928
Anal. Chem. 1988, 60,928-931
Identification of the Position and the Stereochemistry of the Double Bond in Monounsaturated Fatty Acid Methyl Esters by Gas Chromatography/Mass Spectrometry of Dimethyl Disulfide Derivatives Pierre Scribe,*’ J e a n Guezennec? Jacques Dagaut,’ Claude Pepe,’ and Alain Saliot’ Laboratoire de Physique et Chimie Marines, Universite Pierre et Marie Curie, U A CNRS No. 353, tour 24, 4 Place Jussieu, 75252 Paris Cedex 05, France. and Direction de l’Ing6niGrie et de la Technologie, IFREMER-COB, B.P. 337, 29273 Brest Cedex, France
A slnglegtep derlvatlzath procedure Udng dimethyl dlsutlMe (DMDS) Is described for monounsaturated fatty acid esters (MUFAME). The analysls of DMDS adducts by gas chromatography/mass spectrometry Is detalled The mass spectra (electron Impact 70 eV) show molecular Ions (M’) and glve key fragments that petmlt detennlnatlon d the porltlon d the orlglnal double bond. Alkylthlolatlon of Z - and E-MUFAME as a Specnk antladdltlon leads, respecthrely, to the threo and erythro adducts. The two Isomers are well-separated by gas chromatography, permlttlng preclse stereochemlstry of the lnltlal double bond. Nevertheless, for a complex envlronmental mlxtwe d MUFAME, the stereochemkal ldentlfication Is restrlcted to the h e a r series. Thls procedure Is suitable for the analvsls of trace compounds at the nanogram level such as en\ Juntered In the natural environment. An appllcatlon Is presented for the analysis of planktonlc and bacterlally derived fatty aclds In recent marine sedlments.
The applicationof fatty acid methyl esters (FAME) analysis has received special interest for lipid composition of recent sediments leading to an appreciationof the importance of early diagenesis ( 1 , 2 ) and for the taxonomic determination of bacterial communities (ref 3 and 4 and references therein). The FAME distribution patterns of bacterial communitiesare often sufficiently different to enable the various respiratory types to be recognized by their particular fatty acid composition (5). In addition, bacterially derived FAME have been identified in significant amounts to characterize the development of microbial communities in the natural environment, such as in marine sediments (6), marine and estuarine waters (7, 8), and freshwaters ( 9 ) . Structural differences between unsaturated fatty acid methyl esters from bacterial and from other natural sources, e.g., phytoplankton and algae, are merely based on the following three parameters: i, chain length; ii, chain type (straight, branched, cyclopropyl,...); iii, number, position, and stereochemistry of the double bonds. The determination of the length of the carbon chain can routinely be achieved by the length-equivalent-chain (LEC) method (IO). Most difficulties are encountered in determining the position and the geometry of double bonds for monounsaturated and polyunsaturated fatty acid methyl esters (MUFAME and PUFAME). For aliphatic carbon chains, the location of the double bond position cannot be directly achieved by gas chromatography (GC), except if appropriate standards are available, or by gas chromatography/mass Laboratoire de Physique et Chimie Marines, Universite Pierre et Marie Curie. Direction de l’Ing6nibrie et de la Technologie, IFREMER-COB.
