Anal. Chem. 1986, 58, 2429-2433
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Fast Atom Bombardment and Tandem Mass Spectrometry for Determining Structural Modification of Fatty Acids Kenneth B. Tomer, Nancy J. J e n s e n , a n d Michael L. Gross* Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 Previous studies showed that colllsionally activated gasphase carboxylate anlons of saturated fatty acids undergo losses of the elements of CH4, C2H6, C,H6, by way of a highly speclfk 1,4-eUmlnatbn of H,. These CnH2n+2loses begln at the alkyl terminus and progress along the entire alkyl chaln. I n this paper we report that the presence of a substituent such as an alkyl branch, hydroxy group, cyclopropane rlng, cyclopropene ring, or epoxide rlng Interrupts this process in a characteristicfashlon that permits lndentlflcation of the substltuent and location of its position on the acid chaln. This method not only is useful for structural characterlzatlon of pure free fatty aclds but also Is applicable to the analysis of mixtures of carboxylic acids.
...
Many lower forms of organisms such as bacteria, sponges, and certain plants possess the capability of producing a variety of structurally modified fatty acids. The structural modifications include branching, cyclopropane rings, cyclopropene rings, epoxy substituents, hydroxy and alkoxy groups, and unusual unsaturated acids. These acids are of interest because of their uniqueness and their utility for characterizing the organisms (1-5). Moreover, they may have substantial physiological implications for the organisms producing those substances and for organisms with which they come in contact. The problem of studying a specific fatty acid from a natural source is complicated because it must be isolated from a complex mixture of similar compounds. Classical methods of lipid analysis are described in several texts (4,6,7). A wide range of techniques have been used for characterizing the purified compounds. They include comparison of chromatographic retention times with those of known compounds, degradative analysis, derivatization followed by analysis, synthesis of model compounds for comparison, and a variety of spectroscopic methods. Mass spectrometry has been especially useful for studying these compounds because pertinent structural information may be obtained from small samples, and gas chromatography/mass spectrometry (GC/MS) is an effective method for dealing with mixtures (8). The recent characterization of lipids of marine sponges by Djerassi and co-workers exemplifies the practical applicability of the method (ref 9 and 10 and references cited therein). Unfortunately, derivatization is necessary, as free acids are not amenable to GC/MS. A number of mass spectrometry methods have been applied to the study of structurally modified acids (11). Pioneering work in the field was done by Stenhagen and co-workers (12-16) who studied methyl esters of fatty acids by electron ionization (EI) mass spectrometry. Informative high mass ions are of low abundance and if double bonds are present, their location is obscured by rearrangments. Pyrrolidide (17-20) and more recently picolinyl derivatives of fatty acids (21-23) were established to be useful for locating the double bond position and other structural modifications. Chemical ionization (CI) mass spectrometry has been tipplied to the study of unsaturated fatty acids esters (11)and epoxy (24, 25) and hydroxy (25-29) substituted esters. The principal advantages of CI are enhanced molecular weight
information and limited fragmentation, which makes key fragments more apparent. Derivatization is necessary for double bond location and may be accomplished either prior to analysis or in the mass spectrometer source via an ion/ molecule reaction. Fatty acids derived from natural sources are usually obtained as mixtures of free fatty acids. It appears to be possible to deal with these mixtures by using tandem mass spectrometry (MS/MS) and fast atom bombardment (FAB) to desorb carboxylate anions. The general suitability of FAB and tandem methods for mixture analysis is becoming established (30, 31). Recently, we showed carboxylate anions from saturated, unsaturated, and terminally branched acids to be quite amenable to such analysis (32-37). Free fatty acids are readily desorbed as (M - H)- ions without fragmentation (32-37). However, if collisionally activated, the (M - H)- ions undergo losses of the elements of CHI, C2H6,C,H,, ... by way of a highly specific 1,4-elimination of H, (33-35). These CnH2n+2losses begin at the alkyl terminus and progress along the entire alkyl chain unless there is a double bond or a methyl branch to modify the chemistry (32, 34, 35). The resulting fragmentation pattern can be interpreted to identify and locate the structural modification. The methodology appears to be generally applicable except to polyunsaturated fatty acids. However, simple modification of polyunsaturated acids by chemical reduction with N2D, allows for location of the double bond sites (34). We report here the extension of the methodology to the structural determination of fatty acids modified by multiple branching, epoxy rings, cyclopropane rings, cyclopropene rings, and hydroxy functions. Also demonstrated is the applicability of the combination of FAB and MS/MS for analysis of a complex bacterial acid mixture containing a variety of modified fatty acids. EXPERIMENTAL SECTION Materials. Sterculic, dihydrosterculic, malvalic, and dihydromalvalic acids were obtained from Gordon Fisher of the Southern Regional Research Center, ARS, USDA, as the methyl esters and were originally isolated at that facility (38). trans9,lO-Methyleneoctadecanoic acid, 9,10-epoxyoctadecanoic acid, phytanic acid, 2-hydroxytetradecanoic acid, 2-hydroxyhexacosanoic acid, and methyl 3-hydroxytetradecanoate were purchased from the Foxboro Co. (North Haven, CT). 