Veterinary drug residues analytical methods

Chemical analytical methods used in veterinary drug residue depletion studies in target animals constitute a potential source of suitable methods for determining compliance of tissue residues with established MRLs. In some situations. 

National and regional authorities responsible for the protection of public health must consider the concentration of residues of veterinary drug residues, pesticides, and other chemicals that may be in food regardless of whether the substance is allowed for that use. In many regions, in the absence of an approval for the substance, the concentration of residues allowed in food is considered to be zero. In practical terms, this is frequently defined by the technical capability of the analytical method. Attempts to improve on zero include the ALARA (as low as reasonably achievable) approach, which recognizes that absolute zero is unattainable, and describes an approach that considers what is technically achievable, the resources needed to achieve that technical goal, and the benefit gained. 

Any other factors that may define the analytical requirements should also be considered. For example, when dealing with veterinary drug residue analysis, while the target tissue for domestically produced animals may be liver or kidney, these organ tissues are less commonly available as imported products. The majority of imported meat products are muscle tissue. Therefore, although the method has been validated for analysis of kidney for domestic samples, it is not fit for purpose for use on most import samples until it has also been validated for muscle tissue and possibly even some processed meat products. A method validated for the analysis of an aquaculture drug or natural toxin in oysters from domestic production may also, for example, require validation for shrimp or tilapia for application to imports. In addition, the requirement may include development of a... 

The definition of calibration function does not specify that the measurement be made in the presence of potential interferants. This serves as an introduction to a discussion of the appropriate approach to calibration in an analytical method for veterinary drug residues, such as antibiotics. Construction of a calibration curve requires a sufficient number of standard solutions to define the response in relation to concentration, where the number of standard solutions used is a function of the concentration range. In most cases, a minimum of five concentrations (plus a blank, or zero ) is considered appropriate for characterization of the calibration curve during method validation. It is also typically recommended that the curve be statistically tested and expressed, usually through linear regression analysis. However, for LC/ESI-MS analysis of residues, the function tends to be quadratic. The analytical range for the analysis is usually defined by the minimum and maximum concentrations used in establishing the calibration curve. 

Decision 2002/657/EC and CAC/GL 71-200932 contain additional performance requirements for mass spectral methods used in the confirmation of veterinary drug residues in foods. It should be noted that the performance specifications given for GC-MS methods using El spectra are more stringent than for other techniques, including GC-MS with Cl, LC-MS, and LC-MS/MS. It is also recommended that only ions with an intensity >10% of the base peak should be used as analytical peaks for confirmation. The EU and CAC requirements address performance of both the chromatographic system and the mass spectrometer ... 

Obvious questions are how many replicates should be included in the design and also how many analytical runs should be completed. Eurachem guidance suggests a minimum of 10 replicates for recovery (accuracy) and precision. A collaborative smdy design is recommended to include a minimum of five materials (matrices) at three concentrations, in blind duplicate, which means that each participating laboratory produces 10 results for each concentration. However, when one considers the recommendation that at least six different sources of matrix should be used in validation for methods for veterinary drug residues in foods and that the validation should include analyses conducted on multiple days, with inclusion of other variables, such as analyst, equipment, and reagents, it is obvious that 10 replicates will prove insufficient to provide the necessary data for a suitable assessment of... 

The use of pre-extraction spiking is particularly important when the presence of matrix co-extractives modifies the response of the analyte as compared with analytical standards. It is increasingly common in methods for veterinary drug residues in foods to base the quantitative determination on a standard curve prepared by addition of standard to known blank representative matrix material at a range of appropriate concentrations that bracket the target value (the analytical function). Use of such a tissue standard curve for calibration incorporates a recovery correction into the analytical results obtained. 

It can be assumed that with the development and study of new methods, the ability to determine M (S), the method bias component of uncertainty, cannot be done given that it can be evaluated only relative to a true measure of analyte concentration. This can be achieved by analysis of a certified reference material, which is usually uncommon, or by comparison to a well-characterized/accepted method, which is unlikely to exist for veterinary drug residues of recent interest. Given that method bias is typically corrected using matrix-matched calibration standards, internal standard or recovery spikes, it is considered that the use of these approaches provides correction for the systematic component of method bias. The random error would be considered part of the interlaboratory derived components of uncertainty. 

In many cases, maximrun residue limits (MRLs) exist for veterinary drugs and the analytical methods must be capable of measming below such MRLs. Since MRLs vary considerably, the sensitivity requirements of the various methods also vary widely. Typically, MRLs for veterinary drug residues vary from Imgkg (ppm) to Ipgkg (ppb) (Table 1) or, in the case of banned substances, to no detectable residue . Where no detectable residue is specified, it is common to declare an action limit or a limit of decision for use by regulatory agencies this is not strictly scientifically based and takes into account other aspects such as the sensitivity of a suitable confirmatory technique and policy requirements. 

