Separation and detection

A survey of the literature with a key phrase tissue residue analysis yielded a distribution of separation and detection techniques as outlined in Table 2. LC with either UV or fluorescence detection was the most common separation and detection technique, representing 61% of the citations. The results are an indication of the maturity of LC as a common, well-understood technique. The second most commonly used technique cited in the literature (13%) was GC with either a mass-selective or electron capture detector. GC is also a mature technology and a good choice owing to the 

Once the analyte has been separated from the matrix in LC, the best approach to the detection of the molecule must be determined. This section will discuss the detection techniques of ultraviolet/visible (UVA IS), fluorescence (FL), and electrochemical (EC) detection, with MS being addressed separately in Section 4.2. When deciding on the most appropriate detector for an LC separation, the appropriate chemical data on the analyte should be collected by using a spectrophotometer, fluorimeter, and potentiometer. 

For FL detection, maximum emission and excitation wavelengths are determined using a fluorimeter. Stoev used fluorescence detection to analyze for closantel (excitation at 335 nm, emission at 510 nm) residues in animal tissue. 

Electrochemically active compounds can be evaluated using a potentiometer to generate a cyclic voltammogram for the analyte. Cyclic voltammetry will allow the analyst to determine whether the compound can be oxidized or reduced, to choose the appropriate potential to use in the electrochemical detector, and to establish whether oxidation or reduction is irreversible. Irreversible oxidation or reduction of the analyte could be predictive of problems with electrode poisoning and reduced sensitivity of the electrochemical detector over time. Turberg et al. used EC detection at an applied potential of -1-600 mV to analyze for ractopamine. 

At this point, the anaiyte may not be amenabie to UV, FL, or EC detection. In this case, the best course of action may be to choose LC/MS (see Section 4.2). However, one other option is to use a pre- or post-coiumn derivatization step to increase the detectabiiity of the anaiyte with respect to FL or UV. Fluorescent or UV labels are available for carboxylic acids, amines, phenols, and thiols. The decision to use pre- or post-column derivatization is predicated upon the functionality of the analyte available for derivatization and the rate and extent of the reaction between each derivatizing agent and the analyte. 

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Section 13 22 Mass spectrometry exploits the information obtained when a molecule is ionized by electron impact and then dissociates to smaller fragments Pos itive ions are separated and detected according to their mass to charge (m/z) ratio By examining the fragments and by knowing how classes of molecules dissociate on electron impact one can deduce the structure of a compound Mass spectrometry is quite sensitive as little as 10 g of compound is sufficient for analysis... 

The instrumentation for SSIMS can be divided into two parts (a) the primary ion source in which the primary ions are generated, transported, and focused towards the sample and (b) the mass analyzer in which sputtered secondary ions are extracted, mass separated, and detected. 

Supercritical fluid extraction (SFE) has been extensively used for the extraction of volatile components such as essential oils, flavours and aromas from plant materials on an industrial as well as an analytical scale (61). The extract thus obtained is usually analysed by GC. Off-line SFE-GC is frequently employed, but on-line SEE-GC has also been used. The direct coupling of SEE with supercritical fluid chromatography (SEC) has also been successfully caried out. Coupling SEE with SEC provides several advantages for the separation and detection of organic substances low temperatures can be used for both SEE and SEC, so they are well suited for the analysis of natural materials that contain compounds which are temperature-sensitive, such as flavours and fragrances. 

Post-column on-line derivatisation is carried out in a special reactor situated between the column and detector. A feature of this technique is that the derivatisation reaction need not go to completion provided it can be made reproducible. The reaction, however, needs to be fairly rapid at moderate temperatures and there should be no detector response to any excess reagent present. Clearly an advantage of post-column derivatisation is that ideally the separation and detection processes can be optimised separately. A problem which may arise, however, is that the most suitable eluant for the chromatographic separation rarely provides an ideal reaction medium for derivatisation this is particularly true for electrochemical detectors which operate correctly only within a limited range of pH, ionic strength and aqueous solvent composition. 

For single-carbon-number AOS samples, analyses can be performed satisfactorily using just the GC method. For multicarbon-number AOS samples that have high sultone content (about 50 ppm), the LC method provides adequate resolution and sensitivity. However, for multicarbon-number AOS samples containing normal sultone levels and for AES samples, the combined LC-GC method is necessary to obtain the required separation and detection levels. Additionally, the combined method is advantageous in eliminating interferences in the LC method that are sometimes observed with AOS samples that have been bleached. 

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. 

HPLC has been recommended as a cleanup and fiactionation procedure for food samples prior to analysis by GC/ECD (Gillespie and Walters 1986). The advantages over the AOAC-recommended Florisil colunrn are that it is faster, requires less solvent, and gives better resolution. HPLC coupled with various detectors MS, MS/MS, UV/electrochemical detector, or UV/polarographic detection has been tested as a rapid, simplified separation and detection system to replace GC (Betowski and Jones 1988 Clark et al. 1985 Koen and Huber 1970). Recoveries, detection limits, and precisions were generally good, but further work is needed before the techniques are adopted for general use. 