12Hydroxydodecanoic, 16-hydroxyhexadecanoic,and 12-hydroxystearic acid were purchased from Sigma Chemical Co., St. Louis, MO. The bacterial acid mixture was purchased from Supelco, Inc. (Bellefonte, PA). All methyl esters were hydrolyzed overnight in cold concentrated ethanolic KOH. Vaccenic epoxide was prepared from 3-chloroperoxybenzoicacid (Aldrich, Milwaukee, WI) and vaccenic acid (Sigma, St. Louis, MO) following the basic epoxidation procedure of Swern et al. (39). Mass Spectrometry. Mass spectra were obtained with a Kratos MS-50 triple analyzer tandem mass spectrometer, which was described previously ( 4 0 ) . This instrument consists of a high-resolution MS-I of Nier-Johnson geometry followed by an electrostatic analyzer used as MS-11. The resolution of MS-I1 is limited t o approximately 100 by the peak broadening caused by the kinetic energy released upon fragmentation. Fast atom bombardment (FAB) (41) was used to ionize desorptively the preformed conjugate bases from a triethanolamine or glycerol
0003-2700/86/0358-2429$01.50/0 Q 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
PHYTANIC
ACID
MI2 311
O I
/I I
-cz0l e
i
d c
b o
C
I1 I i I
I (El
Figure 1. Spectrum of the daughter ions produced by activating the (M - H)- ion of m l z 31 1 of phytanic acid.
matrix. Each sample (-2 wg) was dissolved in ca. 1 WL of the matrix placed on the copper target of the FAB direct inlet insertion probe. Previous studies showed that recognizable CAD spectra for samples as little as 25 ng applied to the probe tip may be obtained (34). Mass spectra of the fragments from (M - H)ions of the fatty acids were taken by collisionally activating the ions in the third field free region by using helium gas (sufficient helium was added to suppress the ion beam by 50%) and by scanning MS-11. Each CAD spectrum is an average of 15-30 20-5 scans processed with a standard DS-55 data system using software written in this laboratory (42).
RESULTS AND DISCUSSION Mass spectra of the negative ions of branched, cyclopropane, cyclopropene, epoxy, and hydroxy acids desorbed by FAB resemble those seen for saturated acids discussed elsewhere (33, 36, 37). Namely, only the (M - H)- ion and associated isotopic ions for the acid are observed as a result of the FAB desorption process. Obviously the preponderance of the molecular ion is useful for obtaining molecular weight information but nearly uniformative with regard to structure. However, the lack of fragmentation makes selection of the (M - H)- ion a simple process even for unknowns and mixtures and facilitates structure determination by collisional activation because the ionization is highly concentrated in one species. Negative ion chemical ionization is potentially another method of generating these ions directly from the acids, which can be thermally desorbed into the high vacuum ion source. Branched Fatty Acids. The positions of the methyl branches in phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) are readily discerned from the CAD spectrum of the (M - H)- ion (Figure 1) . Remote-charge-site fragmentation is observed for this compound with enhanced fragmentation at the branch point and nearly total suppression of fragmentation involving the simultaneous losses of the methyl and alkyl branches. The ability to locate methyl branching on alkyl chains from suppression of remote-charge-site fragmentations pertains to singly branched fatty acids such as iso- and anteiso-acids (35) and to other materials such as steroids (43). We previously reported this observation for the branching of cholesterylhemisuccinate (33)and cholestanol3-sulfate (43). The methyl branching on the chain of vitamin E (a-tocopherol) may also be determined from the CAD spectrum of the (M - H)- ion (44). Cyclopropane/Cyclopropene Substituted Acids. The CAD spectra of the (M - H)-anions of dihydrosterculic (cis-9,lO-methyleneheptadecanoic acid), trans-9,10methyleneoctadecanoic acid (Figure 2, parts A and B, respectively), and dihydromalvalic acid are representatives of cyclopropane-containing acids. Remote-charge-site frag-
l@&S.9,1D-METH’fLENE
sb
OCTADECANOIC ACID
1 bo
I50
MI2
295
200
II
I
250
M/Z
1 (c)
STERCULIC
ACID
-m
W 2 293
a
z m W i
U
50
100
150
200
250
300
MIZ
Figure 2. Spectra of the daughter ions produced by collisionally activating the (M - H)- ions of cyclopropane and cyclopropene substituted fatty acids: (A) dihydrosterculic acid (cis-9,lO-methylenoctadecanoic acid), (M - H)- ion is of m l z 295; (B) trans-9,lO-methylenoctadecanoic acid, (M - H)- ion is of rnlz 295; (C) stercuiic acid, (M - H)- ion is of m l z 293.
