Structural Characterization of Unusual Fatty Acid Methyl Esters with Double and Triple Bonds Using HPLC/APCI-MS2 with Acetonitrile In-Source Derivatization

Double and triple bonds have significant effects on the biological activities of lipids. Determining multiple bond positions in their molecules by mass spectrometry usually requires chemical derivatization. This work presents an HPLC/MS method for pinpointing the double and triple bonds in fatty acids. Fatty acid methyl esters were separated by reversed-phase HPLC with an acetonitrile mobile phase. In the APCI source, acetonitrile formed reactive species, which added to double and triple bonds to form [M + C3H5N]+• ions. Their collisional activation in an ion trap provided fragments helpful in localizing the multiple bond positions. This approach was applied to fatty acids with isolated, cumulated, and conjugated double bonds and triple bonds. The fatty acids were isolated from the fat body of early-nesting bumblebee Bombus pratorum and seeds or seed oils of Punicum granatum, Marrubium vulgare, and Santalum album. Using the method, the presence of the known fatty acids was confirmed, and new ones were discovered.

This work deals with the localization of double and triple bonds in FAMEs. The conversion of lipids or lipid mixtures to FAMEs is frequently used in lipidomics workflows because the GC or LC analysis of FAMEs provides quick and valuable information on the fatty acyl chains. Here, FAME standards and FAMEs obtained by the transesterification of the TGs from biological samples were analyzed by HPLC/APCI-MS/MS using an acetonitrile mobile phase. Isolated, cumulated, and conjugated double bonds and triple bonds were localized using the fragmentation of [M + C 3 H 5 N] +• adducts generated in the ion source. To the best of our knowledge, the localization of triple bonds in FAMEs by RP-HPLC with MS detection is reported here for the first time.

Results and Discussion
The chromatographic separation of FAMEs was achieved on the Develosil RP-Aqueous C30 column using isocratic elution with acetonitrile. The mobile phase in the APCI source formed reactive species, which added to double and triple bonds. The adducts were isolated and activated in the ion trap to generate ions bearing information on the original double or triple bond position. The diagnostic ions formed by the cleavages of adjacent C-C bonds were marked α if they carried the ester moiety or ω if they contained the terminal-carbon end without the ester group. The diagnostic peaks corresponding to cleavages before the first and after the last unsaturated bond in polyunsaturated FAMEs tended to be more abundant than the others. This phenomenon was used for deducing the arrangement of the double and triple bonds in polyunsaturated chains. A parameter named "multiple bond region" (MBR) was calculated and tabulated for various theoretically possible arrangements of multiple bonds ( Table 1). The MBR value was calculated using theoretical m/z values of the adduct (precursor) and α and ω fragments corresponding to cleavages before the first and after the last unsaturated bond as follows:

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One triple bond -C≡C-81 One double bond -CH=CH-93 Two cumulated double bonds -CH=C=CH-103 Two conjugated triple bonds -C≡C-C≡C-105 One double bond and one triple bond, conjugated -CH=CH-C≡C-107 Two conjugated double bonds -CH=CH-CH=CH-119 One double bond and one triple bond, methylene-interrupted -CH=CH-CH 2 -C≡CH-121 Two methylene-interrupted double bonds -CH=CH-CH 2 -CH=CH-133 Three conjugated double bonds -CH=CH-CH=CH-CH=CH-161 Three methylene-interrupted double bonds -CH=CH-CH 2 -CH=CH-CH 2 -CH=CH-14n + 107 Two double bonds interrupted by several methylenes (-CH 2 -) n The experimental MBR values calculated for the adduct and the most abundant α and ω fragments in the spectra were then compared to theoretical MBRs. For instance, the MS/MS spectrum of [M + 55] +• adduct of unknown FA at m/z 347.0 provided the most abundant α and ω peaks at m/z 290.2 and m/z 190.2, respectively. The calculated MBR value (290 + 190 − 347 = 133) suggested FAME with three conjugated double bonds (Table 1). Diagnostic ions were accompanied by less abundant satellite peaks differing from α and ω ions by 14 or 15 Da. These fragments representing cleavages at more distant C-C bonds were important for distinguishing double and triple bonds. The elemental composition of the major fragments in the spectra of FAME standards was confirmed by Orbitrap high-resolution data (Supplementary Materials Table S1).

