GUIDELINES FOR THE VALIDATION OF ANALYTICAL METHODS FOR ACTIVE CONSTITUENT, AGRICULTURAL AND VETERINARY CHEMICAL PRODUCTS
October 2004
APVMA
PO Box E240 KINGSTON 2604 AUSTRALIA
v.au
ph : +61 2 6272 5852
Table of Contents
1.0Introduction________________________________________________________________3 1.1Scope______________________________________________________________________3 1.2Data Requirements__________________________________________________________3 1.3Parameters for Method Validation_____________________________________________3 1.3.1Selectivity (Specificity)____________________________________________________3 1.3.2Linearity_______________________________________________________________4 1.3.3Range__________________________________________________________________4 1.3.4Accuracy_______________________________________________________________4 1.3.5Precision_______________________________________________________________5 1.3.6Limit of Detection (LOD)_________________________________________________6 1.3.7Limit of Quantitation (LOQ)______________________________________________6 1.4Validation Characteristics and Requirements____________________________________6 1.5General Notes_______________________________________________________________6 1.5.1Regulatory Analytical Methods____________________________________________6 1.5.2Alternative Analytical Methods____________________________________________7 1.5.3Typical Characteristics for Nuclear Magnetic Resonance (NMR) Data___________7 1.5.4Analytical Reference Standards____________________________________________7 1.5.5G
ood Laboratory Practice_________________________________________________7 1.6Revalidation________________________________________________________________7 1.7Definitions and Abbreviations_________________________________________________8 1.8References__________________________________________________________________8
1.0 INTRODUCTION
In order to generate the Part 2 Chemistry and Manufacture data for the approval of active constituents and registration of agricultural and veterinary chemical products, robust, accurate and preci analytical methods are required. Methods are required for the identification, batch analysis, and storage stability data for active constituents and agricultural and veterinary chemical products, and for post-registration compliance purpos.
1.1 SCOPE
The objective of validation of an analytical method is to demonstrate that the procedure, when correctly applied, produces results that are fit for purpo. The guidelines describe the procedures to be carried out to validate the analytical procedures included as part of an application for approval of an active constituent and registration of an agricultural and veterinary chemical product, including
丝竹指代什么tho ud in storage stability studies. They are not intended to apply to analytical methods for residue analysis, biological and biotechnological products. Approaches other than tho t forth in this guideline may be acceptable provided they are supported by adequate scientific justification.治疗便秘最快的方法
In general, non-chromatographic analytical methods are not typically expected to comply with this guideline. However, the APVMA may require that a non-chromatographic method demonstrate some form of validation in order to satisfy itlf that the method is fit for purpo [e.g. Nuclear Magnetic Resonance (NMR) methods are typically required to demonstrate certain validation parameters].
1.2 DATA REQUIREMENTS
The following is a list of information that should typically be included in support of the adequacy of the analytical procedures:
• Method description – this ction should contain a full description of the analytical method. The description should include details of all-important operational parameters, such as sample preparation, including method of extraction of the active constituent from the product, details of the reference standards and reagents preparation. Documentation confirming the purity of the reference materials should also be provided.
• Validation data - all relevant data collected during validation should also be provided. Relevant data are considered to be: copies of chromatograms that are clearly labelled with peak identity and peak integration data; NMR spectra clearly showing chemical shifts and coupling constants;
formulae and calculations ud for calculating validation characteristics.
1.3 PARAMETERS FOR METHOD VALIDATION
To be fit for the intended purpo, the method must meet certain validation characteristics. Typical validation characteristics, which should be considered are:lectivity (specificity), linearity, range, accuracy, precision, limit of detection and quantitation.
1.3.1 Selectivity (Specificity)
Selectivity of a method refers to the extent to which it can determine particular analyte(s) in a complex mixture without interference from other components in the mixture. The terms lectivity and specificity have often been ud interchangeably. The term specific generally refers to a method that produces a respon for a single analyte only, while the term lective refers to a method that provides respons for a number of chemical entities that may or may not be distinguished from eac
h other. If the respon is distinguished from all other respons, the method is said to be lective. Since very few
analytical methods respond to only one analyte, the u of the term lectivity is more appropriate than specificity. The International Union of Pure and Applied Chemistry (IUPAC) has expresd the view that “Specificity is the ultimate of Selectivity’. The IUPAC discourages u of the term specificity and instead encourages the u of the term lectivity.
