Analysis of sterol esters by capillary gas chromatography-electron impact and chemical lonization-mass spectrometry

Synthetic mixtures of C₄₀ to C₄₇ sterol esters in groups of 7 esters were effectively separated and analyzed by capillary gas chromatography-mass spectrometry. Ammonia chemical ionization of all 20 sterol esters analyzed at a source block temperature of 120 C yielded (M+NH₄)⁺ and (M+H-RCO₂H)⁺ ions of high abundance or as base peak, thereby indirectly indicating the molecular weights of the ester and the sterol and acid moieties. Ammonia CI spectra of all esters containing a Δ⁵-sterol moiety exhibited in addition to the above 2 ions an M+NH₄-RCO₂ H fragment. At a source block temperature of 150 C, M+H-RCO₂ H fragment was the base peak for all esters, and there was little or no indication of an (M+NH₄)⁺ adduct ion. Protonated molecules were not observed for any esters analyzed by methane or isobutane CI. Molecular ions of 3–14% intensity were obtained for only 3 of the esters analyzed by electron impact; they contained a Δ⁷-bond in the sterol nucleus, and the acid moiety was either saturated normal or branched chain or contained a single double bond. The base peak was a function of both the acid and sterol moieties of the sterol ester. The esters containing both saturated straight chain acid and saturated sterol moieties exhibited a base peak at m/z 215. The Δ⁵-sterol esters with saturated branched or straight chain acid moieties exhibited base peaks at M-RCO₂ H. Other ions also were of diagnostic value.     

Fatty acids of unusual double-bond positions and chain lengths found in rat skin surface lipids

The fatty acids of rat skin surface lipids comprise four main skeletal types of chains which occur both as saturates and monoenes and range from C₁₂ to C₃₈: straight even, straight odd, iso and anteiso (the latter two identified by GC retention data only). Two unidentified series of branched monoenes also occur in trace amounts. Reductive ozonolysis of monoenes reveals two characteristic double-bond position patterns, one for the straight even chain series and the other for the straight odd chain series. The straight even chain pattern comprises four series, of which ω7 ≫ω9>ω5>ω11; the straight odd chain series in contrast shows a large number of ω series with irregular distribution. The biosynthesis of the even chain fatty acid monoenes can be thought of as occurring in two stages: synthesis of 14∶Δ9, 16∶Δ9, 18∶Δ9 and 20∶Δ9, with 16∶Δ9 predominating; elongation of these chains mostly by 1, 2, or 3 C₂ units but up to the unusually long lengths by 11 C₂ units. For the formation of the former, two schemes by known pathways are proposed. Iso and anteiso chains which are nearly all saturated comprised 1/3 the total fatty acids. Special terms and abbreviations: Normal even=a straight chain with an even number of carbon atoms, normal odd=a straight chain with an odd number of carbon atoms, ω=terminal carbon atom, iso=a straight chain with a methyl group at the ω−1 position, anteiso=a straight chain with a methyl group at the w−2 position, Δn=a double bond between the n^th and the (n+1)^th carbon atom from the carbonyl group of the fatty acid or ester, ωn=a double bond between the ω∩n^th and the ω-(n−1)^th carbon atom where n is an integer, aldester=aldehyde methyl ester, Me=methyl, GLC=gas-liquid chromatography, TLC=thin-layer chromatography.     

The fatty acids of wax esters and sterol esters from vernix caseosa and from human skin surface lipid

