Extraction Methodology

In the following description and discussion of our results, various criteria derived from the literature are used to determine whether or not each sample is of adequate preservation to allow it to be confidently included in a stable isotope study. The values applied in the various criteria have been found to be associated with archaeological bone collagen that retained an isotopic composition that was reflective of its diet, while the majority of samples that had values outside of the criteria did not retain an isotopic composition that reflected diet. The values for these criteria may vary slightly depending upon the collagen extraction methodology used, and such criteria are not exact. In this study samples that fall within the criteria values are deemed acceptable, and those that do not are deemed unacceptable. ... 

Maharjan, R. P. Ferenci,T. Global metabolite analysis the influence of extraction methodology on metabolome profiles of Escherichia coli. Anal. Biochem. 2003,313, 154-154. 

OTHER TISSUE EXTRACTION METHODOLOGIES FOR FFPE TISSUE... 

Nirmalan NJ, Harnden P, Selby PJ, et al. Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting. J. Pathol. 2009 217 497-506. 

One particular method is designed to characterize Ce to C28+ petroleum hydrocarbons in soil as a series of aliphatic and aromatic carbon range fractions. The extraction methodology differs from other petroleum hydrocarbon methods because it uses n-pentane, not methylene chloride, as the extraction solvent. If methylene chloride is used as the extraction solvent, aliphatic and aromatic compounds cannot be separated. 

Geraniin was easily detected in tissue which had been extracted at room temperature, while no geraniin was detected in heated samples of similar tissue. Apparently, the large amounts of free (unbound) ellagic acid detected, at least for JG. viscosissimum var. viscosissimum, were artifacts of the extraction methodology employed. 

In order to accelerate sample preparation, new extraction methodologies such as accelerated solvent extraction (ASE) and MAE, based on the use of elevated temperature and pressure to heat the mixture sample-solvent, have been recently developed and applied for PAH extraction from meat [695] and vegetables [696-698]. Garda Falcon et al. [699] used microwave treatment with hexane to accelerate PAH extraction from freeze-dried foods. The fat extracted in this way underwent microwave assisted saponification with ethanolic KOH. Hernandez-Borges et al. [700] combined microwave-assisted hydrolysis and extraction to isolate organic pollutants from mussels, while... 

Typical extraction methodologies using lipophilic silica gels... 

Cu2+ and Zn2+ pheophytin analysis is based on Schwartz (1984). Together, these methods allow the analyst to set up a chromatographic system based on a single stationary phase that can easily be modified for flexible and complete analysis of relevant chlorophyll derivatives. When combined with the appropriate extraction methodology, these protocols can be applied to any application relevant to chlorophyll analysis. 

Ionophores, or polyether (PET) antibiotics, produced by various species of Streptomyces, possess broad spectrum anticoccidial activities. They are chemically characterized by several cyclic esters, a single terminal carboxylic acid group, and several hydroxyl groups. Representative members of this class include salinomycin (SAL), monensin (MON), lasalocid (LAS), narasin (NAR), maduramicin (MAD), and semduramicin (SEM). The main chemical properties of interest in the extraction methodology are their low polarities and instability under acidic conditions. They are able to form stable complexes with alkaline cations. All of these compounds, with the exception of LAS, bind monovalent cations (e.g., Na+ and K+). Lasalocid has a tendency to form dimers and can form complexes with divalent cations such as Mg2+ and Ca2+. The formation of metal complexes results in all of these compounds adopting a quasi-cyclic formation consequent to head-to-tail hydrogen bonding. No MRLs have yet been set by the EU for any of the carboxylic acid PETs (98). 

Extraction methodologies obviously need to be further compared and optimised. Clean-up of extracts has been applied but this may generate losses as observed for TBT in biological materials (Astruc et al., 1989). Supercritical extractions offer good possibilities for extracting the species without alteration as demonstrated for butyltins (Bayona, 1996) and MeHg (Quevauviller et al., 1998a). Much work... 

An interesting, and somewhat radical, departure from traditional extraction methodology was proposed by Cave and Wragg (1997). They demonstrated that, with an appropriate chemometric mixture resolution procedure, a simple, nonspecific extraction could provide information on metal binding in soil SRM 2710 similar to that obtained by a Tessier sequential extraction. The method used a central composite design, with extraction time, nitric acid concentration and sample extractant ratio as variables, together with PCA. 

Of course, in addition to the requirement for an unbiased extraction methodology, it is imperative that all enzymic activity is quenched throughout the extraction and sample preparation. Typically, this is assumed to be the case when extracting in organic solvents, although numerous enzymes may retain activity in such environments. The physical nature of the samples themselves has an impact upon the efficiency of extraction of metabolites. For example, eukaryotic and prokaryotic samples behave very differently during the several steps of classic sample preparation methods. Even within the eukaryotes alone, there is a vast diversity of cellular structures that would seem to make it imprudent to blindly adopt protocols that were devised for other organisms or tissues. 

Extraction techniques are universal, standardized, and commonly applied sample preparation methods (see for example [1 ]). Development of extraction methodology in the past 20 years has been eventful and impressive. Universalism of these techniques is associated with applicability to analyses of a great diversity of organic compounds (including DNA and RNA) and elements and their speciation forms, which are present in different sample matrices both in trace and macro quantities. 

Cichoric acid is believed to contribute to the immunostimulatory activity, but the compound tends to decompose through enzymatic degradation during extractions (Bauer, 1997) thus, the variability in cichoric acid levels found by many researchers may be due to the extraction methodology and not to the plant species or origin. Bauer (1999b) evaluated the cichoric acid content of six commercially available expressed juice preparations of E. purpurea. The thermally treated preparations had higher cichoric acid than ethanol-preserved preparations. The inactivation of polyphenol oxidase by heat may account for the difference found between heated and nonheated preparations. 

All comparisons of the elemental composition of soil humates with aquatic humates must be viewed with caution. In recent years, the extraction methodology has changed rapidly. A soil sample extracted with base in the classical manner may have up to 20% carbohydrate and up to 10% ash, while a sample extracted with XAD resins may have less than 2% carbohydrate and 2% ash. To compare two samples extracted by two different methods would be akin to comparing apples and oranges. Fortunately, a body of literature is accumulating which reports the properties of humates extracted by a single, well-defined method. 

As the applications described in this book demonstrate, these modem extraction techniques are not only better for the environment but are also faster, easier to automate, more sensitive, more selective, and more robust than classical organic solvent-based extraction techniques. These qualities are demonstrated in Chapter 8, regarding online coupling of extraction and analysis techniques, and in Chapter 9, on extraction methodologies with integrated cleanup steps. 

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