Marker residue

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. 

Accuracy (systematic error or bias) expresses the closeness of the measured value to the true or actual value. Accuracy is usually expressed as the percentage recovery of added analyte. Acceptable average analyte recovery for determinative procedures is 80-110% for a tolerance of > 100 p-g kg and 60-110% is acceptable for a tolerance of < 100 p-g kg Correction factors are not allowed. Methods utilizing internal standards may have lower analyte absolute recovery values. Internal standard suitability needs to be verified by showing that the extraction efficiencies and response factors of the internal standard are similar to those of the analyte over the entire concentration range. The analyst should be aware that in residue analysis the recovery of the fortified marker residue from the control matrix might not be similar to the recovery from an incurred marker residue. 

For the confirmatory procedure, it is recommended that the sponsor develop spectral data based on at least three structurally specific ions that completely define the marker residue molecule. These ions may or may not include the molecular ion. The use of water loss and isotopic ions is usually unacceptable and CVM concurrence should be sought when water loss ions or isotopic ions are selected for the confirmatory analysis. The proposed fragment ion structures should be consistent with the fragmentation pattern, and justification for specificity of selected ions or scan range should be included. All confirmation criteria should be specified in the standard operating procedure. 

Structure and full spectrum of the marker residue should be included. 

The final difference is that the FDA analyst alone makes the recommendation based on the data for the acceptance of the confirmatory procedure. The conclusion of the analyst stating the suitability of the procedure for confirming the presence of the marker residue is sent directly to the CVM method trial coordinator in the Office of New Animal Drug Evaluation (ONADE) and not back to the sponsor as with the determinative procedure. 

For confirmatory methods, the confirmatory procedure criteria described previously should be met. All negative control samples should fail to meet the confirmation standard established in the procedure. All samples fortified at or above the tolerance and all incurred residue samples at or above the tolerance should meet the confirmation standard (to confirm) described in the SOP. It has been argued that it is not necessary for incurred samples containing the marker residue at a concentration below the tolerance to meet established confirmatory criteria. However, failure to confirm the marker residue in these samples may indicate a lack of robusmess of the procedure. Any procedure that had this problem would be closely examined to ensure that the method would meet the needs of the Agency. 

The most critical decision to be made is the choice of the best solvent to facilitate extraction of the drug residue while minimizing interference. A review of available solubility, logP, and pK /pKb data for the marker residue can become an important first step in the selection of the best extraction solvents to try. A selected list of solvents from the literature methods include individual solvents (n-hexane, " dichloromethane, ethyl acetate, acetone, acetonitrile, methanol, and water ) mixtures of solvents (dichloromethane-methanol-acetic acid, isooctane-ethyl acetate, methanol-water, and acetonitrile-water ), and aqueous buffer solutions (phosphate and sodium sulfate ). Hexane is a very nonpolar solvent and could be chosen as an extraction solvent if the analyte is also very nonpolar. For example, Serrano et al used n-hexane to extract the very nonpolar polychlorinated biphenyls (PCBs) from fat, liver, and kidney of whale. One advantage of using n-hexane as an extraction solvent for fat tissue is that the fat itself will be completely dissolved, but this will necessitate an additional cleanup step to remove the substantial fat matrix. The choice of chlorinated hydrocarbons such as methylene chloride, chloroform, and carbon tetrachloride should be avoided owing to safety and environmental concerns with these solvents. Diethyl ether and ethyl acetate are other relatively nonpolar solvents that are appropriate for extraction of nonpolar analytes. Diethyl ether or ethyl acetate may also be combined with hexane (or other hydrocarbon solvent) to create an extraction solvent that has a polarity intermediate between the two solvents. For example, Gerhardt et a/. used a combination of isooctane and ethyl acetate for the extraction of several ionophores from various animal tissues. 

Several extraction techniques have also been described that use enzymatic or chemical reactions to improve extraction efficiency. A technique that has been used to increase the overall recovery of the marker residue is enzymatic hydrolysis to convert specific phase II metabolites (glucuronides or sulfates) back into the parent residue. Cooper etal used a glucuronidase to increase 10-fold the concentration of chloramphenicol residues in incurred tissue. As an example of a chemical reaction, Moghaddam et al. used Raney nickel to reduce thioether bonds between benomyl and polar cellular components, and as a result achieved a substantially improved recovery over conventional solvent extraction. In choosing to use either of these approaches, thorough characterization of the metabolism in the tissue sample must be available. 

When selecting the most appropriate SPE cartridge to use, several factors may influence the decision. One consideration is the chemical properties of the marker residue such as polarity and Polar analytes will be retained to a greater ex-... 

Another subset of SPE is immunoaffinity extraction, in which an antibody specific to the analyte is incorporated into the SPE sorbent. This technique is very selective to the analyte and would be very effective in separating the marker residue from tissue-related matrix components. Disadvantages of immunoaffinity extraction are the need to develop a specific antibody-based SPE for each analyte. This approach holds promise for the future as the development of antibody-based methods becomes more commonplace. 

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. 

Figure 1. Typical depletion curve for total residues and a marker residue in an edible tissue.

Residue depletion studies in chickens and turkeys orally dosed with 10 mg enrofloxacin/kg bw for 7 days showed that the sum of enrofloxacin and ciprofloxacin, which has been designated as the market residue for regulatory purposes, in the chicken liver declined from 42 ppb at 3 day withdrawal to 11 ppb at 15 day withdrawal in turkeys, the level of the marker residue in liver... 

When cattle were subcutaneously injected with a single dose of 7.5 mg enrofloxacin/kg bw, the levels of the marker residue in liver, kidney, muscle, and fat fell rapidly from about 30 ppb, 20 ppb, less than 10 ppb, and less than 10 ppb, respectively, at day 3 after dosing to less than 10 ppb in all tissues at 7 day withdrawal. When enrofloxacin was injected intravenously to cows, ciprofloxacin could be detected at higher concentrations and for a longer period than the parent enrofloxacin (153). When enrofloxacin was administered parenterally to dairy cows at 5 mg/kg bw, the levels of the marker residue in milk remained above... 

The marker residue for triclabendazole is 5-chloro-6-(2, 3 -dichlorophe-noxy)-benzimidazole-2-one, a compound formed when common fragments of triclabendazole-related residues are hydrolyzed under alkaline conditions at 90-100 C. Since the marker residue does not represent total residues, the marker residue levels are converted to triclabendazole equivalents using a conversion factor of 1.09 (31). 

Residue depletion studies in cattle showed that liver, kidney, muscle, and fat contained 109, 103, 104, and 40 ppb of the marker residue at 28 day withdrawal. Residue depletion studies in sheep showed that liver, kidney, muscle, and fat contained 440, 260, 180, and 40 ppb of the marker residue at 10 day withdrawal. 

Residue depletion studies (1) using melengesfiol acetate as marker residue and fat as marker tissue have demonstrated that residues in fat remained well... 

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... 

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