Application of Chemical Ionization Mass Spectrometry of Epoxides to the Determination of Olefin Position in Aliphatic Chains J. H. Tumlinson, R. R. Heath, and R. E. Doolittle lnsect Attractants, Behavior and Basic Biology Research Laboratory, Agricultural Research Service, USDA. Gainesville, Fla. 32604
Chemical ionization (CI) mass spectrometry is a relatively new technique ( I ) that is being used with increasing frequency in the elucidation of organic structures. In this technique, an ionizing reagent gas reacts with the sample molecule to produce spectra that are often quite different from those produced by electron impact (EI) mass spectrometry. Thus, certain information about a compound is derived from CI spectra that is often not available from E1 spectra. For example, the ionizing reagent gas reacts most frequently with polar functional groups in a molecule and, so, often gives useful information about their location or identity. Additionally, the reagent gas may be varied considerably (2-7) so a particularly suitable reactant for a class of compounds may be chosen, or a compound may be subjected to various ionizing plasmas to yield more structural information. Since the location and configuration of olefinic and epoxide groups that abound in natural products, particularly in insect hormones (8) and pheromones (9, I O ) , are crucial to biological activity, considerable effort has been expended on the analysis of these groups. Ozonolysis of olefins ( 1 1 ) has been a particularly useful technique though the products are sometimes difficult to analyze when small molecules are produced. Also, epoxide position and configuration may be determined in many cases by reaction thin-layer chromatography ( 2 2 ) .However, mass spectrometry of olefins, even with the CI technique ( I 3 ) ,will not usually locate the double bond. Olefin position may often be located by E1 mass spectrometry of derivatives prepared by addition and oxidation reactions. These include deuteration (14-16), formation of ketones (17), hydroxyamines ( I c i ) , 0-isopropylidene derivatives (19), methyl F. H . Field, Accounts Chem. Res.. 1, 42 (1968) F H . Field. J , Amer. Chem. Soc.. 92, 2672 (1970). D F. H u n t , C. N . McEwen, and R. A . Upham. Anal. Chem.. 44, 1292 ( 1972)
D F. H u n t and J F. Ryan I l l , Anal. Chem.. 44, 1306 (1972) D F H u n t and J F. Ryan I l l . J. Chem. Soc. Chem. Commun., 620 (1972).
D F. H u n t and J . F. Ryan I l l , Tetrahedroniett.. 4535 (1971). D F. H u n t , C. N . McEwen, and R. A . Upham. Tetrahedron Left., 4539 (1971).
H . Roller, K . H . Dahm, C. C. Sweeley. and B. M . Trost, Angew. Chem.. 6 , 179 (1967) B. A. Bierl, M . Beroza, and C . W. Collier, Soence, 170, 8 7 (1970) M . Jacobson, "Insect Sex Pheromones." Academlc Press, New York. N . Y . , 1972. M . Beroza and B. A . Bierl, Anal. Chem., 39, 1131 (1967) B . A . Bierl, M Beroza, and M . H Aldridge. Anal. Chem.. 43, 636 (1971 ) . F t i Fieid. J. Amer Chem. Soc , 90, 5649 (1968) N . Dinh-Nguyen, R Ryhage. and S. Stallberg-Stenhagen. A r k . Kemi. 15, 433 (1959) E Seike, C. R. Scholfield, C D. Evans, and H . J. D u t t o n , J. Amer 011Chem. Soc.. 38, 614 (1961 j K . K . Sun, H . W . Hayes, and R T. Holman, Org. Mass Spectrom.. 3, 1035 (1970) G . W Kenner and E . Stenhagen. Acta Chem. Scand.. 1 8 , 1551 (1964) H Audier. S. Bory, M . Fetizon, P Longevialle. and R. Toubiana. Bull. Soc C h m Fr.. 1964, 3034. J A. McCloskey and M . J McClelland, J. Amer. Chem. Soc.. 87, 5090 (1965)
ethers (20), silyl ethers (21-23), and epoxides ( 2 4 ) . However, most of these reactions usually require a t least 100 yg of sample. Moreover, though E1 mass spectrometry may be useful in many cases in elucidating the structure of simple epoxides ( 2 4 ) , many difficulties may be associated with interpretation of low resolution spectra of epoxides (25, 26). In our continuing investigation of biologically active compounds in insects, we have sought techniques that would permit us to locate and determine the configuration of double bonds and epoxides with 1 Fg or less of a compound. Thus, as a preliminary step in the development of a generally useful analytical technique, we recorded the CI spectra of a series of unbranched aliphatic epoxides. Included in the series were epoxydecanes and acetates of epoxidized long chain unsaturated alcohols. Methane and isobutane were employed as reagent gases. Easily distinguishable characteristic ions were formed that greatly facilitate location of the epoxy groups.
