Simple Extraction Methods

Extraction methodology, including rapid, simple extraction methods and more complex fractionation procedures, will be considered initially, followed by mutagenicity assay methods with emphasis on the Salmonella histidine-reversion assaytogether with aspects of their applicable procedures. 

If the purpose of an evaluation of mutagenicity of a complex mixture is to ascertain whether activity is present, it may suffice to use a simple extraction method. The extraction can be designed to remove and concentrate organics in general or to perform a preliminary separation in preparation for the isolation of specific classes of compounds. Organic solvents of choice are dimethyl-sulfoxide (DMSO), ethanol, benzene, methylene chloride, hexane, methanol, isopropanol, cyclohexane, or combinations of various solvents. Examples of such extraction procedures utilized as environmental bioassays of potential mutagens (carcinogens ) contained on or within particulate matter have been 

A generalized procedure would include extraction of a weighed amount of material (e.g., in a Soxhlet extractor or with several successive portions of a solvent in a shaking apparatus) for a number of hours, separation of the extract, and evaporation to reduce volume or to dryness. The sample could then be weighed, reextracted, or solubilized directly for short-term bioassay. One 

TABLE 1. Summary of Materials Utilized in Short-Term Mutagenicity Tests 

Automotive emissions Organic extraction Class fractionation 34,35 

---

Rezk et al. [74] developed and validated a reversed-phase HPLC assay method for the simultaneous quantitative determination of omeprazole and its three metabolites in human plasma. The method provides excellent chromatographic resolution and peak shape for the four components and the internal standard within a 17-min run time. The simple extraction method results in a clean baseline and relatively high extraction efficiency. The method was validated over the range of 2-2000 ng/ml. The resolution and analysis for the four analytes omeprazole, hydroxyome-prazole, omeprazole sulfone, and omeprazole sulfide and the internal standard utilized a Zorbax C18 (15 cm x 3 mm, 5 /im) with a Zorbax C18 (12.5 cm x 4.6 mm) guard column. The mobile phase consisted of two components. Mobile phase A was 22 mM phosphate monobasic, adjusted to a pH of 6 with diluted sodium hydroxide. This solution was filtered through a 0.45-/im membrane filter, then mixed as 900 ml buffer to 100 ml methanol. Mobile phase B was composed of 100 ml of the phosphate buffer as mobile phase A, mixed with 800 ml of acetonitrile, 100 ml of methanol, and 100 /A of trifluoroacetic acid with an initial flow-rate of 0.55 ml/min and detection at 302 nm. 

Mochizuki T, Kuge Y, Zhao S et al (2003) Detection of apoptotic tumour response in vivo after a single dose of chemotherapy with " Tc-annexin V. J Nucl Med 44 92-97 Mu M, Kung MP, Plossl K et al (1999) Quantitation of Tc-TRODAT in human plasma samples by a simple extraction method. J Labelled Comp Radiopharm 42 213-215... 

Rapid, simple, quaUtative methods suitable for determining the presence of benzene in the workplace or surroundings have been utilized since the 1930s. Many early tests offered methods for detection of aromatics but were not specific for benzene. A straightforward test allowing selective detection of benzene involves nitration of a sample to y -dinitrobenzene and reaction of the resultant ether extract with an ethanoHc solution of sodium hydroxide and methyl ethyl ketone (2-butanone), followed by the addition of acetic acid to eliminate interferences from toluene and xylenes. Benzene imparts a persistent red color to the solution (87). The method is claimed to be sensitive to concentrations as low as 0.27 ppm benzene from 10 mL air samples. 

The concentrations of nitrosamines were reduced to undetectable levels by ultraviolet treatment of the amine solutions and were not increased by addition of 2 ppm NaN02> indicating that the nitrosamines were present originally in the amines and were not formed in the GC injection port. Similar concentrations were found when the amine samples were analyzed using the column extraction method. Direct injection is appropriate for analysis of relatively simple mixtures, if adequate precautions are taken ( ), but can result in significant artifact formation in more complex systems (42). 

We will give here a short overview of the most common XRPD techniques used to study the microstructure of materials, starting from the most used and simple Scherrer method to the quite complex Warren-Averbach method, which is able to extract all the information available on sample microstructure and defects. 

Sample preparation techniques vary depending on the analyte and the matrix. An advantage of immunoassays is that less sample preparation is often needed prior to analysis. Because the ELISA is conducted in an aqueous system, aqueous samples such as groundwater may be analyzed directly in the immunoassay or following dilution in a buffer solution. For soil, plant material or complex water samples (e.g., sewage effluent), the analyte must be extracted from the matrix. The extraction method must meet performance criteria such as recovery, reproducibility and ruggedness, and ultimately the analyte must be in a solution that is aqueous or in a water-miscible solvent. For chemical analytes such as pesticides, a simple extraction with methanol may be suitable. At the other extreme, multiple extractions, column cleanup and finally solvent exchange may be necessary to extract the analyte into a solution that is free of matrix interference. 

Chromatographic methods were extensively used to characterize the allelopathic agents. Because a great majority of the suspected toxins were already known (and several of them are ccrmercially available), simple chromatographic methods were thought to be sufficient for their characterization from the extracts. However, no detailed investigations were ever undertaken to characterize those inhibitors that may be present in minute amounts. 

