The use of antibiotics in livestock farming has always been controversial; in the EU the levels of antibiotic residues in food for human consumption is under strict regulatory control. This article describes the various methods used for the analysis of antibiotic residues in food of animal origin, the validation of such methods and the principal issues in the quality control of the resulting analytical data.
Since the 1950s antimicrobial agents have been used in livestock farming as prophylactic and therapeutic agents and as feed additives for growth promotion. The addition of sub-therapeutic amounts of antimicrobials to feedstuff in order to enhance its efficiency has always been controversial, particularly since the mechanisms by which the use of antimicrobials actually leads to growth promotion still remain incompletely understood .
In the light of this, it is not surprising that over the last twenty years a vigorous debate has taken place regarding the use of antibiotics in food-producing animals. The main issue is whether the resistance generated in animals to antimicrobials of human importance can spread to humans . Reflecting this concern, all growth-promoting antibiotics were banned in Sweden in 1986 and in Denmark in 1998. In an application of the "Precautionary Principle", the EU started banning the glycopeptide antibiotic avoparcin in 1997. Regulation (EC) 1831/2003 prohibited the use of all antibiotics other than coccidiostats and histomonostats as feed additives from 1st January 2006 . However, on the other hand antimicrobial growth promoters continue to be authorised in USA under FDA regulation and control on a case-by-case basis .
The use of antibiotics in animal farming means that there is a risk of residues of the antibiotic occurring in the final food product. To protect the consumer from this risk, European regulatory authorities have introduced several legislative initiatives such as the establishment of maximum residue limits (MRL) and the development of other controls on food, be it produced in the internal market or imported from countries outside the EU . Such regulatory stipulations mean that analytical chemists are being continually required to make a large effort to develop and validate new methods that can satisfy the requirements of the food safety authorities.
In this paper, we briefly review the process of the validation of the methods used for the analysis of antibiotic residues in food of animal origin within the framework of the regulations established by the EU and the Codex Alimentarius. Some significant issues related to regulatory or organisational aspects of the quality control of residues of veterinary drugs in food are also addressed.
EU food safety policy has resulted in great pressure being placed on official laboratories, since they have to assay a large number of analytes in a huge number of samples (animal tissue, milk, eggs, honey, etc.). As a first step, laboratories usually employ screening methods that cover whole families of compounds. Such screening methods should have a low rate of false non-compliant samples and also be capable of being carried out at high throughput. Cost is however also an important issue. For screening antibiotics, the principal methods used are microbiological assays, ELISA, biosensors and liquid chromatography (LC). Samples found to be non-compliant by these methods must then be analysed by confirmatory methods, which provide information that allows the unequivocal identification of the analytes and their quantification. How this is done depends on whether the analyte is classified— as defined by EU Directive 96/23 — as a group A compound (i.e. banned) or group B compound (i.e. one with an MRL) . Application of the EU decision 2002/657/EC means that three identification points (IP) are required to confirm the identity of a group B compound whereas four IPs are needed for group A substances . The number of IPs that can be obtained for an individual compound depends on the technique used for its analysis; in practice, MS detection is almost mandatory to get the number of IPs required by the regulations. Thus, confirmatory methods in the analysis of antibiotic residues are mainly based on LC with MS detection, with triple quadrupole MS instruments currently being the most commonly used [7, 8]. The performance of the new generation of high resolution MS detectors makes them very attractive for residue analysis in food, but their high price is still an obstacle to their widespread use in control laboratories. As for the chromatographic system, the new small particle size LC columns used in ultra-high pressure liquid chromatography allow short run times and high throughputs. Methods using capillary electrophoresis with MS detection are another alternative, but are much less common because of lower sensitivity.
In both screening and confirmatory testing, the analytes must usually first be extracted from the sample matrix and frequently the extracts themselves then need a clean-up step . A wide range of organic solvents or hydro-organic mixtures are generally used for extracting the analytes from the sample and ultrasound-assisted extraction, microwave-assisted extraction or pressurised liquid extraction are also sometimes employed to reduce extraction time or improve recoveries. Solid-phase extraction is the most common approach for clean-up, with conventional silica or polymer-based sorbents being most often used, Solid phases using restricted access materials, highly specific immunosorbents or molecularly imprinted polymers are also employed.
Multiclass and multimatrix methods that are suitable for a large number of analytes belonging to different chemical classes of antibiotics in different sample matrices are particularly useful for optimising laboratory resources and increasing efficicency, but it is important that such methods used have good recoveries for the whole set of analytes and matrices.
The EU Commission Decision 2002/657/EC is a key document describing the requirements for separation and detection techniques and validation of the analytical methods used for veterinary drug residues . Whether the method is confirmatory or screening determines the analytical parameters to be evaluated. For quantitative confirmatory methods, in addition to the criteria for identification, validation requirements also include specificity, linear range, precision (repeatability and within-laboratory reproducibility), trueness, recovery, decision limit (CC)
Recovery is the mass of analyte in the sample which is present in the final extract of the sample and so is a measure of the efficiency of the sample treatment process. In quantitative residue analysis, recoveries must be used to correct the results, except when the calibration method already provides recovery-corrected data. Although the best approach to determine recoveries is via the analysis of a certified reference material (CRM), the scarcity of suitable CRMs has led to the use of spiked samples, under the assumption that the added analyte behaves in the same way as the analyte originally present in the sample (incurred analyte). It should be noted that the term recovery is often improperly or wrongly used — to avoid any confusion, some authors use the terms absolute recovery or extraction recovery instead.
