Removal of Interfering Compounds

Polysaccharides are another class of macromolecules that can interfere with cellular and enzyme assays, as demonstrated during the NCI campaign to discover anti-HIV agents from natural sources. Anionic polysaccharides show anti-HIV activity and, to avoid a too high hit rate, had to be removed by 50% aqueous ethanol precipitation.72 

For these considerations, the selective removal of certain constituents from plant extracts can be a double-edged sword. On the one hand, it reduces the 

5 Selection Strategies for Plant-Based Natural Product Drug Discovery 

The discovery of several important drugs can be traced back to the medicinal or ritual use of specific plants in a non-Western culture. Alkaloids like quinine, emetine, physostigmine and tubocurarine were all discovered because their plant producer had been identified for a particular activity by a native culture.74 The World Health Organization (WHO) has estimated that 80% of the world s population rely mainly on plant-based traditional medicine for their primary health.75 There is, therefore, an enormous past and current literature on the human use of plants. 

But despite these limitations, the supplementation of phytochemical data with ethnopharmacological information has been demonstrated to lead to higher hit rates in both the virtual and the actual screening of natural product libraries.76 

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Anthocyanin purification steps are important for anthocyanin characterization. Removal of interfering compounds allows for more reliable HPLC separation, spectral information, mass spectra, and NMR spectra during the identification of anthocyanins in plant extracts. 

A sample preparation is often necessary to isolate the components of interest from a sample matrix. Removal of interfering compounds prevents blocking of the HPLC column in spite of the... 

A simple sample dilution is sufficient for removal of interfering compounds. IPCR is still able to detect the antigen in sample dilutions [66], whereby the loss in sensitivity limits the usefulness of additional dilution in conventional ELISA. 

Solid-phase extraction objectives, apart from the removal of interfering compounds and the preconcentration of the sample, include ... 

Even though the interest in microwave-assisted extraction (MAE) has increased during the last 10 years, this technique has not been utilized much in food and feed applications. Only a few papers can be found with the combination of POPs and food/feed samples. This may be because MAE applications frequently require laborious and tedious clean-up of the extracts before final analysis. In some cases, only a simple filtration or centrifugation may be sufficient to separate the solid matrix from the extract but since MAE most often is more exhaustive than selective, extensive clean-up procedures based on for example solid-phase extraction is commonly needed for removal of interfering compounds (73-75). Other techniques that have been used for clean-up of MAE extracts are gel permeation chromatography (75), solid-phase micro extraction (77, 78), and liquid-liquid extraction (79). 

Sample preparation procedures are determined by target analytes, their concentrations, the sample matrix, and the analytical instrumentation used for measurement. Procedures can range from a simple filtration to a sequence of steps which may include filtration, centrifugation, extraction, concentration, removal of interfering compounds, and derivatization. Simple uncomphcated sample preparation procedures are preferred since they are less prone to errors, less susceptible to contamination, and less time consuming. Sample preparation can be quite labor-intensive although on-line procedures have also been developed." ... 

Amino acids should be as free from impurities as possible, since they exhibit a pronounced capacity for binding metal ions. The analysis of amino acids in natural fluids or extracts requires the removal of interfering compounds prior to chromatographic separation, in order to prevent tailing and deformation of the spots (e.g., high salt concentrations are found in urine samples and hydrolysates of proteins or peptides). 

Phillips, F., Kaczor, K., Gandhi, N., Pendley, B.D., Danish, R.K., Neuman, M.R., Toth, B., Horvath, V., Lindner, E., 2007. Measurement of sodium ion concentration in undiluted urine with cation-selective polymeric membrane electrodes after the removal of interfering compounds. Talanta 74, 255-264. 

Trace enrichment and sample clean-up are probably the most important applications of LC-LC separation methods. The interest in these LC-LC techniques has increased rapidly in recent years, particularly in environmental analysis and clean-up and/or trace analysis in biological matrices which demands accurate determinations of compounds at very low concentration levels present in complex matrices (12-24). Both sample clean-up and trace enrichment are frequently employed in the same LC-LC scheme of course, if the concentration of the analytes of interest are Sufficient for detection then only the removal of interfering substances by sample clean-up is necessary for analysis. 