mentation is seen with relatively more facile cleavages (as CnH2n+2 loss) a t points a and b (see Figure 2A,B). This behavior corresponds to the prominent allylic cleavages observed for unsaturated acids (32). Also notable is the reduced abundance of the CnH2n+2 loss resulting from fragmentation at a C-C bond one bond removed from that which is readily cleaved (labeled a’ in Figure 2A,B). The low abundance loss of C9Hmresults from a fragmentation not yet understood and possibly involves several steps. Comparison of the spectrum of dihydrosterculic (the cis isomer) with that of the trans isomer (Figure 2A,B) shows sufficient differences to permit distinction of the two isomers. One key difference is the substantially greater relative abundance of the a” fragmentation of the cis isomer. That process involves breaking of a bond to the cyclopropane ring, which may be facilitated by relief of steric crowding in the cis isomer (compare the abundances of a” relative to a for the two geometric isomers). The CAD spectra of the (M - H)- anions of malvalic (not shown) and sterculic acids (Figure 2C), two cyclopropenecontaining acids, also show reduced fragmentation in the ring region. These compounds are very unstable in the acid form. Consequently, the spectra have poorer signal-to-noise ratios than those of the cyclopropane acid anions, possibly because of decomposition during hydrolysis and isolation of the acids. The ion formed by cleavage @ to the cyclopropene moiety on the hydrocarbon terminus side and the ion formed by the @-cleavageon the carboxylate side of the cyclopropene are of enhanced abundance. However, the cleavage of the C-C bond of the hydrocarbon side is enhanced only a small amount whereas the fragmentation of the carboxylate side is substantially increased. In general, the presence and location of cyclopropane and cyclopropene rings in fatty acids are identified by a set of four adjoining low-intensity ion peaks in the CAD spectra of these
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
2431
r (n) 2 -
WVDROXY
TLIIADECAWOIC
ACID
MI2 243
-41
d 3 2
m 4
i W
K
____~___
.
EPOXIDE
M/Z 297
_1
U
50
I00
I50
M/Z
2oo
250
300
Figure 4. Characteristic fragmentation of epoxy-substituted fatty acid: (A) fragmentations that yield key diagnostic daughter ions; (B) spectrum of fragment ions produced by activating the vaccenic acid (M - H)- ions of m l z 297.
i W K
A
I"""
so
1 50
200
Figure 3. Spectra of the daughter ions produced by collisionally activating the (M - H)- ions of hydroxy-substituted acids: (A) 2hydroxytetradecanoic, (M - H)- is of m l z 243; (B) 3-hydroxytetradecanoic, (M - H)- is of m / z 243; (C)12hydroxystearic, (M - H)is of m l z 299; (D) 12-hydroxydodecanoic, (M - H)- is of m l z 243.