However, compared with the traditional analytical methods, the adoption of chromatographic methods represented a signihcant improvement in pharmaceutical analysis. This was because chromatographic methods had the advantages of method specihcity, the ability to separate and detect low-level impurities. Specihcity is especially important for methods intended for early-phase drug development when the chemical and physical properties of the active pharmaceutical ingredient (API) are not fully understood and the synthetic processes are not fully developed. Therefore the assurance of safety in clinical trials of an API relies heavily on the ability of analytical methods to detect and quantitate unknown impurities that may pose safety concerns. This task was not easily performed or simply could not be carried out by classic wet chemistry methods. Therefore, slowly, HPLC and GC established their places as the mainstream analytical methods in pharmaceutical analysis. 

The use of high performance liquid chromatography (HPLC) for the study of paralytic shellfish poisoning (PSP) has facilitated a greater understanding of the biochemistry and chemistry of the toxins involved. HPLC enables the determination of the type and quantity of the PSP toxins present in biological samples. An overview of the HPLC method is presented that outlines the conditions for both separation and detection of the PSP toxins. Examples of the use of the HPLC method in toxin research are reviewed, including its use in the determination of the enzymatic conversion of the toxins and studies on the movement of the toxins up the marine food chain. 

DALLUGE J J, NELSON B c, THOMAS J B, WELCH M J and SANDER L c (1997) Capillary liquid chromatography/electrospray mass spectrometry for the separation and detection of catechins in green tea and human plasma , Rapid Commun Mass Spectrom, 11, 1753-6. 

Separation and detection methods There exists an array of methods for the separation of the Sb species, ranging from liquid-liquid extraction to the formation of complexes with immobilized reagents, followed by HPLC or reversed phase chromatography, and eletroanalytical methods. Detection is based on spectrophotometric measurement, AAS, ICP-OES and ICP-MS. 

Separation and detection methods Ion chromatography is routinely used for the isolation of bromate. Diverse detection methods are mentioned in the literature, including isotope dilution analysis (Creed and Brockhoff 1999), conductivity measurement (Jackson et al. 1998), fluorimetric determination (Gahr et al. 1998), ICP-MS (Seubert and Nowak 1998) and spectrophotometry (Achilii and Romele 1999),... 

Separation and detection methods The common methods used to separate the Cr(III)/(VI) species are solvent extraction, chromatography and coprecipitation. In case of Cr(VI) from welding fumes trapped on a filter, a suitable leaching of the Cr(VI) from the sample matrix is needed, without reducing the Cr(VI) species. The most used detection methods for chromium are graphite furnace AAS, chemiluminescence, electrochemical methods, ICP-MS, thermal ionization isotope dilution mass spectrometry and spectrophotometry (Vercoutere and Cornelis 1995)- The separation of the two species is the most delicate part of the procedure. 

Separation and detection methods Very interesting hyphenated techniques have been developed for the separation and measurement of organic and inorganic lead spe-... 

Separation and detection methods A survey on determination of tin species in environmental samples has been published by Leroy et al. (1998). A more detailed overview of GS-MS methodology has been published by Morabito et al. 1995) and on sample preparation using supercritical fluid extraction has been described by Bayona (1995)- The techniques are now under control, so that routine procedures are available at a relatively low cost (Leroy et al. 1998). 

Separation and detection methods Very refined chromatographic and electrophoretic separation techniques have been developed for metallothioneins. The detection is commonly based on the retention time and UV detection. Other researchers measured the element with e.g. ICP-MS to quantify the compotmd. Combination with electrospray MS-MS leads to the unequivocal identification of the species. 

Validation of the complete analytical procedure (induding solvent extraction, cleanup of the extracts, isolation of the analytes of interest, and chromatographic separation and detection) requires the use of CRMs with matrices similar to those... 

The most significant differences (i.e. independence) in the analytical methods are provided in the final chromatographic separation and detection step using GC/ MS and LC-FL. GC and reversed-phase LG provide significantly different separation mechanisms for PAHs and thus provide the independence required in the separation. The use of mass spectrometry (MS) for the GC detection and fluorescence spectroscopy for the LG detection provide further independence in the methods, e.g. MS can not differentiate among PAH isomers whereas fluorescence spectroscopy often can. For the GC/MS analyses the 5% phenyl methylpolysiloxane phase has been a commonly used phase for the separation of PAHs however, several important PAH isomers are not completely resolved on this phase, i.e. chrysene and triphenylene, benzo[b]fluoranthene and benzofjjfluoranthene, and diben-z[o,h]anthracene and dibenz[a,c]anthracene. To achieve separation of these isomers, GC/MS analyses were also performed using two other phases with different selectivity, a 50% phenyl methylpolysiloxane phase and a smectic liquid crystalline phase. 

Table 2 Survey results of separation and detection methods used in tissue residue analysis...

It is often difficult to define where sample extraction ends and cleanup procedures begin. Sample extracts may be injected directly into a gas or liquid chromatograph in certain cases, but this will be dependent on the analyte, sample matrix, injection, separation and detection system, and the limit of determination (LOD) which is required. It is also more likely that matrix-matched calibration standards will be needed in order to obtain robust quantitative data if no cleanup steps are employed. 

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