Separation of sterol esters from wax esters in the lipids of vernix caseosa and adult human skin surface was accomplished by column chromatography on MgO. The fatty acids of the sterol esters and wax esters of both samples were separated into saturates and monoenes, and examined in detail by gas liquid chromatography (GLC). The saturated fatty acids of the wax esters of vernix caseosa and of adult human skin surface were remarkably similar. They ranged in chain length from at least C₁₁ to C₃₀, six skeletal types being present: straight even, straight odd, iso, anteiso, other monomethyl branched and dimethyl branched. A large number of patterns of monoenes were observed, each pattern consisting of desaturation of a specific chain at Δ6 or Δ9 plus its extension or degradation products. The mole per cent of the total Δ6 and Δ9 patterns of wax ester fatty acid monoenes of vernix caseosa were 87% and 12%, respectively, and 98% and 1%, respectively, for adult human skin surface lipid. The sterol ester fatty acids of vernix caseosa were much different from those of adult human skin surface: vernix caseosa saturates were largely branched and of lengths greater than C₁₈, whereas the saturates of adult human surface lipid resembled the wax ester fatty acids. Of the vernix caseosa monoene patterns, the mole per cent was 30% Δ6 and 70% Δ9, whereas of the adult human skin surface sterol ester fatty acids 89% were Δ6 and 11% Δ9. Chain extension was particularly pronounced in the sterol ester fatty acid monoenes of vernix caseosa amounting to 7–8 C₂ units in some cases. The fatty acids of the sterol esters of both vernix caseosa and adult human skin surface appear to be derived from the sebaceous gland and from the keratinizing epidermis, but those of the wax esters are from the sebaceous glands only.     

A new conjugated linoleic acid isomer, 7 trans, 9 cis-octadecadienoic acid, in cow milk, cheese, beef and human milk and adipose tissue

The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis-octadecadienoic acid (18∶2) was confirmed in milk, cheese, beef, human milk, and human adipose tissue. The 7 trans, 9 cis-18∶2 isomer was resolved chromatographically as the methyl ester by silver ion-high-performance liquid chromatography (Ag⁺-HPLC); it eluted after the major 9 cis, 11 trans-18∶2 isomer (rumenic acid) in the natural products analyzed. In the biological matrices in-vestigated by Ag⁺-HPLC, the 7 trans, 9 cis-18∶2 peak was generally due to the most abundant minor CLA isomer, ranging in concentration from 3 to 16% of total CLA. By gas chromatography (GC) with long polar capillary columns, the methyl ester of 7 trans, 9 cis-18∶2 was shown to elute near the leading edge of the major 9 cis, 11 trans-18∶2 peak, while the 4,4-dimethyloxazoline (DMOX) derivative permitted partial resolution of these two CLA isomers. The DMOX derivative of this new CLA isomer was analyzed by gas chromatography-electron ionization mass spectrometry (GC-EIMS). The double bond positions were at Δ7 and Δ9 as indicated by the characteristic mass spectral fragment ions at m/z 168, 180, 194, and 206, and their allylic cleavages at m/z 154 and 234. The cis/trans double-bond configuration was established by GC-direct deposition-Fourier transform infrared as evidenced from the doublet at 988 and 949 cm⁻¹ and absorptions at 3020 and 3002 cm⁻¹. The 7 trans, 9 cis-18∶2 configuration was established by GC-EIMS for the DMOX derivative of the natural products examined, and by comparison to a similar product obtained from treatment of a mixture of methyl 8-hydroxy-and 11-hydroxyoctadec-9 cis enoates with BF₃, in methanol. Contribution number S010 from the Food Research Center, Guelph, Ontario, Canada.     

Characterization of epoxy carotenoids by fast atom bombardment collision-induced dissociation MS/MS

The characterization and structure of epoxy carotenoids possessing 5,6-epoxy, 5,8-epoxy and 3,6-epoxy end groups conjugated to the polyene chain were investigated using highenergy fast atom bombardment collision-induced dissociation MS/MS methods. In addition to [M-80]⁺, a characteristic fragment ion of an epoxy carotenoid, product ions resulting from the cleavage of C−C bonds in the polyene chain from the epoxy end group, such as m/z 181 (b ion) and 121 (c ion), were detected. On the other hand, diagnostic ions of m/z 286 (e-H ion) and 312 (f-H ion) were observed, not in the 5,6-epoxy or 5,8-epoxy carotenoid but in the 3,6-epoxy carotenoid. These fragmentation patterns can be used to distinguish 3,6-epoxy carotenoids from 5,6-epoxy or 5,8-epoxy carotenoids. The structure of an epoxy carotenoid, 3,6-epoxy-5,6-dihydro-7′,8′-didehydro-β,β-carotene-5,3′-diol (8), isolated from oyster, was characterized using FAB CID-MS/MS by comparing fragmentation patterns with those of related known compounds.     

