A novel polymeric activated ester reagent has been developed that improves final detectability and chromatographic performance in high-performance 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 linkage 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. Percent 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 approach, 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.

Optically active and detector-sensitive polymeric reagents have been synthesized, loadings determined, derivatizations/separations/detection optimized, and applications to simple amines and amino alcohols described. Such reagents have been designed to contain different chiral centers, usually amino acids, leashed via an activated ester attachment to an insoluble, structurally rigid, organic polymer backbone. 9-Fluorenylmethyl (FMOC) moieties chemically bonded to the amino acids were used as ultraviolet (UV) and fluorescence (FL) sensitive detector probes to the final diastereomers of enantiomer substrates. Such diastereomers can be readily separated by isocratic or gradient elution normal-phase methods. The kinetics for diastereomer formation have been determined, and final UV/FL responses for known mixtures of enantiomers have been compared to demonstrate overall validity of the method. Minimum detection limits, linearity of calibration plots, dual detector responses, and linear diode array spectra and absorbance ratios have also been demonstrated. In some cases, authentic standards have been prepared to calculate absolute percent derivatizations for specific enantiomer pairs. The overall approach permits, for the very first time, off-line or on-line precolumn derivatization for the formation of diastereomers having unique detector properties. It has been proven that the rates and rate constants for such formations are identical for at least those pairs of enantiomers studied. Separations are base-line or near-base-line, permitting accurate and precise quantitative determinations, by both UV and FL, of enantiomer/optical purity and chemical purity.