Base extraction cleanup procedure

Broad Spectrum Analysis of Resin Extracts A Base Extraction Cleanup Procedure... 

The base extraction cleanup procedure changed the initial GC profile data contained in the original ethyl ether extract. Specifically,... 

All radioimmunoassays published thereafter, except those described by Hock and Liemann (38), Freebairn and Crosby (39), and Pohlschmidt et al. (40), were based on a similar procedure (Table 28.2). However, Hock and Liemann (38) applied a more simplified extraction/cleanup procedure for the analysis of chloramphenicol residues in animal tissues, milk, urine, and plasma. In this assay, competitive inhibition between chloramphenicol labeled with " C and antibody has been demonstrated. 

The quantification of PAHs on a routine basis is often conducted using liquid chromatography (LC) in conjunction with a luminescence technique since they exhibit fluorescent properties. In general, the algorithms of analysis include extraction, cleanup procedure, concentration, chromatographic separation, and determination. Liquid-liquid, solid-phase and cloud point extractions have been interfaced with flow-based methodologies. 

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. 

A cleanup procedure is usually carried out to remove co-extracted matrix components that may interfere in the chromatographic analysis or be detrimental to the analytical instrument. The cleanup procedure is dependent on the nature of the analyte, the type of sample to be analyzed, and the selectivity and sensitivity of the analytical instrument used in the analysis. Preliminary purification of the sample extracts prior to chromatographic separation involves liquid-liquid partitioning and/or solid-phase extraction (SPE) using charcoal/Celite, Elorisil, carbon black, silica, or aminopropyl-silica based adsorbents or gel permeation chromatography (GPC). 

Diethylstilbestrol is particularly difficult to quantitate below 1.0 ppb in bovine tissues, especially in liver, which is among the last tissues to contain diethystilbestrol after cattle are withdrawn from receiving tire drug (101, 102). Interferences from tissue matrix constitute a major problem that might be due to nonspecific interference of lipids and fatty compounds (103, 104). In addition, problems with false-positive results often appear in urine analysis unless a chromatographic step such as a solid-phase extraction cleanup (105, 106) is introduced. Simple sample preparation procedures such as those based on solvent extraction and liquid-liquid partitioning do not usually give satisfactory results (107, 108). 

Since many interferences with diethylstilbestrol analysis are caused by undefined sources, use of a highly specific antibody would probably not correct the problem. Nevertheless, several attempts to prepare a drug-specific antibody have been made without success. Thus, more emphasis was finally placed on developing more efficient extraction and cleanup procedures to ensure selectivity. Current methods combine several cleanup steps based on different isolation principles to provide adequately purified sample extracts for the determination of diethystilbes-trol by radioimmunoassays (Table 28.4). 

Unlike clenbuterol, salbutamol is a difficult compound to analyze due to its particular chemical attributes. It is a basic compound subjected to protein binding poor recoveries are obtained especially when protein precipitation techniques are used to prepare the extracts (145). In addition, salbutamol is charged at all pH values and does not readily lend itself to simple, specific back-extracting procedures. This severely restricts the options of sample cleanup. However, a Subtilisin protease digestion step followed by acid clarification and solid-phase extraction has been suggested (146) as an adequate extraction and cleanup procedure prior to the end-point determination of salbutamol by an enzyme immunoassay (139) based on the cross-reactivity of anticlenbuterol antibodies. 

A specific cleanup procedure based on immunoaffinity chromatography with polyclonal antibodies has been described by Gude et al. (50) for the gas chromatographic determination of chloramphenicol in swine tissues. In this method, tissue sample is extracted with acetonitrile/4% sodium chloride (1 1) Following centrifugation, the supernatant is purified with n-hexane, and chloram-... 

Detection in liquid chromatography is mostly performed by fluorescence and/or ultraviolet absorption. In a few instances, electrochemical detection has also been employed (357, 368). For compounds that exhibit inherent intense fluorescence such as albendazole and metabolites (319, 320, 338, 355), closantel (344), and thiabendazole and metabolites (378), fluorometric detection is the preferred detection mode since it allows higher sensitivity. Compounds that do not fluoresce such as eprinomectin, moxidectin, and ivermectin, are usually converted to fluorescent derivatives prior to their injection into the liquid chromatographic analytical column. The derivatization procedure commonly applied for this group of compounds includes reaction with trifluoroacetic anhydride in presence of A-methylimidazole as a base catalyst in acetonitrile (346, 347, 351, 352, 366, 369, 372-374). The formation of the fluorophore is achieved in 30 s at 25 C and results in a very stable derivative of ivermectin and moxidectin (353) but a relatively unstable derivative of eprinomectin (365). However, the derivatized extracts are not pure enough, so that their injection dramatically shortens the life of the liquid chromatographic column unless a silica solid-phase extraction cleanup is finally applied. 

This chapter describes the simpler cleanup approach of base extraction for XAD resin extracts. Quality assurance procedures statistically define the benefits of this analytical approach for broad spectrum capillary GC. The extraction procedure was optimized by studying UV absorbance of the base extractant. 

Bean et al. (10) used size-exclusion chromatography to remove materials of MW >800. This method of cleanup was rejected because size-exclusion chromatography might introduce new artifacts and cause additional delays in sample analysis time. Because the matrix problem appeared to have acidic components, it was decided that a base extraction procedure might remove these materials. 

Optimization of Base Extraction. The base extraction, sample cleanup procedure was optimized by taking into account the following facts ... 

The cleanup procedure used was discussed in the section entitled Resin Elution Concentration. The extraction procedure was not the optimum procedure that would result from the observations. The data indicated that a base extraction at pH >12 and a base-to-solvent ratio of 15 1 with multiple extraction steps were needed to maximize removal of the matrix interferences. However, the extraction efficiency must be balanced against minimal sample destruction and operational ease. 

Liquid-liquid extraction has also been employed as a cleanup step, with separations being made between an acid or alkaline aqueous phase and an organic solvent (48). This procedure takes advantages of differences in the physical and chemical characteristics between the carbamate and the substrate. Another commonly used procedure is based on the generally high solubility of carbamates in polar solvents and their low solubility in saturated hydrocarbons. Table 5 summarizes the use of the various cleanup procedures in the determination of carbamate pesticides. 

Extraction and cleanup procedures usually require solid-phase extraction based on commercially available Cjg cartridges, for instance, after liquid extraction with common organic solvents (methylene dichloride. 

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