Analytical methodology

This guideline is based on the International Co-operation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products guideline (VICH GL) 49, which was intended to provide a general description of the criteria that have been found by the European Union, Japan, the United States, Australia, New Zealand and Canada to be suitable for the validation of analytical methods used in veterinary drug residue depletion studies.

VICH GL49 is one of a series developed to facilitate the mutual acceptance by national or regional regulators of residue information for veterinary drugs used in food-producing animals. It was originally prepared after consideration of the current national or regional requirements and recommendations for evaluating veterinary drug residues in the European Union, Japan, the United States, Australia, New Zealand, and Canada.

While VICH GL 49 covers most of the Australian recommended considerations in terms of analytical methodology used to quantify veterinary drug residues, there are some additional considerations that are unique to Australia. These additional methodological considerations are detailed in this document.

1. Guidance on residue analytical methodology

This guideline is only intended to apply to analytical procedures that have been developed for the evaluation of veterinary drug residue methods (assays developed to determine residues in marker residue depletion studies). It is not intended to define the criteria needed for validation of regulatory monitoring assay procedures.

1.1. Introduction

Residues data are used to establish maximum residue limits, demonstrate compliance with existing maximum residue limits, determine appropriate withholding periods and determine export slaughter intervals. In addition to the residues data that support registration of veterinary products that are used in or on food-producing species, you should provide a description of the analytical methods used to generate the residues data.

When developing analytical methods for a veterinary drug substance, the method(s) should:

  • have the ability to determine (identify, quantify and confirm) all the components included in the residue definition or marker residue
  • be specific enough that interfering substances never exceed 30 per cent of the limit of analytical quantitation
  • have demonstrated repeatability
  • cover all tissues or commodities that may be obtained from treated animals

1.2. Types of analytical methods

Analytical methods currently available for determining residues of veterinary drugs are as follows:

  • Bioassays: Broad-spectrum bioassays are used extensively in Australia to routinely screen urine, milk and kidney for the presence of antimicrobial agents. They continue to be important in the screening of large numbers of samples for the presence of inhibitory substances. These methods are generally not suitable for generating quantitative data for registration purposes. Specific bioassays or immunoassays are used for quantifying residues of veterinary drugs, especially antibiotics.
  • Instrumental methods: Methods such as gas liquid chromatography or high-performance liquid chromatography, in conjunction with various detectors, can be used both for the routine analyses of most veterinary drugs and to confirm and quantify their residues. Instrumental quantitation of new antimicrobial drugs is preferred to quantitation by bioassay or immunoassay, provided the specificity and sensitivity of the instrumental method are adequate.
  • Other instrumental methods: Methods including scanning thin layer chromatography and spectroscopic methods are less commonly used to quantify residues of veterinary drugs.

The choice between bioassays, immunological tests and instrumental methods will depend on the suitability of the respective methodologies for the task at hand. Circumstances will dictate which of the method types is appropriate. The APVMA’s preference is for an instrumental method that is specific and quantitative.

1.3. Objectives of analytical methods

When developing instrumental analytical methods for veterinary drugs, it is important the method(s) should:

  • have a substantiated and acceptable extraction efficiency
  • have the ability to determine (identify and quantify) all the components included in the residue definition or marker residue
  • be specific so that interfering substances never exceed 30 per cent of the limit of quantitation
  • have acceptable precision and accuracy
  • cover all animal species that are proposed to be the subject of the registration application
  • apply to tissues and commodities (such as muscle, liver, kidney, fat, injection sites, eggs, milk, and honey) that are relevant to the registration application.

In the case of bioassays and immunoassays, you only have to meet those objectives listed above that are relevant.

1.4. Development of analytical methods

The laboratory should demonstrate that the performance characteristics of the analytical method meet the criteria outlined in the below section Guidance on validation of residue analytical methodology.

