PDF(Pubmed)
Abstract：
Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.
摘要：
河豚毒素(TTX)是引起河豚食物中毒的代表性天然毒素,这在东亚和东南亚尤其突出，包括日本。TTX已通过柱后衍生高效液相色谱（HPLC）进行分析，离子对LC-MS(/MS)，和亲水相互作用液相色谱（HILIC）-MS（/MS）作为小鼠生物测定方法的替代方法。然而,柱后衍生化需要一个在线衍生化反应系统，用离子对LC-MS方法，很难去除残留在设备中的残留离子对试剂。此外，与反相(RP)HPLC相比,HILIC-MS提供差的分离并且需要长时间来达到平衡。因此,我们决定开发一种使用柱前衍生和RPHPLC的TTX分析方法，用于快速评估爆发样本，包括食物残留物。在这项研究中,我们专注于TTX的vic-diol部分，并设计了一种新的衍生化试剂，编码为NBD-H-DAB。这种NBD-H-DAB是由4-肼基-7-硝基-2,1,3-苯并恶二唑（NBD-H）和3-氟-2-甲酰基苯基硼酸（FFPBA）通过简单的反应系统合成的，并迅速转化为其硼酸酯形式，编码NBD-H-PBA,在水性反应溶液中。NBD-H-PBA显示出保留在RP分析柱上的适当的疏水性，并用UV光谱仪成功检测。它容易与TTX的vic-diol部分(C6和C11)反应以合成硼酸酯。衍生的TTX可以使用RPHPLC-UV检测，鱼肉样品的检出限为0.06mg/kg。这种用NBD-H-PBA对TTX的新型柱前衍生化证明能够分析TTX。
