2018
Anal. Chem. 1985, 57,2018-2021
Collisional Activation Decomposition Mass Spectra for Locating Double Bonds in Polyunsaturated Fatty Acids Nancy J. Jensen, Kenneth B. Tomer, and Michael L. Gross* Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588
It was established previously that gas-phase carboxylate anlons of saturated fatty aclds undergo losses of the elements of CH,, C,H,, C,H,, by way of a hlghiy speclflc 1,4-elimination of Hp These CnH2,+, losses begin at the alkyl terminus and progress along the entlre alkyl chain. Introduction of a double bond causes a dlsruptlon In the CnH2"+, loss pattern whlch can be interpreted readlly to locate the double bond position. However, locatlng double bonds in polyunsaturated fatty aclds Is more dllficult and becomes lmposslble for aclds contalnlng four or more carbon-carbon double bonds. These highly unsaturated fatty acid anions fragment upon coilisional actlvatlon by ellmlnatlng 45 mass unHs which preempts the C,H,+,-ioss process. It is demonstrated In this paper that the hlghly specllic mechanlsm for C, HW+, loss and a dlhnlde (N,4) reduction provlde the opportuntty to develop a method of determlnlng double bond locatlon In poiyunsaturated fatty aclds. Accordlngiy, a serles of polyunsaturated fatty aclds was reduced and the resulting deuterated saturated acids were desorbed as Carboxylate anions. Interpreting the m a s shMs caused by lncorporatlon of deuterlum into the departing C, H,, +, neutral fragments permits the double bonds In the orlglnal acid to be located.
...
The location of double bonds in olefins or other doublebond-containing compounds by using mass spectrometry has been an interesting and challenging problem (1-3). Unsaturated fatty acids are of particular interest because of their importance in biochemistry. Unfortunately, they and their simple ester derivatives suffer rearrangement of the double bond under electron ionization conditions and give mass spectra which cannot be interpreted to locate the doubIe bond. Negative ion chemical ionization (CI) mass spectra of these substances are often not useful for determining the location of the double bond due to lack of fragmentation. Hence two methods commonly used are prior derivatization of the double bond followed by either electron ionization (EI) (4-11) or chemical ionization (12-15) and direct derivatization within the mass spectrometer source under CI conditions (16-20). A third approach is the modification of the acid function to give more complex derivatives such as pyrrolidides (22-23) and picolinyl esters (24). These molecules fragment more selectively than the fatty acids or simple fatty esters upon electron ionization possibly because the derivatizing group is better able to localize the charge or radical sites. However, this method works less well for highly unsaturated fatty acids because the diagnostic ions are of low abundance (24). A solution to this problem which involves two derivatization steps was proposed by Kawaguchi et al. (25). Polyunsaturated fatty acids esters were reduced with diimide and the ester function was converted into a pyrrolidide. Reasonable results are obtainable by using this approach provided the incorporation of deuterium is nearly complete and the fatty acid is reasonably pure. The development of fast atom bombardment (FAB) (26) for effectively desorbing preformed ions such as carboxylate anions and tandem mass spectrometry (MS/MS) for colli-
sional activation (27,223provides new possibilities for locating double bonds in unsaturated acids. In previous work (29), we showed that collisional activation of FAB-desorbed (M H)-ions of monounsaturated fatty acids results in highly specific fragmentation which may be interpreted readily to locate the double bond. However, the collisionally activated decomposition (CAD) spectra for acids containing two and three double bonds are not interpreted as easily as those of the monounsaturated acids (29). Moreover, the CAD spectra of fatty acid anions containing four or more double bonds are totally uninformative because the anion undergoes loss of only 45 amu. An alternate approach is described in this paper and involves deuterium labeling of the double bonds coupled with collisional activation of the saturated fatty acid carboxylates. Unsaturated fatty acids can be reduced readily with diimide-dz (N2D.J to form saturated acids with deuterium labeling at the original unsaturated sites. The use of diimide reduction followed by E1 mass analysis was proposed previously. The earlier method (10, 11) is confounded by low abundance of diagnostic ions, as mentioned above, whereas the more recent proposal required a two-step derivatization (25). These problems are alleviated by taking CAD spectra of carboxylate anions which show abundant high mass fragments formed without H/D scrambling (30). Moreover, only one derivatization step is required.
