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MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION

GENERAL PHARMACOPEIAN AUTHORIZATION

Validation of analytical procedures OFS.1.1.0012.15

Introduced for the first time

Validation of an analytical technique is the experimental proof that the technique is suitable for solving the intended problems.

This General Pharmacopoeia Monograph regulates the characteristics of analytical methods determined for the purpose of their validation, and the corresponding criteria for the suitability of validated methods intended for quality control of medicinal products: pharmaceutical substances and medicinal products.

Methods of quantitative determination, including methods for determining impurities and methods for determining the limit of content, are subject to validation. Authentication methods are validated if necessary to confirm their specificity.

During validation, the analytical method is evaluated according to the characteristics listed below, selected taking into account the typical recommendations given in the table:

• specificity;
• detection limit;
• limit of quantitative determination (quantitation limit);
• analytical area (range);
• linearity (linearity);
• correctness (trueness);
• precision (precision);
• stability (robustness).

Table 1 — Characteristics of methods determined during validation

*) can be determined if necessary;

**) the lack of specificity of one analytical method can be compensated for by using another analytical method.

Revalidation (re-validation) of methods is carried out when:

• technologies for obtaining the object of analysis;
• composition of the medicinal product (object of analysis);
• previously approved analysis methodology.

1. Specificity

Specificity is the ability of an analytical procedure to unambiguously evaluate an analyte in the presence of concomitant components.

Evidence of the specificity of a validated technique is usually based on consideration of data obtained using it from the analysis of model mixtures of known composition.

The specificity of a validated technique can also be proved by appropriate statistical processing of the results of analyzes of real objects performed using it and, in parallel, using another, obviously specific, technique (a technique whose specificity has been proven).

1.1 For identity test methods

A validated method (or a set of methods) should provide reliable information about the presence of a given active substance in a substance or dosage form if it contains the components provided for in the formulation, which is subject to experimental confirmation.

The authenticity of the active substance in the pharmaceutical substance or medicinal product is established in comparison with a standard sample or by physicochemical or chemical properties that are not characteristic of other components.

1.2 For quantitation and impurity test procedures

For a validated quantification method and testing for impurities, the same approaches are used - its specificity in relation to the analyte must be evaluated, i.e. it must be experimentally confirmed that the presence of accompanying components does not affect the result of the analysis in an unintended way.

It is allowed to assess the specificity of the validated method both by analyzing model mixtures of known composition containing the analyte, and by comparing the results of analyzes of real objects obtained simultaneously with the use of the validated and other, obviously specific, methods. The results of the relevant experiments should be statistically processed.

The lack of test specificity can be compensated for by another additional test(s).

When validating methods, if appropriate, samples of medicinal products subjected to extreme conditions (light, temperature, humidity) or chemically modified in any suitable way can be used to accumulate impurities in them.

For chromatographic techniques, show the resolution between the two most closely eluting substances at the respective concentrations.

1. LIMIT OF DETECTION

The detection limit is the smallest amount (concentration) of an analyte in a sample that can be detected (or approximated) using a validated technique.

The limit of detection in the cases indicated in the table is usually expressed as the concentration of the analyte (in % relative or parts per million - ppm).

Depending on the type of technique (visual or instrumental), different methods are used to determine the detection limit.

2.1 For methods with a visual assessment of the result of the analysis

Test samples with various known amounts (concentrations) of the analyte and establish the minimum value at which the result of the analysis can be evaluated visually. This value is an estimate of the limit of detection.

2.2 For methods with instrumental evaluation of the analysis result

2.2.1 By signal-to-noise ratio

This approach is applicable to methods for which baseline noise is observed. Compare the signal values ​​obtained for the control experiment and for samples with low concentrations of the analyte. Set the minimum amount (concentration) of the analyte in the sample, at which the ratio of the analytical signal to the noise level is equal to 3.

The value found is an estimate of the detection limit.

2.2.2 By the value of the standard deviation of the signal and the slope of the calibration curve

The limit of detection (LO) is found by the equation:

ON = 3.3 S/b,

where S

b is the sensitivity coefficient, which is the ratio of the analytical signal to the determined value (the tangent of the slope of the calibration curve).

S and b

S S a free term of the equation of this graph. The obtained value of the detection limit, if necessary, can be confirmed by direct experiment at amounts (concentrations) of the analyte close to the found value of the detection limit.

As a rule, if there is evidence of the suitability of a method for the reliable determination of a substance at concentrations both above and below its content limit established by the specification, it is not required to determine the actual limit of detection for such a method.

1. LIMIT OF QUANTIFICATION

The limit of quantitation is the smallest amount (concentration) of a substance in a sample that can be quantified using a validated method with the required accuracy and within-laboratory (intermediate) precision.

The limit of quantitation is a necessary validation characteristic of procedures used to assess small amounts (concentrations) of substances in a sample and, in particular, to assess the content of impurities.

Depending on the type of technique, the following methods are used to find the limit of quantitation.

3.1 For methods with a visual assessment of the result of the analysis

Test samples with various known amounts (concentrations) of the analyte and establish the minimum value at which the result of the analysis can be obtained visually with the required accuracy and intralaboratory (intermediate) precision.

3.2 For methods with instrumental evaluation of the analysis result

3.2.1 Signal-to-noise ratio

Set the minimum concentration of the analyte in the sample, at which the ratio of the analytical signal to the noise level is about 10:1.

