Analysis of drug residues

Table 20.2 Applications of SPE Cleanup in the Analysis of Drug Residues in Edible Animal Products... 

The limit of quantification is more relevant than the limit of detection in the analysis of drug residues in foods. In these applications, the limit of quantification can be more practically defined as the lowest drug concentration in food samples that can be measured with a desired level of accuracy and precision. It is usually determined by reducing the analyte concentration until a level is reached where the precision of the assay becomes unacceptable. If the required precision of the method at the limit of quantification has been specified, a number of samples with decreasing amounts of the analyte are analyzed 6 times at minimum, and the calculated RSD% of the precision is plotted against the analyte amount the amount that corresponds to the previously defined required precision is equal to the limit of quantification. 

Analysis of drug residues in foods is a challenging task and numerous methods have been developed for the direct screening of meat, milk, eggs, and honey. The analyst has a wide range of extraction, enrichment, and instrumental techniques to choose from. There is no best method, and the analyst s choice will depend on the nature of the sample matrix, whether it is solid or liquid, fatty or nonfatty, and the expected range and levels of the analytes. The instruments available for the confirmation and quantification of the individual residues will also influence the choice of enrichment and quantification method. 

E. Davoli, R. Fanelli and R. Bagnati, Purification and analysis of drug residues in urine samples by on-line immunoaffinity chromatography/high-performance liquid chro-matography/continuos-flow fast atom bombardment mass spectrometry , Anal. Chem. 65 2679-2685 (1993). 

Ramsey, E.D., Perkins, J.R., Games, D.E., and Startin, J.R. 1989. Analysis of drug residues in tissue by combined supercritical-fluid extraction-supercritical-fluid chromatography-mass spectrometry-mass spectrometry. Journal of Chromatography, 464 353-64. 

Turnipseed SB, Analysis of drug residues in food, in Hui YH, Bruinsma BE, Richard Gorham J, et al., eds.. Food Plant Sanitation. Food Science and Technology, CRC Press, Boca Raton, PL, 2003. 

Regulatory requirements in the field of drug residues analysis are limited, in most cases, to the identification of only the major metabolites. Quantitative selection of major over minor metabolites is certainly devoid of rational biological ground, since several studies have shown that the toxic metabolites are usually transitory and often present in small quantities. However, isolation and identification of all these metabolites are difficult and proper assessment of the toxicity of drug residues is still a real challenge. 

Validation attempts in tlie field of drug residue analysis have demonstrated that die requirement for a full collaborative trial at the ideal level, while desirable, is sometimes impractical. Limiting factors for completing ideal multilaboratory validation studies are usually the high cost, lack of sufficient expert laboratories willing to participate in such studies, and overall time constraints. Hence, a three-laboratory validation study is often applied (16). 

Sample preparation procedures commonly used in the field of drug residues analysis are briefly described below. Since some of these overlap and cannot be strictly separated, the intent of the authors is to attempt to outline the complexity of die sample preparation issue rather than to give a comprehensive listing of the relevant literature. 

Selectivity in lAC depends on the specificity of the immobilized antibody and, thus, monoclonal antibodies are preferentially used. In that case, a large amount of sample can be subjected to immunoaffinity cleanup without any retention of matrix components. This opens the possibility to determine very low concentrations of drug residues in edible animal products. For example, 20 ng chloramphenicol in 1 L milk can be determined with a recovery of 99% when 1 L of defatted milk is submitted to immunoaffinity cleanup. The chromatograms obtained after LC analysis were as clean as those obtained when 10 ml milk containing the same amount of chloramphenicol was also submitted to immunoaffinity cleanup (170). 

Enzymatic derivatization offers major advantages over alternative procedures. The selectivity of an enzyme for its substrate along with the sensitivity offered by modern analytical instrumentation makes enzymatic derivatization a powerful tool in the field of drug residue analysis. 

Enzymes can be used in several ways in chromatographic applications to improve selectivity or to enhance the detector response. Applications may involve enzymes with either a broad specificity toward a group of related compounds or a high specificity toward a particular compound. In the field of drug residue analysis, most current applications concern enzymatic reactions taking place in separate reactors incorporated in LC systems before or after the analytical column. Reactors with immobilized enzymes have proven to be suitable in such continuous flow systems. 

When a PTV instead of a classic injector was utilized in the analysis of penicillin residues, the sensitivity and the precision of the analysis were markedly improved (45). With the cooled PTV injector, some microliters could be injected, and the split-splitless mode allowed solvent venting at low injector temperatures with open slit in a first step, and quantitative transfer of volatile or derivatized drugs by a freely selected linear heat-up rate between 2-12 C/s in the splitless mode in the second step. Sensitivity could be enhanced by multiple injections before heat-up. Nonvolatile components of a sample did not contaminate the chromatographic system, since they accumulated in the glass vaporization tube, which could be changed easily. 

Validation procedures and key analytical parameters usually examined in common validation practice for both inhouse-developed and standard methods in the field of drug residue analysis are briefly discussed below. 

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