EXPERIMENTAL SECTION Reagents. Linoleic acid, linolenic acid, arachidonic acid, and docosahexaenoicacid were obtained from Sigma Chemical Co. Eleostearic acid was obtained from commercial Danish oil furniture finish. Deuterated hydrazine (Cambridge Isotopes) and D20 (Aldrich Chemical) were used for reducing the acids. Preparation of Deuterated Acids. Octadecanoicacid, eicosa9,10,12,13-d4acid, octadecanoic-9,10,12,13,15,16-d6 noic-5,6,8,9,11,12,14,15-d8 acid, and docosanoic4,5,7,8,10,11,13,14,16,1 7,19,2O-dl2acid were prepared from linoleic, linolenic, arachidonic, and docosahexaenoicacid, respectively,by reacting deuteriodiimide (NzD2)with the unsaturated acids to add deuterium to the double bond (31,32). For each reduction, 15 mg of the unsaturated acid was placed in 1 mL of HzO and neutralized by adding 0.1 F LiOH. The water was evaporated, and the residue was washed with acetone and then dried. This residue was heated at reflux for 3 to 4 days with 0.5 mL of DzO and 0.5 mL of hydrazine-dl. The completion of the reaction was signaled by the disappearance of white foaming caused by liberation of hydrogen gas (presumablyas Dz). The solvent was then evaporated, and the remaining residue acidified with 1 F HC1. The product was extracted into ether and recovered after evaporating the solvent. The molecular weights of the products were determined using negative ion FAB mass spectrometry. The spectrum scanned over a narrow mass range showed an isotopic distribution of 30% d,, 29% d7, 16% d6, 8% d5, and 7 % d4 for reduced arachidonate. The latter could be a compound containing one double bond and six deuteriums. The extent of complete deuteration for the other acids was 55%, 49%, and 38% for linoleic, linolenic, and docosahexanenoicacids, respectively. Since eleostearic acid is present as the methyl ester (Tung oil) in the commercial furniture finish, initial steps of the reaction procedure were modified for this compound. Tung oil (25 pL) was hydrolyzed by heating in 1.5 mL of methanol with 2 mg of LiOH for 15 min at reflux. The solution was saturated and cooled
0003-2700/85/0357-20 18$01.50/0 0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985 STEARIC ACID
M/Z
283
-coo-
50
l
100
200
I50
250
- Id M/Z
O L E I C ACID
M/Z
!I 1
281
coo-
I
50
/I i
h"
100
I50 M/Z
200
250
Flgure 1. Spectra of the daughter ions produced from collisionally activating the (M - H)- ions of stearic acid (A) and oleic acid (B). The term i refers to Ion abundance.
with COz by adding a small amount of pulverized dry ice. The solvent was removed by evaporation, and the residue was treated in the same fashion as described above for the lithium salts of the free acids. The isotopic distribution of the product was 28% ds, 10% dg,17% d4,9% dB,and 24% d,. The ion of 24% relative abundance could also represent an acid containing one double bond and four deuteriums. Instrumentation. The mass spectra were obtained with a Kratos MS-50 triple sector mass spectrometer (33)comprised of a high-resolution MS-I (a standard Kratos MS-50) followed by an electrostatic sector MS-11. This instrument is equipped with a standard Kratos FAB source and an Ion Tech gun. Each sample was dissolved in triethanolamine, and a drop of the resulting solution was placed on the copper target of the FAB direct insertion probe for analysis. The samples were bombarded with 8-keV Xe atoms, and the ions produced were accelerated through 8 keV. Full scan negative ion mass spectra were used to verify the identity of reduced compounds, and the spectra were acquired at a resolution of ca. 2000 by scanning MS-I and leaving MS-I1 fixed to pass all ions. CAD spectra were obtained by selecting the appropriate ion in MS-I, introducing sufficient helium into the collision cell, located between MS-I and MS-11, to give a 50% reduction of the intensity of the signal for the selected ion, and scanning MS-11. Each CAD spectrum reported here was an average of several 20-9 scans processed with a standard DS-55 data system using software written in this laboratory (34).
