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

Abstract

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.

Chiral determination of amphetamine and related compounds using chloroformates for derivatization and high-performance liquid chromatography

“…[16][17][18][19][20] A solid-phase reagent containing the 9-fluorenylmethyl chloroformate-l-proline (FMOC-l-Pro) tag has also been used for the resolution of amphetamine enantiomers on a C 18 analytical column. 21,22 However, to our knowledge, FMOC-l-Pro has not yet been used for the solution derivatization of amphetamines.…”

“…For elution under normal-phase conditions, different mixtures of acetonitrile-acetic acid-TEA, hexanepropan-2-ol and acetonitrile-ethyl acetate were tested. Several isocratic and gradient elution programs were tested, the flow rates ranging between 0.25 and 1.0 mL min 21 . In all instances, solvents were filtered through 0.45 mm nylon membranes (Teknokroma) and de-gassed with helium before use.…”

Mixed‐bed polymeric o‐nitrobenzophenone reagents for the on‐line derivatization of amines in high performance liquid chromatography

“…comparing the temperature increase from 50 to 60 "C, keeping solvent conditions constant, and was consistent with results obtained from the earlier temperature study. Acetonitrile solvent was determined in a previous study to give optimum percent derivatizations (Gao and Krull, 1990). Other solvents would be expected to show similar trends of increasing derivatization with increasing percent organic, although the final percent derivatization with 100% organic would be less than the derivatization with 100% acetonitrile.…”

“…Polymeric activated ester reagents for the derivatization of amine compounds to amides or carbamates have recently been developed for improved detection in high performance liquid chromatography (HPLC) (Bourque and Krull, 1991a;Chou et al, 1989;Gao and Krull, 1989a,b;Gao et al, 1989;Gao and Krull, 1990;Gao, et al, 1991). Because primary and secondary amines often show poor chromatographic performance and detectability, chemical derivatization is employed to enhance both chromatographic behaviour and sensitivity.…”

Derivatization of Amphetamine and Methamphetamine With 1,2-Naphthoquinone 4-Sulfonic Acid Into Solid-phase Extraction Cartridges. Determination of Amphetamine in Pharmaceutical and Urine Samples

“…Gao et al 5 studied a polymeric reagent containing an activated ester linkage to a 9-fluorenyl group. Zhou et al 6 described the preparation of a covalently bound, fluorescencesensitive tag, 9-fluoreneacetyl, on a pore size-controlled, rigid polystyrene-divinylbenzene resin.…”