Total synthesis of 2-methoxy-14-methylpentadecanoic acid and the novel 2-methoxy-14-methylhexadecanoic acid identified in the sponge <Emphasis Type="Italic">agelas dispar</Emphasis>

The phospholipid FA composition of the Carbbean sponge Agelas dispar was revisited and 40 different FA were identified. Among these a novel 2-methoxylated FA, namely, the anteiso methyl-branched 2-methoxy-14-methylhexadecanoic acid, was identified together with the recently discovered iso methyl-branched 2-methoxy-14-methylpentadecanoic acid and the normal-chain 2-methoxytetradecanoic acid. The structures of the iso and anteiso methyl-branched 2-methoxylated FA were confirmed by total syntheses, which were accomplished in seven steps and in 45–48% overall yields. Other phospholipid FA identified in A. dispar include the unusual methyl-branched 10, 130dimethyltetradecanoic acid, 3,7,11,15-tetramethylhexadecanoic (phytanic) acid, and the 11-methyloctadecanoic acid. In addition, the Δ^5,9 FA (5Z, 9Z)-15-methyl-5,9-hexadecadienoic acid and (5Z, 9Z)-5,9-octadecadienoic acid were characterized. These findings establish alternative FA biosynthetic possibilities for these marine organisms.     

Mass fragmentographic determination of docosenoic acid in rapeseed oils

A highly sensitive and accurate reference method for determination of docosenoic acid (mainly erucic acid, 22∶1n−9) in different rapeseed oils is described. A fixed amount of [1-¹⁴C]erucic acid methyl ester (about 1 μg) is added to a fixed amount of oil. After treatment with sodium methoxide/methanol reagent and extraction with hexane, the amount of unlabeled erucic acid is determined from the ratio between the recordings at m/e 320 and m/e 322 obtained after analysis with a combined gas chromatograph-mass spectrometer equipped with an MID (multiple ion detector). The two ions used correspond to the M-32 peak in the mass spectrum of unlabeled and [1-¹⁴C]labeled erucic acid methyl ester. The relative standard deviation of the method is about 1.8%. The method was compared with a gas chromatographic method for determination of erucic acid.     

The occurrence of long chain α,ω-diols in the lipids of steer and human meibomian glands

A group of long chain α,ω-diols (C₂₉ to C₃₄) has been identified in the lipids of steer and human meibomian gland excreta (meibum). These new lipids were isolated from the steer meibum unsaponifiables. Proof of structure was provided by 1) the column chromatographic behavior and TLC of the diols and their diacetates; 2) GLC on glass capillary columns; 3) fragmentation patterns in GC-MS; 4) NMR data, and 5) ozonolysis studies of the unsaturates. Chain types for the steer sample were 51% straight monoenes, 8.5% straight saturates, 39% iso and anteiso saturates and 1.5% iso and anteiso unsaturates. GC for the human sample gave straight monoenes 83%, straight saturates 8%, and iso plus anteiso saturates 9%. Close correspondence of the α,ω-diol chain lengths and types with meibum ω-hydroxy fatty acids suggests a biochemical precursor relationship.     

Identification and analysis of wax esters by mass spectrometry

Several ions in the mass spectra of wax esters were related to the molecular structures. Assigned structures of ions were confirmed by deuterium labeling. A simple, direct method for quantititive analyses of mixtures was developed. The method involved a comparison of sets of three ions, RCO₂H⁺, RCO₂H₂ ⁺ and [R′−1]⁺ from all compounds in the mixture. The method was found applicable for mixtures of unsaturated wax esters after reduction with tetradeuterio hydrazine. Presented in part before the ISF-AOCS World Congress, Chicago, September 1970. Part III of a series of mass spectrometry of lipids. For VI see Lipids.     