RESULTS AND DISCUSSION High-energy collisional activation of the (M - H)- ions (presumably RCOO-) of fatty acids yields a distinctive, highly reproducible fragmentation pattern. A unique feature of this fragmentation is a series of losses of the elements CflH2fl+2 from the alkyl terminus of the (M - H)- ion (29, 30). The CAD spectrum of stearic acid (Figure 1A) is characteristic in both the spacing (Le., ions separated by 14 amu) and relative abundances of ions formed from a saturated carboxylate anion containing 10 or more carbon atoms. Elimination of the elements of C2Hs,C3H8, and C4H10 gives rise to the ions m / z 253, 239, and 225, respectively. The decompositions also pertain to carboxylateswith as few as six to ten carbon atoms, but the pattern is not as well-defined. The only other ions formed from carboxylate anions are those resulting from HzO loss from the (M - H)- ion and m/z 58,71, and 86 (Figure 1A). The latter three ions are produced in all cases and are of relatively low abundance for acids having longer chains. Monounsaturated acids such as oleic acid (Figure 1B) fragment in a fashion similar to their saturated counterparts. However, transfer of vinylic hydrogens and cleavage of double
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bonds are not accomplished readily; hence the position of the double bond is quite apparent from the interruption in the remote-charge-site fragmentation pattern. The spectral feature of two abundant ions (labeled A and A' in Figure 1B) followed by three low abundance ions at lower mass and then a third abundant ion (labeled B) is a general characteristic of monounsaturated fatty acids (29). While the CAD spectra of multiply unsaturated acids such as linoleic (Figure 2A) contain much information, they are not interpreted as easily as those of monounsaturated acids. Furthermore, for acids such as arachidonic and docosahexaenoic with four and six double bonds, respectively, the remote-charge-site fragmentation pattern essentially disappears. The only fragmentation exhibited by the (M - H)- of these polyunsaturated acids is loss of 45 mass units which may actually be an unresolved doublet of losses of 44 and 46 amu. Determination of Double Bond Position in Polyunsaturated Acids. Reduction of a fatty acid containing multiple unsaturation sites with deuteriodiimide (N2D2)results in the formation of a saturated acid with deuterium labeling at the original double bond sites. If the anion of this acid is collisionally activated, it will undergo CflH,+, losses. However, since deuterium is present only at sites of the original double bonds, the ion series in the CAD spectrum will not be regularly spaced at 14 amu as shown above, but instead spacing will be altered where deuterium loss occurs. The application of this approach is exemplified by considering the case of 9J2-0ctadecadienoic acid (linoleic acid). Reduction of this compound with NzDzyields octadecanoic-d4 acid. Since the negative ion FAB spectra of carboxylate anions show few ions except for a cluster of ions in the (M - H)region, the (M - H)-ion of this reduced acid can be selected by using MS-I of the tandem mass spectrometer and collisionally activated without interference by unreduced or partially reduced material. For an unknown acid, the FAB spectrum of the unreduced material would serve as a guide for choosing, after reduction, the correct ion for collisional activation. Examination of the resulting CAD spectrum (see Figure 2B and scheme) shows that losses of the elements of CHI, CzHs,and C3H8occur without deuterium involvement. However, losses of C4H9Dand C5HllD are seen along with those of C4H10 and C5H12, respectively. These losses do not give rise to doublets because the energy released in the decomposition broadens the peaks such that the C4H9D/C4H10 and the C5H11D/C5HlZ losses are each represented as a single peak centered between the expected masses. In accordance with the mechanism that CflH2fl+2 losses occur from the alkyl terminus and probably involve a 1,4-elimination of H2/HD (see eq 1))it can be determined that there is a double bond at carbon 12. Similarly losses of C7Hl4DZ/C7Hl3D3 and C8H1,D2/C8Hl6D3are interpreted to locate the second double bond at position 9.
