View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Double Bond Migration to Methylidene Positions During Electron Ionization Mass Spectrometry of Branched Monounsaturated Fatty Acid Derivatives Jean-François Rontani,a Nathalie Zabeti,a and Claude Aubertb a Laboratoire de Microbiologie de Géochimie et d’Ecologie Marines (UMR-CNRS 6117), Centre d’Océanologie de Marseille (OSU), Marseille, France b Laboratoire de Pharmacocinétique et Toxicocinétique (UPRES 3286), Faculté de Pharmacie, Marseille, France Electron ionization mass spectra of several monounsaturated methyl-branched fatty acid methyl and trimethylsilyl esters were examined. These spectra exhibited some intensive fragment ions, whose formation could be explained after double-bond migration to methylidene position. This preferential migration (substantiated by deuterium labeling) acts significantly in the case of monounsaturated fatty acid methyl and trimethylsilyl esters possessing a methyl branch localized between the penultimate and the C4 positions (relative to the ester group), whatever the position of the double-bond. Allylic cleavage and ␥-hydrogen rearrangement of the ionized methylidene group thus formed afforded very interesting fragment ions, which could be particularly useful to determine branching positions of monounsaturated methyl-branched fatty acid methyl and trimethylsilyl esters without additional treatment. (J Am Soc Mass Spectrom 2009, 20, 1997–2005) © 2009 American Society for Mass Spectrometry as chromatography/electron ionization mass double bonds. In these last cases, the carboxyl group is spectrometry of methyl (for a review see [1]) derivatized with a reagent containing a nitrogen atom. Gand trimethylsilyl [2] esters constitutes a partic- When the molecule is ionized in the mass spectrometer, ularly powerful technique for the identification of fatty the nitrogen atom, not the alkyl chain, carries the charge, acids. Unfortunately, the mass spectra of methyl and and double-bond migration is minimized. trimethylsilyl esters of monoenoic fatty acids have no Methyl-branched monounsaturated fatty acids have information that helps to locate the position of double been detected in several bacteria [10–14]; they are also bonds. While there have been suggestions that such present in some fish [15] and sponge [16, 17]. Careful information can be obtained from close examination of examination of EI mass spectra of methyl and trimeth- certain minor peaks in the spectrum, the value of such ylsilyl derivatives of these compounds suggested to us techniques seems doubtful. There is no feature that that the presence of branching strongly favors the permits location of the double-bond, because this can migration of the double-bond to the methylidene posi- migrate to any position when the alkyl chain is ionized tion. Such a “specific” migration, which could lead to in the mass spectrometer. To get around the problem of misinterpretation of mass spectra of these compounds, location of double bonds, it is possible to prepare would be, in contrast, very useful to indicate the specific derivatives of unsaturated fatty acids that ‘fix’ position of branching on their alkyl chain without the double-bond. Very many have been described. The additional treatment. In the present work, we thus: more commonly employed are dimethyldisulfide ad- (1) examined EI mass spectra of numerous methyl- ducts (which have excellent mass spectrometric prop- branched monounsaturated fatty acids formally iden- erties and are prepared in a simple one-pot reaction) tified, and (2) carried out deuterium labeling to try to [3, 4] and vicinal trimethylsilyl ethers arising from confirm this assumption. stereospecific OsO4 oxidation of double bonds [5]. Al- ternatively, picolinyl esters [6, 7] or DMOX [8] or pyrrolidine [9] derivatives can be utilized to locate Experimental Fatty Acids Address reprint requests to Dr. J.-F. Rontani, Laboratoire de Microbiologie de Géochimie et d’Ecologie Marines (UMR 6117), Centre d’Océanologie de Marseille (OSU), Campus de Luminy – case 901, 13288 Marseille, France. C15-C18 iso- and anteiso-methyl-branched monoun- E-mail: [email protected] saturated fatty acids, 11-methyloctadec-12-enoic and Published online August 12, 2009 © 2009 American Society for Mass Spectrometry. Published by Elsevier Inc. Received April 10, 2009 1044-0305/09/$32.00 Revised July 24, 2009 doi:10.1016/j.jasms.2009.07.020 Accepted July 27, 2009 1998 RONTANI ET AL. J Am Soc Mass Spectrom 2009, 20, 1997–2005 Figure 1. EI mass spectra of (a) 11-methyloctadec-12-enoic, (b) 11(D3)-methyloctadecanoic, and (c) 11(D3)-methyloctadec-12-enoic acid methyl esters. J Am Soc Mass Spectrom 2009, 20, 1997–2005 EIMS OF BRANCHED MONOUNSATURATED FATTY ACID DERIVATIVES 1999 12-methyloctadec-11-enoic acids, were obtained from form (4:1, vol/vol), dried over anhydrous Na2SO4, bacterial lipid extracts [12, 14]. filtered, and concentrated using rotary evaporation. Deuterium Labeling Formation of Pyrrolidide Derivatives It was recently demonstrated that the formation of 11-methyloctadec-12-enoic acid in bacteria resulted Methyl esters were dissolved in 1 mL pyrrolidine. Then, from the methionine-mediated methylation of cis- 0.1 mL of acetic acid was added and the mixture was vaccenic acid [14]. 