Polymeric benzotriazole reagent for the off-line high-performance liquid chromatographic derivatization of polyamines and related nucleophiles in biological fluids

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.