Issues to consider during method development are:

  • The method should cover all relevant tissues (such as muscle, liver, kidney and fat) and animal commodities (such as eggs, milk and honey) in accordance with the registration proposal. Where milk is involved, the analytical method should apply to whole milk. When use of the drug involves administration to laying hens, eggs should be included in the method development. While partitioning of the residue between the yolk and the white of eggs may be determined, the method development should occur using the whole egg (minus shell).
  • The analytical method should address the residue definition or marker residue and be able to determine (identify and quantify) all the components included in the residue definition. If the analytical method does not address the residue definition, it may not be suitable for generating residue data.
  • Whereas bioassays are acceptable for screening large numbers of samples for residues, they are generally unacceptable for maximum residue limit–setting purposes. When using instrumental methods, you should base the residue definition or marker residue on the moiety or moieties measured; that is, the parent compound and/or one or more metabolites. In certain circumstances, the residue definition or marker residue will require the chemical conversion of parent and/or metabolites to another derivative that is measured.
  • The method should be fully validated. You should provide validation data for all matrices for which the method is to be used. In addition, you should provide data for control and fortified samples. Method validation is discussed in detail in the below section Guidance on validation of residue analytical methodology.
  • Extraction efficiency of the method should be determined with incurred residues, as it is a critical component for measuring the true analyte concentration. The method should adopt an extraction procedure that has been proven by analysis of samples for radiolabelled studies or by an exhaustive series of extractions that utilise different solvent and/or buffer combinations for the successive extractions of incurred residues. Linking method development to metabolism studies that utilised radiolabelled drug is one means of achieving the former.
  • The extraction efficiency of methods used in submissions pertaining to generic veterinary drugs should also be considered and demonstrated. You can accomplish this by demonstrating equivalence to the existing method through proficiency testing programs such as the National Residue Survey programs. If you need to develop methods for generic veterinary drugs, we advise you to use, where possible, the same extraction procedure and solvents as you used for establishing the maximum residue limits in the first instance. Alternatively, you could use a method published by the Codex Alimentarius Commission, or other published methods as guidance. These may involve exhaustive extractions and/or comparisons of extraction efficiencies when different solvents are used. You should also consider using other means to release residues from the matrix (for example, the use of perchloric acid releases neomycin from tissues; the use of glucuronidase for freeing residue conjugates; and the use of protease for releasing protein bound residues).
  • The method should also demonstrate stability of the residue in the extracts, especially where the method cannot be completed within one work session or where extraction or clean-up occurs overnight. This is more critical with some classes of compounds than others.
  • The sensitivity of the method should be acceptable for the intended purpose. It is noted that liquid chromatography with tandem mass spectrometry detection methods are routinely used in monitoring and surveillance laboratories these days, so contemporary analytical methods need to have comparable selectivity and sensitivity. Where the data are being used to support a change in either the maximum residue limit or the withholding period, or for the establishment of an export slaughter interval, the method should be able to detect residues to levels at the method’s limit of quantitation/limit of detection. Further, laboratories are asked to quantify and report values between the limit of detection and limit of quantitation of the method, to assist in the statistical analyses of the residues data.
  • You should generate recovery data across the range of residue concentrations that occur in the trial samples, and should, as a minimum, include recoveries at the limit of quantitation and the proposed maximum residue limit. Single-point recovery data are not sufficient when validating a method. For assays that utilise an internal standard, you should provide recovery data for both the analyte and the internal standard.
  • You should compare bioassays and immunoassays with an instrumental method to determine specificity and to demonstrate comparable quantitation of the residue definition. If bioassays or immunoassays are not able to quantify the residue definition, they are unsuitable for generating residues data for registration purposes.

You should perform all residue studies that are conducted in Australia, including both the animal and laboratory phases, in accordance with good laboratory practice (GLP) standards. For studies conducted in Australia, only those undertaken by facilities that are accredited under the Australian GLP compliance monitoring program can claim to be GLP compliant. For overseas studies to claim GLP compliance they must be conducted by facilities accredited in accordance with that country's GLP compliance monitoring program.