spectrometry (GC/MS), because positional and geometrical isomers show almost identical mass spectra. This problem has been tentatively solved by using chemical degradation methods such as ozonation (11) or by preparing suitable derivatives capable of labeling the original position of the double bonds. Extensive reviews of the various methods have been published on this subject (12, 13). In the monounsaturated fatty acid series several methods have been proposed: (i) The first method is the conversion of MUFAME via stereospecific oxidation with Os04 followed by condensation with acetone into 1,3-dioxolanecompounds (14) or derivatization into trimethylsilyl ethers (15-18). Fragment ions obtained do permit the unambiguous identification of the position of the double bond. However, as might be expected, the mass spectra and the retention times of erythro and threo isomers of TMS derivatives are very similar. Only weak differences are observed for o-isopropylidene derivatives (14). (ii) Another method is a Diels-Alder reaction of olefins with ketals of tetrachlorocyclopentadiene, which permits determination of both the position and the stereochemistry bond (19). In the last few years several methods using chemical ionization NO (20)and methyl vinyl ether (21)have been proposed. Although these procedures are commonly used in determining individual chemical structures, they cannot be used for complex mixtures of unsaturated compounds at the nanogram level in concentration. We report here the use of a one-step derivatization technique which can be carried out on a few nanograms of complex mixtures of monounsaturated fatty acids. This method consists of the iodine-catalyzed addition of dimethyl disulfide (Figure l), which has been previously reported for n-alkenes (22,23) and for monounsaturated acetates (24). This step, followed by the GC/MS analysis of the derivatives in the electron impact mode, allows us to identify the position and, with aid of the retention time of the erythro or threo DMDS derivatives, the stereochemistry of the initial double bond. Application is presented for a relevant environmental problem: the study of planktonic and bacterial markers in recent marine sediments. EXPERIMENTAL SECTION Isolation of Fatty Acids from Sediment. The sediment analyzed was cored during the MISEDOR oceanographic cruise on the R/V Jean Charcot, in the Mahakam Delta in December 1984. Station 17 (Oo 20‘ 85” S 117’ 51’ 22” E) was chosen. Water depth was 85 m. The surficial sediment (0-2 cm) was composed of a brown mud and contained 0.18% dry weight of organic carbon. Lipids were extracted from the freeze-driedsediment in a Soxhlet apparatus using a mixture of chloroform and methanol (41 (v/v)). Fatty acids were isolated from the lipids as follows: the lipid extract was taken up into toluenemethanol-water (2:41 (v/v/v) 6 mL) which was 1.5 N in KOH and refluxed for 2 h (under an
0003-2700/88/0360-0928$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
c H ,-(c H ,) n - ~H = c H - (CH ) c o o - c H,
lA3
i'
c H ,-(CH ,) ,- c H - c H - (CH ,;) c o 0-c H , I I
7
s\ CHf
CH,
Figure 1. Reaction scheme of dimethyl disulfide on monounsaturated
fatty acid methyl esters.
CH,-(CH ,),-cH-cH
I
2 CH3
I
I
- (CH, ;1
COO-CH?
929
io
'F1
'
'
lb0
200
'
240
'
26-14:l
M+ 334
a I
+*
60
LI 280
360
320
400
440
480
8,
l 'gr
A+
CH3
173 d
CH,-(cH,)
n
-
II
~
~
C H -(CH ,);COO
It
I
40
80
I
,I.
120
7
E + + A* +'\CH,
I
I
-CH,
I1
-CH,-OH
II 100
%
Figure 2. Major fragment ions obtained In mass spectrometry (E1 mode) of MUFAME DMDS adducts.
inert atmosphere of argon in order to avoid autooxidation of polyuhsaturated fatty acids). After the solution was cooled, 5 mL of distilled water was added, and the mixture was acidified up to pH 2 with a solution of HC14 N, the total lipids were extracted three times with a mixture of hexane/ether (9:l (v/v)). Extracts were evaporated to dryness under a pure nitrogen stream and separated by adsorption chromatography on a column (50 mm X 5 mm i.d.) filled with 1 g of SO2. The fractions eluted successively by 6 mL of hexane, 8 mL of hexane/ethyl acetate (50:1, (v/v)), 20 mL of ethyl acetate, and finally 10 mL of methanol contained, respectively, nonaromatic hydrocarbons, aromatic hydrocarbons, alcohols, sterols, and fatty acids, and the fourth fraction remaining unidentified. The fatty acids contained in the third fraction were converted into their methyl esters by heating with a solution of BF3, 14% in methanol, for half an hour and the methyl esters purified by adsorption chromatography of the same type as described above. FAME were eluted in the second fraction. Methyl (2)and (E)-9-octadecenoate (2and E-9-18:l) were obtained from Supelco,Inc. (France), and dimethyl disulfde was obtained from Fluka (Switzerland). Derivatization. Total FAME fraction (1-100 pg) was treated in 100 pL of hexane by addition of 100 pL of DMDS and 20 pL of iodine solution (60 mg of I2 in 1 mL of diethyl ether). The reaction was &ed out in a 10-mL tube closed with a Teflon-lined cap and kept over 48 h at 50 "C. Samples were diluted with 200 pL of hexane and the iodine in excess was reduced by treatment with 200 pL of NalSzOBsolution (5% in distilled water). The organic phase was removed with a syringe and the aqueous phase extracted a second time with 200 p L of hexane. The two organic extracts were joined and concentrated to 50 p L under a pure nitrogen stream. An aliquot was analyzed immediately by GC/FID (flame ionization detection) or GC/MS in order to avoid the decomposition of DMDS adducts which has been found to occur within a few days. The reaction yield has been determined on pure methyl (2)and (E)-9-octadecenoate by GC peak integration of the initial esters and, respectively, the erythro and threo adducts using an internal standard (deuteriated tricosanoate methyl esters).
