ORIGINAL ARTICLE Identification of C18 Intermediates Formed During Stearidonic Acid Biohydrogenation by Rumen Microorganisms In Vitro S. P. Alves • M. R. G. Maia • R. J. B. Bessa • A. J. M. Fonseca • A. R. J. Cabrita Received: 1 August 2011 / Accepted: 23 September 2011 / Published online: 30 October 2011 Ó AOCS 2011 Abstract In vitro batch incubations were used to study the rumen biohydrogenation of unsaturated fatty acids. An earlier study using increasing supplementation levels of stearidonic acid (18:4n-3), revealed that the rumen micro- bial population extensively biohydrogenates 18:4n-3 after 72 h of in vitro incubation, though several intermediates formed were not completely characterized. Therefore, in the present study, samples were reanalyzed in order to identify the 18:2, 18:3 and 18:4 biohydrogenation inter- mediates of 18:4n-3. Gas–liquid chromatography coupled to mass spectrometry was used to characterize these intermediates. The acetonitrile chemical ionization mass spectrometry of the fatty acid methyl esters derivatives enabled the discrimination of fatty acids as non-conjugated or conjugated biohydrogenation intermediates. In addition, the acetonitrile covalent adduct chemical ionization tandem mass spectrometry yielded prominent ions indica- tive of the double bond position of the major 18:3 isomers, i.e. D5,11,15 18:3. Furthermore, the 4,4-dimethyloxazoline derivatives prepared from the fatty acid methyl esters enabled the structure of novel 18:2, 18:3 and 18:4 biohy- drogenation intermediates to be elucidated. The interme- diates accumulated in the fermentation media after 72 h of incubation of 18:4n-3 suggest that similar to the biohy- drogenation pathways of linoleic (18:2n-6) and a-linolenic (18:3n-3) acids, the pathway of the 18:4n-3 also proceeds with the formation of conjugated fatty acids followed by hydrogenation, although no conjugated dienes were found. The formation of the novel biohydrogenation intermediates of 18:4n-3 seems to follow an uncommon isomerization pattern with distinct double bond migrations. Keywords Biohydrogenation intermediates Á Stearidonic acid Á Gas–liquid chromatography Á Mass spectrometry Á 4,4-Dimethyloxazoline Á Covalent adduct chemical ionization Abbreviations CACI Covalent adduct chemical ionization CACIMS/MS Covalent adduct chemical ionization tandem mass spectrometry CI Chemical ionization DHA Docosahexaenoic acid DM Dry matter DMOX 4,4-Dimethyloxazoline EI Electron impact EPA Eicosapentaenoic acid FAME Fatty acid methyl ester(s) GLC Gas–liquid chromatography MS Mass spectrometry PFI Polyenoic fatty acid isomerase S. P. Alves (&) Á R. J. B. Bessa INRB, Instituto Nacional dos Recursos Biolo ´gicos, Unidade de Produc ¸a ˜o Animal, 2005-048 Fonte-Boa, Vale de Santare ´m, Portugal e-mail: susana_alves@sapo.pt S. P. Alves Á M. R. G. Maia Á A. J. M. Fonseca REQUIMTE, ICBAS, Instituto de Cie ˆncias Biome ´dicas de Abel Salazar, Universidade do Porto, Rua Padre Armando Quintas, 4485-661 Vaira ˜o, Portugal R. J. B. Bessa Faculdade de Medicina Veterina ´ria, Universidade Te ´cnica de Lisboa, CIISA, Po ´lo Universita ´rio do Alto da Ajuda, Av. da Universidade Te ´cnica, 1300-477 Lisbon, Portugal A. R. J. Cabrita REQUIMTE, Departamento de Geocie ˆncias, Ambiente e Ordenamento do Territo ´rio, Faculdade de Cie ˆncias, Universidade do Porto, Rua Padre Armando Quintas, 4485-661 Vaira ˜o, Portugal 123 Lipids (2012) 47:171–183 DOI 10.1007/s11745-011-3621-6