Further studies of the saturated methyl branched fatty acids ofVernix caseosa lipid

By the method of capillary gas chromatography-mass spectrometry, we have identified 35 monomethyl and 46 dimethyl branched acids in the saturated acids of vernix caseosa lipid with chain lengths ranging from C₁₁ to C₁₈. Many other mono-, di-, and trimethyl branched acids have been partially identified. All methyl branches were found to be on the even numbered C-atoms except for some terminal iso methyl groups. Three types of dimethyl branched acids were found: those with a terminal iso structure, those with a terminal anteiso structure, and those with neither iso nor anteiso structures. The 4-methyl branch predominated for all types of branched acids. Equivalent chain length data for di- and trimethyl branched acids were determined on a Pentasil (nonpolar) wall coated capillary column and checked by calculation from monomethyl branched acid data. Mass spectral identification was performed with and without the aid of a data system. A possible mode of formation of these acids is discussed.     

Detection of hydroxy fatty acids in biological samples using capillary gas chromatography in combination with positive and negative chemical ionization mass spectrometry

The most common method for use in the structural analysis of hydroxy fatty acids in biological samples is the gas chromatography-mass spectrometry (GC-MS) analysis of trimethylsilyl ethers of the methyl esters using electron impact ionization. A comparison of electron impact (EI) with chemical ionization mass spectrometry (CI-MS) shows that CI-MS is the superior technique. All ions necessary for structural analysis are observed at sufficiently high levels of intensity when methane or isobutane are used as reactant gases. The molecular weight can be determined from the ion group M+H, M-15 and M+H−90. The ionic series M+H−n×90 enables one to determine the number of hydroxyl groups. The position of the hydroxyl groups can be derived from the fragments of the α-cleavage of the fatty acid chain. The application of heptafluorobutyrates as derivatives for hydroxy fatty acid methyl esters shows advantages in the trace analysis of these compounds. Heptafluorobutyrates exhibit useful mass fragmentation patterns in the positive as well as in the negative CI mode. With methane as the reactant gas, M+H usually is base peak in positive mass spectra. The ionic series M+H−n×214 leads to the number of hydroxy groups in the molecule. In the negative mass spectra, M and M-20 are indicative for the molecular weight. The ion group m/z 213, 194 and 178 at high levels of intensity is typical for heptafluorobutyrates. The advantage of the application of heptafluorobutyrates is the high sensitivity which can be obtained in trace analysis using negative MS. Heptafluorobutyrates of hydroxy fatty acids gave a 20-fold higher response in the negative scan mode compared to that of the positive. The detection limit for heptafluorobutyrates in negative CI-MS was on the order of 1 fg (10⁻¹⁵ g).     

Characterization of sterols by gas chromatography-mass spectrometry of the trimethylsilyl ethers

The utility of combined gas chromatography-mass spectrometry in the analysis and characterization of sterols has been explored. Methylene unit (MU) values and principal mass spectrometric data are presented for trimethylsilyl ethers of 28 sterols, including the major natural sterols. The diagnostic value of the fragmentation of trimethylsilyl ethers of Δ⁵-3 β-hydroxysteroids has been confirmed. Characteristic fragmentations of Δ⁴-3 β-trimethylsilyloxysteroids, and of Δ^5,7-3 β-trimethylsilyloxysteroids were also found. Location of side-chain hydroxyl groups is facilitated by the α-cleavages typical of the trimethylsilyl ethers. Fragmentations of saturated sterols, and of Δ⁷, Δ⁸⁽⁹⁾ and Δ⁽¹⁴⁾ stenols, are less influenced by trimethylsilyl ether formation, but the derivatives still afford satisfactory mass spectra. The combination of gas chromatographic and mass spectrometric information allows positive identification of any of the sterols examined, whereas application of either technique alone may give inconclusive results.     

The structures of the branched fatty acids in the wax esters of vernix caseosa

By combined gas liquid chromatography-mass spectrometry a series of monomethyl branched fatty acids was found in the fatty acid moiety of the wax esters of vernix caseosa. The methyl branch occurred on the even C-atoms of chains ranging from C₁₁ to C₁₇ (some 43 compounds in all). Except for the iso acids and possibly some of the anteiso acids, these could be formed by replacement of malonyl CoA with a molecule of methyl malonyl CoA at the point of the branch. Smaller amounts of fatty acids also were found with two methyl branches occurring on the even C-atoms of chains ranging from C₉ to C₁₅.     