compounds compared to the spectra of analogous saturated fatty acids. The identification of cis and trans isomers appears to be possible on the basis of the relative abundances of ions formed by fragmentation adjacent to the ring. In a similar fashion, sites of unsaturation are located in monounsaturated fatty acids by identifying a spectral region having reduced intensity for three adjoining ion peaks (32). Hydroxy Acids. The CAD spectra of 2-hydroxy acids such as 2-hydroxytetradecanoic acid (Figure 3) and 2-hydroxyhexacosanoic acid are dominated by an abundant ion formed by the loss of 46 u. We presume the loss to be of the elements of formic acid (HCOOH), but this remains to be proved. The reaction may occur to give an enolate anion as
-
RCH2CH2CHzCH(OH)COORCH2CHz=CHO-
+ HCOzH (1)
Remote-site fragmentation by losses of the elements CnH2n+2 from the anion and from the (M - H - 46)- ion still occurs but to much smaller extent than the production of the daughter ion formed by the loss of 46 u. The loss of 46 u also occurs for a 3-hydroxy acid such as 3-hydroxytetradecanoic acid as for the the 2-hydroxy acids, but the resulting product ion is of relatively low abundance (see Figure 3B). The ion of m / z 59 dominates the daughter ion spectrum and is probably C2H302-,but this remains to be proved. The remote-site fragmentation involving losses
of CnH2n+2 still occurs and is much more apparent for the 3-hydroxy acids than for the 2-hydroxy acids. 12-Hydroxystearic acid is an example of a mid-chain hydroxy-substituted acid (Figure 3C). Water loss is the principal CAD fragmentation. However, the position of the hydroxy substituent may be readily located by enhanced fragmentation on either side of the point of substitution and by suppressed fragmentation at the substituted carbon. The decompositions are very similar to those seen for methyl-branched acids (see above), but the hydroxy-substituted acid may be distinguished by the abundant HzO loss as well as the mass difference between CHOH and CH-CH3. Acids that bear hydroxy functions on the terminal carbon of the alkyl chain such as 12-hydroxydodecanoic (Figure 3D) and 16-hydroxyhexadecanoicacids show water loss as the most facile fragmentation and also relatively facile losses of the elements of CH30H and CzH50H. These reactions are regarded as occurring remote from the charge site; however, because of the position of the OH substituent, the fragments lost have the general formula of C,H2,+20 instead of CnH2n+2. Epoxy-Substituted Acids. Vaccenic epoxide (Figure 4) and 9,lO-epoxyoctadecanoic acid undergo fragmentations representative of carboxylate anions that have epoxide substituents. The ion formed by water loss is the most abundant. However, rearrangement and cleavage a t the epoxide ring create fragment ions that are diagnostic for location of the epoxide. The scheme shown in Figure 4 is an explanation of the rearrangement (shown by arrows and designated a) and cleavage (shown by the cleavage arrow across the ring and designated b). 9,10-Epoxyoctadecanoic acid fragments in a similar fashion, but for this compound, fragment a is CsH18 and fragment b is C9H18because the epoxide is in the 9,lO rather than the 11,12 position. Detailed mechanisms of the fragmentations of the epoxy and other acids reported here are unknown. Elucidation of mechanism will require deuterium and possible 13C labeling of the various acids. Application to a Bacterial Acid Mixture. A commercially available mixture of methyl esters of 22 bacterial fatty acids (see Table I) was hydrolyzed with cold alcoholic KOH and analyzed. Ions formed by desorption of the carboxylate anions (M - H)- and associated isotopic ions are the only ions of note observed in the mass spectrum of negative ions desorbed by FAB (Figure 5A). The near exclusive production of molecular ions was determined for the known mixture by comparing the reported mixture composition with the ions of the spectrum. The only ion of significant abundance not
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 198 ?"