EXPERIMENTAL Equipment. All mass spectra were measured on a Finnigan Model 1015C quadrupole mass spectrometer equipped with a CI source. Methane and isobutane were used as reagent gases at source pressures (measured by a thermocouple gauge) of about 1 and 0.4 Torr, respectively. The source temperature was held between 70 and 100 "C. Samples were admitted to the source uia a Varian Mdel 1400 gas chromatograph that was interfaced to the mass spectrometer with lh,j-in. (0.d.) stainless steel tubing and equipped with a 2-m X 2.3-mm (i.d,) stainless steel column packed with 3% OV-1 on 100/120 mesh Varaport 30. The reagent gas was used as the carrier gas, and the total effluent from the column entered the mass spectrometer. Sample size ranged from 0.5 to 1 pg. Data acquisition and reduction were accomplished with a Systems Industries Disc System 150 computer interfaced to the mass spectrometer. Peaks derived from background (column bleed etc.) were subtracted from all spectra. Test Compounds. The decenes were purchased from Chemical Samples Company and used without further purification. The unsaturated esters were synthesized as a part of' other programs. Epoxidation was carried out by adding the olefin to dichloromethane that contained a molar excess of 99+70 m-chloroperbenzoic acid (27) and allowing the mixture to stand at room temperature for 30 min to several hours. When compounds containing two ethylenic bonds were epoxidized. the reaction was monitored by gas chromatography so we could determine the reaction time necessary to produce monoepoxides rather than diepoxides. The time necessary for completion of the reaction varies considerably (20) W G Niehaus and R Ryhage Terrahedron Lett 1967, 5021 (21) P Capellaand C M Zorzut Ana/ Chem 40, 1458 (1968) (22) G Eglinton D H Hunneman and A McCormick Org Mass Specfrom 1 , 593 (1968) (23) C B Johnson N Z J Sci 12, 27 (1969) (24) R T Aplin and L Coles Chem Commun 858 (1967) ( 2 5 ) P Brown J Kossanyi and C Dlerassi Tetrahedron Suppiement 8 , Part 1 241 (19661 (26) H Budzikiewicz C Djerassi and D H W lliams Mass Spectrom
etry of Organic Compounds 1967 pp449-466 (27) N N Schwartz and J (1964)
H
Holden Day San Francisco Calif Blumbergs J Org Chem
A N A L Y T I C A L C H E M I S T R Y , V O L . 46. NO. 9. A U G U S T 1974
29. 1976
1309
~
Table I. Principal Isobutane Ionization Mass Spectrometric D a t a for the Epoxydecanes and 5-Decene PrinciDal Ions in Mass SDectrum (Relative abundance in parentheses) M f l
5,6-Epoxydecane 4,5-Epoxydecane 3,4-Epoxydecane 2,3-Epoxydecane 1,2-Epoxydecane
157 (98) 157 (100) 157 (100) 157 (100) 157 (100)
139 139 139 139 139
M + l
5-Decene
(98) (99) (99) (99) (12)
87 (100) 101 (25) 115 (23) 129 (z) 143 (2)
73 59 45 31
C
69 (32) 83 (88) 97 (54) 140 (38). 83 (83) 140 (24) 113 (12) 85 (12) (18) 97 (51) 83 (89) 140 (31) 99 (22) 83 (64) 97 (28) 140 (21) (6) 111 (5) 83 (40) 97 (17) 69 (9) (z) 125 (z)
x (37)
99 71 69 69 155
(34) 85 (27) (12) 69 (12) (15) (9) 113 (9) (7)
Other significant ions
M
M - 1
141 (85)
Other significant ions
b'
b
a
139 (66) 140 (100) 99 (9)
113 (68)
85 (55)
71 (46) 127 (40)
83 (27)
97 (16)
x. b' is the same as b in this case. z. This fragment does not occur in the spectrum.