Fatty acid desaturase specificity in tetrahymena

The ciliate,Tetrahymena, was provided a supplement of the fatty acid [1-¹⁴C] 18∶2Δ6,9. After a period of growth the cells were claimed, the lipids extracted, the polar lipids recovered and the mild alkali-labile fatty acid methyl esters generated. The fatty acids were resolved by high pressure liquid chromatography (HPLC), the 18∶3Δ6,9,12 (γ-linolenic acid) was recovered and its identity verified by high pressure liquid chromatography (HPLC), gas liquid chromatography (GLC), hydrogenation and oxidation. Fifty-three percent of the cell-associated label was found in γ-linolenic acid; thus, a Δ12 fatty acid desaturase converts the 6,9 octadecadienoic acid to the 6,9,12 derivative. No carboxyl or methyl terminus restriction appears on Δ9 monoenoic or dienoic fatty acid desaturation in this cell as is found in higher plants and animals.     

Mass spectra of TMS esters of deuterated decanoic acids and of TMS ethers of deuterated decanols

Mass spectra of trimethylsilyl esters of nine specifically deuterated decanoic acids and of trimethylsilyl ethers of the corresponding deuterated decanols and of tri[²H₃] methylsilyl derivatives of most of the acids and of decanol have been measured. The fragmentation patterns have been used to examine the formation of ions m/z 117, 129, 131, 132, 145, 159, 171, 185 and 201 in the spectrum of the TMS ester of decanoic acid. Mechanisms of breakdown of one ion to another have been proposed. Similarities and differences in ion formation between spectra of TMS esters and methyl esters have been examined. It has been shown that ion m/z 132 is produced by the McLafferty rearrangement and that ion m/z 117 is formed from it by loss of a silyl methyl group. Ion m/z 145 is formed by hydrogen transfer from C-5, C-6 or C-7 to the carbonyl oxygen and γ-cleavage. Ion m/z 129 is formed from ion m/z 145 by loss of the previously transferred hydrogen and a silyl methyl group. A mechanism of formation of ions m/z 115, 129, 143 and 157, which contained only one silyl methyl group, observed in the mass spectrum of the TMS ether of decanol, has been proposed. Ions in spectra of the derivatives can be used to locate the position of deuterium, or other substituents, at a number of the carbons of decanoic acid or decanol.     

Selected ion monitoring gas chromatography/mass spectrometry of 1,2-diacylglyceroltert-butyldimethylsilyl ethers derived from glycerophospholipids

Selected ion monitoring was used in conjunction with gas chromatography/mass spectrometry to analyzetert-butyldimethylsilyl ethers (tert-BDMS) of 1,2-diacyl-sn-glycerols derived from naturally occurring glycerophospholipids, including those ofEscherichia coli, soybean, egg yolks and porcine liver. First, the fatty acid composition of the unknown glycerophospholipid was determined by gasliquid chromatography (GLC) and, based on that, the fatty acids (mostly >0.5 wt%) were selected for monitoring the characteristic fragmentation ions produced from the fatty acid residues of the correspondingtert-BDMS derivatives of 1,2-diacyl-sn-glycerols. Next, thetert-BDMS derivatives were separated by GLC on a 65% methylphenylsilicone gum wall-coated open-tubular (WCOT) column according to the degree of unsaturation and the chain length of the fatty acid residues, and then directly introduced into the ion source of the mass spectrometer. The selected fragmentation ions, [RCO+74]⁺ representative of the fatty acid residues, and [M-57]⁺ indicative of the molecular weight of the derivatives, were monitored simultaneously. It thus became possible to determine the molecular species of thetert-BDMS derivatives by measuring a specific combination of two [RCO+74]⁺ and an [M-57]⁺ ion with the same retention time on the selected ion monitoring (SIM) profile. High background noise caused by volatilization of stationary phase at high temperature was largely overcome by selected ion monitoring. However, the fragmentation ion peaks produced fromtert-BDMS derivatives of highly unsaturated glycerophospholipids showed a distorted SIM profile, which was attributed to interaction between thetert-BDMS derivatives and the methylphenylsilicone phase of the column. Use of a WCOT column with a more polar liquid phase is therefore recommended for the analysis of highly unsaturated molecular species.     