The two components of accuracy, namely trueness and precision, are important parameters for quantitative methods. Decision 657 from the EU specifies the minimum requirements of precision and trueness that quantitative methods should meet but the criterion for precision is quite broad. Trueness should be established by means of a certified reference material CRM, but if no CRMs are available it can be calculated from the recovery of spiked blank matrices. In both cases the data — duly corrected for the mean recovery— must fall within the established range. Trueness is often used in the literature to refer to the agreement between the true value and the mean result, but some authors use recovery or corrected recovery instead of trueness. Whereas the term "corrected recovery" is quite clear (especially if it is properly explained), the term recovery on its own is often ambiguous, being sometimes used for results that actually correspond to recovery-corrected data.
Specificity is an important parameter for confirmatory methods; there are two different approaches to establish specificity. In the first one, representative blank samples are analysed to detect any signal from endogenous substances and matrix constituents. In the second approach, the effect of other constituents likely to be present in the matrices of interest is evaluated. Since in practice the number of substances that can be tested is quite limited, it is usually only substances that are closely related to the analytes, such as metabolites, that are checked. Specificity is highly dependent on the measuring technique. Whereas the risk of interferences in LC-MS/MS methods is quite low, other analytical techniques would require more detailed study. Although ruggedness is a validation parameter included in Decision 657, it is seldom reported in the literature.
a), detection capability (CCb) and ruggedness. In contrast, for screening methods the most important parameter is CCb. a and CCb are the two analytical limits that replace the limits of detection and quantification, respectively. The decision limit, CCa is defined as being the limit at and above which it can be concluded with an error probability of a (usually 5%) that a sample is non-compliant, and is a crucial parameter for confirmatory methods. The detection capability, CCb is defined as the smallest content of the substance that can be detected and/or quantified, with an error probability of b in a sample. b is 1% for banned substances and 5% for group B substances. For authorised substances, the level of interest is the MRL and so CCa is calculated from the results of analysis of blank samples spiked with the analytes at their MRL (concentration and standard deviation). CCb is calculated from the CCa value and the standard deviation at this concentration level. For substances with no established MRL, the so-called zero tolerance levels should be applied, which can cause some ambiguity from a practical viewpoint. The use of different methodological approaches may result in different decision limits CCa and detection capability CCb.
In order to increase the reliability of results, all laboratories involved in the analysis of official food samples should establish, implement and maintain a management system that fulfils the requirements of the ISO/IEC 17025:2005 international standard . Among the quality control activities cited in this standard, the most important are the use of reference materials (RM) and regular participation in proficiency testing programmes.
Matrix reference materials (spiked or incurred materials that contain the analytes of interest in natural matrices) are the best option of the different types of reference materials because they allow the assessment of the whole analytical process, from extraction to the final measurement step, with samples that are similar to those to be analysed in routine. The main drawbacks of such matrix reference materials are the difficulties of their preparation, preservation and characterisation, which explains their scarcity. The international data base COMAR  provides free of charge information on the availability and characteristics of CRM for use in several fields of application. The three major European producers of RMs, namely the Institute for Reference Materials and Measurements (IRMM), the Bundesanstalt für Materialforschung und -prüfung and the Laboratory of the Government Chemist (LGC), have created a consortium to produce a new brand of RM called European Reference Materials (ERM) . About a dozen certified reference materials related to the analysis of antimicrobial agents in food can be found in the ERM catalogue.
Inter-lab proficiency testing
Regular participation in interlaboratory comparisons or in proficiency tests (PT) is a stringent requirement of all laboratory accreditation schemes with the result that over the last decade the number of providers of proficiency test schemes has increased significantly, although the absolute number of accredited PT providers is still small. Proficiency testing is particularly difficult in the field of veterinary drug analysis, mainly because of the problems caused by the complexity and limited stability of the samples and analytes of interest. The European Proficiency Testing Information System (EPTIS)  is a useful tool for getting information on the available proficiency testing schemes that are regularly carried out in several fields of testing.
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Prof R. Companyó, Dr M. Granados,
Dr J. Guiteras and Dr M.D. Prat
Department of Analytical Chemistry.
Universitat de Barcelona.
Martí I Franquès 1-11.
08028 Barcelona, Spain
The current article is a summary version of the more complete article published by the authors: "Antibiotics in food: Legislation and validation of analytical methodology" Anal Bioanal Chem 2009 July 28th DOI 10.1007/s00216-009-2969-4.
by Prof R. Companyó, Dr M. Granados, Dr J. Guiteras and Dr M.D. Prat