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BIOMEDICAL AND ENVIRONMENTAL MASS SPECTROMETRY, VOL. 18, 328-336 (1989) Characterization of Epoxides of Polyunsaturated Fatty Acids by Mass Spectrometry via 3-Pyridinylmethyl Esters Michael Balazy and Alan S. Niest University of Colorado Health Sciences Center, Division of Clinical Pharmacoiogy, Box C-237, Denver, Colorado 80262, USA The isomeric epoxides of linoleic, arachidonic and docosahexaenoic acids were prepared by reaction with mchloroperoxybenzoic acid and, after separation by normal-phase high-performance liquid chromatography, were esterified with 3-pyridylcarbinol via the unstable imidazolide generated by the reaction with 1 ,l'carbonyldiimidazole. The electron impact mass spectra of these derivatives showed a molecular ion and a sequence of peaks with two characteristic abundant ions that resulted from formal cleavage of the carbon-carbon bonds at the oxirane ring. Both these ions retained the ester group. This fragmentation pattern allowed the unequivucal identification of the separate epoxide isomers. INTRODUCTION Oxygenation via cytochrome P450 linked monoxygenase is recognized as an important metabolic pathway of polyunsaturated fatty acids such as linoleic, arachidonic and docosahexaenoic acids. Among several types of products of the NADPH-dependent oxygenation, unsaturated epoxides have attracted attention because of their diverse biological actions, although their physiological or pathophysiological role is unclear. A variety of cell preparations has been reported to produce these epoxides. For example, both expoxides of linoleic acid have been found in incubates of leukocytes from hyperoxic lung,* and one of them, 9,10-epoxy-12octadecenoic acid (leukotoxin), causes cardiac failure in dogs and is a smooth muscle r e l a ~ a n t .Arachidonic ~ acid epoxides, epoxyeicosatrienoic acids (EETAs), are found in microsomal preparations of rabbit liver4 and kidney' and also in human Isomeric EETAs show a broad spectrum of biological activity including inhibition of platelet aggregation and cyclooxygenase a ~ t i v i t y ,inhibition ~ of vasopressin-stimulated osmotic water f l o ~ relaxation ,~ of rabbit pulmonary artery,' release of somatostatin," leutinizing hormone,' ' insulin and glucagon,'2 and mobilization of microsomal c a l ~ i u m . ' ~Epoxides derived from docosahexaenoic acid, although not isolated, appear to be intermediate products in the formation of vicinal diols that have been extracted from rabbit liver microsomal incuba te^.'^ Structural differentiation of the regioisomeric epoxides by electron impact (EI) or chemical ionization (CI) mass spectrometry is difíicult with derivatives commonly used for gas chromatography (GC) such as methyl or trimethylsilyl esters because of extensive fragmentation in the low-mass range and similarity of spectra.'j4 Several methods have been proposed to identify the isomeric epoxides. Charge-exchange ionization with ' t Author to whom correspondence should be addressed 0887-61 34/89/050328-09 $05.00 (C 1989 by John Wiley & Sons, Ltd. carbon disulfide as the moderating reagent provides m a s spectra of methyl arachidonate epoxides with characteristic peaks at low masses and relatively weak molecular ions.15 Another method involves fast atom bombardment (FAB) desorption of lithiated fatty acid epoxide molecular ions, followed by collisional activation.I6 Although pentafluorobenzyl esters of epoxides produce an abundant M - 181 ion in negative ion Ci and are useful for their quantification,6 the structural information is lost because such derivatized isomeric epoxides display similar spectra and are not well resolved by capillary GC.' Other methods are based on the acidic aqueous hydrolysis of the epoxide with subsequent gas chromatography/mass spectrometry (GC/MS) of the silylated vicinal dio1 or permethylation of products after reaction with phenyltrimethylammonium hydroxide.' We report here a rapid, mild and quantiative derivatization method for preparation of 3-pyridinylmethyl (picolinyl) esters of epoxides derived from linoleic, arachidonic and docosahexaenoic acids. These