Sample preparation immunoaffinity cleanup

Immunoaffinity cleanup was first applied in drug residue analysis for the determination of chloramphenicol in swine muscle tissue by LC (113). The lAC column was prepared using monoclonal antibodies originally developed for an enzyme-linked immunosorbent assay (ELISA) method (171) specific for chloramphenicol. Meat samples were extracted with water, and a concentrated phosphate buffer was added to the filtered extracts before immunoaffinity cleanup. A phosphate buffer was used in the washing process, whereas chloramphenicol was eluted from the column with a glycine/sodium chloride solution of pH 2.8. For subsequent LC analysis, this eluate was extracted with ethyl acetate, evaporated, and reconstimted in the mobile phase. The same analytical scheme was later successfully applied for the determination of chloramphenicol in eggs and milk as well (170, 172). 

Starting with a description of the analytical challenge in Chapter 19, the third part, which is devoted to analytical attitudes, proceeds with a detailed description in Chapter 20 of modern sample preparation procedures including solid-phase extraction, matrix solid-phase dispersion, use of restricted-access media, supercritical fluid extraction, and immunoaffinity cleanup. Flexible derivatization techniques including fluorescence, ultraviolet-visible, enzymatic, and photochemical derivatization procedures are presented in Chapter 21. 

Immunoaffinity (lA) methods are based on the principle of molecular recognition via very selective antigen-antibody interactions. Their use for sample preparation prior to TLC has been mostly for determination of toxins. The following are selected examples the steroid animal drug trenbolone and its metabolite in bovine urine " fumonisin B1 in corn with methanol-water (80 20) extraction followed by lA column cleanup " aflatoxins Bl, B2, Gl, and G2 in foods regulated within... 

Proper sampling procedures have to be applied to obtain representative samples from analyzed biological material since mycotoxins are not homogenously distributed in contaminated grain stocks. Sample preparation usually involves a few cleanup steps to eliminate compounds present in the matrix that might coelute with the mycotoxins. The cleanup methods may involve either liquid-liquid extraction (LLE), solid-phase extraction (SPE) on columns that contain various types of solid phases, or immunoaffinity columns [39,42,43]. The recovery achieved with a cleanup method is critical for the final results. 

Immunoaffinity chromatography cleanup has also been applied as an ideal and reliable strategy for residue analysis. Immunoaffinity columns prepared by coupling the antibodies to a cyanogen bromide-activated support were used to analyze avermectin BI residues in cattle tissues (359) and ivermectin in sheep serum (376). An immunoaffinity column prepared by an alternative activation/ coupling procedure with carbonyl diimidazole was also employed to analyze ivermectin residues in swine liver (361) since the earlier-reported methods did not work well in the analysis of this matrix. This recent work demonstrated the high specificity of tire antibody-mediated cleanup, but also showed that the immunoaffinity procedures could not always or completely eliminate matrix interference of samples. Therefore, application of additional cleanup steps before or after these procedures is often inevitable. 

A recent approach to producing highly selective sorbents for SPE is based on molecular recognition technology and utilizes antibodies immobilized by covalent reaction onto solid supports such as silica (Figure 2.30). Preparation of immunoaffinity sorbents for SPE was reviewed by Stevenson [101] and Stevenson et al. [102], Using immunosorbents, efficient cleanup is achieved from complex biological and environmental samples. 

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