~~~~
(A] B I C T F R l A L
Table I. Components of Bacterial Acid Mixture m / z of (M - H)- of free acid methyl undecanoate methyl 2-hydroxydecanoate methyl dodecanoate
185 187 199
methyl tridecanoate methyl 2-hydroxydodecanoate methyl 3-hydroxydodecanoate methyl tetradecanoate methyl 12-methyltetradecanoate methyl pentadecanoate methyl 2-hydroxytetradecanoate methyl 3-hydroxytetradecanoate methyl cis-9-hexadecenoate methyl hexadecanoate methyl 14-methylhexadecanoate
213
methyl cis-9,lO-methylenehexadecanoate
267
methyl methyl methyl methyl
269
heptadecanoate 2-hydroxyhexadecanoate cis-9-octadecenoate octadecanoate
A C I D MIXTURE
NEGIllVE
ION
70
215
215
18)
EICOSANOIC
ACID
M I 2 311
227 241 241
I
243 243
253 255 269 271
281 283
methyl 9,lO-methylenoctadecanoate
295
methyl nonadecanoate methyl eicosanoate
297 311
accounted for by this comparison is the ion of m / z 186. The even mass of this ion distinguishes it from the carboxylate anions and suggests that it is an impurity. The analysis of an unknown acid mixture would be expected to be straightforward because little fragmentation occurs; the ions seen in the mass spectrum of desorbed negative ions are attributed to carboxylate anions. Although the mass spectral data are useful, selection of a given ion with MS-I followed by collisional activation and analysis by MS-I1 is expected to allow for specific structural characterization of the fatty acids. The CAD spectra of the ions of m / z 311, 271, and 267 shown in Figure 5 are representative of the spectra obtained for the various components of the mixture. The distinctive, uninterrupted, and smooth envelope pattern of daughter ions is readily interpreted to identify the ion of m / z 311 as a straight chain, unsubstituted acid carboxylate of eicosanoic acid (Figure 5B). The principal fragment ion formed by collisionally activating the ion of m / z 271 is an ion of m / z 225 (formed by loss of 46 u). The expulsion of 46 u corresponds to the expected fragmentation behavior of 2-hydroxyhexadecanoic acid (compare Figures 3A and 5C). Fragment ions labeled a, a', and b in the CAD spectrum of the m / t 267 ion (Figure 5d) are interpreted to identify the presence of a cyclopropane ring a t the 9,lO position of acid (compare to Figure 2B). The ion labeled a" is the key diagnostic feature for establishing the cis geometry of the ring. Hence the presence of cis-9,lO-methylenehexadecanoic acid in the mixture is confirmed. The signalto-noise (S/N) ratio decreases for the labile cyclopropane- and cyclopropene-substituted acids. The lower S / N is apparent in mixture analyses; nevertheless, identification is possible. Most of the other components of the mixture yielded the expected spectra; however, three exceptions are noted. The ion that should correspond to 2-hydroxydecanoate shows a distinctly different behavior than the %hydroxy standards. Little fragmentation is seen for this ion. A low abundance ion formed by the loss of H 2 0 is the principal fragment and no loss of 46 u is observed. A possible explanation is that chain length is a factor. Previously, we noted in our study of saturated fatty acids that a minimum chain length of ten carbons is necessary to observe the typical CnH2n+2 loss pattern (33). A second exception is the spectrum of the ion of m / z 281, which was expected to be oleate on the basis of the specified
.50
n
-
0
1
o
I
io
hvz
c
b
3
do
m
200
250
0
do u
W A
50
IO0
150
wz
200
250
Figure 5. (A) Mass spectrum of negative ions produced by FAB desorption of the mixture as 22 bacterial acids. Spectra of fragment ions produced by collisionally activating the ions labeled B, C, and D are shown in B, C, and D: (B) (M - H)- of eicosanoic acid at m l z 31 1; (C) (M - H)- of 2-hydroxyhexadecanoic acid at m l z 271; (D) (M - H)of cis-9,lO-methylenehexadecanoic acid at m l z 267.
mixture composition. However, abundances of several ions were altered in a fashion that could be accounted for if the ion of m/z 281 arose from a mixture of oleic and vaccenic acids (32, 34). The third problem is for the expected mixture of 12methyltetradecanoic acid and its linear isomer, pentadecanoate. No branched isomer could be detected. The distinction of isomers present in a mixture might be expected to be a serious problem in this method of analysis, but often the diagnostic fragmentation is so evident that identification is possible. For example, the presence of both 2- and 3hydroxydodecanoate and 2- and 3-hydroxytetradecanoate was confirmed to make up the ions of m / z 215 and 243, respectively, of the mixture. The presence of the ion of m / z 59 identifies the 3-hydroxy component whereas an abundant product ion formed by the loss of 46 u confirms the presence of the 2-hydroxy isomer. Previously, it was established that terminally branched acids may be distinguished from their straight-chained isomers readily in 1:1 mixtures with identification becoming difficult for mixtures greater than 1:10 (35). Thus, the inability to distinguish 12-methyltetradecanoate in the mixture may be because its concentration is less than 10% of that of the straight chain isomer. Suitable information was not supplied with the bacterial acid mixture to determine concentration dynamic range. However, it should be noted that, because no prior separation
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
was performed on the mixture, each sample of the acid solution contained all 22 acids. It was established earlier that the limit of detection for MS/MS analysis of FAB desorbed carboxylate anions is 25-50 ng of sample (34). Therefore, if 2-5 yg of the mixture were loaded on the probe, we might expect the concentration dynamic range for acids that are not isomeric to be ca. 1OO:l.