picolinyl esters provided very characteristic spectra that allowed differentiation of the isomeric epoxides at nanogram levels. A previously reported method for preparation of picolinyl esters via fatty acid chlorides'8 could not be used for derivatization of these epoxides because of their instability in acidic solutions. ' EXPERIMENTAL Materials Epoxides were synthesized by the reaction of metachloroperoxybenzoic acid (1.5 mEq, 50 mM) with linoleic. arachidonic or docosahexaenoic acid (Nucheck) iri methylene chloride for 8 h at room temperature as d e ~ c r i b e d . After ~ the solvent was evaporated, the epoxides were injected in hexane onto a highperformance liquid chromatography (HPLC) column Receiued 16 Septemher 1988 Revised 22 Novemher 1988 329 EPOXIDES OF UNSATURATED FATTY ACIDS a AA C o MCPBA , i, r L Y 0 - 2 O N c, a m u c a n ii O UI n a O 10 20 retention time 30 C m i n l Figure 1. Normal-phase HPLC separation of epoxidec (EETA) synthesized from arachidonic acid (AA) and rn-chloroperoxybenzoic acid (MCPBA) as described in the text. (Altex, Ultrasphere Si, 250 x 4.6 mm, 5 pm particles) and eluted with hexane-2-propanol-acetic acid (500:0.5:0.25 v/v/v) at a ñow rate of 1 ml/min with ultraviolet (UV) detection at 200 nm. In some experiments 0.1 pCi of (l-14C)arachidonic acid was added to the reaction mixture in order to quantify the epoxides and to determine the yield of the subsequent derivatization. tions of the sample dissolved in hexane were made with a splitless injector. Mass spectra were recorded with a VG Micromass 16 spectrometer interfaced with a VG 2000 data system. Operating conditions were : electron energy, 70 eV; trap current, 200 pA; acceleration voltage, 4 kV; scan 3 s per decade in exponential downfield mode. Some epoxide esters were also analyzed with a 15 m DB-1 column interfaced with a VG 7070H mass spectrometer and a Teknivent data system. Derivatization To 0.15-1.5 nmol of epoxide dissolved in 100 p1 dry methylene chloride, 10 p1 of freshly prepared solution (50 pg/ml) of 1,l'-carbonyldiimidazolewas added. After 1 min, 10 pl of 1% 3-pyridylcarbinol solution was added along with 10 p1 of triethylamine. The reagent solutions were prepared with dry methylene chloride. After 10 min at 37°C the solution was washed with 1 N sodium hydroxide saturated with carbon tetra~ h l o r i d e , ' washed ~ with water and evaporated under nitrogen. The residue was redissolved in hexane-ethyl acetate (3: 1) and eluted from a 2 cm silica CC4 (Mallinckrodt) column. Epoxide esters with N , N dimethylethanolamine, 1-methyl-3-piperidinemethanol and 3-hydroxypyridine were prepared in the same way. Al1 reagents were purchased from Aldrich. Analysis by thin-layer chromatography (TLC) was performed on silica gel G plates (Analtech) with hexane-ethyl acetate (1 : 1 v/v) as eluent, and radioactivity was detected by using a computerized Berthold scanner. GC/MS A 30 m DB-1 column (J&W Scientific) was temperature programmed from 220 "C to 300 "C at 16 "C/min. Injec- RESULTS AND DISCUSSION Epoxides of the three unsaturated fatty acids-linoleic, arachidonic and docosahexaenoic acids-were prepared with good yield (6&70%) via the reaction with mchloroperoxybenzoic acid. The crude reaction mixture was analyzed by normal-phase HPLC, and a representative chromatogram for the epoxides obtained from arachidonic acid is shown in Fig. 1. The retention times for al1 prepared epoxides are reported in Table 1. Each of the separately collected epoxides was subsequently derivatized in a two-step esterification. First, by reaction with 1,l'-carbonyldiimidazole the unsaturated epoxy acid was converted into the reactive imidazolide which, without isolation, was reacted with 3pyridylcarbinol in the presence of triethylamine. The mild and basic conditions stabilized the epoxide throughout the derivatization, and the pyridine ring in the ester protected the epoxy group against acidic impurities during chromatography. The radioactivity added was quantitatively recovered in about 4 ml of eluate from the silica column chromatography. The crude reaction mixture applied directly to a TLC plate showed a single radioactive peak corresponding to the M. BALAZY AND A. S . NIES 330 Table 1. Normal-phase HPLC retention times (RT) for epoxides synthesized from linoleic, arachidonic and docosahexaenoic acids, and GC relative retention times expressed as carbon numbers (CN) for the picolinyl esters of the epoxides RTb (min) Relative peak area CNC 12.7 16.1 1 .o 1.3 27.8 14,15 11,12 8. 