Extraction techniques immunoaffinity chromatography

Higher-specificity techniques such as immunoaffinity chromatography have been also found widespread acceptance for the determination of chloramphenicol residues in edible animal products. The first pertinent reports concerned the determination of chloramphenicol residues in swine muscle (60) and milk and eggs (21). Alternative immunoaffinity chromatography procedures were suggested later for exiraction/preconcentration of chloramphenicol residues from swine tissues (50), or for online immunoaffinity extraction for the determination of chloramphenicol in milk and swine muscle by an automated column-switching system (22). 

Ultrafiltration (278, 279) and immunoaffinity chromatography (282) have also been described for removal of matrix components from milk extracts, while online trace enrichment has been reported for isolation/purification of tetracycline, oxytetracycline, demeclocycline, and chlortetracycline residues from animal tissues and egg constituents (305). The latter technique involves trapping of the analytes onto a metal chelate affinity preconcentration column (Anagel-TSK Chelate-5PW), rinsing of coextracted materials to waste, and finally flushing of the concentrated analytes onto the analytical column. 

The analyte binding efficiency is matrix dependent. Some matrices, such as urine and tissue extracts, can be directly loaded onto the column, other matrices such as milk may need sample processing prior to loading onto an immunoaffinity column. The simplest sample preparation method is dilution this method has been applied to serum, liver, and kidney extracts after removal of particulates. Sometimes dilution alone is not sufficient to eliminate the matrix effect and classical sample preparation techniques (solvent/solvent extraction, solid phase extraction, etc.) will be necessary prior to immunoaffinity chromatography. We found milk often needs this type of treatment. 

Which is better, GC-MS or immunoassay This is a question often asked about plant hormone quantification. GC-MS, which is now more widely available since the Introduction of bench-top instruments, has the advantage that it not only provides quantification of the hormone by the isotope dilution method, but also confirms the identity of the compound concerned by comparison of its spectrum with that of a standard. However, once validated for a particular tissue, immunoassay has the advantage that many samples can be analysed very quickly. Both techniques require sample pre-purification, often by the same methods. A more recent development is a powerful combination of the two technologies which uses the antibody immobilised on a polymer support as a method of affinity-purifying the hormones (together with interfering substances) from plant extracts prior to analysis by GC-MS. Immunoaffinity chromatography is discussed in the next section. 

The first publication that reported the use of LC—MS for quantification of IsoPs in urine used reversed-phase LC coupled with ESI/MS. The method used only 1 mL urine and the clean-up procedure using solid phase extraction (SPE) columns gave quantitative recovery of the IsoP. The chromatographic runs and SPE purification methods are short, and this results in a very easy and user-friendly procedure (Li et al., 1999). A comprehensive review by Tsikas et al. describes sample preparation techniques and compares GC—MS methods with the most recent LC—MS/MS methods (Tsikas et al., 2003). The focus is primarily on 8-f5o-PGF2ci and highlights the difficulty to detect only one IsoP isomer without immunoaffinity chromatography preparation. The large concentration differences for the various IsoP classes in urine are also addressed. 

Analytical techniques Charcoal column chromatography Chromatography Coleus forskohlii Lorskolin ELISA GLC HPLC HPTLC Hydrotropic extraction Immunoaffinity chromatography Microwave-assisted extraction Molecular simulation TLC... 

The fundamentals of affinity chromatography can be taken from the literature [2-5]. Since affinity-based analytical methods are extremely dependent on the analytes and their respective binding molecules, it is not possible to go into much detail with regard to practical advice. It has to be mentioned that affinity chromatography is rarely a true chromatographic technique, and in most cases has to be considered to be a selective extraction method. Hence, the term affinity extraction or immunoaffinity extraction should be preferred where appropriate. 

The majority of reports have used electrospray ionization mass spectroscopy (ESI-MS) as an analytical detection method because of its sensitivity and the soft namre of its ionization procedure, which generally only leads to the detection of the molecular ions of the positive library members. Many separation techniques have been coupled to ESI-MS, including affinity chromatography (49), size exclusion chromatography (50, 51), gel filtration (52), affinity capillary electrophoresis (53-58), capillary isoelectric focusing (59), immunoaffinity ultrafiltration (60), and immunoaffinity extraction (61). ESI-MS has also been used alone (62) to screen a small carbohydrate library. Other examples reported alternative analytical techniques such as MALDI MS, either alone (63, 64) or in conjunction with size exclusion methods (65), or HPLC coupled with immunoaffinity deletion (66). 

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. 

With the exception of mold culture extracts, which can be analyzed directly after extraction, the mycotoxin from a test sample must be concentrated and purified. Various techniques, such as column chromatography on silica gel, octadecyl-bonded silica gel, alumina, magnesium silicate, size-exclusion gels, charcoal, ion-exchange bonded phases, or immunoaffinity packing, are used. The most commonly used column packing is silica gel. Lipid material is eluted first with a nonpolar solvent such as hexane. The mycotoxin of interest is eluted with a solvent that will remove all of the mycotoxin but leave other material on the column. Solvent partition, metal complexation, ion-pairing, and precipitation are also used for purification. Smaller cleanup columns, which use less solvent, have... 

Immunoaffinity extraction is a technique often adopted in clinical chemistry. Immu-noaffinity supports contained polyclonal antibodies covalently immobilized on sephar-ose, agarose, or silica supports. Two different kinds of supports, crushed sol-gel monoliths and sol-gel-coated highly porous silica particles, were applied to the extraction of 16 sulfonylureas in food and natural water followed by high-performance liquid chromatography-ultraviolet/diode-array detection (LC-UV/DAD) [32]. 

---