Target Marker Residue

Free residues and conjugates can be easily extracted after dialysis, proteolysis, or denaturation of proteins by heat or acid treatments. In general, the bound residues are 

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NADA methods should be capable of reliably measuring an analyte (i.e., the marker residue) that has a defined quantifative relationship to the total residues of toxicological concern in the tissues of interest, namely the target tissue and muscle. The target tissue is generally the last tissue in which total residues deplete to the permitted maximum safe concentration. When the marker residue is at the tolerance, a defined unique concentration, the total residues have depleted to the respectively established safe concentrations in the target tissue and muscle. 

By definition, the determinative procedure must be able to quantify the concentration of the marker residue. For compounds with a tolerance, it is critical that the analysis be able to determine accurately if the concentration of the marker residue is above or below the tolerance in the target tissue. The CVM guidelines for determinative procedures call for an average recovery >80% with a coefficient of variation (CV) of <10% for marker residue tolerances of lOOpgkg or greater and an average recovery of >60% with a CV of <20% for marker residues with a tolerance below 100 ppb. 

Specificity is a measure of how selectively the analytical method measures the marker compound in the presence of other compounds. The descriptors used to establish specificity differ depending upon the guideline (see Table 3), but the purpose behind them is the same. In all cases, the method must be demonstrated to have no interference from several (at least five) confrol animals that represent variation in sex, age, and breed. Further, incurred residue samples or authentic metabolite standards must demonstrate no interference with the marker residue detection. The method must be tested with other approved dmgs for the target species to show that no interference exists if these compounds are also present. 

Antibiotics Marker residue Animal species MRL (ppm) EC US FDA Target tissues... 

To ensure compliance with the withdrawal period, an assay is needed to monitor total residues in the edible tissues. Because it is impractical to develop assays for each residue in each of the edible tissues, the concept of a marker residue and a target tissue is introduced. The marker residue is a selected analyte whose level in a particular tissue has a known relationship to the level of the total residue of toxicological concern in all edible tissues. Therefore, it can be taken as a measure of the total residue of interest in the target animal. The information obtained from studies of the depletion of the radiolabeled total residue can be used to calculate a level of the marker residue that must not be exceeded in a selected tissue (the target tissue) if the total residue of toxicological concern in the edible tissues of the target animal is not to exceed its safe concentration. 

Since it is difficult, in practical terms, for a monitoring plan to measure analytically a series of residues with widely differing chemical structures, control exigencies require that MRL values be expressed in terms of a single chemical entity, know as the marker residue. It is important that die contents of this marker residue evolve in the different tissues of treated animals in proportion to all targeted residues, if it is to reflect them. For obvious practical reasons, this marker residue must also satisfy two requisites it must permit a practical dosage and must be commercially or otherwise available for tire purposes of official controls. 

Having determined the target tissue, the parent drug and/or one or more of the metabolites in the target tissue are chosen to be the marker residue. The proportion of the marker residue to total residues is obtained at the point on the total residue depletion curve where this line crosses its permitted safe concentration. The level of the marker residue at that point represents the tolerance since it is specified in the Code of Federal Regulations, Title 21, Part 556. 

The objective of the Residue File is to allow the elaboration of MRLs taking into account the ADI calculated in the Safety File in conjunction with the pharmacokinetics, residues depletion data, and a knowledge of target tissues and marker residues. The individual MRLs in different tissues should be a function of the amount of the food items consumed, and should also reflect the kinetics of the depletion of the residues to be consistent with the established withdrawal periods. MRLs should be proposed in such a way that the total amount of residues ingested with 500 g meat or 500 g poultry or 300 g fish, plus 1500 g milk, plus 100 g egg, plus 20 g honey does not exceed the ADI. The EU uses the daily intake values presented in Table 11.5. After an MRL has been established for a... 

For residue monitoring purposes, it is frequently useful to define MRLs for a particular marker residue. A specific quantitative analytical method for measuring the concentration of the residue with the required sensitivity must be available. The MRL establishes the concentration of the marker residue permitted in the target tissue. Marker residue and target tissue are selected in such a way that total residues in each edible tissue are at or below its safe concentration if the marker residue is at or below the MRLs. For milk or eggs, it may be necessary to select a marker residue different from the marker residue selected for the target tissue representing the edible carcass. 

