Immunoaffinity-based solid-phase extraction

The low selectivity of the SPE columns currently in use can be increased with more selective sorbents such as the immunosorbents, which have been quite extensively used in SPE-LC (72). Immunoaffinity-based solid-phase extraction (lASPE) sorbents have also been used in coupled gas chromatography for determining... 

Figure 13.20 GC-FID chromatograms of an extract obtained by (a) SPE and, (b) IASPE of 10 ml of municipal waste water, spiked with 1 p,g l-1 of seven s-triazines (c) represents a blank run from IASPE-GC-NPD of 10 ml of HPLC water. Peak identification is as follows 1, atrazine 2, terbuthylazine 3, sebuthylazine 4, simetryn 5, prometryn 6, terbutryn 7, dipropetryn. Reprinted from Journal of Chromatography, A 830, J. Dalltige et al., On-line coupling of immunoaffinity-based solid-phase extraction and gas chromatography for the determination of s-triazines in aqueous samples , pp. 377-386, copyright 1999, with permission from Elsevier Science.

J. Dalluge, T. Hankemeier, J. J. Vreuls and U. A. Th Brinkman, On-line coupling of immunoaffinity-based solid-phase extraction and gas chromatography for the determination of s-triazines in aqueous samples , J. Chromatogr. 830 377-386 (1999). 

For reliable identification of a residue, detailed information about the molecular structure of the analyte is essential. The total information about the molecular structure of the analyte is the sum of the information derived from each individual analytical step of tire method. Frequently used selective analytical steps based on chromatography or immunoaffinity, provide more or less general indirect information. For example, solid-phase extraction (SPE) cleanup followed by liquid chromatography/ultraviolet detection (LC/UV) has been suggested for screening and quantification of ivermectin residues in liver, but presumptive positive samples can be confirmed by derivatizing an aliquot of the SPE eluate and reanalyzing the fluorescent derivative of ivermectin in an LC-fluorescence system (17). 

Cleanup An additional separation of the mycotoxin from lipids and other components of the matrix is accomplished through the cleanup step. Most procedures include solid-phase extraction on stationary phases such as silica, C,8, florisil, and phenyl. Prepacked columns are largely used, with the variations between lots being recently ameliorated. Alternatively, the use of cleanup by immunoaffinity, based on the formation of mycotoxin-protein conjugate, is on the increase, since this is very rapid, selective, and usefully employed in various food matrices. One disadvantage is that the cost is still rather high, and cross-contamination phenomena (false-positive) can occur (30). 

The cleanup step is generally based on two different approaches (a) the use of solid-phase extraction (SPE) cartridges, (b) the use of immunoaffinity (IA) columns. 

High-performance affinity chromatography has recently been reported with trypsin-modified avidin supported on 5 pm silica. While the separations were successful and a wide range of foods were studied, elution times were 80 minutes and ADAM post-column reactions were still required (Hayakawa et al. 2009). However, such affinity columns within a solid-phase extraction (SPE) platform make realistic choices for sample preparation, whereby the biotin can be purified and concentrated prior to reversed-phase HPLC. R-Biopharm has recently developed a commercially available antibody-based immunoaffinity column to bind biotin from aqueous extracts, providing an excellent technique to clean up complex samples. 

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