Enforcement methods

A special case that falls under the present definition of risk assessment methods is provided by the methods for analysis of dioxins and related compounds included in the calculation of toxic equivalent quantity (see Section 11.4.1). 

Result minus expanded uncertainty above limit 

Result above limit limit within expanded uncertainty interval 

Result plus expanded uncertainty below limit 

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The purpose of this article is to clarify the assessment of residue analytical methods in the context of Directive 91/414/EEC. After discussing the legal and historical background, requirements for enforcement methods as well as data generation methods are reviewed. Finally, an outlook over further developments in the assessment and validation of analytical methods is provided. 

Requirements for post-registration and monitoring (enforcement) methods... 

To avoid different interpretations, a list of analytical techniques, regarded as commonly available, is given in the guidance document SANCO/825/00. Other techniques may also be powerful tools in residue analysis the acceptance of these additional techniques as part of enforcement methods will be discussed at appropriate intervals by governmental experts. Therefore, whilst not wishing to prevent... 

The submitted enforcement method must be described in detail along with specifying equipment, materials and conditions. The following points must be... 

The extent of validation of confirmatory techniques is currently under consideration. Qne approach is that the extent of validation may be smaller than for the enforcement method. In principle, validation in triplicate at the relevant concentration level (LOQ or MRL) is sufficient. In the case where an MRL is set for multiple crops, a single validation in all representative crop groups is sufficient. A confirmatory method for residues in air is not required if a corresponding method was submitted for the other sample matrices. This approach is realized in Germany. ... 

The enforcement method must be suitable for the determination of all compounds included in the residue definition in order to enable Member States to determine compliance with MRLs. It is not feasible to validate a method for all commodities if a wide range of MRLs are set. Therefore, a concept of crop groups was developed in SANCO/825/00. The following crop groups with representative crops are presented ... 

The second requirement is that enforcement methods for food must be validated by an independent laboratory [independent laboratory validation (ILV)]. The sample set is identical with the general sample set (see Section 4.1). If the method is identical for all four crop groups (mentioned at the beginning of the section), it may be sufficient to perform the ILV for plant materials with a minimum of two matrices, one of them with a high water content. In the case of food of animal origin, the ILV should be performed with at least two of the matrices milk, egg, meat, and, if appropriate, fat. 

The prerequisite that the laboratory chosen to conduct the ILV trials must not be involved in the method development and/or in its subsequent use is not applicable for multi-methods. If the applicability of a multi-method is published in an official manual, an ILV is not obligatory for this particular a.i. ILV is always required for single methods. Communications between the chosen laboratory and the method developers must be reported, provided that these communications were required to carry out the analysis successfully. Also, any subsequent amendments or modifications to the original method must be reported. Furthermore, the ILV report must contain a statement as to the applicability of the method. In contrast, it is not necessary to confirm fhe resulfs of fhe enforcement methods for soil, water, body fluids, tissues, and air by an independent laboratory validation. 

From the analytical point of view there is no essential difference between drinking water and groundwater. Therefore, it is sufficient if the enforcement method is validated only for either drinking water or groundwater. The LOQ for drinking water/ groundwater must be <0.1 qgL (EU drinking water limit). 

The majority of validation data required for analytical methods supporting authorization purposes are common to those described for enforcement methods (see Section 4). However, some of the requirements such as minimum cost and commonly available equipment do not apply to methods supporting pre-registration studies (e.g., the use of GC/MS/MS technology). 

In contrast to the requirements for enforcement methods, validation of a previously collaboratively tested method, which is used to generate data, should be validated for new laboratory conditions. Also, where published methods are submitted, validation is required, when applied to the relevant sample matrix and laboratory conditions. 

Contrary to the enforcement methods, additional confirmatory analysis is not necessary where it is demonstrated that the primary residue method is specific to the analyte(s) and the source of the analyte(s) is known. 

The use of immunoassay methodology for residue trial analysis is in principle just as acceptable as for enforcement methods, provided that the method has been adequately validated. Because the validation of such methods requires a different approach, as opposed to chromatographic and spectrometric methods, some important points to be aware of in the use are explained in SANCO/3029/99. The authors do not go into detail on this subject here, since on the one hand very few methods have been submitted up to the present, and on the other it would go beyond the scope of this article. 