The lectivity of the analytical method must be demonstrated by providing data to show the abnce of interference peaks with regard to degradation products, synthetic impurities and the matrix (excipients prent in the formulated product at their expected levels).
The lectivity of chromatographic methods may be assd by examination of peak homogeneity or peak purity test (e.g., diode array, mass spectrometry) to show that the analyte chromatographic peak is not attributable to more than one component.
1.3.2 Linearity
The linearity is the ability of analytical procedure to produce test results which are proportional to the
concentration (amount) of analyte in samples within a given concentration range, either directly or by means of a well-defined mathematical transformation. Linearity should be determined by using a minimum of six standards who concentration span 80 –120% of the expected concentration range. The linearity of a method should be established by visual inspection of a plot of analytical respon as a function of analyte concentration. If there is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of the regression line by the method of least squares. In some cas, the test data may need to be subjected to a mathematical transformation prior to regression analysis.
Reports submitted must include, the slope of the line, intercept and correlation coefficient data. The measured slope should demonstrate a clear correlation between respon and analyte concentrations. The results should not show a significant deviation from linearity, which is taken to mean that the correlation coefficient, r > 0.99, over the working range (80 –120%). If this is not the ca (i.e. r is < 0.99), the submitter must provide an explanation of how accurate calibration is to be maintained. In cas where a non-linear respon is deliberately ud, an explanation must be provided.
1.3.3 Range
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The specified range is normally derived from the linearity studies. The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical method has suitable levels of precision, accuracy and linearity.
The following minimum specified ranges should be considered:
• For the assay of the active constituent or an agricultural/veterinary chemical product: normally from 80 –120% of the test concentration/label concentration; and
• For the determination of an impurity: from the specification level of the impurity to 120% of the specification.
数学家高斯的故事1.3.4 Accuracy
The accuracy of an analytical method is defined as the degree to which the determined value of analyte in a sample corresponds to the true value. Accuracy may be measured in different ways and the method should be appropriate to the matrix. The accuracy of an analytical method may be determined by any of the following ways:
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• Analysing a sample of known concentration and comparing the measured value to the ‘true’ value. However, a well characterid sample (e.g., reference standard) must be ud.
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• Spiked – placebo (product matrix) recovery method. In the spiked – placebo recovery method, a known amount of pure active constituent is added to formulation blank [sample that contains all other ingredients except the active(s)], the resulting mixture is assayed, and the results obtained are compared with the expected result.
• Standard addition method. In the standard addition method, a sample is assayed, a known amount of pure active constituent is added, and the sample is again assayed. The difference between the results of the two assays is compared with the expected answer.
In both methods (spiked – placebo recovery and standard addition method), recovery is defined as the ratio of the obrved result to the expected result expresd as a percentage.
The accuracy of a method may vary across the range of possible assay values and therefore must be determined at veral different fortification levels. The accuracy should cover at least 3 concentrations (80, 100 and 120%) in the expected range.
Accuracy may also be determined by comparing test results with tho obtained using another validated test method.
Acceptance criteria: the expected recovery depends on the sample matrix, the sample processing procedure and on the analyte concentration. The mean % recovery should be within the following ranges:
% Active/impurity content Acceptable mean recovery
≥ 10 ≥ 1 0.1 – 1 < 0.1 98 –102% 90 –110% 80 – 120% 75 – 125%
1.3.5 Precision
金元券The precision of an analytical procedure express the cloness of agreement (degree of scatter) between a ries of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. For the guidelines, a simple asssment of repeatability will be acceptable. The precision of an analytical procedure is usually expresd as the variance, standard deviation or coefficient of variation of a ries of measurements. A minimum of 5 replicate s
ample determinations should be made together with a simple statistical asssment of the results, including the percent relative standard deviation. If considered appropriate, a suitable test for outliers (Dixon’s or Grubbs Test) may be applied to the results. Where outliers have been discarded, that fact must be clearly indicated. An explanation as to the reason for the occurrence of individual outliers must be attempted.
The following levels of precision are recommended.
Component measured in sample Precision
>
10.0% ≤ 2%
1.0 up to 10.0% ≤ 5%
0.1 up to 1.0% ≤ 10%
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0.1% ≤ 20%