(1)
Although mass assignment is the most effective parameter for locating the position of the deuterium in the labeled acid, the position is often recognized easily due to perturbation of the typical CAD pattern. In the spectrum of reduced linoleic acid, the peaks labeled d, e, g, and h are the broadened doublets due to both H and D transfer. They are of reduced intensity with respect to their counterparts in spectra of unlabeled acids (compare Figures 1A and 2B). The perturbed CAD spectral patterns are more apparent at the high mass end of the spectrum. Reducing the unsaturated acid and obtaining the negative ion CAD spectrum are effective for locating double bonds in
ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985
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REDUCED ARACHIDONIC ACID LINOLEIC ACID (A 1
IM-H)-
REGION
-0oc
h
I
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l o s s e s of d
e
f
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C
F
REDUCED LINOLEIC ACID
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o
Figure 4. Narrow scan FAB mass spectrum of the (M - H)- ions formed from the reduction of arachidonic acid.
I Ii
Figure 2. Spectra of the daughter ions produced from collisionally activating the (M - H)- ions of linoleic acid (A) and reduced linoleic acid (B). Perturbation of the remote site fragmentation pattern for linoleic acM Is apparent In spectrum A, but correlation to double bond locations is not obvious. However, location of the original double bond sites is accomplished readily for the deuterium-reduced acid (B and scheme). /II
R E D U C E D L I N O L E N I C ACID
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M / Z 289
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1
REDUCED E L E O S T E A R I C A C I D
u 50
I do
M/Z 289
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01
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250
Flgure 3. Spectra of the daughter ions produced from collislonally actlvatlng the (M - H)- ions of linolenic acid (A) and eleostearic acid
(6).
more highly unsaturated acids. Reduced linolenic acid (octadecanoic-9,10,12,13,15,16-d6 acid) and reduced eleostearic acid (octadecanoic-9,10,11,12,13,14-d, acid) are isomers and may be distinguished by comparing their CAD spectra (see Figure 3). Mass assignments, as determined from expanded portions of the spectrum to allow for greater accuracy,permit the conclusion that reduced linolenic acid undergoes losses of CH3D and C2H5D(al, a2),C4H7D3and C5H& (b1, bz), and C7HllD5and C8HI3D5(cl, c2)which are diagnostic of double
bonds at positions 15,12, and 9, respectively, in the original unsaturated acid. Similarly, reduced eleostearic acid gives fragment ions corresponding to the losses of C3H7Dand C4H$ (81, a2), Ct310D2 and CsHllD3 (bl, bJ, and C7H12D4and C8H13D5 (cl, c2) which can be used to identify the double bond positions at 13, 11, and 9 for the original acid. The method proved successful for locating the double bonds in arachidonic (vide infra) and docosahexanoic acids. Other Experimental Considerations. Although remote site fragmentations occur for only about 1%of the selected ion beam of the (M - HI- ion, the near absence of other fragmentations allows for straightforward interpretation of the CAD spectra. Moreover, recognizable CAD spectra can be obtained for sample sizes as little as 25 ng applied to the probe tip. The limit of detection for an original sample processed through both steps was not determined although the percent yield for reducing 40 mg has been reported to range from 40 to 90% depending on the acid with a typical value of about 50% (12). The reduction method has also been reported to give deuterated acids with isotopic purity of 70430% for a series of acids (12). For arachidonic acid, approximately 30% of the molecular ions were fully labeled; Le., d8 (Figure 4). Nevertheless, the arachidonic-d8 acid (M - H)-was sufficiently abundant that it could be selected by using MS-I of the tandem mass spectrometer