In contrast to the requirements for enforcement methods and to ensure sufficient quality of the generated data, validation data should be submitted for all types of crop samples to be analyzed. However, matrix comparability and a reduced validation data set may be considered where two or more very similar matrices are to be analyzed (e.g., cereal grain). A reduced sample set may also be acceptable (two levels, at least three determinations and an assessment of matrix interference) provided that the investigated samples belong to the same crop group as described in SANCO/825/00 (see also Section 4.2.1). 

The principles of validation of residue methods for food, water and soil are generally the same. However, not all procedures and requirements are identical. From the public s point of view, the information on residues in food is probably the most important task. Compared with the other two areas (water and soil), the food sector is characterized by the largest number of regulations and legal limits. Therefore, this overview of validation requirements of enforcement methods will focus on methods for pesticide residues in food. 

Until 1991, manufacturers seeking authorizations for pesticides had to fulfil country-specific requirements of validation of enforcement methods. The term enforcement method means analytical methods which are developed for post-registration control and monitoring purposes. The harmonization of these requirements was initiated with the European Economic Community (EEC) Council Directive 91/414/EEC and temporarily finalized with the Guidance Document on Residue Analytical Methods SANCO/825/00 rev. 6, dated 20 June 2000 [Santd et Protection des Consommateurs (SANCO)]. The evaluation of validation studies by the competent authority is conducted by comparison of these European Union (EU) requirements with the study results and most often without any practical experience of the method. Some details of this evaluation are discussed below. 

Evaluation of enforcement methods for food provided by manufacturers... 

On the other hand, single-residue methods developed by the applicants give basic information about appropriate cleanup steps and specific determination procedures. In addition, not many laboratories other than those from the applicants are able to test the real solvent extraction efficiency. The reason is that extraction studies need radio-labeled incurred residues instead of fortified samples. Hence enforcement methods provided by the manufacturers accelerate the development of methods which meet the needs of (official) food control laboratories. 

Enforcement methods provided by the manufacturer are not generally tested in the laboratories of the European regulatory authorities. Very often, proposed methods are evaluated by assessing the logic of proposed procedures and only for the completeness of validation data. For this theoretical review process, as much information as possible should be available. Recovery data from many validation experiments with different kinds of matrices and the resulting chromatograms of control and fortified samples provide the confidence needed by the referee. In the following sections, the most important aspects of this evaluation will be considered. 

Prior to registration, an agreed commitment to the residue component(s) which should be analyzed does not exist. This is contrary to the situation with residue methods, which are developed after MRL setting. Therefore, to establish an acceptable residue definition is the first step necessary prior to any method development. This residue definition for enforcement methods is based on the results of metabolism studies and may cause serious difficulties, especially if the metabolic pathways of the parent compound are very complex, generating a large number of metabolites. 

However, there is no general requirement that enforcement methods need to monitor all metabolites of an active ingredient. The primary purpose of enforcement methods is to detect violations of good agricultural practice. For this purpose, residue levels found in samples from the market (so-called Market Basket Surveys) have to be compared with MRLs, which are derived from residue concentrations found in supervised trials. It is not necessary for this comparison to be based on the total pesticide residue. Most often the choice of a single compound (e.g., parent or primary metabolite) as a marker of the total pesticide residue is more feasible. Method development and the later method application are much easier in that case. Only for intake calculation purposes, e.g., when the daily intake of pesticide residues (calculated from the results... 

Although we speak generally of validated methods , only the performance of a method applied to a particular range of materials (matrices) is reported. The possibility of matrix interferences or the efficiency of cleanup steps may vary with matrix type. For that reason, methods should be validated in all matrix types, which differ significantly. In the context of the validation of enforcement methods by applicants, significant difference is not a well defined term. To avoid any dispute about completeness of validation, five material types had been selected for crops, which usually... 

On the other hand, if only specific GC detectors, e.g. the electron capture, nitrogen-phosphorus or flame photometric detectors, are tested, the argument of lack of GC method sensitivity is not acceptable. In most cases mass spectrometric detectors provide the sensitivity and selectivity needed. Unfortunately, tandem mass spectrometry (MS/MS) or MS" detectors for GC are still not widely used in official laboratories, and therefore these techniques are not always accepted for enforcement methods. 