9 5. 6 11.4 12.2 16.2 24.4 3.1 1.4 1 .o 1.1 30.2 EDP 16,17 13,14 19,20 7, 8 10.8 11.7 13.5 17.1 1.1 1 .o 2.0 1.1 33.2 Epoxide" EOE 12,13 9, 10 EET EOE, epoxyoctadecenoic acid; EET, epoxyeicocatrienoic acid; ED P. epoxydocosapentaenoic acid. 1 mijmin hexane-2-propanol-acetic acid Conditionc: (500:0.5:0.25, v/v/v); column, Ultracphere Si, 250 x 4.6 rnm, 5 pm particles. "Conditionc: 15 m DE-1 column programmed from 200°C by 15 "C/min with helium as a carrier gas at linear flow of 40 crn/e. a esterified epoxide ( R , = 0.37). No spot was observed for underivatized epoxide at R, = 0.20. G C analysis produced single, symmetrical peaks for al1 expoxide derivatives studied, but the isomeric epoxides were not resolved with our G C conditions. It is possible that the described esterification could be applied differently. The biological extract, containing epoxides of polyunsaturated fatty acids, could be derivatized first, followed by separation of the picolinyl esters by HPLC. Although we have not tried to analyze the picolinyl epoxides by HPLC, we expect that a reasonable separation should be achieved, at least to the same extent as the quite satisfactory separation we have accomplished with the isomeric methyl arachidonate epoxides (not shown). This procedure would be more eficient, since only a single derivatization step would be required, and might be particularly useful when only small amounts of biologicai material are available. This approach also would offer the interesting possibility that a liquid chromatographic/mass spectrometric system could be used for an on-line separation and analysis of picolinyl epoxides. In our case, however, the amount of prepared epoxides was suficient to allow us to analyze the separate HPLC fractions in order to provide the unambiguous standard spectrum for each isomeric epoxide. Spectra of arachidonate epoxides Figure 2 shows the E1 m a s spectra of the picolinyl arachidonate epoxides. General features of these spectra in the low-mass range were similar to those observed previously by Harvey for picolinyl esters of polyunsaturated fatty acids." The m a s spectra of these compounds as well as of epoxides studied here showed abundant ions at m/z 92 (base peak), 93, 108, 151 (resulting from McLafferty rearrangement) and 164, indicating an analogous fragmentation pattern for these ions.18 A series of ions was also produced by radical-induced cleavage at each single carbon-carbon bond following a remote hydrogen abstraction from the unsaturated chain. However, the spectra of the epoxides showed severa1 distinct features that made them valuable for analytical purposes. The molecular ion (m/z 411) was observed in al1 spectra with a relative abundance of 1.5-8.0%. This ion was found at the odd mass because the derivative contained one nitrogen atom. The other high-mass ion that was frequently observed was M - 18, corresponding to loss of a molecule of water-a typical neutral loss found in the spectra of many epoxides.20.21Its abundance was about the same as that of the molecular ion. The most characteristic and diagnostic feature of the spectra of the picolinyl epoxides studied was the presence of two approximately equally abundant ions (called here a and b) resulting from the formal cleavage of the carbon-carbon bonds located at the oxirane ring. These ions are separated by 42 daltons. Both ions retained that part of the molecule containing the 3pyridinylmethyl ester function. Ion type b also formally retained the epoxide group. This type of fragmentation suggested the involvement of radical-induced cleavage from the remote pyridyl group. Adopting a similar pattern of fragmentation to that proposed by Harvey," the most plausible mechanism for the formation of ions a and b requires the initial rearrangement of