-
100-
3
-
80-
I I
w
8
0CCH3
*
:,lo 15
+
-
/O\
'-C4Hs
/O\ CH, ICH21 CH-CHlCH2i 2
CH, 4
CH,ICH,i
CH-CHKH,) 2
z
IM t I )
', m I 2
jL
OH
o w
I
B
40-
CH, 4
I ,
e
I" 167-Mi
60-
B
g
+ H
+
20
0
t-l
H
H--t
157
I YH
" w
W
'
II
1
85
61 0,
Ill
97
20-
,
#,/I
.)I
5 ;
I25
(.I.., ,,,I,
,
165 I
'"1 i?
2 60
,.,
,.
r
0
o , m h 139
CH,(CH21
2o
CUOH 2
I
-OCCH3
c, r n h
C
83
Scheme I 60 MIE
80
100
120
140
160
80
200
220
240
260
Figure 1. lsobutane mass spectra of (Z)-7-dodecen-l-ol acetate
and (Z)-7,8-epoxy-l-dodecanol acetate depending on the relative concentration of the m-chloroperbenzoic acid. All the epoxides were purified by preparative gas chromatography. The reaction mixture was injected on a 2-m X 2.3mm (i.d.) column that contained 3% OV-210 on 100/120 mesh Varaport 30 with a helium carrier flow of 20 ml/min. The epoxides were collected in glass capillaries cooled in Dry Ice as they eluted from the column. This column adequately separated all the epoxides except the monoepoxides produced from (Z,Z)-9,12octadecadien-1-01acetate (linoleylacetate).
RESULTS AND DISCUSSION Epoxides may be easily prepared from many olefins using m-chloroperbenzoic acid. Also, the reaction may be sampled a t any stage by preparative gas chromatography. If only a very small amount of olefin is available, the reaction mixture may be injected directly into the gas chromatograph-mass spectrometer. Thus, the reaction may be examined by mass spectrometry a t the halfway point, allowing direct comparison of olefin and epoxide spectra. Linoleyl acetate, which has 2 methylene interrupted cis double bonds, produced monoepoxides that co-chromatographed on the OV-210 column. We could not separate these epoxides on any gas chromatographic column we employed. No differences between any of the cis (2)and trans ( E ) isomers were observed in any of the CI mass spectra we obtained. 1310
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, N O .
The spectra of the epoxydecanes are straight forward. Scheme I illustrates the proposed ring cleavage in the 3,4-, 4,5-, and 5,6-epoxydecanes. All the ions in this scheme are very prominent in the isobutane spectra (Table I) and somewhat less intense in most methane spectra. Also, the fragmentation pattern of the epoxydecanes is essentially the same with both reagents though the relative abundances differ with each gas. When methane is the reagent gas, (M + l)+ and a[(M 1) - HzO]' are relatively less abundant as expected, and more fragmentation occurs ( I , 28). Ring clevage provides the diagnostic peaks that locate the position of the epoxide in the chain. The other significant ions listed in Table I apparently arise by further fragmentation of the remaining hydrocarbon moiety and are comparable with the ions produced by isobutane ionization of 5-decene. When the epoxide function is in the 1,2 or 2,3 position, a different pattern emerges. The hydrogen shift from the terminal methyl group is apparently not favored in 2,3-epoxydecane since the ion b was not observed. A comparison of the relative abundance of [(M 1) - H2OI-t from 1,2- and 2,3-epoxydecanes showed a very low yield of ion a from the 1,2-epoxydecane analog. Therefore, the relative abundance of ions b and b' is indicative of a 1,2- or 2,3-epoxydecane. Also the low relative abundance of ion a is strong evidence for the 1,2-epoxydecane analog. The spectra of the epoxyalkyl acetates are more com-
+
+
(28) B Munson, Ana/ Chem , 43 (13). 28A (1971)
9, AUGUST 1974
Table 11. Principal Isobutane Ionization Mass Spectrometric Data for the Epoxyalkyl Acetates Principal ions in mass spectrum (Relative abundance in parentheses) M + 1
2,3-Epoxy-l-hexadecanol acetate
f
e
d
h
L?
