Anal. Chem. 1989, 6 1 , 1564-1571
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Dimethyl Disulfide Derivatives of Long Chain Alkenes, Alkadienes, and Alkatrienes for Gas Chromatography/Mass Spectrometry David A. Carlson*
U S D A , ARS, Insects Affecting M a n and Animals Research Laboratory, P.O. Box 14565, Gainesville, Florida 32604 Chin-Shyan Roan a n d R i c h a r d A. Yost
Chemistry Department, University of Florida, Gainesville, Florida 32611 J u l i o Hector
U S D A , A R S , Insects Affecting M a n and Animals Research Laboratory, P.O. Box 14565, Gainesville, Florida 32604
Natural and synthetic long-chain alkenes, aikadienes, and alkatrienes (C25-C37) were readily derivatized to stable adducts with dimethyl disulfide (DMDS). This technique was extended to mixtures of unseparated alkanes, alkenes, alkadienes, and aikatrienes, but limited here to the types found in two-winged insects including African honey bees, frult flies, and stable flies, which included several unreported compounds. Capillary column gas chromatography/eiectronionization mass spectrometry (GC/EI-MS) of stable DMDS adducts allowed location of both internal and terminal unsaturated sites. Derivatlration of the total hydrocarbon fraction gave excellent results after only 4 h of reaction time. Alkanes eluted first, followed by the alkene adducts, then the diadducts of dienes, with each group well separated from the others. Molecular ions of di- and triadducts were observed, together with ions showing losses of 48, 94, 141, and 188; cleavage at carbons bearing SCH,, then losses of 48 and 94 located both double bonds. The C, and C,, dladducts were well separated during GC/MS or GC, wlth no thermal cleavage observed. Homologous C, C24,and C26triene diadducts and the CZ6triene triadduct survived GC/MS and displayed characteristic cleavage patterns. Major differences were found in proportions of honey bee alkenes that differentiated European and African races, as the C31, C,, and C, alkenes, mentioned as diagnostic for the races, were present in the diagnostic proportion when viewed as adducts. Also, a 9isomer in the C31 homologue indicated the presence of African genetic material in worker bees and wax. The variety of dienes glving linear adducts was limited in that the double bonds were separated by four or more methylenes; otherwlse cyclic derivatives of up to 29 carbons were observed.
INTRODUCTION Electron impact (EI) mass spectra of positional isomers of unsaturated aliphatic compounds are usually indistinguishable, rendering gas chromatography/mass spectrometry (GC/MS) useless in determining double bond positions. This has been generally attributed to lack of cleavage between carbon-carbon double bonds or to extensive and facile hydrogen rearrangement (Le., double bond migration) along the chain after molecular ion formation and before fragmentation (1). Remote-site fragmentation using tandem mass spectrometry for determination of double bond location via allylic cleavage has been described (2). However, this indirect method requires an instrument that is not available to most potential users
and appears difficult to apply to mixtures of isomers that are often present in natural samples. Classical approaches to location of double bonds employ isolation followed by ozonolysis and GC but require tedious separation steps that can be difficult with trace quantities (3). Alternatively, derivatization before GC/MS includes: osmium tetroxide oxidation to the vicinal diol followed by silylation (4),epoxidation of acetates and aldehydes (5) and alkadienes (6). Methoxy derivatives cleave at the methoxy group (7-9); however the former workers could not locate double bonds in beeswax alkadienes despite excellent results with alkenes. Contributing to the difficulty of locating the second double bond is the additional cleanup needed for separation of polymethoxy derivatives before analysis. Such difficulties are worse when more than one double bond is present in more than one isomer, as in dienes (10) or trienes where multiple combinations of methoxy-substituted ethers are formed uncontrollably. The derivatization of alkenes with dimethyl disulfide (DMDS) was described for GC (11). The capillary GC/EI-MS analysis of the DMDS adducts of monounsaturated acetates (12) was extended to monounsaturated fatty acids from four moth species (13). The results of DMDS derivatization of diunsaturated acetates and alcohols of up to 18 carbons that formed linear or cyclic polythioether adducts during 60-h reaction times were recently reported (14). We describe the first derivatization of long chain alkenes (CZ3to C37) to form noncyclic linear adducts with DMDS reagent. Also, we describe the first DMDS derivatization of long chain alkadienes and alkatrienes and extension of the method to the elucidation of structures in mixtures of alkanes, alkenes, alkadienes, and alkatrienes. The utility and the limitations of this technique are described. EXPERIMENTAL SECTION Biological Materials. Male tephritid fruit flies, Anastrepha suspensa, and mixed sex Drosophila melanogaster were laboratory-reared on a standard synthetic diet and collected at 3 days of age for extraction in hexane (15,16). Laboratory colony stable flies, Stomoxys calcitrans (L.), were reared on citrated bovine blood and collected at 3 days of age for extraction. African honey bee workers, Apis mellifera scutellata, were collected from colonies near Harare, Zimbabwe. European/Florida bees, A. m. mellifera, were collected from colonies in Gainesville,FL. Africanized bees were obtained from Panama and also from Panama City, FL. Extracts of all samples were obtained by soaking intact specimens in hexane for 2-10 min. The total hydrocarbon fraction was obtained by elution from a 2-cm column of silica gel with hexane only ( 1 7 ) . Wax samples were collected from colonies at the same
0003-2700/89/0361-1564$01.50/00 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 14, JULY 15, 1989
1565
H
a
i ,
1.4,
300 500
400
500
600
640
020
1000
700 11:40
800 1320
900 1500
Scan Time (mi".)
