A mixed‐bed, multi‐derivatization approach using polymeric reagents for derivatizations of amines in high performance liquid chromatographic detection

Abstract

Immobilized, polymeric reagents containing covalently attached tagging groups have been synthesized and reacted individually, off-line or on-line, pre-column in high performance liquid chromatographic (HPLC) detection. These reagents have also been combined into a single, mixed-bed reactor, useful for simultaneously preparing several derivatives from a single analyte, all at the same time. Each derivative possesses different chromatographic and detection properties, dependent on the nature of the original polymeric reagent containing the immobilized, tagging species. These particular reagents were designed to impart Ultraviolet/fluorescence, Ultraviolet/electrochemistry (oxidative/reductive or oxidative-hv-electrochemistry) to the final derivatives. Variations in the amounts/ratios of polymeric reagents contained in a single mixed-bed reactor will lead to varying ratios of the final derivatives. These can be predicted knowing the approximate reactivity of each polymeric reagent, percent derivatizations, and overall rates for each reagent towards a given substrate. In this first example of mixed-bed, polymeric reagents for improved derivatization approaches in chromatography, emphasis has been placed on simple amines or amine-like analytes. Multiple derivatives can be effectively used to improve the identification of an unknown analyte in a complex sample matrix, as well as to improve the detectability of that analyte. As one real world application, amphetamine in human urine was quantitated via on-line derivatizations with a mixed-bed reactor. With the least sampling work-up, the resulting sample solutions were directly injected into the on-line derivatization HPLC system for quantitation. The method was validated by single blind spiking experiments. The precision and accuracy were acceptable.

Analytical Aspects of Amino, Quaternary Ammonium, Nitro, Nitroso and Related Functional Groups

“…The objective of multiple pre-column tagging was to aid identification of unknown analytes in a complex matrix. The method was demonstrated for amphetamine (28) in human urine with acceptable accuracy and precision 244 .…”

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

“…Percent derivatization of propylamine was determined by comparison of derivative peak areas t o those of authentic standards. Preparation and characterization of the authentic standards has been described (Gao et al, 1991).…”

“…Another important advantage of solid phase reagents is that two or more reagents can be used in a single reactor to form multiple derivatives simultaneously (Gao et al, 1991;Bourque and Krull, 1991b). These derivatives are formed precolumn and separated via reversed phase HPLC, so that the peak height or area of each derivative can be used for quantitation.…”

“…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.…”

“…These solid-phase reagents have been applied both offand on-line with an HPLC system for improved detection of primary and secondary amines , aliphatic amines in air samples (Gao and Krull, 1989a) and determination of chiral and achiral drugs in urine . Two or three of these reagents have been used in the same reactor, both off-line * Author to whom correspondence should be addres\ed and on-line, for the simultaneous derivatization of analytes precolumn to form multiple products (Gao et al, 1991).…”