Preparation, separation, and confirmation of the eight geometrical cis/trans conjugated linoleic acid isomers 8,10-through 11,13–18∶2

Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or I₂ catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8,10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18∶2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag⁺-HPLC) and gas chromatography (GC). Ag⁺-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12–18∶2. However, one of the 8,10 isomers (8cis, 10trans-18∶2) coeluted with the 9trans,11cis18∶2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag⁺-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13–18∶2 geometric isomers (trans,cis before cis,trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by GC. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18∶1 18∶2, and 18∶3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.     

GC/MS analysis of some long chain esters,ketones and propanediol diesters

Identification of radiolysis products which are formed in lipids in the range of molecular weights from 400-600 has been established on the basis of gas chromatography/mass spectrometry (GC/MS) studies of long chain authentic samples of alkyl esters, ketones and propanediol diesters. This paper describes the GC/MS behavior of these compounds. Double hydrogen rearrangement was found to be the predominant ion in the spectrum of long chain saturated esters whereas in the unsaturated esters, a peak corresponding to the loss of alcohol from the molecular ion was more pronounced. On the contrary to short chain ketones, McLafferty rearrangement did not appear to be the major fragmentation in the spectrum of saturated and unsaturated long chain ketones. α-Cleavage was found to be the predominant fragmentation in the spectrum of these ketones. The “McLafferty + 1” rearrangement peak was more pronounced for the long chain ketones than those found in the spectrum of smaller ketones. Fragmentation patterns of propanediol diesters were shown to be similar to those in triglycerides, giving rise to predominant peaks corresponding to acylium ion [RCO]⁺ and parent minus acyloxy ion [R-COO]⁺.     

Isolation and characterization of the monounsaturated long chain fatty acids ofMycobacterium tuberculosis

The positions of double bond in the monounsaturated C₁₅−C₃₂ fatty acids ofMycobacterium tuberculosis H37Ra were established by gas chromatography/mass spectrometry of the ozonized esters and their pyrrolidide derivatives. The monounsaturated C₁₅−C₂₁ fatty acids had the double bond primarily at the Δ⁹ position while the monounsaturated longer chain fatty acids (C₂₂−C₃₂) had the double bond in several positions. Many of the latter acids, especially the odd-numbered series, were very complex isomeric mixtures. Quantitation showed the most abundant even-numbered long chain fatty acid isomers to be as follow: C₂₂, Δ⁴; C₂₄, Δ⁵; C₂₆, Δ⁷ and Δ⁹; C₂₈, Δ⁹; C₃₀, Δ¹¹ and Δ¹³; C₃₂, Δ¹³ and Δ¹⁵.     

Synthesis and Characterization of Diepoxyalkenes Derived from (3Z,6Z,9Z)-Trienes: Lymantriid Sex Pheromones and Their Candidates

All stereoisomers of three diepoxyalkenes derived from (3Z,6Z,9Z)-trienes with a C₂₁, C₁₉, or C₁₈ straight chain, lymantriid sex pheromones and their candidates, were synthesized by MCPBA oxidation of optically active epoxyalkadienes. Their chromatographic behaviors were examined with GC and LC equipped with achiral and chiral columns. Detailed inspection of the mass spectra of these epoxides indicated the following diagnostic ions for determining the chemical structures: m/z 128, 167, M-87 and M-85 for (Z)-cis-3,4-cis-6,7-diepoxy-9-enes; m/z 111, M-125 and M-69 for (Z)-cis-6,7-cis-9,10-diepoxy-3-enes; and m/z M-125 and M-139 for (Z)-cis-3,4-cis-9,10-diepoxy-6-enes. Mass chromatographic analysis that monitored these fragment ions revealed the existence of a new pheromonal compound with a C₂₁ chain in an extract from virgin females of a lymantriid species, Perina nuda F. The three diepoxyalkenes were converted into the corresponding DMDS adducts, which showed characteristic ions from fragmentation between the two thiomethyl groups, reflecting the position of an original double bond.