Mass Spectra of Standards with Conjugated Double Bonds
The system with two conjugated double bonds was investigated using standards of FAME 18:2n-7t,9t (Mangold's acid methyl ester) and FAME 18:2n-7c,9c (ricinenic acid methyl ester). The fragments in the MS/MS spectrum for FAME 18:2n-7t,9t ( Figure 1) were rationalized as follows: α n-7 peak at m/z 264.1, α n-9 peak at m/z 238.2, ω n-7 peak at m/z 166.1, and ω n-9 peak at m/z 192.1. The MBR value calculated from the two most intense fragments in the spectrum (i.e., m/z 192.1 and m/z 264.1) was 107. Despite the presence of satellite fragments differing by 14 Da from the diagnostics peaks, the spectrum provided clear evidence of two conjugated double bonds in the n-7 and n-9 positions. The spectrum of FAME 18:2n-7c,9c having the opposite geometry on both double bonds looked similar ( Figure S1), which confirmed the negligible effect of double bond geometry on the adduct fragmentation documented earlier [19].
The MS/MS spectrum of punicic acid methyl ester with three conjugated double bonds (FAME 18:3n-5c,7t,9c) is shown in Figure 2. The major fragments in the spectrum were formed by cleavages before and after the series of double bonds. They were easily distinguishable from the other ions. The most abundant fragments α n-5 at m/z 290.2 and ω n-9 at m/z 190.2 delimited the group of conjugated double bonds and corresponded to an MBR value of 133. The fragments formed by the cleavages between conjugated double bonds α n-7 (m/z 264.3), α n-9 (m/z 238.2), ω n-7 (m/z 164.2), and ω n-5 (m/z 138.2) were of low intensities but discernable in the spectrum. The same diagnostic fragments and MBR value could theoretically be expected for a FAME with two cumulated double bonds separated by one methylene group from the third double bond. Such an arrangement of double bonds would be, however, clearly distinguishable because the system of cumulated . Such an ion (m/z 251 or m/z 291 in this case) is not present in the spectrum. Therefore, the spectrum in Figure 2 can be unambiguously interpreted as FAME 18:3n-5,7,9. The MS/MS spectrum of punicic acid methyl ester with three conjugated double bonds (FAME 18:3n-5c,7t,9c) is shown in Figure 2. The major fragments in the spectrum were formed by cleavages before and after the series of double bonds. They were easily distinguishable from the other ions. The most abundant fragments α n-5 at m/z 290.2 and ω n-9 at m/z 190.2 delimited the group of conjugated double bonds and corresponded to an MBR value of 133. The fragments formed by the cleavages between conjugated double bonds α n-7 (m/z 264.3), α n-9 (m/z 238.2), ω n-7 (m/z 164.2), and ω n-5 (m/z 138.2) were of low intensities but discernable in the spectrum. The same diagnostic fragments and MBR value could theoretically be expected for a FAME with two cumulated double bonds separated by one methylene group from the third double bond. Such an arrangement of double bonds would be, however, clearly distinguishable because the system of cumulated double bonds manifests itself by abundant α + 1 Da ion (Section 2.3.3.). Such an ion (m/z 251 or m/z 291 in this case) is not present in the spectrum. Therefore, the spectrum in Figure 2 can be unambiguously interpreted as FAME 18:3n-5,7,9.   The MS/MS spectrum of punicic acid methyl ester with three conjugate bonds (FAME 18:3n-5c,7t,9c) is shown in Figure 2. The major fragments in the were formed by cleavages before and after the series of double bonds. They w distinguishable from the other ions. The most abundant fragments α n-5 at m/z ω n-9 at m/z 190.2 delimited the group of conjugated double bonds and corresp an MBR value of 133. The fragments formed by the cleavages between conjugate bonds α n-7 (m/z 264.3), α n-9 (m/z 238.2), ω n-7 (m/z 164.2), and ω n-5 (m/z 138.2 low intensities but discernable in the spectrum. The same diagnostic fragments value could theoretically be expected for a FAME with two cumulated double b arated by one methylene group from the third double bond. Such an arrangemen ble bonds would be, however, clearly distinguishable because the system of cu double bonds manifests itself by abundant α + 1 Da ion (Section 2.3.3.). Such an 251 or m/z 291 in this case) is not present in the spectrum. Therefore, the spectru ure 2 can be unambiguously interpreted as FAME 18:3n-5,7,9.   Figure 3 shows the MS/MS spectrum of FAME 18:1n-9 TB (stearolic acid methyl ester) [M + 55] +• adduct. The abundant fragments m/z 236.2 (α n-9 TB ) and m/z 192.2 (ω n-9 TB ) clearly indicated a triple bond in the n-9 position. Unlike FAMEs with double bonds, the satellite fragments differed by +15 Da from α TB and ω TB (m/z 207.1 and m/z 251.1, respectively). The intensities of the diagnostic fragments and their +15 Da satellites were similar, allowing us to recognize these peaks in the spectrum easily. Such a pattern distinctly indicated a triple bond. Satellite fragments differing by +14 Da, typical for double bonds, were present at significantly lower intensities. satellite fragments differed by +15 Da from α TB and ω TB (m/z 207.1 and m/z 251.1, tively). The intensities of the diagnostic fragments and their +15 Da satellites were s allowing us to recognize these peaks in the spectrum easily. Such a pattern distin dicated a triple bond. Satellite fragments differing by +14 Da, typical for double were present at significantly lower intensities. The satellite fragment ions made it also possible to characterize FAMEs with bination of double and triple bonds. For instance, crepenynic acid methyl ester w double bond and one triple bond (FAME 18:2n-6 TB ,9c) provided a spectrum with th abundant peak at m/z 150.1 ( Figure 4). This signal is a diagnostic fragment for tripl (ω n-6 TB ) because its satellite appears at a 15 Da higher m/z value (m/z 165.0). Analo the m/z 276.1 with its satellite at m/z 291.1 is the triple bond diagnostic peak (α Fragment m/z 190.1 indicates a double bond (ω n-9) because its satellite peak app m/z 204.1.

Analysis of Natural Samples
The fragmentation of FAME standards with various arrangements of double ple bonds helped us characterize the FAMEs isolated from biological samples. Th

FAMEs from the Fat Body of Bombus pratorum
The early-nesting bumblebee Bombus pratorum is widespread in Europe. It is the earliest bumblebee species to emerge from hibernation each year. The fat bod The satellite fragment ions made it also possible to characterize FAMEs with a combination of double and triple bonds. For instance, crepenynic acid methyl ester with one double bond and one triple bond (FAME 18:2n-6 TB ,9c) provided a spectrum with the most abundant peak at m/z 150.1 (Figure 4). This signal is a diagnostic fragment for triple bond (ω n-6 TB ) because its satellite appears at a 15 Da higher m/z value (m/z 165.0). Analogously, the m/z 276.1 with its satellite at m/z 291.1 is the triple bond diagnostic peak (α n-6 TB ). Fragment m/z 190.1 indicates a double bond (ω n-9) because its satellite peak appears at m/z 204.1. pratorum males contains TGs with long, diunsaturated fatty acyls, which are structurally related to its marking pheromone [41]. The chromatogram of B. pratorum FAMEs is shown in Figure 5. The MS/MS spectra of diunsaturated FAMEs ( Figure 6) provided abundant and recognizable α and ω fragments interpreted as FAME 24:2n-7,17, FAME 25:2n-7,17, and 26:2n-7,17. The double bond positions were in excellent agreement with previous work, where the positions of the double bonds were established using dimethyl disulfide derivatization [41].

Analysis of Natural Samples
The fragmentation of FAME standards with various arrangements of double and triple bonds helped us characterize the FAMEs isolated from biological samples. The The early-nesting bumblebee Bombus pratorum is widespread in Europe. It is one of the earliest bumblebee species to emerge from hibernation each year. The fat body of B. pratorum males contains TGs with long, diunsaturated fatty acyls, which are structurally related to its marking pheromone [41].
The chromatogram of B. pratorum FAMEs is shown in Figure 5. The MS/MS spectra of diunsaturated FAMEs ( Figure 6) provided abundant and recognizable α and ω fragments interpreted as FAME 24:2n-7,17, FAME 25:2n-7,17, and 26:2n-7,17. The double bond positions were in excellent agreement with previous work, where the positions of the double bonds were established using dimethyl disulfide derivatization [41].
pratorum males contains TGs with long, diunsaturated fatty acyls, which are structurally related to its marking pheromone [41].

FAMEs from Pomegranate Seed Oil
Pomegranate (Punicic granatum) seed oil (PSO) is a rich source of FAs with conjugated double bonds. Cold-pressed PSO was transesterified, and the resulting mixture was analyzed by HPLC/MS. Many isomeric species with similar retention times tended to coelute. Still, the partial separation of the peaks allowed us to identify most of these lipids (Figure 7).

FAMEs from Marrubium vulgare Seeds
White horehound (Marrubium vulgare) is a perennial, aromatic herb native to Europe, northern Africa, and southwestern and central Asia. Like other plants of the Lamiaceae family, it contains FAs with cumulated double bonds (allenic FAs). TGs from white horehound seeds were transesterified, and the resulting mixture of FAMEs analyzed by HPLC/MS (Figure 9). FAMEs with 18 to 21 carbons and up to three double bonds were detected.