299 (100) 281 (8) 239 (32) 221 (3)
103 (19) 213 (z)
k
j
2
87 (1)
43 (y)
27 (y)
4,5-Epoxy-l-tetradecanol
acetate 5,6-Epoxy-l-tetradecanol acetate 6,7-Epoxy-l-tetradecanol acetate 7,8-Epoxy-l-dodecanol acetate 9,10-Epoxy-1-tetradecanol acetate
271 (19) 253 (4) 211 (100) 193 (22) 131 (10) 157 (1)
115 (1)
71 (15) 55 (y)
271 (35)
253 (15) 211 (100) 193 (67)
145 (31) 143 (8) 129 (18) 85 (70)
271 (65)
253 (40) 211 (33) 193 (100) 159 (83) 129 (23) 143 (31) 99 (62)
69 (7) 83 (30)
243 (19) 225 (15) 183 (14)
165 (100) 173 (33) 87 (9)
157 (13) 113 (8)
271 (83)
193 (100) 201 (39) 87 (25)
185 (11) 151 (8) 125 (33)
253 (20) 211 (12)
97 (29)
y. Not recorded; fragments in this region are masked. z. This fragment does not occur in the spectrum.
Table 111. Principal Isobutane Ionization Mass Spectrometric Data for the Epoxyalkenyl Acetates Principil ions in mass spsctrum (Relative abundance in parentheses)
M + 1
9,lO-Epoxy-12-'tetradecen-l-o1 acetate 269 (85)
d
f
e
i
i
h
L?
251 (21) 209 (21) 191 (11) 201 (100) 85 (15) 185 (23) 141 (8) [Other significant ions: 199 (52),157 (13),139 (10)]
12,13-Epoxy-9-tetradecen-l-o1 acetate 269 (100) 251 (73) 209 (22) 191 (29) 241 (z) 45 (2) 225 (13) 9,10-Epoxy-12-octadecen-l-ol acetate 325 (100)"307 (23) 265 (1) 247 (1) 201 (10) 141 (2) 185 (2) [Other significant ions: 199 (S),157 (5),139 (2)] 12,13-Epoxy-9-octadecen-l-o1 acetate 325 307 (23) 265 (1) 247 (1) 241 (2) 101 (2) 225 (2) [Other significant ion: 99 (13)] 7,8-Epoxy-ll-hexadecen-l-ol acetate 297 (59) 279 (48) 237 (33) 219 (19) 173 (100) 141 (25) 157 (21) 11,12-Epoxy-7-hexadecen-l-o1 acetate 297 (100) 279 (85) 237 (11) 219 (13) 227 (11) 87 (3) 211 (3)
k
125 (37)
181 (2)
165 (59)
141 (x)
125 (3)
181 (1)
165 (10)
113 (19)
97 (33)
167 (2)
151 (16)
" Since these two compounds could not be separated, all the ions in these two spectra were actually derived from one spectrum of a mixture of the two monoepoxides of linoleyl acetate. x. J has the same mass as h in this spectrum. z. This fragment does not occur in the spectrum.