methylene hydrogen to give a distonic cation in which the charge and radical sites are separated. For example, in the picolinyl ester of 11,12EET the migration of allylic hydrogen at carbon 7 (Fig. 2(b); Scheme 1) to the high hydrogen afinity pyridine ring results in movement of the site of the unpaired electron to that carbon. The cleavage of the bond between carbons 10 and 11 yields the ion m/z 258 (type a). This ion may have the structure of a terminally unsaturated fatty acid picolinyl cation. The second abundant ion was found at m/z 300 (type b) and may originate from radical abstraction of hydrogen at carbon 10 followed by a rearrangement in the oxirane ring and cleavage of the bond CI2-Cl3 (Scheme 1). This ion might have the structure of an even-electron aldehyde. Such an aldehyde ion may readily lose a neutral carbon monoxide, also producing an ion at m/z 272 (type c). In the spectra of derivatives of 5,6-EET and 8,9-EET, in addition to ions a and b, peaks at mfz 208 and 248 were found, respectively. Corresponding ions (type d), possibly of the structure of an aldehyde (Scheme 2) might result from carbon-carbon and carbon-oxygen bond cleavage within the epoxide ring. These ions were not observed in the two other epoxides. The relative preference of formation of ion type d versus c reflects the differences in the bond polarities between CO- and C-C bonds in the epoxide ring that are additionally influenced by the inductive effect of the ester group in the derivative of $6-, and 8,9-EET. In these two epoxide esters the ion at m/z 300 corresponded to the M - 11 1 fragement resulting from clevage of CI3-C,, bond. The ion M - 111 has been observed in E1 spectra of 33 1 EPOXIDES OF UNSATURATED FATTY ACIDS 92 1O0 l O 340 70 60 50 40 30 20 10 O 50 1O 0 150 200 250 300 350 400 450 400 450 M / Z 90 O -1 ""11 I 70 50 1O0 150 200 250 M / 300 350 Z of the double bonds certainly contributed to the shortsevera1 silyl ether and methyl ester derivatives of lipoxyening of the distance between the hydrogens and the genase metabolites of arachidonic acid, e.g. 12-HETEZ2 nirogen by bending the unsaturated chain towards the or LTB423 and also in the picolinyl derivative of arachiester group. This also implies that some hydrogens are donic acid. more favored for abstraction than others. The formation of distonic cations, as exemplified in The relations between the thermochemical and Scheme 1, followed by fragmentation to ions a and b, stereochemical factors has a clear impact on the spectra comprises two factors. The first, thermochemical, indistudied. For example, although the BDE of the allylic cates that the hydrogen(s) of the lowest C-H bond dishydrogens at C , and C in the picolinyl derivative of sociation energy (BDE) will be most easily abstracted as a radical by the positively charged picolinyl g r o ~ p . ~ ~11,12-EET (Fig. 2(b)) should be the same and lower than that of hydrogens at C , , the abstraction of hydroSecond, a certain spatial arrangement (stereochemical gen from the more remote C,, did not occur (the ion factor) is also necessary for hydrogen abstraction. It was found at m / z 354 was very small). Rather, an abstraction shown that in the primary, unbranched alkylamine radof the closer hydrogen at C , actually took place icals the energy barrier for hydrogen shift decreased producing ion b (m/z 258), aithough the BDE of the with increasing distance, and the 1 3 shift was the most C,-H bond was less favorable. Thus the stereochemif a v o ~ r a b l e In . ~ ~the compounds studied here the epoxy cal factor contributed to the higher abundance of ion a group lowered the dissociation energy of the adjacent over ion b in the epoxides studied: the distonic cation carbon-hydrogen methylene bonds, and the 2 geometry , M. BALAZY AND A. S. NIES 332 92 1O 0 90 80 70 60 50 40 30 20 1 0 O 50 I 1 O8 I 164 151 1 218 354 395411 L + 1 O0 150 250 200 350 300 400 450 M / Z 92 1 O0 208 90 80 70 60 50 40 30 20 10 O 50 1O 0 150 250 200 M / 300 350 400 450 Z Figure 2. Mass spectra (70 eV) of picolinyl esters of epoxides originated from arachidonic acid. About 400 ng of epoxide was derivatized to obtained each