ACKNOWLEDGMENT We thank Gordon Fisher of the Southern Regional Research Center, ARS, USDA, for the sterculic, dihydrosterculic, malvalic, and dihydromalvalic methyl esters. Registry No. H02CCH(OH)(CH2),,CH3,2507-55-3;H02CCHZCH(OH)(CHz),oCH3, 1961-72-4; HOzC(CHz)1oCH(OH)(CH2)5CH3, 106-14-9;HOZC(CH2)110H, 505-95-3;HOZCCH(0H)(CH?),CH,, 5393-81-7; HOZC(CHz)IoCH3, 143-07-7; HO&(CH,)l,CH,, 638-53-9; HO2CCH(OH)(CH,)&H3,2984-55-6; H02CCHZCH(OH)(CH,)&H,, 1883-13-2;H02C(CH2)&H3, 544-63-8; HO&(CHZ)loCH(CH3)Et,5502-94-3;H02C(CHJ&H3, 1002-84-2; c~s-HO&(CH&CH=CH(CH~)&H~, 373-49-9; H02C(CH2)14CH3, 57-10-3;HO&(CH2)&H(CH3)Et, 5918-29-6;H02C(CH2)15CH,, 506-12-7; HOZCCH(OH)(CH2)13CH3, 764-67-0; c ~ s - H O ~ C (CH2)7CH=CH(CH2)7CH3, 112-80-1;HOZC(CHz)&H3,57-11-4; H02C(CHp)I;CH,, 646-30-0; HO&(CH2)&H3,506-30-9; H02C(CH2)&H3, 112-37-8; phytanic acid, 14721-66-5; cis-dihydrosterculic acid, 4675-61-0; trans-dihydrosterculic acid, 29203-99-4;sterculic acid, 738-87-4;vaccenic acid, 693-72-1;cis9.10-methylenehexadecanoate,4675-60-9.
LITERATURE CITED Katayma-Fujirnura, Y ; Tsuzaki, N.; Kutaishi. H. J. Gen. Microbioi 1982, 128, 1599. B0e, B.; Gjerde, J. J. Gen. Microbiol. 1980, 116, 41. Goodfellow, M.; Colllns, M. D.; Minnikin, D. E. J. Appl. Microbiol. 1980, 48. 269. Asseiineau, J. The Bacterial Lipids ; Holden-Day: San Francisco, CA, 1966. Asseiineau, J. Bull. Inst. Pasture (Paris) 1983, 8 1 , 367. Christie, W. W. Lipid Analysis; Pergamon Press: New York, 1982 Lipid Biochemical Preparations, Bergelson. L. D., Ed.; Elsevier: Arnsterdam. 1980. Gas ChromatographylMass Spectrometry Applied to Microbiology ; Odharn, G.. Lakson, L., Maardh, P.-A,, Eds.: Plenum: New York, 1984. ~. Ayanoglu, E.; Kurtz, K.; Kornprobst, J. M.; Djerassi, C. Lipids 1985, 2 0 . 141. Wijekoon, W. M. D.; Ayanoglu, E.; Djerassi, C. Tetrahedron Lett. 1984, 2 5 , 3265. Jensen, N. J.; Fross, M. L. Mass Spectrom. Rev., in preparation. Ryhage. R.; Stenhagen, E. J. LipidRes. 19 0 , 1 , 361. Abrahamsson, S.;Stallberg-Stenhagen, S:; tenhagen, E. The Higher Saturated Branched Chain Fatty Acids; Holman, R. T., Malkin, T., Eds.; Pergamon: Oxford, 1963, pp 41-59. Ryhage, R.; Stenhagen, E. Ark. Kemi 1960, 15, 291.
