Analysis of oxylipins by high-performance liquid chromatography with evaporative light-scattering detection and particle beam-mass spectrometry.
The metabolism of 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid was investigated in a crude enzyme extract from mung bean seedlings (Phaseolus radiatus L.). Hydroperoxide-metabolizing activity was mainly due to a hydroperoxide lyase and, to a lesser extent, to an allene oxide synthase and a peroxygenase. Oxylipins originating from hydrolysis and cyclization of the allene oxide synthase product 12,13-epoxy-9Z,11,15Z-octadecatrienoic acid and from peroxygenase catalysis were identified by high-performance liquid chromatography (HPLC) particle beam-mass spectrometry (PB-MS) and quantified by normal-phase HPLC with an evaporative light-scattering detector (ELSD). An advantage of this methodology was the possibility to avoid extensive derivatization procedures commonly used for the gas chromatographic analysis of oxylipins. Owing to a comparable sample inlet system, the ELSD served an important analytical pilot function for the PB-MS: Qualitatively identical chromatographic patterns were obtained with both detection systems. The HPLC system enabled the separation of methyl 12-oxo-phytodienoate, methyl 11-hydroxy-12-oxo-9Z,15Z-octadecadienoate, methyl 12-oxo-13-hydroxy-9Z,15Z-octadecadienoate, methyl 9-hydroxy-12-oxo-10E,15Z-octadecadienoate, methyl 13-hydroxy-9Z,11E,15Z-octadecatrienoate, methyl 15,16-epoxy-13-hydroxy-9Z,11E-octadecadienoate, and methyl 13-hydroperoxy-9Z,11E,15Z-octadecatrienoate on a Lichrospher DIOL column within 33 min. Compared with a diode array detector, the ELSD proved to be more sensitive, in the case of methyl 12-oxo-13-hydroxy-9Z, 15Z-octadecadienoate by a factor of about 15. In addition, volatile metabolites were analyzed by capillary gas chromatography. The yield of the hydroperoxide lyase product 2E-hexenal was 49%, whereas the sum of oxylipins reached about 15%.
Rehbock
B
B
Institut für Lebensmittelchemie, Universität Hannover, Germany.
Gansser
D
D
Berger
R G
RG
D000446:Aldehyde-Lyases / Q000378:metabolism*; D000447:Aldehydes / Q000032:analysis / Q000378:metabolism; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology; D019746:Intramolecular Oxidoreductases / Q000378:metabolism; D008042:Linolenic Acids / Q000138:chemical synthesis / Q000378:metabolism; D008054:Lipid Peroxides / Q000138:chemical synthesis / Q000378:metabolism*; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000378:metabolism; D015394:Molecular Structure; D010946:Plants, Medicinal; D012542:Scattering, Radiation; D012639:Seeds / Q000201:enzymology
D000447:Aldehydes; D008042:Linolenic Acids; D008054:Lipid Peroxides; C057375:13-hydroperoxy-9,11,15-octadecatrienoic acid; C051750:2-hexenal; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C022333:peroxygenase; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase