DEVELOPMENT OF COMBINED HPLC/ESR/MS FOR RADICAL STRUCTURE DETERMINATION

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Identification of the radical adducts has been performed using ESR spectra. However, sometimes, information from ESR spectra were not enough for the identification of the radical adducts. To overcome the difficulty we have been developing the combined liquid chromatography/electron spin resonance spectrometry/electrospray ionization mass spectrometry.First, we isolated the radical adducts formed in the reaction mixtures using HPLC/ESR (high performance liquid chromatography/electron spin resonance spectrometry. And then the isolated radical adducts were applied to CI mass spectrometry. In the reaction of soybean lipoxygenase with unsaturated fatty acids such as linoleic acid, linolenic acid, and arachidonic acid, we identified the 4-POBN radical adducts of pentyl radical and pentenyl radical which are considerably stable. However, CI mass measurements were not suitable for unstable radical adducts.Secondly, LC/MS (liquid chromatography/mass spectrometry) was successfully applied for the identification of the relatively unstable radical adducts. We identified the nitrosobenzene and MNP radical adducts of 12,13-epoxy-linoleic acid, 12,13-epoxy-linolenic acid, and 14,15-epoxy-arachidonic acid radicals in the reactions of soybean lipoxygenase with unsaturated fatty acids such as linoleic acid, linolenic acid, and arachidonic acid using LC/MS.Thirdly, We have developed the combined liquid chromatography/electron spin resonance spectrometry/electron ionization mass spectrometry (HPLC/ESR/MS). The system is consist of HPLC, ESR, and Mass spectrometer. These equipments are directly combined using stainless tubes. This system allowed us to detect and identify the radical adducts simultaneously. We have identified several radical adducts of hydrazine-derived radicals with spin trap reagents such as 4-POBN, DMPO, PBN, and MNP using the HPLC/ESR/MS.