.
1,
I.
200
240
M* 390 280
C+
Ib
29-16: 1
I
c H -(c H l)my, c H=c o
I
160
320
360
400
440
480
1 '*
217
]
A+ 173
100-
% E9 - 1 8 : l
M+ 390
c
I
k
l 280
360
,
,
400
,
,
,
,
480
440
cc J J
100
4
320
43
I 173
213
2 11- 2 0 : 1
M+ 418
d 280
320
360
400
440
480
Figure 3. Mass spectra of DMDS adducts of (a)26-14:l (b) 29-18:1, (c) E9-18:1, and (d) 211-2O:l.
Gas Chromatography Analysis. GC analyses were performed on a Girdel 32 gas chromatograph. The column employed was a fused silica capillary (50 m X 0.25 mm i.d.) coated with CP Si1 88 (Chrompack, France). The column temperature was programmed from 100 to 195 "C at a rate of 2 "C/min. Mass Spectrometry. GC/MS analyses were performed on a R 10 10 C Nermag quadrupole mass spectrometer coupled with a Girdel 32 gas chromatograph. The column employed was a fused silica capillary (50 m long, 0.25 mm i.d.), coated with CP Si1 88 phase (Chrompack). The samples were injected in the splitless
930
ANALYTICAL CHEMISTRY, VOL. 60,
NO. 9,MAY 1, 1988 C02-CH3
13
1
IS
!
I
C0,-CH,
2000
-
3000
"IO
1 *( 1
4000 5000 SCANS
7000
9000
Flgure 6. Reconstructed gas chromatogram from GC/MS of DMDS derivatized MUFAME (see legend of Figure 5);16a, unidentified compound. \
25
1
26
TIC
CH,
Figure 4. Stereochemistry of the addltlon of the DMDS on the double
bond of
MUFAME. IS
1
RIG-334
5
__
-
30
I -~ -
MINUTES
00
F I p e 5. GC/FID detector of F A M before ckivatization: (1)is0 13:0, (2)13:0,(3)is0 14:0,(4)14:0,(5)2 6 - 7 4 : 7 (6) br 15:0,(7)is0 15:0, (8)ante 15:0,(9) br 6 - 7 5 : 7 (10)15:0,(11)26-15:l (12)is0 16:0,(13) 160,(14)Z6-76:7 (15)2 9 - 1 6 : 7 (16)2 7 7 - 7 6 : 7 (17)is0 17:0,(18)ante 17:0,(19)17:0,20)26-77:7 (21)29-17:l (22)211-17:1, (23)CY 17:0, (24)18:0, (25)29-18~1, (26)211-18:l (27)29,26-18:2,(28)212, (32)20:0, 29-18~2, (29)19:0,(30)Cy 19:0,(31)215, 212,29-18~3, (33)21 1-20:1,(34)21:0,(35)22:0,(36)(internal standard) 23:0,(37) 24:O. Saturated FAME are denoted as N : M where N is the number
2 R ICz362
I
II
J
22
R IC r 3 7 6
of carbons and M is the number of double bonds: unsaturted FAME are denoted 2 or EL-N:M, where L specifies the position of the double bond according IUPAC nomenclature; br = branched: cy = cyclopropyl. mode (1-2 wL)and the column temperature was programmed from 100 to 220 OC at a rate of 3 OC/min. The mass spectrometer was operated from 31 up to 550 amu with one scan per second. Electron energy was 70 eV. MS data were acquired and processed by using an on-line PDP 11/23 PLUS computer with SIDAR 111 data system.
RESULTS AND DISCUSSION The electron impact (EI, 70 eV) mass spectra show easily recognizable molecular ions (M+)and give key fragments that allow us to clearly identify the position of the original double bond. The major fragment ions A+ and B+ derived fro= the cleavage of the carbon-carbon bond between the two methyl suKde substituents for the typical FAME are shown in Figure 2. Fragment m/z 61 (CH3S'+=CH2)originating from the key fragment A+ via a H rearrangement is encountered in all derivatives studied, as well as in alkenes (20) and acetates (22). Key fragment B+ decomposes further and gives C+ via loss of MeOH (B+- 32). Fragments leading to ions in a lower mass range were not identified. Typical spectra are shown in Figure 3.
I RIG-418 33
i
i
5000
6000
7000
8000
9000
SCANS
Figure 7. Fragmentograms of DMDS adducts of MUFAME from sedimentary lipids.
Because straight-chain and branched MUFA in the E configuration such as Ell-18:1, Ell-17:1, and E9-16:l are considered as potential bacterial markers (61,we checked with special attention for the stereospecificity of the addition of DMDS on the double bond of MUFA, as illustrated in Figure 4, and the GC efficiency with respect to the stereoisomers
ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
obtained in the erythro and/or threo configuration. These two points have been studied on methyl (2)and (E)-g-octadecenoate (Zand E9-181). DMDS reacts with each isomer to give a unique adduct in both cases with a 95% yield. Mass spectra are identical for the two adduct compounds obtained, but GC retention times are different on the CP sil88 column. The adduct obtained from the 29-18:l shows a shorter retention time than that obtained from the E isomer. As far as the olefinic reaction is concerned, we can reasonably assume that the alkylthiolation is an antiaddition (20-22). Thus DMDS adduct I (Figure 4) would be identified as racemic methyl threo-9,10-bis(methylthio)octadecanoate and adduct I1 as racemic methyl erythro-9,10-bis(methylthio)octadecanoate (Figure 4). As found for DMDS adducts of acetates (20), the threo isomer is observed to elute prior to the erythro isomer. This fact provides an interesting ability for deducing the stereochemistryof the double bond from the GC retention times as far as series of linear homologues are concerned.
APPLICATION TO THE ANALYSIS OF A RECENT MARINE SEDIMENT In order to investigate the biogeochemistry of organic matter deposited in equatorial deltaic environment (Mahakam, Borneo), analysis of fatty acids has been realized on solvent-extracted lipids from surface sediments separated as a function of particle size. To illustrate the application of the DMDS method, Figures 5 and 6 show chromatograms of FAME before and after derivatization with DMDS. No residual MUFAME were detectable after a reaction time of 48 h at 50 "C. Reaction yields are 95%. The samples were injected after removal of the excess of Iz with Na2S203.No further cleanup was necessary for the present sample, but DMDS adducts can be purified by chromatography on silica if required. From data obtained (Figure 6) it is possible to determine with absolute confidence the double bond locations in an extremely complex mixture of FAME (Figure 5) such as encountered in sedimentary environments (6, 25). The linear and branched saturated FAME from 13 to 24 carbon atoms, the MUFAME 29-16:l and 29-181, and the PUFAME 29,26-18:2,212,29-18:2,and Z15,212,29-183 have been previously identified by comparison of the retention times with external standards on a Silar 5CP column (Chrompack, France). From the GC/MS analysis of DMDS adducts of FAME, a search of molecular ions m/z 334,348,362,376,390,
931
and 418 revealed the presence of 12 MUFAME located between scan 5000 and lo000 (Figure 7). Examination of their mass spectra allowed us to identify unambigously MUFAME indexes: 5,9,11,14,15, 16,20,21, 22,25,26, and 33 (see Figures 5 and 7 and legends). Any E isomers has not been found in this particular sample. The DMDS derivatives of MUFAME containing more than 20 carbon atoms and of PUFAME could not be detected by GC/MS because they were not sufficiently volatile or decomposed thermally.