FAMEs from Santalum album Seeds
Indian sandalwood (Santalum album) is a tropical tree native to southern India and Southeast Asia. The oil from its seeds and seeds of other Santalaceae species is a rich source of acetylenic FAs. [96]. FAMEs obtained by the transesterification of the TGs from Santalum album seeds provided chromatogram shown in Figure 12.
The most abundant peak t R 10.3 min corresponded to FAME with 18 carbons and either three double bonds or a double and a triple bond. The MS/MS spectrum ( Figure 13A) revealed the latter possibility, i.e., an acetylenic acid methyl ester. Diagnostic fragment m/z 190.1 and its satellite ion m/z 205.1 indicated a triple bond in the n-9 position (ω n-9 TB ). The corresponding α fragment (α n-9 TB ) at m/z 236.1 was not accompanied by a significant satellite ion at m/z 251.1, likely because of the triple bond conjugation with the n-7 double bond. The α fragment m/z 262.0 and its satellite m/z 276.1 indicated a double bond in the position n-7. Low-intensity fragment ω n-7 was detected at m/z 166.1. The MBR value of 105 corresponds to a conjugated system of one double and one triple bond. The compound was identified as FAME 18:2n-7,9 TB , most probably santalbic acid methyl ester.

FAMEs from Santalum album Seeds
Indian sandalwood (Santalum album) is a tropical tree native to southern India and Southeast Asia. The oil from its seeds and seeds of other Santalaceae species is a rich source of acetylenic FAs. [96]. FAMEs obtained by the transesterification of the TGs from Santalum album seeds provided chromatogram shown in Figure 12.   The MS/MS spectrum of a peak in 8.6 min revealed another acetylenic FAME with two triple bonds ( Figure 13B). The ω fragment m/z 188.1 and its satellite peak m/z 203.1 indicated the triple bond at the position n-9 TB , and the α fragment m/z 260.1 and its satellite m/z 275.0 the triple bond in n-7 TB . The complementary α (n-9 TB ) and ω (n-7 TB ) fragments The MS/MS spectrum of a peak in 8.6 min revealed another acetylenic FAME with two triple bonds ( Figure 13B). The ω fragment m/z 188.1 and its satellite peak m/z 203.1 indicated the triple bond at the position n-9 TB , and the α fragment m/z 260.1 and its satellite m/z 275.0 the triple bond in n-7 TB . The complementary α (n-9 TB ) and ω (n-7 TB ) fragments m/z 236.1 and m/z 164.1, respectively, were of low abundance. The MBR value calculated from the most abundant fragments (m/z 188.1 and m/z 260.1) equaled 103 and was consistent with two conjugated triple bonds. The compound was identified as FAME 18:2n-7 TB ,9 TB .

Extraction and Transesterification of Lipids
The samples were treated with organic solvents to obtain total lipid extracts. Briefly, peripheral fat bodies of three B. pratorum males were dissected and extracted with CHCl 3 /CH 3 OH (1:1, v/v) containing di-tert-butyl-4-methylphenol at a concentration of 25 mg/mL (500 µL each) and sonicated for 15 min. The extract was collected using a Pasteur pipette. M. vulgare seeds (approx. 240 pieces; 0.25 g) or S. album seeds (5 pieces; 0.94 g) were crushed and extracted in methanol/chloroform (2:1 v/v, 10 mL) for 30 min. After filtration, 5 mL of 0.9% NaCl was added, shaken for few seconds, and the aqueous (upper) phase was removed. The cleaning step was repeated three more times with 2 mL of 0.9% NaCl solution.
Total lipid extracts or seed oil were separated by semipreparative TLC to isolate TGs. Pre-cleaned, in-house made silica-gel glass TLC plates (60 mm × 76 mm) and hexane/diethyl ether (80:20, by vol.) mobile phase were used. TLC zones were made visible by spraying Rhodamine 6G solution (0.05% in ethanol). A zone corresponding to TGs (B. pratorum R f = 0.36-0.55, pomegranate R f = 0.20-0.55, M. vulgare R f = 0.33-0.55, S. album R f = 0.30-0.55) was scraped off the plate and extracted with 10 mL freshly distilled diethyl ether. The solvent was evaporated to dryness under a nitrogen stream.
While TGs from B. pratorum, pomegranate seed oil, and M. vulgare seeds were transesterified in acidic conditions [98], base-catalyzed transesterification [99] was required for S. album lipids containing triple bonds. FA standards were methylated by diazomethane (synthesized in-house from Diazald). Diazomethane in diethyl ether was added dropwise to the FA solution in chloroform (10 mg/mL) until the color of the reaction mixture turned light-yellow. Unreacted diazomethane was deactivated by formic acid.