As discussed before, the efficiency of the extraction step is one of the fundamental performance characteristics of an analytical method. Unfortunately, the provisions regarding extraction efficiency in Council Directive 91/414/EEC (amended by Directive 96/68/EG) are listed in the metabolism section of the directive (Annex IIA 6.1 and 6.2). Nevertheless, results obtained in these studies are essential for the development of enforcement methods and must be reflected in this context. 

The enforcement methods provided by the applicants give basic information about appropriate cleanup steps and specific determination procedures. Typically, direct use of this developmental work occurred when a GC multi-residue method was found appropriate. Owing to the recent developments in the field of MS/MS with atmospheric pressure ionization, an alternative approach for those compounds that can be analyzed by liquid chromatography (LC) will soon be possible. It is important that some fundamental considerations for such method(s) should be agreed at the outset. Considerations include the most suitable extraction solvents and cleanup steps and some standard HPLC conditions. 

This validation typically requires samples with radiolabeled analytes. However, alternative approaches are proposed which involve (i) comparison with extraction of samples using a procedure which has been previously validated rigorously, (ii) comparison with extraction of samples by a very different technique or (iii) analysis of a certified reference material. Generally, this validation should be performed with samples containing analyte incurred by the route by which residues would normally be expected to arise. The simplest option (i) requires fully validated and documented enforcement methods provided by the manufacturer of a pesticide. 

In Europe, very different concepts of method validation are in use. The extent of validation depends upon legal requirements (e.g., for enforcement methods provided by the applicant), upon the required level of acceptance (e.g., for CEN methods) and upon national resources. Undoubtedly, the best method validation is performed with the help of inter-laboratory studies of performance, but such studies can be uneconomic, too slow to reach completion or restricted in scope. 

This enforcement method has been validated on the (raw agricultural commodities) (RAC) and processed parts of various crops. The method limit of quantitation (LOQ) was validated at 0.05 mg kg and the method limit of detection (LOD) was set at 0.01 mgkg for all of the crop matrices. The method flow chart is presented in Figure 1. 

A brief summary of EPA method requirements for tolerance enforcement methods is given in Table 1. Taken in total, these requirements ensure that the means to conduct the method are available to laboratories and that experimental evidence to establish method performance, on a substrate-by-substrate basis, is generated prior to analysis of samples and as part of each analytical set. Thus, an analyst who must generate data to support method performance in his or her hands can obtain whatever is required to reproduce the method. 

Enforcement method to undergo independent laboratory validation study... 

The only published immunoassay method submitted to date to EPA OPPTS as an enforcement method for a range of substrates (water, sediment, crops, processed crop fractions, and animal tissues) is the spinosad method, developed by Young et al. This method uses the spinosad RaPID Assay (Strategic Diagnostics) for determination of total spinosad residues (TSR). This discussion will be limited to crop and animal tissues, because the water and soil analyses are analogous to the triasulfuron method. The extraction, cleanup, and method parameters are summarized in Table 2. 

Full acceptance of HPLC/MS methods by the US EPA OPP as enforcement methods occurred between 1998 and 2001. For example, in 1998, the EPA OPP accepted HPLC/MS (without MS/MS) methods as primary enforcement methods, and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) only was suitable for confirmatory methods. However, in 2001, HPLC/MS/MS methods also became acceptable for primary enforcement. Table 4 summarizes the types of methods that were validated by the EPA OPP method validation program, for both food tolerance enforcement methods and environmental chemistry methods. 

Other ways to minimize matrix effects include improving the sample cleanup, diluting the sample, using labeled internal standards, using standard addition, or using matrix-matched standards. The last approach, however, is not permitted for enforcement methods at present by the US EPA or the US Pood and Drug Administration... 

FQ. Bramble, S. Frizzell, S.W George, B.A. Peterson, D.D. Ranken, J.J. Stry, and A.R. Sumpter, DuPont Project Identification AMR 3954-96 Proposed Analytical Enforcement Method For the Determination of Pyiithiobac Sodium in Cotton Gin Trash Using ASE Extraction and LC/MS/MS Analysis, Dupont, Wilmington, DE (1997). 

The official enforcement method is a GC method, but the method described here is quicker and more robust. Approximately 36 samples can be analyzed within 24 h. 

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