1.5. Reporting of analytical methods

You should provide complete details of the validated analytical method(s) used:

  • in pharmacokinetic and residue kinetic studies
  • for the determination of marker residue(s) in the trials conducted for the estimation of withholding periods.

The following is a list of information that you should typically include in support of the adequacy of the analytical procedures you are using:

  • a full description of the analytical method, including:
    • purpose and scope
    • reagents
    • equipment and instrumentation
    • collection of samples
    • storage of samples
    • stability of residues during storage
    • preparation of the laboratory sample
    • preparation and clean-up of tissue extracts
    • procedure for the determination of the residues
    • calculation of results, for example method of standardisation, use of calibration curves (mathematical model, parameters, working range)
    • quality control (internal)
    • documentation confirming the purity of the reference materials.
  • full details of the validation results, including raw data (refer to the below section Guidance on validation of residue analytical methodology)
  • details of studies into extraction efficiency, including the systems examined and/or solvents used, and the results
  • representative chromatograms—the minimum information that should be submitted includes a standard, an untreated sample, a fortified untreated sample and a sample for each matrix from a treated animal.

2. Guidance on validation of residue analytical methodology

The objective of validation of an analytical method is to demonstrate that the procedure, when correctly applied, produces results that are fit for purpose. The purpose of this document is to describe the procedures to be carried out to validate the analytical procedures used to conduct residues analysis and metabolism studies. These procedures are not intended to apply to analytical methods applied to the chemistry and manufacture of veterinary drugs or products containing veterinary drugs.

2.1. Performance characteristics

In general, there are specific performance characteristics of a method validation. Those performance characteristics are defined as follows:

  • standard calibration
  • linearity
  • accuracy
  • precision
  • limit of detection
  • limit of quantitation
  • selectivity or specificity
  • stability in matrix
  • conduct of storage stability trials
  • process sample stability
  • robustness.

Each of these characteristics will be described below as they apply to the validation of methods intended for use in veterinary drug residue depletion studies.

2.1.1. Standard calibration

Reference compounds should be of known purity. You should determine the stability of reference. For some antimicrobial compounds fresh dilutions should be prepared daily. You should assess linearity using at least five standard concentrations ranging from the limit of quantitation to the maximum concentration expected to be found in the sample extract, or the proposed maximum residue limit, whichever is higher.

2.1.2. Linearity

A calibration curve should be generated in which the linear relationship is evaluated across the range of the expected matrix (tissue, milk, egg or honey) concentrations. Calibration standard curves can be generated in three formats, depending upon the methodology: standards in solvent or buffer, standards fortified into control matrix extract and standards fortified into a control matrix and processed through the extraction procedure. You should describe linearity by a linear, polynomial or other (as appropriate) regression plot of known concentration versus response, using a minimum of five different concentrations. Acceptability of weighting factors should be determined by evaluation of the residuals across three runs to determine if the residuals are randomly distributed. You should evaluate the residuals across at least three separate runs.

The recommended acceptance criterion for a standard curve is dependent upon the format of the standard curve. Calibration standard curves generated by fortification of a control matrix and processed through the procedure are subject to the same acceptance criteria as the samples (see Precision). Calibration standard curves generated by standards in solvent/buffer or by fortification of control matrix extract would require more stringent acceptance criteria (repeatability of 15 per cent or less at all concentrations, except at or below the limit of quantitation, where it can be less than or equal to 20 per cent).

Some assays (for example, microbiological assays) could require log transformations to achieve linearity where other assays (for example enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays) could require a more complicated mathematical function to establish the relationship between concentration and response. Again, you should verify acceptability of the function selected by evaluating the residuals generated when that function is used.

2.1.3. Accuracy

Accuracy refers to the closeness of agreement between the true value of the analyte concentration and the mean result that is obtained by applying the experimental procedure a very large number of times. Accuracy can also be determined by recovery experiments using fortified blank matrices (mutually independent replicates). For example, 18 blank test portions could be selected and six fortified at each analyte level. Accuracy is closely related to systematic error (analytical method bias) and analyte recovery (measured as per cent recovery). The recommended accuracy for residue methods will vary depending upon the concentration of the analyte. The accuracy should meet the range listed in Table 1.

