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.

New polymeric benzotriazole reagents for off-line derivatizations of amines and polyamines in HPLC.

New polymeric reagents are synthesized, based on a polystyrene-bound benzotriazole containing an o-acetylsalicyl or 9-fluorenyl labelling moiety. This is used in an off-line mode, prior to high performance liquid chromatography (HPLC), for derivatizations of trace primary and secondary amines, polyamines, and related compounds in connection with HPLC. Standards are prepared, characterized by physical and spectral properties, and then used as external standards to determine percent derivatizations. The polymeric reagents are characterized by elemental analyses and loading determinations. The feasibility and applicability of this reagent for derivatization of nucleophiles is confirmed with a number of amines under optimized conditions. The activated labelling moiety, bonded to the polymeric support, makes the derivatization reactions extremely rapid and efficient under mild reaction conditions. This alone provides significant advantages over the analogous solution derivatizations for the same amines. A comparison of solution and solid phase derivatization reactions is reported. The limits of detection are 1 to 2 pmol for polyamines, such as cadaverine, putrescine, and 1,7-diaminoheptane, using the benzotriazole fluorenyl reagent followed by fluorescence detection.