LITERATURE CITED (1) Voikman, J. K.; Johns, R. B.; Gillan, F. T.; Perry, G. J. Geochim. Cosmochlm. Acta 1880, 44. 1133. (2) Cranweli, P. A. Geochim. Cosmochlm. Acta 1878, 42, 1523. (3) Lechevaller, M. P. Crlt. Rev. Microblo/. 1977, 7 , 109. (4) Mallory, L. M.; Sayier, G. S. Mlcroblol. Ecol. 1884, 70, 283. (5) Shaw, N. A&. Appl. Mlcroblol. 1874, 77, 63. (6) Gillan, F. T.; Johns, R. 6.; Verheyen, T. V.; Nlchois, P. D.; Esdallle, R. J.; Bavor, H. J. I n Advances In Organlc Geochemlstry; Bjtdroy, M., et ai., Eds.; Wiley: New York, 1883; pp 198-206. (7) Goutx,M.; Sallot, A. Mar. Chem. 1880, 8 , 299. (8) Tronczynski, J.; Marty, J. C.; Scribe, P.; Lorre, A.; Sallot, A. Oceanis 1885, 7 7 , 399. (9) Letoile, R.; Blalson, N.; Chesterlkoff, A.; Grateloup, I.; Scribe, P.; Barouxls, A.; Marty, J. C.; Sallot, A. Colioque sur les Nitrates, Paris, Octobre 1985. (10) Tranchant, J. Manvelpratique de chromatogrephieen phase gazeuse, 3rd ed.; Masson: Paris, 1982; pp 301-337. (11) Beroza, M.; Bieri, B. Anal. Chem. 1887, 3 9 , 1131. (12) KIWRR,D. R. Hendbodc of AnaMlcd Derlvatlzatbn Reactlons; Wiley: New York, 1979; p 229. (13) Mulier, K. Functlonal Group Determlnatbn of Oleffnic and Acetylenlc Unsafufatlon; Academic: London, 1975; p 73. (14) McCloskey, J. A.; McClelland, M. J. J . Am. Chem. Soc. 1885, 8 7 , 5090. (15) Niehaus, W. 0.;Ryhage, R. Tetrahedron Lett. 1887. 49, 5021. (16) Argoudells, C. J.; Perklns, E. G. UpMs 1888. 3 , 379. (17) Capella, P.; Zorcut, C. M. Anal. Chem. 1888, 40, 1458. (18) Eallnton. Q.; Hunneman. D. H.; McCormick. A. Ora. - Mass SDectrom. 1088, 7 , 593. (19) Kldwell, A. D.; Biemann, K. Anal. Chem. 1882, 54, 2462. (20) Brauner, A.; Budzlkiewicz, H.; bland, W. Org. Mass Spectrom. 1882. 17. 161-164. . - . . - .. (21) Chai, R.; Harrison, A. G. Anal. Chem. 1881, 53. 34-37. (22) Francis, G. W.; Veland, K. N. J. Chromatogr. 1881, 279, 379. (23) Caserio, M. C.; Fisher, C. L.: Kim, J. K. J . Org. Chem. 1885, 50, 4390. (24) Buser, H. R.; Arn, H.; Guerlain, P.; Rauscher, S. Anal. Chem. 1883, 55, 818. (25) Plllon, P.; Jocteur-Monrozier, L.; Gonzalez, C.; Saliot, A. Org. Geochem. 1988, 70, 711.
.
RECEIVED for review August 3,1987. Accepted December 28, 1987.
