Chou T Y, Gao C X, Colgan S T, Krull I S, Dorschel C, Bidlingmeyer B
Department of Chemistry, Barnett Institute, Northeastern University, Boston, MA 02115.
J Chromatogr. 1988 Nov 11;454:169-83. doi: 10.1016/s0021-9673(00)88611-2.
A polymeric benzotriazole reagent containing a 9-fluorenylmethyleneoxycarbonyl (FMOC) group has been synthesized, characterized, and its derivatizations, off-line, for three polyamines, have been optimized with regard to solvent, time, and temperature. An authentic FMOC derivative of cadaverine has been prepared, characterized, and used as the external standard for quantitation of off-line derivatizations and identification of final derivatives. Actual percent derivatizations have been determined, rather than just percent disappearance of starting material. The polyamines in urine or other biological fluids can be derivatized without organic solvent or solid phase extraction, but rather in situ by the simple addition of the polymeric reagent to the fluid, incubation for a few minutes at room or elevated temperature, filtration and direct injection. Derivatizations could also be performed by transferring a small volume of the hydrolyzed and filtered biological fluid to a disposable pipette containing the polymeric reagent. Derivatization was then followed by elution, filtration, and direct injection onto the high-performance liquid chromatographic (HPLC) system. Automation of the overall polymeric derivatization, filtration, HPLC injection, separation, detection, quantitation, and data acquisition-interpretation is suggested. The polymeric reagent has been utilized for the qualitative and quantitative determination of cadaverine and putrescine, normally occurring polyamines, in human urine. These levels were compared with the corresponding literature values for healthy human subjects, and the values were found to be in excellent agreement. This novel derivatization approach, though off-line, provides for a much simpler, more rapid, and more efficient conversion of these and related polyamines or nucleophiles to derivatives having vastly improved chromatographic detection properties in HPLC. The final derivatives contain the FMOC group, making them extremely chromophoric and fluorophoric, and providing trace detection at ppb (microgram/l) and sub-ppb levels. The overall approach is recommended for these and other biologically occurring polyamines, in fluids and tissues, as well as related bioorganic and biologically active nucleophiles, including drugs and their metabolites.
一种含有9-芴甲氧羰基（FMOC）基团的聚合型苯并三唑试剂已被合成、表征，并针对三种多胺进行了离线衍生化，在溶剂、时间和温度方面进行了优化。已制备并表征了尸胺的纯FMOC衍生物，并将其用作离线衍生化定量和最终衍生物鉴定的外标。已确定实际衍生化百分比，而不仅仅是起始原料的消失百分比。尿液或其他生物流体中的多胺无需有机溶剂或固相萃取即可进行衍生化，而是通过简单地向流体中添加聚合试剂、在室温或高温下孵育几分钟、过滤并直接进样进行原位衍生化。衍生化也可以通过将少量水解并过滤后的生物流体转移到含有聚合试剂的一次性移液器中来进行。然后进行洗脱、过滤，直接注入高效液相色谱（HPLC）系统。建议对整个聚合衍生化、过滤、HPLC进样、分离、检测、定量以及数据采集-解释过程进行自动化操作。该聚合试剂已用于定性和定量测定人尿中通常存在的多胺尸胺和腐胺。将这些水平与健康人类受试者的相应文献值进行比较，发现两者非常吻合。这种新颖的衍生化方法虽然是离线的，但能更简单、快速且高效地将这些及相关多胺或亲核试剂转化为在HPLC中具有大大改善的色谱检测特性的衍生物。最终衍生物含有FMOC基团，使其具有极强的发色和荧光特性，并能在ppb（微克/升）和亚ppb水平进行痕量检测。对于流体和组织中的这些及其他生物多胺，以及相关的生物有机和亲核生物活性物质，包括药物及其代谢物，推荐采用这种整体方法。