spectrum. Fragments formally corresponding to ions a and b are schematically indicated in the chemical structure. resulting in ion a was produced by abstraction of a hydrogen closer to the picolinyl group. Also in the spectrum of the picolinyl derivative of 5,6-EET (Fig. 2(d)) the peaks a and b were very small, reíiecting the very unfavorable conformation for hydrogen abstraction, especially for ion a ( m / z 178). The fragmentation of the picolinyl derivatives of arachidonate epoxides seemed to occur through distonic cations formed by the rearrangement of allylic hydrogens at both sides of the double bond adjacent to the epoxide ring. Spectra of linoleate and docosahexaenoate epoxides The general features of these spectra were similar to that observed for the arachidonate epoxides. In both picolinyl linoleate epoxides the intensity of the M - 18 peak (m/z 369) was about five time greater than that of the molecular ion peak. In contrast, the molecular ion was more abundant in picolinyl docosahexaenoate epoxides. Again, two characteristic ions of type a and h were observed (Figs 3 and 4) that helped to indicate the elution order of these epoxides from HPLC. The ion at m/z 164 was not abundant in the docosahexaenoate epoxides, refiecting the presence of the delta 4 double bond that prevented the loss of the allylic radical after cleavage between carbons C , and C,. The epoxidation of docosahexaenoic acid yielded very small amourits of the 4 3 and 10,ll isomers, and these epoxides were riot characterized as picolinyl esters. The picolinyl derivative of 13,14-epoxydocosapentaenoic acid (Fig. 3) showed a sequence of abundant ions 333 EPOXIDES OF UNSATURATED FATTY ACIDS .. H m/z 248 d O 0 H m/z 258 H a H O O f-'. m/z 208 d Scheme 2 H H - 0 - C8HlS " H m/z 300 b Scheme 1 beginning with an ion at m/z 164 and separated from subsequent ions by 40 daitons, which corresponds to a difference of a -CH=CH-CH,unit. Therefore, these fragment ions indicate the position of the double bonds in the unsaturated chain. A gap of 42 daltons was observed only between ions m/z 284 and 326, which indicates the presence of the oxirane ring. This spectrum therefore showed that there were three double bonds between the ester group and oxirane ring, namely delta 4. 7 and 11. The other two double bonds were located at C,, and CL,.The ion at m/z 406 resulted from the loss of a C2H, radical from the molecular ion (m/z 439, indicating that the terminal double bond was at C , , . A small ion at m/z 417 originated from the loss of H 2 0 . Thus the sequence of ions demonstrated the location of the double bonds and the oxirane ring in this compound and other epoxides studied. Interestingly, the intensities of the ion peaks in the major sequence followed approximate Gaussian-type distribution, as compared to about equal intensities in the spectrum of the picolinyl ester of docosahexaenoic acid. * Obviously, introduction of an epoxy group changed the BDE of adjacent carbon-hydrogen bonds favoring abstraction by the pyridinyl group, thereby changing the otherwise random hydrogen abstraction. The fragmentation pattern of a compound can be elucidated by several methods. One is based on the study of spectra of isotopically labeled analogs and/or appropriate derivatives. We expected that the similarity between the spectra of various amino esters of epoxides would indicate a common origin of fragmentation. To verify this assumption the esters of 12,13-epoxy-9-octadecenoic acid with several substituted amino alcohols 92 P r326 'O0 1 l o 284 284 244 I 1 326 366 435 406 d - L 50 1 O0 150 200 250 300 350 J L 400 M / Z Figure 3. Mass spectrum of picolinyl ester of 13.1 4-epoxydocosapentaenoic acid. 4 50 M. BALAZY AND A. S . NIES 334 A 1 O0 0 - 90 O 274 - 316 - O T a7 o0 h. tu, 60 164 - 274 z c w z w 2 4 w IL 50 1O0 150 200 250 300 350 400 450 M / Z 1 o9 B 1O 0 90 80 h. tvi 70 60 c W z 50 W t 3 40 W <z 30 355 20 10 O 50 1O 0 150 200 were prepared. Mass spectra were compared with that for the picolinyl derivative (Fig. 4). Introduction of a functional ester group containing nitrogen which was separated by three or four carbons from carboxylic