Table 1: Analyte concentrations and acceptable ranges for accuracy
Analyte concentration (µg/kg)* Acceptable range for accuracy
<1 μg/kg –50% to +20% (50–120%)
≥1 μg/kg –40% to +20% (60–120%)
≥10 μg/kg <100 μg/kg –30% to +10% (70–110%)
≥100 μg/kg –20% to +10% (80–110%)

* μg/kg = ng/g = ppb (micrograms per kilogram = nanograms per gram = parts per billion)

2.1.4. Precision

The precision of a method is the closeness of agreement between mutually independent test results obtained from homogenous test material under stipulated conditions of use. Results are typically expressed as the percentage-relative standard deviation of replicate analyses (n = 6) at various concentrations of analyte. Analytical variability between different laboratories is defined as reproducibility, and variability from repeated analyses within a laboratory is defined as repeatability. Single-laboratory validation precision should include a within-run (repeatability) and a between-run component.

The within- and between-run precision of the analytical method can be determined as part of the validation procedure. There is generally not a need to determine reproducibility (between-laboratory precision) in order to conduct a residue depletion study, because the laboratory that is developing the method is often the same laboratory assaying the samples from the residue study. Instead of establishing reproducibility of the assay, a within-run precision can be determined. Within- and between-run precision should be determined by the evaluation of a minimum of three replicates at three different concentrations representative of the intended validation range (which should include the limit of quantitation) across three days of analysis.

For the purposes of the residue method validation, acceptable variability is dependent upon the concentration of the analyte. The precision should meet the range listed in Table 2.

Table 2: Analyte concentrations and acceptable within- and between-run precision
Analyte concentration Acceptable within-run precision (repeatability) coefficient of variation (CV) Acceptable between-run precision CV
<1 μg/kg 30% 45%
≥1 μg/kg and <10 μg/kg 25% 32%
≥10 μg/kg and <100 μg/kg 15% 23%
≥100 μg/kg 10% 16%

2.1.5. Limit of detection

The limit of detection (LOD) is the smallest measured concentration of an analyte from which it is possible to deduce the presence of the analyte in the test sample with acceptable certainty. There are several scientifically valid ways to determine LOD. The APVMA’s preferred approach to determining the limit of detection is the definition used by the International Union of Pure and Applied Chemistry (IUPAC), where the LOD is estimated as the mean of 20 control sample (from at least six separate sources) assay results plus three times the standard deviation of the mean.

2.1.6. Limit of quantitation

The limit of quantitation (LOQ) is the smallest measured content of an analyte above which the determination can be made with the specified degree of accuracy and precision. As with the LOD, there are several scientifically valid ways to determine LOQ.

The APVMA’s preferred approach to determining the LOQ is also the IUPAC definition, where the LOQ is estimated as the mean of 20 control sample (from at least six separate sources) assay results plus 10 times the standard deviation of the mean.

Testing of the accuracy and precision at the estimated LOQ provides the final evidence for determination of the LOQ. If the coefficient of variation for the repeatability measurement at that concentration is less than or equal to the accuracy and precision acceptance criteria (see above), then the estimated LOQ is acceptable.

Particular emphasis is placed on determining the ‘true’ LOD and LOQ for the analytical method as, in many instances, the LOQ becomes the target endpoint for export slaughter interval determinations when a major importing country has not set any import standards or tolerances for the veterinary drug. In instances where a veterinary drug is classed as a ‘banned substance’, any detection may be classed as a violation, so surveillance testing may be conducted down to LOD concentrations.

