Toyo'oka T, Ishibashi M, Takeda Y, Nakashima K, Akiyama S, Uzu S, Imai K
Division of Drugs, National Institute of Hygienic Sciences, Tokyo, Japan.
J Chromatogr. 1991 Dec 27;588(1-2):61-71. doi: 10.1016/0021-9673(91)85008-4.
Four new 2,1,3-benzoxadiazole amine reagents having different functional groups at the 4- and 7-positions, [4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ), 4-(N,N-dimethylaminosulphonyl)-7-N-piperazino-2,1,3-benzoxad iazole (DBD-PZ), 4-(N,N-dimethylaminosulphonyl)-7-N-cadaverino-2,1,3-benzoxad iazole (DBD-CD) and ammonium 7-N-piperazino-2,1,3-benzoxadiazole-4-sulphonate (SBD-PZ)] were synthesized as fluorogenic tagging reagents for carboxylic acids in high-performance liquid chromatography. The reagents, except SBD-PZ, reacted with carboxylic acid at room temperature in the presence of activation agents to produce fluorescent adducts. The maximum wavelengths of arachidic acid tagged with DBD-PZ, DBD-CD and NBD-PZ were 569 nm (excitation, 440 nm), 561 nm (excitation, 437 nm) and 541 nm (excitation, 470 nm), respectively. Among various activation agents tested [diethyl phosphorocyanidate (DEPC), diphenyl phosphoroyl azide (DPPA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-pyridine, 2.2'-dipyridyl disulphide-triphenylphosphine (Mukaiyama A) and 2-chloro-1-methylpyridinium iodide-triethylamine (Mukaiyama B)], DEPC and Mukaiyama A were more effective than the others. When the piperazino reagents (DBD-PZ and NBD-PZ) were used as the tagging reagents, the derivatization reaction in the presence of Mukaiyama A was faster than that in the presence of DEPC. Although the reaction in the presence of Mukaiyama A was completed after 30 min, an unknown peak derived from the activation agent appeared on the chromatograms. The fluorescence peak intensities were compared in the presence of DEPC. The order of the fluorescence peak areas obtained after reaction for 6 h in the presence of DEPC was DBD-PZ greater than DBD-CD greater than NBD-PZ. Thirteen saturated free fatty acids (FFAs) derivatized with DBD-PZ (or DBD-CD) and DEPC (or Mukaiyama A) in acetonitrile were separated completely by linear gradient elution on a reversed-phase ODS column. Eight drugs (ibuprofen, indomethacin, dinoprost, prostaglandin E1, dehydrocholic acid, ursodesoxycholic acid, hydrocartisone succinate and prednisolone succinate) were also tagged with DBD-PZ in the presence of DEPC and separated by isocratic elution. The detection limits (signal-to-noise ratio = 3) of FFAs tagged with DBD-PZ were in the range 3.2-4.7 fmol, whereas those of drugs were in the range 3.9-14 fmol.
合成了4种在4-位和7-位具有不同官能团的新型2,1,3-苯并恶二唑胺试剂，即[4-硝基-7-N-哌嗪基-2,1,3-苯并恶二唑（NBD-PZ）、4-(N,N-二甲基氨基磺酰基)-7-N-哌嗪基-2,1,3-苯并恶二唑（DBD-PZ）、4-(N,N-二甲基氨基磺酰基)-7-N-尸胺基-2,1,3-苯并恶二唑（DBD-CD）和7-N-哌嗪基-2,1,3-苯并恶二唑-4-磺酸铵（SBD-PZ）]，作为高效液相色谱中羧酸的荧光标记试剂。除SBD-PZ外，这些试剂在活化剂存在下于室温与羧酸反应生成荧光加合物。用DBD-PZ、DBD-CD和NBD-PZ标记的花生酸的最大波长分别为569 nm（激发波长440 nm）、561 nm（激发波长437 nm）和541 nm（激发波长470 nm）。在测试的各种活化剂[氰基磷酸二乙酯（DEPC）、叠氮磷酸二苯酯（DPPA）、1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDC）-吡啶、2,2'-二吡啶二硫化物-三苯基膦（向山A）和2-氯-1-甲基吡啶碘化物-三乙胺（向山B）]中，DEPC和向山A比其他试剂更有效。当哌嗪试剂（DBD-PZ和NBD-PZ）用作标记试剂时，在向山A存在下的衍生化反应比在DEPC存在下更快。虽然在向山A存在下的反应在30分钟后完成，但色谱图上出现了一个源自活化剂的未知峰。在DEPC存在下比较了荧光峰强度。在DEPC存在下反应6小时后获得的荧光峰面积顺序为DBD-PZ大于DBD-CD大于NBD-PZ。在反相ODS柱上通过线性梯度洗脱，将13种在乙腈中用DBD-PZ（或DBD-CD）和DEPC（或向山A）衍生化的饱和游离脂肪酸（FFA）完全分离。8种药物（布洛芬、吲哚美辛、地诺前列素、前列腺素E1、去氢胆酸、熊去氧胆酸、氢化可的松琥珀酸钠和泼尼松龙琥珀酸钠）也在DEPC存在下用DBD-PZ标记并通过等度洗脱分离。用DBD-PZ标记的FFA的检测限（信噪比 = 3）在3.2 - 4.7 fmol范围内，而药物的检测限在3.9 - 14 fmol范围内。