RP-HPLC/APCI-MS and APCI-MS
The liquid chromatograph consisted of a Rheos Allegro UHPLC pump, Accela autosampler with an integrated column oven, and an LCQ Fleet ion-trap mass spectrometer; the system was controlled by Xcalibur software (all Thermo Fisher Scientific, San Jose, CA, USA). Develosil RP-Aqueous C30 (250 × 4.6 mm, particle size: 5 µm; Nomura Chemical, Seto, Japan) stainless-steel column and isocratic elution with acetonitrile at 0.7 mL/min flow rate [20] were used. The chromatography proceeded at laboratory temperature except for B. pratorum sample separated at 40 • C. The injected volume of samples (standards and biological samples, 1 mg/mL and 10-20 mg/mL, respectively) was 10-20 µL. The APCI vaporizer and heated capillary temperatures were set to 380 • C and 180 • C, respectively; the corona discharge current was 2 µA. Nitrogen served both as the sheath and auxiliary gas at a flow rate of 50 and 20 arbitrary units, respectively. The MS spectra of positively charged ions were recorded in the m/z 180-470 range. The CID MS 2 spectra of [M + 55] +• were collected using a data-dependent analysis with an isolation width of 1.7 Da and normalized collision energy of 28%. The m/z range of MS 2 spectra was set automatically, depending on the precursor ion mass. The masses of the acetonitrile adducts for fragmentation were calculated as higher partners of the base peaks (m/z [M + H] + + 54 Da). The retention times and relative peak areas were obtained from ion chromatograms extracted for [M + H] + . The high-resolution MS data were recorded using an LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an APCI ion source operated at the same conditions as for low resolution. The Orbitrap spectra were acquired at a resolution of 100,000 FWHM.
The standard (1 mg/mL) solutions were also analyzed by direct infusion to the mobile phase flow using the same APCI-MS conditions, as described above.

Fragment Ion Abbreviations and Nomenclature
The diagnostic ions in the MS/MS spectra of [M + 55] +• were denoted "α" if they carried the ester moiety or "ω" if they contained the terminal-carbon end without the ester group. The double bond position was indicated as α n-x and ω n-x, where x is the distance from the terminal end of the hydrocarbon chain. A triple bond was marked by "TB" in superscript.

Conclusions
This work demonstrates the applicability of acetonitrile gas-phase chemistry in APCI for characterizing the structure of polyunsaturated FAMEs. The reaction of C 3 H 5 N +• with double and triple bonds occurs in the ion source, and the reaction products are fragmented to generate diagnostic ions. The method is highly versatile and suitable to many (if not all) arrangements of double and triple bonds in mono-and polyunsaturated chains. It was successfully applied to FAMEs with isolated, cumulated, and conjugated double bonds, triple bonds, and their combinations. The localization of the isolated double and triple bond positions is straightforward because of intense α and ω fragments. Distinguishing a double bond from a triple bond is easy based on the satellite fragments. While the satellite ions appear at +14 Da in the lipids with a double bond, they are found as intense +15 Da fragments in the case of a triple bond. When two or more unsaturated bonds exist in a chain, the spectra predominantly show α and ω fragments related to cleavages of C-C bonds before and after the unsaturated region. This can be utilized for deducing a possible arrangement of unsaturated bonds. A parameter named multiple bond region (MBR) can be calculated using the most abundant fragments and compared to tabulated theoretical values. The type and position of the unsaturated bonds within the unsaturated region can then be inspected in detail after focusing on less intense diagnostic fragments and their satellites. In the case of allenic FAMEs, the α fragment was accompanied by an intense α + 1 fragment, which gave a hint for the cumulated double bonds. When a triple bond was present in a polyunsaturated chain, it manifested itself by the +15 Da satellite peak accompanying the corresponding diagnostic fragment.
The localization of unsaturated bonds by HPLC/APCI-MS/MS with an acetonitrile mobile phase is a simple and convenient method. Since the derivatization occurs in the ion source during ionization, there is no need to perform the chemical modification of the analytes as a separate step before the analysis. Nominal mass resolution spectra were successfully used for the structure elucidation. However, high-resolution MS/MS data could help distinguish α and ω fragments, thus making the interpretation even easier. In this work, unsaturated FAMEs were characterized in Bombus pratorum, Punicum granatum, Marrubium vulgare, and Santalum album. The method's power is illustrated by the fact that, in addition to the known lipids, several new FAMEs were discovered. Although the method can also be applied to complex lipids [19,21,22], spectra interpretation is easier for lipids having only one fatty acyl chain.