Exudate collection in trap solutions usually requires subsequent concentration steps (vacuum evaporation, lyophilization) due to the low concentration of exudate compounds. Depending on the composition of the trap solution, the reduction of sample volume can lead to high salt concentrations, which may interfere with subsequent analysis or may even cause irreversible precipitation of certain exudate compounds (e.g., Ca-citrate, Ca-oxalate, proteins). Therefore, if possible, removal of interfering salts by use of ion exchange resins prior to sample concentration is recommended. Alternatively, solid-phase extraction techniques may be employed for enrichment of exudate compounds from the diluted trap solution (11,22). High-molecular-weight compounds may be concentrated by precipitation with organic solvents [methanol, ethanol, acetone 80% (v/v) for polysaccharides and proteins] or acidification [trichloroacetic acid 10% (w/v), per-... 

Distillation of sample is often necessary for the removal of interfering contaminants. The sample is buffered at pH 9.5 with borate buffer prior to distillation. This decreases hydrolysis of cyanates (CNO ) and organic nitrogen compounds. 

The solvent extract should be subjected to one or more cleanup steps for the removal of interfering substances. The presence of phthalate esters, sulfur, or other chlorinated compounds can mask pesticide peaks. The extract should, therefore, be cleaned up from the interfering substances using a florisil column or by gel permeation chromatography (see Chapter 1.5). The distribution patterns for the pesticides in the florisil column fractions are presented in Table 2.20.2. 

The total phenolic compounds in an aqueous sample can be determined by a colorimetric method using 4-aminoantipyrine. This reagent reacts with phenolic compounds at pH 8 in the presence of potassium ferricyanide to form a colored antipyrine dye, the absorbance of which is measured at 500 nm. The antipyrine dye may also be extracted from the aqueous solution by chloroform. The absorbance of the chloroform extract is measured at 460 nm. The sample may be distilled before analysis for the removal of interfering nonvolatile compounds. The above colorimetric method determines only ortho- and meta-substituted phenols and not all phenols. When the pH is properly adjusted, certain para-substituted phenols, which include methoxyl-, halogen-, carboxyl-, and sulfonic acid substituents, may be analyzed too. 

Investigating metabolism or stability of prostanoids, radiolabeled precursors or analytes are often used. The tritiated or -labeled compounds can easily be detected without any further derivatization using online or off-line liquid scintillation, which is not impaired by any interferences derived from matrix components. Efficient but less sensitive PG analysis is possible by UV detection (190-210 nm) of underivatized substances demanding the remove of interfering contaminants or simple sample matrices like buffers or some cell supernatants. 

The purification procedure should remove potentially interfering compounds and, moreover, fractionate the entire spectrum of cytokinins into groups. It is usually not possible to analyze nucleotides and O-glucosides together with free bases, ribosides and iV-glucosides. Classical liquid-liquid partition steps [274] have been recently replaced by less labor-intensive, rapid and selective solid-liquid extraction. When applied in neutral aqueous solutions, cytokinin nucleotides are retained on weak anion-exchangers (DEA -Sephadex, DEAE-cellulose), while the other cytokinin forms are retained on reversed phase sorbents. Dobrev and Kaminek [275] used mixed-mode solid-phase extraction for step-wise... 

Wine peptides are normally analyzed after a preparation step allowing their separation from the high MW components, mainly proteins and polysaccharides. This is achieved by ultrafiltration on membranes with appropriate cut-off and/or by gel filtration (e.g. on Sephadex LH-20 or G-10 gels) (Desportes et al., 2000 Moreno-Arribas et al., 1996 and 1998) of concentrated samples. This allows the isolation of one or more peptide fractions by removing the interfering compounds with different MW, such as salts, amino acids, phenols, organic acids and sugars. 

More recently lEC has also been employed for the analysis of landfill leachates.Sample pretreatment steps may include centrifugation, filtration, removal of interfering organic compounds, and carbonate removal. An advantage is that these lEC methods analyze for a wider spectrum of organic acids than the GC methods, hence giving a more complete picture of the organic acid composition in landfill leachates. 

A typical biological sample may contain hundreds or even thousands of different compounds while only a small fraction of these is of analytical interest at a given time. It is thus essential to remove the interfering compounds prior to a GC analysis. A selective preconcentration of the substances of interest can ideally be accomplished at the same time. 

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