Another source of concern in food safety control is the increasing amounts of residues of veterinary drugs that can be found in foods. These drugs are mostly antibiotics of different structures, such as tetracyclines, macrolides, quinolones, sulphona-mides, and 6-lactams. The preferred analytical technique for antibiotic residue determination is HPLC, and specific methods for each family of antibiotics can be fovmd in the scientific literature. Most of the published methods use RP columns with gradient elution. Every available detection method can be used, depending on the application, though MS is a powerful technique for the identification and confirmation of veterinary drug residues in food samples, and at the moment, LC-MS is the method of reference. 

An alternative interesting classification approach has been proposed within the Codex Committee for Residues of Veterinary Drugs in Foods (20). In this approach, methods are classified according to their performance attributes. This alternative approach defines methods by the level of analytical detail or information provided concerning the amount and nature of the analyte of interest, and identifies three levels. 

Quality criteria for quantitative analytical methods, in general, have been proposed or arc lo be proposed by several international organizations including the Association of Official Analytical Chemists, the Food and Drug Administration, the Codex Committee for Residues of Veterinary Drugs in Food, the International Dairy Federation, and the European Union. The European Union, in particular, has laid down minimum quality criteria for quantitative drug residue methods,... 

To assist the implementation of these Directives in the EU, the European Commission has defined and published the minimum requirements for analytical methods to confirm the presence of residues of veterinary drugs together with guidelines for screening methods (Heitzman R. J. 1994). The requirements for these methods are rigorous and ensure that the Directives can be effectively implemented. 

BenzylpenicUlin (PCG), phenoxymethylpeniciUin (PCV), oxacillin (MPIPC), cloxaciUin (MCIPC), nafcillin (NFPC), and dicloxacillin (MDIPC), aU of which are representative weakly acidic penicUlins, are widely used as veterinary drugs for livestock. We have reported, in our previous studies, the applicability of sample cleanup with an ion-exchange cartridge, in combination with ion-pair HPLC, for the analysis of ionizable compounds. We therefore applied the same technique to develop an analytical method for the quantitative determination of these residual penicillins in bovine muscle, kidney, and liver. ... 

In summary, when an ADI is established, consideration of the estimated intakes of the relevant foods by human beings allows an assessment to be made of a safe and acceptable residue level for the relevant animal tissue(s). If the levels of residues estimated from supervised trials, when the drug is administered according to good practices in the use of veterinary drugs (only the amount which is necessary to obtain the desired effect is used), are below those considered toxicologically acceptable, then the levels determined by good practice will dictate the acceptable residue level, provided that practical analytical methods are... 

A 2008 paper has described for the first time a dilute and shoot strategy for the simultaneous extraction of wide variety of residues and contaminants (pesticides, myco-toxins, plant toxins, and veterinary drugs) from different foods (meat, milk, honey, and eggs) and feed matrices. Several antimicrobial classes were included (sulfonamides, quinolones, P-lactams, macrolides, ionophores, tetracyclines, and nitroimidazoles) in the analytical method. Sample extraction was performed with water/acetonitrile or acetone/1% formic acid, but instead of dilution of the extracts before analysis by UPLC-MS/MS, small extract volumes (typically 5 til) were injected to minimize matrix effects. Despite the absence of clean-up steps and the inherent complexity of the different sample matrices, adequate recoveries were obtained for the majority of the ana-lyte/matrix combinations (typical values for antimicrobials were in the range of 70-120%). In addition, the use of UPLC allows high-speed analysis, since all analytes eluted within 9 min. 

Stolker et al. " described an analytical method based on TFC-LC-MS/MS for the direct analysis of 11 veterinary drugs (belonging to seven different classes) in milk. The method was applied to a series of raw milk samples, and the analysis was carried out for albendazole, difloxacin, tetracycline, oxytetracycline, phenylbutazone, salinomycin-Na, spiramycin, and sulfamethazine in milk samples with various fat contents. Even without internal standards, results proved to be linear and quantitative in the concentration range of 50-500 (xg/1, as well as repeatable (RSD<14% sulfamethazine and difloxacin <20%). The limits of detection were between 0.1 and 5.2 xg/l, far below the maximum residue limits for milk set by the EU. While matrix effects, namely, ion suppression or enhancement, were observed for all the analytes, the method proved to be useful for screening purposes because of its detection limits, linearity, and repeatability. A set of blank and fortified raw milk samples was analyzed and no false-positive or falsenegative results were obtained. 

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