Residue analytical methods (residues)

Analytical methods are needed to generate the data on which dietary exposure assessments are based and to enforce any statutory maximum residue limits (MRLs) that may be set. Residue analyses are required for all pesticides used on edible crops and animals and resultant produce and for products (such as meat, milk and eggs) from animals that may consume treated crops.

1. Objectives of analytical methods

The following objectives should be met when developing analytical methods for a molecule. The methods should:

• have the ability to determine (identify, quantify and confirm) all the components included in the residue definition
• be specific enough that interfering substances never exceed 30% of the limit of analytical quantitation
• have demonstrated repeatability
• cover all crops and animals that may be treated
• cover animal and crop products if significant residues may occur (that is if the MRL is likely to be above the limit of analytical quantitation)
• cover all edible animal products if animals are likely to consume treated crops
• when possible, identify which multi-residue methods have the capacity to measure the residues.

2. Sensitivity of methods

The Food and Agriculture Organization’s guidelines on residue trials and OECD guidance documents and test guidelines discuss the level of sensitivity required in an analytical method for determining residues of pesticides. The guidelines are applicable to Australia and are largely reproduced below.

The continuing availability of improved clean-up systems and more sensitive and selective detectors has enabled residue chemists to measure smaller and smaller residues in many different samples. However, it may not be essential in some circumstances to measure very low levels of residues.

A residue chemist is frequently involved in measuring residues in samples in order to establish, or monitor, compliance with MRLs of chemicals present in or on commodities moving in international trade. In those cases, residue detection methods should be sufficiently sensitive to establish and monitor the MRL and to determine the residues that are likely to be present in a crop or animal. They do not necessarily need to be so sensitive that they can determine residues 2 or more orders of magnitude lower than the MRL, since that level of sophistication may make monitoring for regulatory purposes difficult.

Methods developed to measure residues at very low levels are usually very expensive and difficult to apply. They can also lead to technical problems in specifying precisely the limit of analytical quantitation of the method.

It may be acceptable to define a lower practical level (LPL) to be determined in any sample. This has the advantage of reducing the technical difficulty of obtaining the data and may also reduce costs. The following proposals for LPLs in various samples could be useful in enabling the residue chemist to devise suitable methods.

For registered active ingredients with agreed MRLs, the LPL can be specified as a fraction of the MRL. For analytical convenience, the fraction will vary and should be as shown in Table 1.

When the MRL is set at the limit of analytical quantitation of the analytical method, the LPL will also be at that level.

For new active ingredients, if the chemical is not yet marketed MRLs will not have been established. It is therefore more difficult to establish LPLs. Two situations can occur, as described below.

2.1. Active ingredients without particular toxicological or ecotoxicological hazard

On the basis of the experience of the applicant (for example, use rates and preliminary residue data), an estimate of a possible MRL should be made at an early stage. The estimated MRL can then be used to derive a provisional LPL as above.

For practical reasons, there should, in general, be no need to measure below the levels given in Table 2. 

¹ Lower levels of analysis may be required for hormones and like compounds.

² To enable residues to be expressed on a pure fat basis, the fat content of the milk should be determined or the milk processed to butter for analyses.

These limits can normally be achieved using commonly available equipment and procedures. The specified limits would allow safety to be addressed in a cost-effective manner.

2.2. Active ingredients with some potentially hazardous aspect

The applicant has to determine adequate LPLs for the types of samples in which the toxic effect may be mediated, based on careful consideration of all the facts available and perhaps in discussion with the Australian Pesticides and Veterinary Medicines Authority.

When establishing LPLs, the validity of the analytical procedures should be checked during the development and use of the method according to the following criteria:

• The normal overall recovery of the method, determined by fortifications of blank samples, should usually be between 70% and 110% at the recommended LPL. Recoveries outside of those limits may be acceptable for occasional batches of analyses. The decision on acceptability should be left to the experience of the supervising analyst.
• The repeatability of the method should be checked by analyses (for example 6 recoveries) of blank samples fortified at the proposed LPL. The relative standard deviation of the results should normally be no more than 20% but may be slightly greater at the lower residue levels. The reproducibility of the method should also be checked wherever possible.

3. Experimental design and reporting requirements

The submission should contain detailed descriptions of methods, including details of equipment, materials and conditions used. It should also identify the chemical entities measured and their relevance to the parent chemical and subsequent metabolism. The methodology used should be included in any subsequent submissions that contain the study results.

For each method, the following parameters must be determined and reported for each relevant substrate: details of extraction techniques, details of clean-up and methods of detection.

The method should also provide the following particulars:

• Residue definition (which compounds have been analysed and how results are expressed).
• Reagents and equipment used.
• Standard calibration. Reference compounds should be of known purity and stable for the duration of the analysis. Standards should be prepared to cover the lowest level of quantitation and the levels of the compound in the extracts of the samples. The response of each standard concentration (minimum of 3 concentrations) should be statistically validated from the limit of analytical quantitation to the concentration found in the sample extract.
• Accuracy of the method. The accuracy of the method is the closeness of agreement between the true value and the mean result as obtained by analysis of fortified samples with known amounts of compound. These ‘checks’ should be carried out at, or just above, the limit of analytical quantitation and at the levels present in the treated samples and should be done at the same time as the analysis of the samples. The deviation of the means from the true value (100%) limits listed in Table 3 can be used as a general guide.

  Table 3: Deviation of means from true value

  True value	Limits
  <1 mg/kg	–50% to +20%
  >1 mg/kg	–30% to +10%

• Limit of analytical quantitation. The limit of analytical quantitation of a method of analysis is the lowest concentration of a pesticide residue that can be quantitatively measured in a sample with an acceptable degree of certainty. It differs from the limit of detection of the method of analysis, which is defined as the lowest concentration of the compound that can be qualitatively detected. Refer to the Maximum residue limit proposals ‘at or about the limit of analytical quantitation’ guideline for further information.
• Specificity of the method. Tests carried out on control cleaned-up extracts should show no interfering substances that exceed 30% of the limit of analytical quantitation.
• Precision of the method (recovery range). The coefficient of variation (defined as the standard deviation divided by the arithmetic mean) of the recoveries may be regarded as a measure for precision. The limits set out in Table 4 can be used as a general guide.

• In general, the recovery data should be in the range of 70% to 110%. However, for compounds that are particularly difficult to analyse and for which methods consistently give low or high recoveries, a range of 60% to 140% may be appropriate. The relative standard deviation of the recovery rates should not exceed 20%.
• Representative chromatograms (minimum of a standard, untreated sample, and a fortified untreated sample). 

4. Analyses of crops and animals for compounds when a natural background level of the same compound exists

If it can be demonstrated, or argued, that residues resulting from treatment with an agricultural or veterinary chemical are indistinguishable from naturally occurring background levels of the same compound, no further residue data will be required.

If it is found that residues resulting from treatment with an agricultural or veterinary chemical result in residues of naturally occurring compounds at levels that are distinguishable from naturally occurring background levels, it will be necessary to demonstrate the maximum level expected under good agricultural practice.

5. Analyses for unstable or rapid-breakdown products

If it can be demonstrated by data or argument that normal analytical procedures cannot be employed due to degradation of fortified samples before quantification, residue data for the unstable compound will not be required.

A typical example would be an oxidising agent, which rapidly decomposes on contact with organic materials.

If the unstable component breaks down to other compounds (metabolites or base materials), residue data will need to be provided on the breakdown product unless it can be demonstrated or argued that there are no resultant residues – for example, the breakdown product is volatile or the resultant compounds are of no toxicological significance.