Compound(s) Marker residue Animal species Target tissues MRLs ( g/kg) Other provisions... 

The United States uses food consumption data and food factors in conjunction with the ADI to calculate the tolerance of residues in edible tissues. The calculation starts with an estimate of the safe concentration of the total drug residues by dividing the ADI by food factors that reflect the contribution of the edible tissues to the daily diet. Following analysis of the depletion of the total residues from the edible tissues, a target tissue is selected for residue monitoring. The residue whose concentration is in known relationship to the total residues in the target tissue is selected as the marker residue. The tolerance is the concentration of the marker residue in the target tissue, which ensures that the total residues in each edible tissue are below their safe concentration. 

Tolerance of the marker residue in the target tissue liver 36 ppb ... 

Fig. 2 Selection of a marker from among the individual residues (metabolites) within the target tissue. The Rra (tolerance) represents the concentration of the marker residue when the total residue achieves its safe concentration. (From Ref. , reprinted with permission from Dairy, Food and Environmental sanitation. Copyright held by the International Association for Food protection.)...

The holder of the generic drug product application uses the existing tolerance, marker residue, target... 

Radiolabeled residue depletion studies in target animals from zero withdrawal time to periods beyond the recommended withdrawal time (these studies should provide information on total residues, including free and bound residues, and major residue components in order to select a marker residue and target tissue). 

Unlabeled drug depletion studies for analysis of marker residue in target animals including muscle, liver, kidney, fat, eggs, milk, and honey as applicable (this should include studies with appropriate formulations, routes of application, and species using up to maximum recommended doses). 

A marker residue is a residue whose level decreases in a known relationship to the level of total residues in tissues, eggs, or milk. In other words, a marker residue is, or is representative of, the residue of toxicological concern in the target tissue and/or milk/ eggs. Identification of a marker residue is important because it is the substance determined for control purposes in the enforcement of MRLs by the national authorities and other parties concerned. 

Metabolism Studies. Having determined the likely choice for a target tissue, the sponsor examines the metabolite profrles during residue depletion to select a marker residue that may serve to monitor the total residue during residue depletion in the target tissue. Metabolic profiles should be examined in tissues other than the target tissue to determine that no additional metabolites are present. One of the residues (metabolite or parent compound) in the target tissue is chosen to be the marker residue and its... 

Analytical Methods for Residues. After the marker residue and target tissue have been identified and a tolerance level or Rm has been set, the sponsor develops determinative and confirmatory methods for the marker residue at the tolerance. The determinative method must be practical and rugged to be useful for routine surveillance monitoring of residues in meat in USDA field laboratories. The confirmatory method is one in which the marker residue is determined unequivocally so that the identity of an above tolerance residue can be supported in a court of law. Methods that are capable of this level of specificity often employ mass spectrometry in one form or another. 

Figure 1. Pharmacokinetic analysis of total residue and metabolite data in a target tissue to determine marker residue and its tolerance (Rm)- Reproduced with permission from Dairy Food and Environmental Sanitation 1992, 12(3), 144-148.

The safe concentration of drug-related residue must be known in order to determine the withdrawal period for a veterinary product. Often the toxicity data is incomplete and an estimate must be made to progress with requisite residue studies. One approach is to conduct a total residue study with sufficiently widely-spaced sacrifice intervals to assess the rate of depletion of total residue over the projected range of probable safe concentrations. A zero-withdrawal sacrifice interval should be included. The target tissue and marker residue are identified and surveillance/confirmatory assays developed. If a major portion of residue is non-extractable (bound) and the marker is undetectable at times when total residue is still significant, a residue bioavailability study may be necessary. To complete the data package, final residue and comparative metabolism studies are conducted. Studies on the metabolism of flunixin in cattle will illustrate this approach. 

Proper conduct of the total residue depletion study is important not only because the results define the depletion of total drug-related residue from the edible tissues of treated animals, but also because this data will be utilized to identify the target tissue (the edible tissue selected to monitor total residue - usually the last tissue in which residues deplete to the safe concentration) and marker residue (residue, i.e. drug and/or metabolite(s), selected to monitor the concentration of the total residue in the target tissue). These results will be utilized to establish the relationship between depletion of total residue and the marker residue in the target tissue. Identification of major metabolites in the urine and feces is important because these products must undergo environmental impact assessment. Tissue and excreta profiles will also be used... 

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