Polymeric activated ester reagents for off-line and on-line derivatizations of amine nucleophiles in high-performance liquid chromatography with ultraviolet and fluorescence detection

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Abstract
A novel polymeric activated ester reagent has been developed that improves final detectability and chromatographic performance in high-peformance liquid chromatography (HPLC) for virtually all primary and secondary amines or amine analogues. This has involved the synthesis, characterization of final reagent, optimization of derivatization and separation conditions, and determination of analytical figures of merit. The polymeric reagent contained an activated ester linakge to the 9-fluorenyl group, which imparted ultraviolet (UV) and fluorescence (FL) detector properties to the final derivatives. Kinetic studies of these solid-phase (heterogeneous) reactions have been conducted, and specific rate constants were compared with those of the analogous solution reaction for the same substrates. Perdent derivatizations have reached 90% and 70% for primary and secondary amines, respectively, under optimized conditions. High reaction reproducibility has been obtained by using the on-line appoach, for more than 50 separate injections of the same amine substrate with a single solid-phase reactor. These solid-phase derivatizations have led to detection limits for typical amines in the low-parts-per-billion range. The final, overall methods can provide rapid, automatable, accurate, and precise detection and quantitation of primary/secondary amines and amine-like compounds in real-world sample matrices. As an illustrative example, amphetamine spiked in urine has been derivatized off-line and on-line, with minimum sample preparation, and detected via HPLC-UV/FL with acceptable accuracy and precision.