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

用ESR波谱对自由基加合物进行了鉴定。然而，有时，来自ESR波谱的信息不足以鉴定自由基加合物。为了克服这一困难，我们一直在开发液相色谱/电子自旋共振光谱/电喷雾电离质谱联用技术。首先，我们利用高效液相色谱/电子自旋共振光谱分离反应混合物中形成的自由基加合物。将分离得到的自由基加合物用于CI-MS。在大豆脂肪加氧酶与亚油酸、亚麻酸和花生四烯酸等不饱和脂肪酸的反应中，我们鉴定出了比较稳定的戊基和戊烯基的4-POBN自由基加合物。其次，成功地将LC/MS(LC/MS)用于鉴定相对不稳定的自由基加合物。在大豆脂肪氧合酶与亚油酸、亚麻酸和花生四烯酸等不饱和脂肪酸的反应中，我们用LC/MS鉴定了亚硝苯和MNP自由基加合物12，13-环氧亚油酸、12，13-环氧亚麻酸和14，15-环氧花生四烯酸自由基。该系统由高效液相色谱、电子自旋共振和质谱仪组成。这些设备直接使用不锈钢管组合。这个系统使我们能够同时检测和鉴定自由基加合物。我们用高效液相/电子自旋共振/质谱仪鉴定了几种肼类自由基与自旋捕捉剂如4-POBN、DMPO、PBN和MNP的加合物。

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.