GPC). The reaction of ozone with polyunsaturated fatty acids can lead to the formation of several products, depending upon the completeness of the ozone reaction and ozonide decomposition rate. Ozone can react with 16:0/18:2-GPC to form three different ozonidessone diozonide and two isomeric monoozonides. The most abundant product observed after exposure of a thin film of 16:0/18:2-GPC to ozone for 2 min was the diozonide, as evidenced by an abundant [M + H]+ at m/z 854; the monoozonide products were substantially less abundant with [M + H]+ ions observed at m/z 806 (Table 1). The abundant ions at m/z 650 and 738 would correspond to [M + H]+ ions from the 1-palmitoyl-2-(9-oxononanoyl)-GPC and 1-palmitoyl-2-(12-oxo-9,10-trioxolandodecanoyl)GPC. Other minor ions were observed at m/z 942, 766, 746, 702, and 658 corresponding to the [M + H]+ or [M + 2H]2+ ions expected for possible cross ozonides formed during the rearrangement of the molozonide to the Criegee ozonide by carbonyl oxide intermediates.11 The tandem mass spectrometry of the [M + H]+ ions of the monoozonide of 16:0/18:2-GPC (Figure 2A) revealed several ions identical to that seen in Figure 1B for the 16:0/18:1-ozonide-GPC, viz. m/z 650, 621, and 537, suggesting that they were derived from the 9,10-ozonide of the linoleic acid residue. The ions at m/z 690 and 706, although of much lesser abundance, would correspond to the 12-oxo and 12-carboxy decomposition products of the 12,13ozonide. Most striking was the abundance of an odd electron ion at m/z 661. This ion could arise by the mechanism outlined in Scheme 4, where the oxygen-centered radical at carbon-12

Figure 2. (A) Product ions obtained from the collision-induced decomposition of m/z 806 [M + H]+ from the monoozonide of 1-palmitoyl-2-linoleoyl-ozonide-GPC. This was a mixture of two isomeric monoozonide species, the 9,10-monoozonide and 12,13monoozonide species. (B) Product ions obtained by collision-induced decomposition of the [M + H]+ from 1-palmitoyl-2-linoleoyl-diozonideGPC observed at m/z 854. Collisional activation was identical to that described in the Figure 1 caption.

Scheme 4

would decompose to the carbon-11-centered radical, which would be resonance stabilized by the remaining double bond at carbons 9 and 10. The formation of this stabilized radical appears to be the preferred pathway for the collision-induced decomposition of the 12,13-monoozonide rather than the decomposition of the ozonide leading to the aldehyde or carboxy functionality at carbon12. The tandem mass spectrometry of the [M + H]+ ions from the 16:0/18:2-diozonide (m/z 854) resulted in ions observed at m/z 754, 738, 666, and 650 (Figure 2B). These ions likely formed Analytical Chemistry, Vol. 68, No. 18, September 15, 1996

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through the previously described mechanism of homolytic cleavage of the peroxide bond and formation of the ω-carboxy and ω-aldehyde species at the carbon atom in the original double bond closest to the phosphocholine charge site. 1-Stearoyl-2-arachidonoylglycerophosphocholine (18:0/ 20:4-GPC). The reaction of ozone with an arachidonatecontaining glycerophosphocholine could lead to the formation of 15 separate ozonides in four separate familiessmonoozonide, diozonide, triozonide, and tetraozonide. The positive ion electrospray mass spectrum of the products obtained following the exposure of ozone to 18:0/20:4-GPC as described in the Experimental Section resulted in a complex mixture of products (Table 1) with most abundant intact ozonide species corresponding to the tetraozonide ([M + H]+, m/z 1002) and substantially lesser quantities of the mono-, di-, and triozonides observed as the corresponding [M + H]+ ions at m/z 858, 906, 954, respectively. The presence of an abundant ion at m/z 810 corresponding to unreacted 18:0/20:4-GPC suggested the ozonation reaction was not as complete under these conditions as that seen for the previously discussed unsaturated glycerophosphocholine lipids. The most abundant ion in the electrospray mass spectrum appeared at m/z 622, corresponding to 1-stearoyl-2-(5-oxo-pentanoyl)-GPC, which would likely be a discreet product from the decomposition of the 5,6-ozonide as well as a fragment ion of the corresponding ozonide during electrospray process. The ions observed at m/z 710, 798, and 886 would correspond to the [M + H]+ ions for the 8-oxo, 11-oxo, and 14-oxo fragments of the 18: 0/20:4-tetraozonide-GPC. Dehydration of these would lead to the ions observed at m/z 694, 780, and 868, respectively. The product ion mass spectrum (Figure 3A) following collisional activation of m/z 1002 ([M + H]+; 18:0/20:4-tetraozonideGPC) yielded a family of fragment ions at m/z 886, 798, 710, and 622 that were diagnostic for the position of the original double bonds in the arachidonoyl moiety at sn-2. These ions corresponded to the terminal aldehyde fragments that would arise following homolytic cleavage of the 14,15-trioxolane, 11,12-trioxolane, 8,9-trioxolane, and the 5,6-trioxolane species, respectively (see Scheme 2). The terminal carboxyl fragments were also observed at m/z 902, 814, 726, and 638. The formation of these four pairs of ions differing by 16 u and each set differing by 88 u revealed the homoconjugated double bond positions found in arachidonic acid with the first double bond at carbon-5. Collisional activation of the [M + H]+ ion corresponding to the 18:0/20:4-monoozonides (m/z 858) revealed a series of abundant odd-electron ions consistent with a mixture of the four possible monoozonides with cleavage of the carbon bond adjacent to the trioxolane ring (Figure 3B). The abundance of these oddelectron ions are a result of the stabilization of the carbon-centered radical site by an adjacent double bond as previously discussed (Scheme 4). In the case of the 5,6-monoozonide, the formation of the resonance-stabilized radical would result in cleavage of the C(7)-C(8) bond with formation of the 5-oxopentanoyl fragment, which retains the charge site. The abundance of this ion at m/z 578 rather than the radical cleavage of C(4)-C(5) (Scheme 4) reinforces the importance of the adjacent double bond in stabilizing the radical site. 1-Palmitoyl-2-(5-hydroxyeicosatetraenoyl)glycerophosphocholine (16:0/5-HETE-GPC). The reaction of ozone with the double bonds present in a structurally diverse fatty acyl group was studied using 16:0/5-HETE as a model compound 3228 Analytical Chemistry, Vol. 68, No. 18, September 15, 1996

Figure 3. (A) Product ions obtained following collision-induced decomposition of the tetraozonide of 1-stearoyl-2-arachidonoyl-GPC [M + H]+ observed at m/z 1002. Collisional activation conditions were identical to that reported in the Figure 1 caption. (B) Product ions obtained following collision-induced decomposition of the [M + H]+ ion for monoozonide mixture obtained from 1-stearoyl-2-arachidonoylGPC. The positions of four different monoozonides are indicated by the structure (inset) with an arrow for the ozone attachment position and the observed fragment ion indicated adjacent to the arrow. For example, the 14,15-ozonide resulted in an abundant product ion at m/z 713 following collision-induced decomposition of m/z 858. The 5-, 8-, and 11-double bonds would be intact in this monoozonide species.

which contained two methylene interrupted double bonds, a conjugated diene, and a hydroxyl substituent on the sn-2 fatty acyl chain. Direct electrospray mass spectrometry of the reaction products revealed the most abundant ion at m/z 798 corresponding to the [M + H]+ of unreacted 16:0/5-HETE-GPC . However, ions were observed at m/z 594 and 624 that corresponded to the terminal aldehyde decomposition products of the ozonides, specifically 16:0/5-oxopentanoyl-GPC and 16:0/6-oxo-5-hydroxyhexanoyl-GPC. Ions of reasonable abundance were observed corresponding to [M + H]+ for the mono-, di-, tri-, and tetraozonides at m/z 846 (19%), 894 (10%), 942 (11%), and 990 (6%), respectively, even though the most abundant species was unreacted starting material at m/z 798 (100%). It would thus appear that the ozonides could be formed with all double bonds in this molecule including the conjugated diene. Collision-induced decomposition of the [M + H]+ ions for monoozonide resulted in product ions corresponding to decomposition by the mechanisms previously described in Schemes 2-4 initiated by homolytic cleavage of the peroxide bridge (Figure 4A). However, the hydroxyl substituent at carbon-5 did play a

Figure 4. (A) Product ions obtained following the collision-induced decomposition of monoozonide of 1-palmitoyl-2-(5-hydroxyeicosatetraenoyl)GPC [M + H]+ observed at m/z 846. The origins of the most abundant ions are suggested in the structure (inset) for the positions of each monoozonide as described in the Figure 3 caption. (B) Product ions obtained following the collision-induced decomposition of the tetraozonide of 1-palmitoyl-2-(5-hydroxyeicosatetraenoyl)-GPC [M + H]+ observed at m/z 990.

