Sample preparation extraction/cleanup

When the analytical laboratory is not responsible for sampling, the quality management system often does not even take these weak links in the analytical process into account. Furthermore, if sample preparation (extraction, cleanup, etc.) has not been carried out carefully, even the most advanced, quality-controlled analytical instruments and sophisticated computer techniques cannot prevent the results of the analysis from being called into question. Finally, unless the interpretation and evaluation of results are underpinned by solid statistical data, the significance of these results is unclear, which in turn greatly undermines their merit. We therefore believe that quality control and quality assurance should involve all the steps of chemical analysis as an integral process, of which the validation of the analytical methods is merely one step, albeit an important one. In laboratory practice, quality criteria should address the rationality of the sampling plan, validation of methods, instruments and laboratory procedures, the reliability of identifications, the accuracy and precision of measured concentrations, and the comparability of laboratory results with relevant information produced earlier or elsewhere. 

The development of a robust analytical method is a complex issue. The residue analyst has available a vast array of techniques to assist in this task, but there are a number of basic rules that should be followed to produce a reliable method. The intention of this article is to provide the analyst with ideas from which a method can be constructed by considering each major component of the analytical method (sample preparation, extraction, sample cleanup, and the determinative step), and to suggest modern techniques that can be used to develop an effective and efficient overall approach. The latter portion emphasizes mass spectrometry (MS) since the current trend for pesticide residue methods is leading to MS becoming the method of choice for simultaneous quantitation and confirmation. This article also serves to update previous publications on similar topics by the authors. ... 

Sample Preparation/Extraction The process of separating potentially interfering components from a sample prior to LC-MS analysis for the purposes of improving sensitivity, specificity, and/or method ruggedness. Variations include solid phase extraction (SPE), liquid-liquid extraction (LLE), and protein precipitation (PPT). Extraction may be performed off-line, in which the cleanup is completely independent from the LC-MS analysis, or on-line, in which the cleanup is integrated directly into the LC-MS analysis. 

Present MS techniques for analysis of organic compounds in complex samples require separation of the sample components prior to MS analysis. The separation is usually accomplished by extracting the samples, separating the extracts into several fractions (cleanup), and analyzing the fractions by GC/MS. These steps, especially the sample preparation and cleanup, are time-consuming and expensive. 

In this section a concise overview of the most widely used analytical procedures for the determination of PCBs in environmental matrices (namely, air, sea water, snow/firn/ice, sediment/soil and biota) is given. Regardless of the nature of the sample, the following steps are generally included in an analytical procedure i) sample collection and storage ii) sample preparation (extraction of the analytes and cleanup of the extract) iii) instrumental analysis iv) data evaluation, including analytical quality control. 

In the analytical scheme, besides the sampling and the final analysis, the sample preparation and cleanup are also crucial. Sample preparation plays an important role in the analysis of FRs in environmental samples because of the complex matrices and only trace levels of analytes. Solid and semisolid samples are usually first dried and homogenized. Then the FRs are extracted from the sample (solid or liquid), and the extract is usually purified, fractionated, and concentrated before the final analysis, which is typically performed with gas or liquid chromatography. The extraction procedure is dependent on the sample matrix different methods are used for sediment, tissue, and liquid samples. After extraction, it will usually be necessary to purify and fractionate the extract, because most extraction methods are insufficiently selective and the separation power of the analytical technique not sufficient. Extracts typically contain several analytes similar to the FRs, which may be present in much higher quantities. The fractionation procedures are similar for the different types of extracts. Typical analytical procedures are given in Tables 31.2 to 31.6. 

The OCs and PCBs were first determined in wastewaters using EPA Method 608 (2). This method originally required packed columns, and because of this, it necessitated extensive sample preparation and cleanup techniques which included liquid-liquid extraction and low-pressure column liquid chromatography. Capillary GC-ECD when combined with more contemporary methods of sample preparation provides for rapid and cost-effective trace environmental analysis. Over the past 10 years, there has been dramatic improvements in sample preparation techniques as this relates to semivolatile and nonvolatile trace analyses. 

The solubility of drugs and ottier solids determines whether tiiey can be placed into (he aqueous phase. For drugs, water solubility is a critical consideration, since it correlates with bioavailability and toxicology. It is also important for sample preparation and cleanup, because most liquid-liquid extractions involve an aqueous phase in contact with an organic phase. The only unique aspect of solubility equilibrium is that, since one component is a solid, it is not expressed, because ttiere is no aqueous concentration. The solubility is referred to as S and is obtained as shown in Example Problem 4.2. 

Environmental samples can be extremely complex and come from a great variety of sources. Each sample requires a sampling and storage strategy. Analytical methods need to include procedures for sample preparation, extraction, and, if necessary, cleanup prior to gas chromatographic analysis. Complex and dirty samples require more difficult sample preparation techniques. Samples can be solids, liquids, gases, or mixtures of these phases. For example, a sample pulled... 

In Table 1, sample preparation and cleanup procedures for vitamin K analysis using HPLC detection are shown, collected in groups of various sample matrices. A remarkable uniformity exists in the preparation of plasma samples. Almost every research group used identical solvents (ethanol in a volume ratio of 1 4) for denaturation of VK transport proteins (lipoproteins of the VLDL fraction) as well as for extraction of the vitamins from plasma (hexane in a volume ratio up to 20, depending on sample volume). MacCrehan et al. (77) and Sakon et al. (90) used isopropanol for denaturation, which is said to provide better extraction recoveries, but coextracted polar compounds may interfere with the vitamins in the final chromatography. The uniformity of this isolation process may be a result of former experiments, using strong acids, alkalines, or different extraction solvents and methods, which are summarized and discussed by Lambert et al. (17) in the second edition of this volume. 