11(D3)-methyloctadec-12-enoic acid heated at 100 °C for 1 h. The amides so formed were was thus obtained after growing of the bacterial strain taken up in dichloromethane and washed with diluted Oceanicaulis alexandrii sp. AG4 in a medium supplemented hydrochloric acid (to remove the excess of pyrrolidine) with [methyl-D3]L-methionine (Aldrich) [14, 18]. and with water. The organic phase was dried over anhydrous Na2SO4, filtered and evaporated to obtain Hydrogenation the required pyrrolidide derivatives. 11(D3)-methyloctadec-12-enoic acid was hydrogenated (under an atmosphere of H2) in methanol with Pd/ Osmium Tetroxide Oxidation CaCO3 (5% Pd, 10–20 mg/mg of extract) (Aldrich) as a catalyst for 12 h with magnetic stirring. After hydroge- Lipid extracts and OsO4 (1:2, wt:wt) were added to a nation, the catalyst was removed by filtration and the pyridine-dioxane mixture (1:8, vol/vol; 5 mL) and filtrate was concentrated by rotary evaporation. incubated for1hatroom temperature. Then, 6 mL of Na2SO3 suspension (8.5 mL of 16% Na2SO3 in water- Methylation methanol, 8.5:2.5, vol/vol) was added and the mix- ture was again incubated for 1.5 h. The resulting Lipid extracts were taken up in 2 mL of anhydrous mixture was gently acidified (pH 3) with HCl and methanolic hydrochloric acid (3N, St. Quentin Fallavier, extracted three times with dichloromethane (5 mL). France, Supelco) and heated at 80 °C for 1 h. After The combined dichloromethane extracts were subse- cooling, an excess of water was added and methyl quently dried over anhydrous Na2SO4, filtered, and esters were extracted three times with hexane-chloro- concentrated. Scheme 1. Proposed formation pathways of ions aϩ,bϩ• and cϩ• involving ionized double-bond migration to methylidene position and subsequent allylic cleavage and ␥-hydrogen rearrangement. force for the formationAs of previously ion proposed a by Boon et al. cule by the ion a respectively; further loss of a neutral methanol mole- 1.5 bar atature 0.04 program bar and min thenwas programmed maintained from at 1.04 1.04 bar bar until to the end of the temper- 130 °C to 300 °C at 4 °C min Indeed, in this⌬ case, allylic cleavagerangement may and be well explained after migration of the ions at methyl ester ( ously described in thetended literature our conclusions to some EI mass spectra previ- EI mass spectra of corresponding branchedhypothesis alkenes was well supported bytion the by lack cyclization of with this the ion ester in group ( afford fragments ions a rearrangement of the alkylester chain would mainly OsO yses of bis-trimethylsilyloxy derivativesthe obtained position after of doublespectra of bonds their involved pyrrolidide derivatives. GC-EIMS Confirmationpresent anal- of work were formallymass determined spectral from EI fragmentationsacid mass are methyl examined andmethyloctadec-12-enoic and in trimethylsilyl 12-methyloctadec-11-enoic derivatives the iso whose EI The double-bond and branchingResults positions and of Discussion C thickness, 0.25 capillary column coatedconditions with SOLGEL-1 were (SGE; employed:a film HP 30 5972 mass m HP spectrometer. 5890 The series following II operative plusspectrometry gas were chromatograph performed connected with to Analyses a by gas Hewlett chromatography/electron impact Packard mass Mass Spectrometry 300 Compounds (1 mg) to be silylatedSilylation were taken up in 2000 from 60 °C to 130 °C at 30 °C min phy/mass spectrometry (GC/MS). (to avoid desilylation) and analyzeddissolved by in ethyl gas acetate chromatogra- (2tion mL/mg) and to BSTFA (0.1 dryness mL) vol) (to and eliminate allowed pyridine), toBis(trimethylsilyl)trifluoroacetamide; react the at residue Supelco) 50 was °C (2:1, for vol/ 1 h. After evapora- 170 °C; mass range,ture, 50–700 250 °C; Th; electron cycle energy, 70 time, eV; 1.5 source s. temperature, formation by allylic cleavage and [12] - and anteiso-methyl-branched monounsaturated, 11- EI mass spectrum of 11-methyloctadec-12-enoic acid 4 ␮ L of a mixture of pyridine and BSTFA ( oxidation and subsequent silylation. We also ex double-bond to methylidene position ( RONTANI ET AL. m/z Figure 1 139, 140, 171, 179, 194, 211, and 226, whose ␮ ϩ a) exhibits