and collisionally activated without interference by partially reduced material. The mass resolving power of MS.11 of the tandem mass spectrometer is approximately 100 due to unavoidable energy release in the decompositions. Mass assignments, from the centroids of the broadened peaks, are valid for a t least two reasons. First, the results are reproducible to within a few tenths of an atomic mass unit. Second, mass measurements both from expanded portions of the spectra and from spectra from narrow scans agree with mass assignments based on full CAD spectra. Because of the limited mass resolution of MS-11, the fragmentations involving both hydrogen and deuterium transfer give corresponding unresolved peaks with centroids that are at intermediate m / z values. A CAD spectrum at unit mass resolution would confirm that it is indeed an unresolved doublet and that H / D exchange does not occur. Such a spectrum was obtained by using a four-sector instrument at National Institutes of Environmental Health Sciences. This tandem instrument has a double focusing MS-I (BE) and double focusing MS-I1 (EB) which has an ultimate resolving power of 10000 for MS-I1 (35). For deuterated arachidonic acid (eicosanoic-d8acid), at least unit mass resolution for the collisionally dissociated daughter ions was obtained (see Figure 5). Since this spectrum represents a single scan and not a signal averaged composite, it is difficult to assess adequately
ANALYTICAL CHEMISTRY, VOL.
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LITERATURE CITED
220
230
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Figure 5. Spectra of the daughter ions in the range of m l z 220 t o m l r 300 produced f r o m collisionally activating the (M - H)- Ions of reduced arachldonic-d, acM. The upper spectrum was obtained with a four-sector instrument; the lower spectrum w a s obtained with the three-sector instrument used In this laboratory. Since the scan modes used to obtain these spectra are different, the upper spectrum cannot be viewed a s a direct, high-resolution verslon of the lower. The t w o spectra cannot be overlaid. Fragment ions a, b, c, d, and e are formed by losses o f CH ,, C3H,, C4H1,/C4H,D, C5HI2/C5HllD, and CBH,,D, respectively.
the contribution of the small satellite peaks and distinguish actual signal from noise. However, it can be determined by examining several such spectra that the satellite peaks have an intensity of 10% or less of the main peaks at a given mass. Thus, such peaks neither interfere with the identification of the singlet/doublet pattern used for double bond location nor diminish the high integrity of the 1,4-eliminationmechanism (30). Moreover, doublets are clearly seen in accord with the prediction of competitive hydrogen/deuterium transfer from labeled sites (peaks c and d, Figure 5), and singlets are seen when only H transfer is expected (peaks a, b, and e, Figure
5). ACKNOWLEDGMENT We thank Ron Hass and the National Institutes of Environmental Health Sciences for the use of the four-sector mass spectrometer and Robert Murphy for the suggestion to study eleostearic acid. Registry No. NzD4,13762-95-3;stearic acid, 57-11-4;oleic acid, 112-80-1;linoleic,60-33-3;linolenic,463-40-1;arachidonic,506-32-1; acid, docosahexaenoicacid, 25167-62-8octadecanoic-9,10,12,13-d4 acid, 96999-38-1; 97011-48-8; octadecanoic-9,10,12,13,15,16-d6 eicosanoic-5,6,8,9,11,12,14,15-d8 acid, 96999-39-2; docosanoic4,5,7,8,10,11,13,14,16,1 7,19,20-dI2acid, 96999-40-5;methyl eleostearate, 29565-44-4;lithium linoleate, 74488-09-8;lithium linolenate, 94138-91-7; lithium arachidonate, 97011-20-6; lithium acid, docasahemenate, 96999-37-0;octadecanoic-9,10,11,12,13,14-d6 96999-41-6.
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RECEIVED for review March 25,1985. Accepted May 10,1985. The NSF is acknowledged for support of this work (Grant No. CHE-8320388). The instrumentation is supported by a NSF Regional Instrumentation Facility Grant to the University of Nebraska (CHE-8211164).