Vandenburg et al. [37,489] have described the use of Hildebrand solubility parameters in a simple and fast solvent selection procedure for PFE of a variety of polymers. Hildebrand parameters for several common solvents and polymers are presented in Tables 3.2 and 3.34, respectively. When the proper solvent mixture for the polymer was determined, PFE resulted in essentially the same recoveries as the traditional extraction methods, but used much less time and solvent. PFE can be used to give very fast extractions and appears to offer the greatest flexibility of solvents and solvent mixtures. The method is ideal for a laboratory which analyses a large number of different polymers. 

The use of the Hildebrand solubility parameter approach to aid solvent selection with a few simple experiments, starting from the liquid solvents used in traditional extraction methods, limits the efforts needed in method development. As for other extraction... 

HPLC allows a quantitative determination with relatively simple extractions. In many cases, extraction only involves a heating of the commodity with water, followed by filtration and injection onto an HPLC column. In the determination of caffeine, theobromine, and theophylline in cocoa, coffee, or tea, as well as in other foods, there is scarcely a month that passes without a new paper on this assay. Kreiser and Martin provide typical conditions for analysis.28 In their studies, samples were extracted in boiling water and filtered prior to injection onto the HPLC column. The HPLC conditions used a Bondapak reversed phase column and a mobile phase of water methanol acetic acid (74 25 1) with detection at 280 nm. This method is accurate, precise, and conserves time. It has also been adopted by the AOAC as an official method for the determination of theobromine and caffeine in cocoa beans and chocolate products.29... 

Albertus, Frater], Simple vacuum extraction method. Alchem Lab Bulls 2, no. 1 (1970). rhttp //www.spagyria.com/alb.zipl. 

Modification of a simple vacuum extraction method. Parachemy 6, no. 1 (Winter 1978) 515-. 

The many technical factors involved in desizing and the need for economy and environmental accountability emphasise the importance of monitoring and analysis. However, only a brief outline can be given here. A review of analytical procedures and simple laboratory methods for size determination is available [193]. Methods are given for size determination directly on the fibre surface, for the extraction of components of low molecular mass and for their subsequent estimation in solution. 

There is significant debate about the relative merits of frequency and time domain. In principle, they are related via the Fourier transformation and have been experimentally verified to be equivalent [9], For some applications, frequency domain instrumentation is easier to implement since ultrashort light pulses are not required, nor is deconvolution of the instrument response function, however, signal to noise ratio has recently been shown to be theoretically higher for time domain. The key advantage of time domain is that multiple decay components can, at least in principle, be extracted with ease from the decay profile by fitting with a multiexponential function, using relatively simple mathematical methods. 

Based on the heat-induced AR principle, DNA/RNA extraction from FFPE tissues can be successfully achieved by a simple heating protocol that allows satisfactory application of molecular analysis using FFPE tissue samples housed in pathology laboratories worldwide. By a combination of improved extraction methods with various innovative techniques of molecular biology, more reliable results of molecular profiling for archival tissue are anticipated. 

Another characteristic to take into account is that ATP is an endogenous component of the cells, both somatic and bacterial. Therefore, an extraction step must to be included in the assay protocol it is very simple and quick to perform. Several extraction methods have been reported, both physical and chemical, such as heating and the use of surfactants, trichloroacetic acid, and organic solvents [89, 120, 121], The chemical methods are generally preferred the addition of a surfactant can be effective in most cases. The use of mild or strong extraction... 

Simple extractions of soil, with or without salts, however, are not the most common methods of extracting soil to determine its composition or chemistry, particularly with regard to aluminum or magnesium [7],... 

Mechanical methods are fairly simple. The methods may be divided into two major groups. One involves the capture of a sample of droplets on a solid surface or in a cell containing a special liquid. The captured droplets are then observed or photographed by means of a microscope, generating information on droplet size. The other involves freeze-up of droplets into solid particles and subsequent sieving to generate droplet size distribution. The major problem is associated with the extraction and collection of representative spray samples. 

To enhance the annotation of TrEMBL, a novel method for the prediction of this information has been developed (Fleischmann et al., 1999). The principle is simple The method tries to find SWISS-PROT entries belonging to the same protein family as the unannotated TrEMBL entry, extracts the annotation shared by all SWISS-PROT entries, assigns this... 

A simple TLC method has been developed for the separation and identification of flavons and flavon glycosides in the extract of Phillyrea latifolia L. The leaves (100 g) were defatted in 11 of chloroform for 24 h and then extracted with 2 X 11 of ethanol-water (80 20, v/v). The collected extracts were concentrated and extracted again with n-hexane to remove chlorophylls and other apolar constituents. Analytes were extracted with ethyl acetate. Both normal phase and RP-TLC have been used for the separation of flavonoids. The results are compiled in Table 2.36. It was concluded from the data that TLC can be successfully applied for the quality control of plant extracts containing various flavone derivatives [124],... 

It was concluded from the measurements that the extraction method is rapid and simple and allows the reproducible and sensitive determination of the dyes by both HPLC-vis andHPLC-MS techniques [101],... 

Comelissen, G. Rigterink, H. ten Hulscher, D.E.M. Vrind, B.A. Van Noort, P.C.M. 2001, A simple Tenax extraction method to determine the availability of sediment-sorbed organic compounds. 

---