Preparation of soil—sediment of water samples for herbicide analysis generally has consisted of solvent extraction of the sample, followed by cleanup of the extract through Uquid—Uquid or column chromatography, and finally, concentration through evaporation (285). This complex but necessary series of procedures is time-consuming and is responsible for the high cost of herbicide analyses. The advent of soUd-phase extraction techniques in which the sample is simultaneously cleaned up and concentrated has condensed these steps and thus gready simplified sample preparation (286). 

The primary method for detecting methyl parathion and metabolites in biological tissues is gas chromatography (GC) coupled with electron capture (BCD), flame photometric (FPD), or flame ionization detection (FID). Sample preparation for methyl parathion analysis routinely involves extraction with an organic solvent (e g., acetone or benzene), centrifugation, concentration, and re suspension in a suitable solvent prior to GC analysis. For low concentrations of methyl parathion, further cleanup procedures, such as column chromatography on silica gel or Florisil are required. 

Analysis of methyl parathion in sediments, soils, foods, and plant and animal tissues poses problems with extraction from the sample matrix, cleanup of samples, and selective detection. Sediments and soils have been analyzed primarily by GC/ECD or GC/FPD. Food, plant, and animal tissues have been analyzed primarily by GC/thermionic detector or GC/FPD, the recommended methods of the Association of Official Analytical Chemists (AOAC). Various extraction and cleanup methods (AOAC 1984 Belisle and Swineford 1988 Capriel et al. 1986 Kadoum 1968) and separation and detection techniques (Alak and Vo-Dinh 1987 Betowski and Jones 1988 Clark et al. 1985 Gillespie and Walters 1986 Koen and Huber 1970 Stan 1989 Stan and Mrowetz 1983 Udaya and Nanda 1981) have been used in an attempt to simplify sample preparation and improve sensitivity, reliability, and selectivity. A detection limit in the low-ppb range and recoveries of 100% were achieved in soil and plant and animal tissue by Kadoum (1968). GC/ECD analysis following extraction, cleanup, and partitioning with a hexane-acetonitrile system was used. 

Until this point, the sample preparation techniques under discussion have relied upon differences in polarity to separate the analyte and the sample matrix in contrast, ultraflltration and on-line dialysis rely upon differences in molecular size between the analyte and matrix components to effect a separation. In ultrafiltration, a centrifugal force is applied across a membrane filter which has a molecular weight cut-off intended to isolate the analyte from larger matrix components. Furusawa incorporated an ultrafiltration step into his separation of sulfadimethoxine from chicken tissue extracts. Some cleanup of the sample extract may be necessary prior to ultrafiltration, or the ultrafiltration membranes can become clogged and ineffective. Also, one must ensure that the choice of membrane filter for ultrafiltration is appropriate in terms of both the molecular weight cut-off and compatibility with the extraction solvent used. 

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. 

Third, the bulk of the items in Table 1 address method performance. These requirements must be satisfied on a substrate-by-substrate basis to address substrate-specific interferences. As discussed above, interferences are best dealt with by application of conventional sample preparation techniques use of blank substrate to account for background interferences is not permitted. The analyst must establish a limit of detection (LOD), the lowest standard concentration that yields a signal that can be differentiated from background, and an LOQ (the reader is referred to Brady for a discussion of different techniques used to determine the LOD for immunoassays). For example, analysis of a variety of corn fractions requires the generation of LOD and LOQ data for each fraction. Procedural recoveries must accompany each analytical set and be based on fresh fortification of substrate prior to extraction. Recovery samples serve to confirm that the extraction and cleanup procedures were conducted correctly for all samples in each set of analyses. Carrying control substrate through the analytical procedure is good practice if practicable. 

Sample preparation consists of homogenization, extraction, and cleanup steps. In the case of multiresidue pesticide analysis, different approaches can have substantially different sample preparation procedures but may employ the same determinative steps. For example, in the case of soil analysis, the imidazolinone herbicides require extraction of the soil in 0.5 M NaQH solution, whereas for the sulfonylurea herbicides, 0.5M NaOH solution would completely decompose the compounds. However, these two classes of compounds have the same determinative procedure. Some detection methods may permit fewer sample preparation steps, but in some cases the quality of the results or ruggedness of the method suffers when short cuts are attempted. For example, when MS is used, one pitfall is that one may automatically assume that all matrix effects are eliminated because of the specificity and selectivity of MS. 

HPLC/MS and HPLC/MS/MS analyses are susceptible to matrix effects, either signal enhancement or suppression, and are often encountered when the cleanup process is not sufficient. To assess whether matrix effects influence the recovery of analytes, a post-extraction fortified sample (fortified extract of control sample that is purified and prepared in the same manner as with the other samples) should be included in each analytical set. The response of the post-extraction fortified sample is assessed against that of standards and samples. Matrix effects can be reduced or corrected for by dilution of samples, additional cleanup, or using calibration standards in the sample matrix for quantitation. 

Principles and Characteristics Because of the limited selectivity of extraction, a chromatographic analysis is almost always needed. Recently, a fair amount of progress has been made regarding the front end of the total analysis procedure, namely the integration of sample preparation (this being the analytical bottleneck) and separation. The idea behind such systems is to perform sample extraction, cleanup and concentration as an integral part of the analysis in a closed system. Scheme 7.2 shows the main procedures related to sample preparation for chromatographic analysis. 

The configuration can be expanded by adding other sample preparation instruments to facilitate automating other preparative steps that may intervene between SFE and the analytical instrument, e.g. solvent exchange, internal standard addition, serial dilutions for calibration curve generation, SPE for further cleanup of the extract output by SFE, derivatisation of components within the SFE extract, and many other (currently) manual-human intervention techniques. 

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