oxygen strongly directed the fragmentation of the epoxide. A general similarity can be noticed among the spectra in Fig. 4. In particular, ions a and b were present but usually in lower relative abundance when compared to the picolinyl derivative. These derivatives also showed quite extensive fragmentation in the low-mass range, reducing the utility of these derivatives for structural determination. The reduced abundance of ions a and b was due in part to the lower hydrogen aEnity of the ionized tertiary aliphatic or alicyclic amine esters (Fig. 4(c) and (d)) relative to the ionized 3-pyridinylmethyl e ~ t e r . *However, ~ these ions were also of low relative abundances in the spectrum of the 250 300 350 400 4 50 3-pyridyl ester (Fig. 4(b)) as compared to the 3pyridinylmethyl ester (Fig. 4(a)), although hydrogen afinities of both ester groups should be similar. Most probably, the missing methylene group in the 3-pyridyl derivative did not permit the proper conformation for hydrogen migration, similar to the mechanism that has been proposed for the interpretation of the spectrum of 2-pyridinylmethyl ester of stearic acid.26 Mass spectra of both picolinyl linoleate epoxides displayed additional fragment ions of abundances comparable to those of fragments a and b. Such fragment ions are: a + 14 (m/z 288) and b + 14 (m/z 330) in the spectrum of picolinyl 12,13-epoxyoctadecenoate (Fig. 4(a)), and also a - 14 ( m / z 220) and a - 28 (m/z 206) in the spectrum of picolinyl 9,10-epoxyoctadecenotate(not shown). This fragmentation pattern is very similar to that of picolinyl derivatives of monounsaturated acids, e.g. picolinyl 335 EPOXIDES OF UNSATURATED FATTY ACIDS 81 C 1O0 90 80 K 70 60 50 W L '4 40 W 349 [L 30 20 10 O 50 1O 0 150 200 250 300 350 400 450 M / Z 110 O 80 - 6: 70 336 I - CH3 128 60 - 50 - W t 5 40 - W n 30 10 zo 336 4 50 294 389 4 0 7 I 1O0 150 200 250 300 350 I 400 450 M / Z Figure 4. Mass spectra of: (a) 3-pyridinylmethyl (picolinyl), ( b ) 3-pyridy1, ( c ) N,N-dirnethylaminoethyl and ( d ) 1 -methyl-3-piperidinemethyl esters of 12.13-epoxyoctadecenoic acid. oieate,26 and can be explained in the same way as the fragmentation of picolinyl arachidonate epoxides. El spectra of amino and picolinyl esters of linoleate epoxide confirmed the formation of ions a and b and provided additionai evidence in favor of the proposed fragmentation pattern. Picolinyl esters produced more intense peaks in the high-mass range and, therefore, were more suitable for qualitative m a s spectrometric analysis of these substances. Analogs of these fatty acids isotopicaily iabeied with deuterium or "0, should confirm the mechanism leading to the production of t hese ions. Fragmentation in E1 spectra of epoxides is usuaiiy controiied by a cleavage following electron abstraction from the epoxide oxygen.20*2'There is no evidence that this mechanism was significant in the derivatives studied. The picolinyl function, with a lower ionization potentiai than the epoxy group, inhibited this mode of fragmentation, and changed the site of primary ionization to the pyridine ring, which directed subsequent fragmentation. It is interesting to note the difference between the mass spectra of picolinyl derivatives of epoxides described here and cyclopropane fatty acids discussed by Harvey.,' The latter spectra are very diagnostic for the determination of the position of the cyclopropane ring, but in a different way when compared to the spectra of the epoxides studied here. Ions that formaily 336 M. BALAZY AND A. S. NIES correspond to epoxide ions a and b are relatively small in cyclopropane spectra; in contrast, ions corresponding formally to ion type c and c + 14 in epoxides are very abundant. We conclude that the unique advantage of the picolinyl esters for mass spectroscopic analysis can be extended to another group of compounds, the epoxides. However, because of the instability of epoxides in acidic environments, the previously reported derivatization procedure had to be modified by initially preparing an imidazolide, which was permitted to react with 3pyridylcarbinol. 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