t
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(15) Ryhage, R.; Stallberg-Stenhagen, S.; Stenhagen, E. Ark. Kemi 1961 18, 179. (16) Ryhage, R.; Stenhagen, E. Ark. Kemi 1960, 75,545. (17) Vetter, W.; Walther, W.; Vecchi, M. Helv. Chim. Acta 1971, 5 4 , 1599. (18) Andersson, B. A.; Heimerrnann, W. H.; Holman, R. T. Lipids 1974, 9 . 443. (19) Andersson, B. A.; Homan, R. T. Lipids 1974, 9 , 185. (20) Andersson, B. A., Christie, W. W.; Holman, R. T. Lipids 1975, 10, 215. (21) Harvey, D. J. Biomed. Mass Spectrom. 1982, 9. 33. (22) Harvery, D. J. Biomed. Mass Spectrom. 1984, 1 1 , 340. (23) Harvey, D. J. Biomed. Mass Spectrom. 1984, 7 1 , 187. (24) Turnlinson, J. H.; Heath, R. D.; Doolittle, R. E. Anal. Chem. 1974, 4 6 , 1309. (25) Piattner, R. D.; Gardner, H. W.; Kieinman, R. J. Am. Oil Chem. SOC. 1983, 6 0 , 1298. (26) Stan, H.; Scheutwinkel-Reich, M. Fresenius' Z . Anal. Chem. 1979, 296, 400. (27) Scheutwinkel-Reich, M.; Stan, H. Biochem. Med. 1980, 6 , 45. (28) Stan., H.; Scheutwinkel-Reich, M. Lipids 1980, 15, 1044. (29) Gardner, H. W.; Weisleder, D.; Nelson, E. C. J. Org. Chem. 1984, 4 9 , 508. (30) Lyon, P. A.; Stebbings, W. L.; Crow, F. W.; Torner, K. B.; Lippstreu, D. L.; Gross, M. L. Anal. Chem. 1984, 5 6 , 8. (31) Lyon, P. A.; Crow, F. W.; Tomer, K. B.; Gross, M. L. Anal. Chem. 1984, 5 6 , 2278. (32) Tomer, K. B.; Crow, F. W.; Gross, M. L. J . Am. Chem. SOC.1983, 105. 5487. (33) Jensen, N. J.; Tomer, K. B.; Gross, M. L. J. Am. Chem. SOC.1985, 107, 1663. (34) Jensen, N. J.; Tomer, K. B.; Gross, M. L. Anal. Chem. 1985, 5 7 , 2018. (35) Jensen, N. J.; Gross, M. L. Lipids 1986, 2 1 , 362. (36) Gross, M. L.; Jensen, N. J.; Lippstreu-Fisher, D. L.; Torner, K. B. Mass Spectrometry in Health and Life Sciences; Burlingame, A. L., Castagnoli, N., Jr., Eds.; Elsevier: Amsterdam, 1985; pp 209-238. (37) Jensen, N. J.; Torner, K. B.; Gross, M.L.; Lyon, P. A. Desorption Mass Spectrometry-Are SIMS and FA8 the Same?; Lyon, P. A,, Ed.; Arnerican Chemical Society: Washington, 1985; p 194. (38) White, J. L., Jr.; Zarins, Z.; Feugo, R. G. J. Am. Oil Chem. SOC. 1977, 5 4 , 335. (39) Swern, D.; Findley, T. W.: Scanlan, J. T. J. Am. Chem. SOC.1944, 6 6 , 1925. (40) Gross, M. L.; Chess, E. K.; Lyon, P. A.; Crow, F. W.; Evans, S.: Tudge, H. I n t . J. Mass Spectrom. Ion Phys. 1982, 42, 243. (41) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. M. J. Chem. Soc.. Chem. Commun. 1981, 325. (42) Crow, F. W.; Lapp, R. L. Paper presented at the 29th Annual Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, May 24-29, 1981; pp 448, (43) Tomer, K. B.; Jensen, N. J.; Gross, M. I; Whitney, J. Biomed. Mass Spectrochim. 1988, 13, 265. (44) Tomer, K. B.; Gross, M. L. Springer Series in Chemical Physics, in press.
RECEIVED for review February 10, 1986. Accepted May 27, 1986. This work was supported by the Midwest Center for Mass Spectrometry, a National Science Foundation Regional Instrumentation Facility (Grant CHE-8211164), and by the National Science Science Foundation (Grant CHE8713918202).