plex than those of the epoxydecanes but, here again, a characteristic fragmentation pattern occurs consistently. Table I1 lists the major peaks of the isobutane spectra of epoxyalkyl acetates, and Figure 1 shows the differences between the isobutane spectra of (2)-7-dodecen-l-o1acetate and its corresponding epoxide. When the spectra of the epoxide and its parent olefin are compared, the peaks resulting from ring cleavage are immediately distinguishable. The unsaturated acetate spectra consist primarily of the (M + 1)+ and [(M 1) - 601- peaks accompanied by characteristic straight chain hydrocarbon fragments in lesser relative abundances. However, protonation of the epoxide oxygen again produces ring cleavage that greatly facilitates location of the epoxide. A plausible fragmentation sequence is depicted in Scheme 11. Apparently. when the epoxide oxygen is protonated, ring cleavage occurs in the same manner as in the epoxydecanes. The major difference is that the fragment containing the hydroxy1 and the acetate groups* correto b, now loses CH3C00H, rather than Hzo, to yield 1. The formation of ion 1 and the loss of both HZO and CH3C00H from the (M ion are not explained. Although there is no definitive evidence for any mechanism, these ions may result from intramolecular reactions between the ester Oxygen and the protonated epoxide in a manner similar to that proposed for
+
f i j
+
1
r+---o I CH2 = C H I C H L l 3 - O
II -
CCH?
-crI,c02r
C+dg
1.129
5.69
Scheme I1 long-chain diols (29) and dimethoxy alkanes This may also explain the formation of ions i and k , which provide diagnostic peaks in most of the epoxyalkyl acetates. The spectra of the epoxyalkyl acetates obtained with methane show considerably more fragmentation than 129) 1 Dzldlc and J A McCloskey J (1971) (30) T H Morton and J L Beauchamp (1972)
Amer J
Chem Soc
93, 4955
Amer Chem SOC 94, 3671
A N A L Y T I C A L C H E M I S T R Y , VOL. 4 6 , NO. 9 , A U G U S T 1 9 7 4
1311
those with isobutane. As an example, the major ions in acetate the methane spectrum of 5,6-epoxy-l-tetradecanol occur in the following relative abundances: (M + 1)+,1%; d, 1%; e, 57%; f, 48%; g, 0%; h. 5%; i, 1%; j , 100%; j-HzO, m l e 69, 44%. Only the isobutane CI spectra of the epoxyalkenyl acetates were recorded (Table 111). Their fragmentation patterns are the same as those of epoxyalkyl acetates when the site of unsaturation is separated from the epoxy group by more than one methylene group. This is illustrated in the spectra of the two monoepoxides formed from ( 2 , E ) 7,11-hexadecadien-1-01 acetate. However, from the relative abundances of the g fragments in the two spectra, the double bond evidently affects the fragmentation somewhat when it is located between the two oxygen-containing functional groups in the molecule. When only one methylene separates the double bond from the epoxy group, the fragmentation is more strongly affected. Thus the spectra of the monoepoxides formed from linoleyl acetate and 9,12-tetradecadien-l-o1 acetate are still very similar to those of the other epoxyalkenyl acetates with most of the fragments proposed in Scheme I1 still present. The g and j peaks are absent in the spectrum of 12.13-epoxy9-tetradecen-1-01 acetate. Additionally, however, unex-
plained prominent peaks occur a t m/e-199, 157, and 139 when the epoxy group and unsaturation are in the 9,10 and 12 positions, respectively. Then, when these two functional groups are reversed in their positions in the 18 carbon chain, a prominent peak appears at mle 99. Peaks of this type did not appear in the spectra of the two monoepoxides formed from (Z,E)-7,11-hexadecadien-l-ol acetate in which two methylenes separate the double bonds. In summary, our results indicate that CI mass spectrometry with isobutane used as a reagent gas provides valuable structural information about epoxyalkanes and related bifunctional compounds. This technique should find considerable use in the analysis of biologically derived olefins and epoxides. ACKNOWLEDGMENT
We thank Helen Su, U.S. Department of Agriculture, Stored-Product Insects Research and Development Laboratory, Savannah, Ga., for the sample of 7,11-hexadecadien-1-01 acetate. Received for review September 24, 1973. Accepted April 4, 1974.