2.1.7. Selectivity or specificity

Selectivity is the ability of a method to distinguish between the analyte being measured and other substances that might be present in the sample being analysed. Details concerning selectivity should relate to any substances that are likely to be present and give rise to a signal when the measuring principle described is used, for example homologues, analogues and metabolic products of the residues of interest. For the methods used in residue depletion studies, selectivity is primarily defined relative to endogenous substances in the samples being measured. Because the residue depletion studies are well controlled, exogenously administered components (that is, other veterinary drugs or vaccines) could either be known or not be allowed during the study.  From the details concerning specificity it should be possible to determine the extent to which the method can distinguish between the analyte and the other substances under the experimental conditions.

2.1.8. Stability in matrix

Samples (tissue, milk, eggs or honey) collected from residue depletion studies are generally frozen and stored until assayed. It is important to determine how long these samples can be stored under the proposed storage conditions without excessive degradation prior to analysis. You should, as part of the validation procedure or as a separate study, conduct a stability study to determine the appropriate storage conditions (for example 4 °C, –20 °C, or –70 °C) and length of time the samples can be stored prior to analysis.

Samples should be fortified with known quantities of analyte and stored under the appropriate conditions. You should periodically assay the samples at specified intervals (for example, initially, one week, one month, three months). If the samples are frozen, you should conduct freeze or thaw studies (three freeze or thaw cycles—one cycle per day at a minimum). Alternatively, you can use incurred samples with initial assays conducted to determine the starting concentrations. The recommended protocol for assessing stability in matrix is the analysis of two different concentrations in triplicate near the high and low end of the validation range. Stability in matrix is considered acceptable if the mean concentration obtained at the specified stability time point agrees with the initial assay results or freshly fortified control sample assay results within the accuracy acceptance criteria established in Accuracy.

2.1.9. Conduct of storage stability trials

You should analyse the samples taken for residue analysis as quickly as possible after collection, before physical and chemical changes take place. If samples are going to be stored for a significant period of time (six months or longer), you should provide either argument or data on the stability of residues during storage conditions you are using.

Studies on the stability of residues in samples, over the time and at the temperature of storage, should be carried out with representative substrates. You should conduct a stability study with sample material subjected to similar sample preparation procedures and storage conditions as those for the proposed magnitude of residue studies. However, you may store the samples being subjected to a storage stability study as homogenates rather than in a whole state. The homogenate represents a worst case, as the homogenisation process can release enzymes, acids and other chemicals that can react with the veterinary drug or its metabolites. This may lead to unacceptable results if the residue degrades under these conditions.

You may conduct experiments on prepared samples with incurred residues. Alternatively, you may spike aliquots of prepared control samples with a known amount of drug before they undergo normal storage conditions.

Where degradation to a metabolite (contained in the residue definition) is likely during storage, it is desirable to conduct stability studies on samples spiked with the metabolite in addition to those for the parent compound.

2.1.10. Process sample stability

Often, test samples are processed one day and assayed on a second day or, because of an instrument failure, they may be stored for additional days, for example over a weekend. The stability of the analyte in the process sample extract might be examined as necessary to determine stability under processed sample storage conditions. Examples of storage conditions would be 4 to 24 hours at room temperature and 48 hours at 4 °C. Other storage conditions might be investigated consistent with the method requirements. The recommended protocol for assessing processed sample stability is the analysis of two different concentrations in triplicate near the high and low end of the validation range. Processed sample stability is considered adequate if the mean concentration obtained at the specified stability time point agrees with the initial assay results or with freshly fortified and processed control sample assay results within the accuracy acceptance criteria established in Accuracy.

2.1.11. Robustness

Robustness should be evaluated, particularly for areas of the method that could undergo changes or modifications over time. These might include reagent lots, incubation temperatures, extraction-solvent composition and volume, extraction time and number of extractions, solid phase extraction cartridge brand and lots, analytical column brand and lots and high-performance liquid chromatography elution solvent composition. During the development, validation or use of the assay, method sensitivity to any or all of these conditions can become apparent and you should evaluate the variations in the ones most likely to affect the method performance.

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