Flgure 2. Reconstructed ion chromatogram of DMDS adducts of Africanized honey bee alkenes. and fitted with a HP-1 capillary column (12 m X 0.20 mm i.d.) operated as above, but with the injector at 320 "C and the interface at 320 "C.
Figure 1. Fragmentation scheme for DMDS adducts of unsaturated hydrocarbons: (a) alkenes; (b) alkadienes; (c) alkatrienes. locations and treated the same as the bees. Reagents. Alkenes and alkadienes were separated from synthetic and natural long-chain hydrocarbons by using silver nitrate thin-layer chromatography (TLC) with 1% ether/hexane or 20% benzenejhexane and checked against the appropriate synthetic standards. Derivatives were prepared from the following standards synthesized in our laboratory, donated, or purchased: (2)-7heptacosene, (Z)-7-pentacosene, (Z)-g-pentacosene (In,1-tetra(6),and (Z,decene, 1-pentadecene, (2,2)-7,1l-pentacosadiene Z)-9,19-heptatriacontadiene(18). Derivatization. Samples (1-10pg) in hexane (200 pL) were treated with 200 pL of neat DMDS (Eastman Kodak Co., Rochester, NY) and 100 pL of iodine solution (60 mg of iodine in 1 mL of diethyl ether) (13). Reaction mixtures were held for 4 h or overnight at 40 "C, diluted with n-hexane, 5% sodium thiosulfate and the organic phase was dried over anhydrous Na2S04. The samples were blown dry, frozen, then redissolved in a minimum of hexane, and analyzed by capillary column GC and GCjMS. Instrumentation. A Tracor Model 540 GC was used for GC analysis with on-column injection (OCI-3,SGE PTY, Austin, TX) using a capillary column of DB-1 (30 m X 0.32 mm i.d., 0.25-pm film,J&W Co., Folsom, CA). The GC oven was programmed from 60 to 200 "C at 30 "Cjmin, then to 320 "C at 7 "Cjmin. A Finnigan MAT (San Jose, CA) triple stage quadrupole GC/MS/MS system equipped with a Finnigan 4500 series ion source that was operated at a pressure of 0.05 Torr and an INCOS data system was used for GC/MS analyses, scanning at 1 cyclejs. The Finnigan 9610 GC was equipped with a splitless Grob capillary injector and a short capillary column (3 m X 0.25 mm i.d., DB-I), with carrier gas (He) at ca. 1 psig. The injection and GCjMS interface temperatures were 320 and 330 "C, respectively. The GC oven was held at 100 "C for 30 s, then ramped to 320 "C at 20 "C/min. Sample introduction via solid probe was also used for high molecular weight derivatives. The amount of sample injected varied from 1 to 10 pg in 1 to 3 pL of hexane. Mass spectra also were obtained on a Hewlett-Packard 5988A GC/MS that was operated in the E1 mode (70 eV), equipped with a HP 9000-300 Chromstation and interfaced with a HP 5890 GC operated in the splitless mode or with an on-column injector as above,
RESULTS AND DISCUSSION Alkenes. Derivatives were formed by the addition of DMDS (mol wt 94) as in Figure la. Only one peak was detected by GC analysis of an alkene adduct. Mass spectra of the derivatives show recognizable molecular ions (M)+under E1 (70 eV) conditions. The double bond is located easily since cleavage of the carbon-carbon bond between the adjacent carbons carrying the methyl sulfide (CH3S)substituents leads to the major fragment ions (A)' and (B)' as previously reported ( 1 1 ) . One of these diagnostic fragments is often the base peak in the spectrum. The m / z values characteristic for these key fragments are identified by the series of m / z = 61 (CH2SCH3+)with addition of 14 (CH,) mass unit intervals. Pairs of (A)+and (B)+ions can be easily recognized because the sums of their m / z values equal (M)+. The derivative of (Z))-g-heptacosenegave fragment ions (A)+ = m / z 173 and (B)+ = m / z 299, showing the double bond to be between carbon 9 and 10, and (M)+at m / z = 472. The E1 spectra of all DMDS adducts showed a significant and diagnostic fragment ion, (M - 47)+,due to loss of SCH, from the molecular ion, and another at (M - 94)+, due to loss of DMDS to regenerate the original compound. In methane CI spectra, derivatives of (Z)-7-pentacosene and (Z)-g-heptacosene both displayed small quasi-molecular ions at m / z 397 and m/z 425 (M - H)+, respectively, as well as base peaks at (M - 47)+. The diagnostic (A)+ and (B)+ions observed by E1 were missing in the CI spectra of the derivative, however. Terminally unsaturated 1-tetradecene and 1-pentadecene were derivatized and showed prominent fragments; (A)+ = m / z 61 and (B)+= m / z 243 for 1-pentadecene, (A)+ = m / z 61 and (B)+= m / z 229 for 1-tetradecene, demonstrating that this method can be applied to terminal (11)as well as internal alkenes. Derivatization was applied to alkene mixtures which may have chemotaxonomic value from three populations of honey bees. Apis mellifera mellifera is the commercial hybrid, now present in the USA that was derived from honey bees imported from Europe, whereas A. mellifera scutellata is a type of Southeast African honey bee. The term "Africanized honey bee" describes the race that has become dominant in South and Central America. DMDS derivatives were analyzed from hydrocarbons extracted from European/Florida (hereafter referred to as European), African, and Africanized worker bees as well as from extracts of comb wax produced by European or Africanized honey bees. Figure 2 displays the reconstructed ion chromatogram of alkene adducts from African honey bees. The major peaks at scan numbers 638, 712, and 792 represented the alkenes of C31,C33,and C35chain length, respec-
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 14, JULY 15, 1989
Table I. Mass-to-Charge Ratio (% Relative Abundance) of DMDS Derivatives and Proportions 01Alkene Isomers in African, Africanized, and European/Florida Honey Bees
m / z (% relative abundance, European only) X
5
7 5
7 5 6 7 6 7 8 6 7 8 10 6 7 8 10 8 10 12
Y
14 12 16 14 18 17 16 19 18 17 21 20 19 17 23 22 21 19 23 21 19
(M)+ 416 (7) 444 (8) 472 (6)
% of each isomerb
(M - 47)+
(M - 94)+
(A)+
(B)+
European
369 (3)
323 (0.5)
145 (11) 173 (100) 145 (11) 173 (100) 145 (16) 159 (11) 173 (94) 159 (100) 173 (23) 187 (15) 159 (75) 173 (25) 187 (100) 215 (4) 159 (7) 173 (7) 187 (100) 215 (15) 187 (69) 215 (100) 243 (19)
271 (7) 243 (88) 299 (9) 271 (87) 327 (11) 313 (4) 299 (74) 341 (79) 327 (21) 313 (11) 369 (64) 355 (21) 341 (96) 313 (4) 397 (5) 383 (4) 369 (89) 341 (14) 397 (48) 369 (78) 341 (18)
9 91 10 90 13 7 80 72 18 10 41
397 (4) 425 (3)
351 (0.5) 379 (0.1)
500 (7)
453 (4)
407 (0.1)
528 (10)
481 (8)
435 (0.4)
556 (6)
509 ( 5 )
463 (0.2)
584 (4)
537 (4)
488 (1)
African
Africanized'
100
-d
100 19 81 10 90 24 76 50 36 14 18 5 77
-
57 2 5 82 13 35 54 11
-
100 -
-
100 12 76 12 50 15 35 13 2 85 60 40
-
60 40 -
-
n-C,; n = position of double bond; m = chain length of alkene; for x and y see Figure 1. Calculated as the average of relative abundance ratios of (A)+ and (B)+for each positional isomer of the particular carbon chain length. cFrom DMDS plus total hydrocarbon sample run on HP GC/MS. d - indicates less than 1%.
tively, with C23 to C29 present as minor components. The retention index relative to n-alkanes (KI) of C31 DMDS derivatives was about 630 KI units (6.3 carbon equivalents or ECL) longer than that for an internally unsaturated C31 alkene (KI 3075). Because resolution on short capillary columns was inadequate to resolve the positional isomers, mass spectra were averaged over several scans in each peak, and the relative abundance of each isomer estimated based upon the abundance of the (A)+and (B)+ions. For illustration, Figure 3 is the spectrum of the C35derivative obtained from European bees, as seen by the molecular ion (M)+ (mlz 584) and the (M - 47)+ ion (mlz 537). Three sets of characteristic (A)+ and (B)+fragment ions were observed: the (A)+ ions at mlz 187, 215, and 243, and the corresponding (B)+ions at m/z 397,369, and 341. These characteristic fragments suggest that the positions of unsaturation were at carbon number 10,12, and 14, with a relative abundance of approximately 35, 54, and 11% . It was not possible to separate adducts of these positional isomers even with a 30-m DB-1 capillary column, although other laboratories have reported the separation of DMDS derivatives of positional isomers (12, 13). This probably is because the monoolefins analyzed in this study have much longer carbon lengths (>C,) than those in previous studies (