Detection of Aliphatic N-Nitrosamine Compounds by Plasma Chromatography F. W. Karasek and D. W. Denney Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada
The carcinogenic activity of nitrosamines was recognized as early as 1956 ( I ) . More recently attention has been focused on the nitrosamines as environmental carcinogens (2, 3). Their presence appears to be ubiquitous ( 4 ) ; however, nitrosamines are very difficult to detect at low concentrations and a group analysis is of limited value for not all nitrosamines are carcinogenic. Automatic colorimetric analysis ( 5 ) ,pyrolysis after GC separation ( 6 ) ,formation of electron capturing derivatives by reaction with fluorinated anhydrides and pyridine ( 7 ) , mass spectrometry (8),gas chromatography (9j and combined GC/MS (10)are some of the methods that have been applied to the analysis of N nitroso compounds. Identification and analysis of trace organic compounds by their ionic mobility spectra observed in plasma chroma-
( 1 ) J. M. Barnes and P. N. Magee, Brit. J. Cancer, 10, 114 (1956). (2) W. Lijinsky and S. S. Epstein. Nature(London), 225, 21 (1970). (3) Von G. Osske, Arch. Geschwulstforch, 39, 62 (1972). (4) G. Neurath, fxperentia, 23, 400 (1967). (5) S. R. Tannenbaum and Tsai-Yi Fan, J. Agr. Food Chem., 19, 1267
(1 97 I). (6) J. W. Rhoades and D. E. Johnson, J. Chromatogr. Sci., 8, 616 (1970). (7) J. B. Brooks, C. A. Allen, and R. Jones, Ana/. Chem., 44, 1881 (1972). (8) J. W. Pensabene, W. Fiddler, C. J. Dooley. R. C. Doerr, and A. E. Wasserman, J. Agr. FoodChem., 20, 274 (1972). (9) W. Lijinsky, W. T. Rainey, and W. H. Christie, "Identification of N-Nitroso Compounds by Mass Spectrometry," presented at the 21st Annual Conference on Mass Spectrometry and Allied Topics, San Francisco, Calif., May 20-25, 1973, Paper 19. (10) C. J. Dooley, S. F. Osman, G. P. Martin, and W. Fiddler, "Detection and Confirmation of Trace Amounts of N-nitrosamines in Meats by Gas Chromotography-High Resolution Mass Spectrometry," /bid., Paper Q8.
1312
A N A L Y T I C A L C H E M I S T R Y , VOL. 4 6 ,
NO.
tography (PC) has been previously described (11-14). Studies of the PC mobility spectra of the dimethyl, diethyl, and dibutyl N-nitrosamines were undertaken as part of a general exploration of the applicability of the PC technique to detect and identify specific compounds (15).The results indicate that these compounds can be specifically detected at gram or less through their mobility spectra. EXPERIMENTAL The Beta VI plasma chromatograph (Franklin GNO Corp.. West Palm Beach, Fla. 33402) used in this study has been described previously (11-13). The experimental conditions for this study were: PC Tube and inlet temperature, 136'; Carrier gas flow rate, 100 ml/min; drift gas flow rate, 450 ml/min; electric field, 250 V/cm; injection and scan gate widths, 0.2 msec; time base, 20 msec; recorded scan time, 2 min; pressure, 728-735 Torr. The carrier and drift gases were nitrogen (Linde High Purity 99.996%) passed through individual stainless steel traps of 2.25-1. capacity packed with Linde Molecular Sizve 13X to remove impurities. The aliphatic N-nitrosamines, dimethyl, diethyl, and dibutyl nitrosamine were obtained as reagent grade chemicals from Eastman Kodak Co., Rochester, N.Y. 14650. Samples were prepared as gram/ml solutions in ethanol and introduced to the plasma chromatograph using either a gas chromatograph coupled to the plasma chromatograph or a clean platinum wire to which 0.1 microliter of solution is added (14). Both techniques give comparable results. ( 11 ) (12) (13) (14)
F. W. Karasek and 0. S. Tatone, Anal. Chem.. 44, 1758 (1972). F. W. Karasek and D. M. Kane, Anal. Chem., 45, 576 (1973). F. W. Karasek and M. J. Cohen, J, Chromatogr. Sci., 9, 390 (1971). F. W. Karasek, 0. S. Tatone, and D. W. Denney, J. Chromatogr., 87,
137 (1973). (15) D W. Denney, "Ionic Species in Plasma Chromatography," M.Sc. Thesis, University of Waterloo, March 1974.
9. AUGUST 1974
