On-line immunoaffinity sample pre-treatment for column liquid chromatography: evaluation of desorption techniques and operating conditions using an anti-estrogen immuno-precolumn as a model system

Abstract

An immunoaffinity precolumn (immuno-precolumn) containing an immobilized antibody directed against estrogen steroids, was used as a model system for the evaluation of different desorbing techniques, suitable for on-line coupling to column liquid chromatography (LC). Desorption of estrogen analytes from the immuno-precolumn proved to be impossible with the buffers and chaotropic solutions frequently used in affinity desorption. Micellar solutions are effective in obstructing the antibody-antigen reaction, but their use as desorbing solutions was not found to be practical because of the large interferences introduced into the chromatograms. Desorption with aqueous solutions at elevated temperature, created by microwave action or conventional heating, although effective is not practical in this instance, because the agarose used in this study as the stationary phase for the immuno-precolumn is prone to heat decomposition. The most effective and practical approach is desorption with a methanol-water mixture (95 + 5 v/v). On-line dilution of the eluate allows the concentration of the desorbed analytes using a reversed-phase LC system with subsequent separation and ultraviolet detection. The performance of the system with spiked urine and plasma samples, which were introduced directly into the system, was evaluated and the results were compared with immunoselective desorption.

Fast detection of atrazine in corn using thermometric biosensors

“…Gly-HCl (pH 2.3) buffer that contains 1% DMSO (v/v) is a highly efficient dissociation buffer, which enables multiple reuse of the immobilized Ab for biosensor applications. 11 In this study, we observed that the combination of Gly-HCl (pH 2.3) buffer that contains 1% DMSO (v/v) with 0.1% Triton X-100 (v/v) showed a comparatively better dissociation for the ATZ Ag-Ab complex.…”

Affinity Chromatography: A Review of Clinical Applications

“…Clinical applications of on-line immunoextraction in HPLC have been developed for such analytes as ␣ 1antitrypsin (104 ), cortisol (105 ), digoxin (106 ), estrogens (107,108 ), human epidermal growth factor (109 ), lysergic acid diethylamide, lysergic acid diethylamide analogs and metabolites (110,111 ), phenytoin (112 ), propranolol (110 ), ⌬ 9 -tetrahydrocannabinol (113 ), and transferrin (104,114 ). Additional details on these methods are provided in Ref.…”

Immunoaffinity Extraction with On-Line Liquid Chromatography—Mass Spectrometry

“…Most of the applications are for preconcentration of drugs from urine, plasma, and milk and only very few deal with preconcentration of environmental pollutants from water or aqueous extracts. IAC has been used to preconcentrate propanolol and LSD from urine samples (7), nortestosterone and related compounds from urine samples (6,8), various estrogen steroids and their glucuronide derivatives from urine samples (9,20), albuterol, digoxin, and PTH (parathyroid hormone) from plasma (10,11,12), aflatoxin M t and zearalenone from milk (15,17) and Cortisol and flumethezone from urine and serum (18,19). Environmental applications of IAC were limited to carbofuran (2), atrazine (73), carbendazim (14), and chlortoluron (16).…”