significant directing role in the formation of certain ion products. This was exemplified by the detailed investigation of the decomposition products of m/z 846, which was likely a mixture of the [M + H]+ ions from all four isomeric monoozonides of 16:0/5HETE-GPC. A minor product ion observed at m/z 661 would likely arise from the cleavage of the 11,12-ozonide, according to the mechanism outlined in Scheme 4, but after a further loss of the 5-hydroxy group as water, the most abundant ion at m/z 643 was formed. In a similar manner, the ion at m/z 701 would result from cleavage of the 14,15-ozonide species and further yield the ion at m/z 683 following the loss of water. The mechanism in Scheme 4 does not appear to operate for ozonides formed at the original conjugated diene position, the 6,7- or 8,9-ozonides, since no stabilized allylic radical would be formed with such a cleavage reaction. The 8,9-ozonide could undergo homolytic fragmentation to form a stabilized radical species at carbon-10 stabilized by resonance, but the resulting oxygen-centered radical at carbon-8 would most likely decompose to the 8-oxo species at m/z 650. The ion at m/z 594 can be derived from the 6,7-ozonide as shown in Scheme 5 with the loss of a neutral aldehyde and a neutral molecule of formic acid. Each of the four monoozonide isomers appeared to fragment to ions indicative of the position of the

Scheme 5

ozonide, but the presence of the hydroxy substituent and the presence of a conjugated diene alter the fragmentation pathways observed for the methylene-interrupted double bonds observed with other phospholipids (vide supra). Taken together, all four Analytical Chemistry, Vol. 68, No. 18, September 15, 1996

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double bond positions could be ascertained from the collisioninduced decomposition of the monoozonide mixture. Chromatographic separation of each isomer followed by collision-induced decomposition of the individual species would be necessary to confirm these postulated fragmentation pathways. The collision-induced dissociation mass spectrum from the tetraozonide of 16:0/5-HETE-GPC ([M + H]+, m/z 990) is shown in Figure 4B. The most abundant fragment ions appear at m/z 594, 624, and 184. Although fragmentation leading to m/z 594 likely arises from the mechanism shown in Scheme 5, this 5-oxo product ion would also arise from ozonides of 5,6-unsaturated species. The ion at m/z 624 however is unique, indicating the insertion of the hydroxy moiety at carbon-5 with a carbon-6,7 double bond. Ions at m/z 537, 550, and 565 arise from the mechanisms in Scheme 4. No ions were observed to indicate the positions of the other double bonds within the molecule, in contrast to the spectrum obtained from the monoozonide mixture shown in Figure 4A. CONCLUSIONS Ozone rapidly reacts with the double bonds present in glycerophosphocholine lipids containing unsaturated fatty acyl groups to form relatively stable ozonides that can be analyzed directly by electrospray ionization mass spectrometry. The maximal number of ozonides observed was identical to the total number of double bonds, even when conjugated double bonds were present in the fatty acyl group. Collision-induced decomposition of the ozonides of unsaturated phospholipids gave rise to fragment ions indicative of the position of the double bonds within these compounds. The major mechanism for decomposition appeared to involve charge remote fragmentation of the 1,2,4trioxolane (Criegee ozonide) ring with homolytic cleavage of the

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Analytical Chemistry, Vol. 68, No. 18, September 15, 1996

peroxide bridge followed by rearrangement to form various aldehyde and carboxylic acid species. Charge remote fragmentation appears to be particularly facilitated in phospholipids because the charge site is localized on the polar head group of the phospholipid, quite removed from the unstable 1,2,4-trioxolane ring. For the most common polyunsaturated fatty acids that contain methylene-interrupted double bonds, partial ozonide formation leads to a facile pathway of decomposition involving formation of a resonance-stabilized radical species as a result of cleavage of the carbon-carbon bond immediately adjacent to the ozonide ring. Furthermore, additional structural functionality such as a hydroxyl group adjacent to a double bond strongly influenced the decomposition pathway of the ozonide through transfer of a radical site to the oxygen-centered radical of the carbinol and loss of the ozonide oxygen atoms as small neutral molecules. Electrospray and tandem mass spectrometry offers a unique advantage in characterizing the polyunsaturated fatty acyl groups esterified to the glycerol backbone of naturally occurring glycerophosphocholine lipids, and furthermore, the reaction with ozone adds a useful method to probe the position of double bonds present in unknown structures of phospholipids through analysis of the resultant intact ozonides. ACKNOWLEDGMENT This work was supported, in part, by a grant from the National Institutes of Health (HL34303). Received for review March 26, 1996. Accepted July 9, 1996.X AC960302C X

Abstract published in Advance ACS Abstracts, August 15, 1996.