3.2.2 By the value of the standard deviation of the signal and the slope of the calibration curve

The limit of quantification (LOQ) is calculated using the equation:

FSP = 10 S/b,

where S is the standard deviation of the analytical signal;

b is the sensitivity coefficient, which is the ratio of the analytical signal to the determined value.

In the presence of experimental data in a wide range of measured values S and b can be estimated by the least squares method.

For a linear calibration plot, the value S taken equal to the standard deviation S a free term of the equation of this graph. The obtained value of the limit of quantitative determination, if necessary, can be confirmed by direct experiment at amounts (concentrations) of the analyte close to the found value of the limit of quantitative determination.

if there is evidence of the ability of a method to reliably detect an analyte at concentrations above and below its specification limit, it is usually not necessary to determine the actual value of the limit of quantitation for such a method.

1. ANALYTICAL FIELD OF THE METHOD

The analytical area of ​​the technique is the interval between the upper and lower values ​​of the analytical characteristics of the determined component in the object of analysis (its quantity, concentration, activity, etc.). Within this range, the results obtained using the method being validated should have an acceptable level of accuracy and within-laboratory (intermediate) precision.

The following requirements are imposed on the size of the analytical area of ​​the methods:

– methods of quantitative determination should be applicable in the range from 80 to 120% of the nominal value of the determined analytical characteristic;

- methods for assessing the uniformity of dosing should be applicable in the range from 70 to 130% of the nominal dose;

- the quantitation methods used in the dissolution test should generally be applicable within the range of 50 to 120% of the expected concentration of the active substance in the dissolution medium;

- test methods for purity should be applicable in the range from the "Limit of Quantification" or "Limit of Detection" to 120% of the allowable content of the determined impurity.

The analytical domain of the technique can be set by the range of experimental data that satisfies the linear model.

1. LINEARITY

The linearity of the technique is the presence of a linear dependence of the analytical signal on the concentration or amount of the analyte in the analyzed sample within the analytical area of ​​the technique.

When validating a method, its linearity in the analytical region is verified experimentally by measuring analytical signals for at least 5 samples with different amounts or concentrations of the analyte. Experimental data is processed by the least squares method using a linear model:

y = b · x + a,

X- the amount or concentration of the analyte;

y is the magnitude of the response;

b- angular coefficient;

a- free term (OFS "Statistical processing of the results of a chemical experiment").

Values ​​must be calculated and presented. b, a and correlation coefficient r. In most cases, linear dependencies are used that meet the condition 0.99, and only when analyzing trace amounts, linear dependencies are considered, for which 0.9.

In some cases, the possibility of linear approximation of experimental data is provided only after their mathematical transformation (for example, by taking logarithms).

For some methods of analysis, which in principle cannot be based on a linear relationship between experimental data, the determination of the concentration or amount of a substance is carried out using non-linear calibration graphs. In this case, the plot of the dependence of the analytical signal on the amount or concentration of the analyte can be approximated by a suitable non-linear function using the least squares method, which is feasible with the appropriate validated software.

1. RIGHT

The correctness of the technique is characterized by the deviation of the average result of the determinations made with its use from the value taken as true.

A validated technique is recognized as correct if the values ​​taken as true lie within the confidence intervals of the corresponding average results of analyzes obtained experimentally using this technique.

The following approaches are applicable to evaluate the validity of quantitation procedures:

a) analysis using a validated method of standard samples or model mixtures with a known content (concentration) of the analyte;

b) comparison of the results obtained using the validated method and the exemplary method, the correctness of which has previously been established;

c) consideration of the results of studying the linearity of the validated methodology: if the free term in the equation given in section 5 does not statistically significantly differ from zero, then the use of such a methodology gives results free from systematic error.

For approaches "a" and "b" it is possible to present the obtained data in the form of a linear dependence equation (regression) between experimentally found and true values. For this equation, the hypotheses about the equality of the tangent of the slope angle to unity are checked b and on the equality to zero of the free term a. As a rule, if these hypotheses are recognized as true with a degree of reliability equal to 0.05, then the use of a validated methodology gives correct, i.e., free from systematic error, results.

1. PRECISION

The precision of a technique is characterized by the dispersion of the results obtained with its use relative to the value of the average result. A measure of such dispersion is the value of the standard deviation of the result of a single determination, obtained for a sample of a sufficiently large size.

Precision is assessed for any quantification procedure by at least three determinations for each of the three levels of analytes (low, middle, and high) that lie within the analytical range of the method. Repeatability can also be assessed for any assay technique from at least six determinations for samples with near-nominal analyte content. In many cases, the assessment of precision can be carried out on the basis of the results of processing experimental data using the least squares method, as indicated in the GPM "Statistical processing of the results of a chemical experiment."

Precision should be tested on homogeneous samples and can be evaluated in three ways:

– as repeatability (convergence);

– as intralaboratory (intermediate) precision;

– as interlaboratory precision (reproducibility).

The results of the evaluation of the analysis method for each of the options for precision are usually characterized by the corresponding value of the standard deviation of the result of a separate determination.

Usually, when developing an original technique, the repeatability (convergence) of the results obtained with its use is determined. If it is necessary to include the developed method in the regulatory documentation, its intralaboratory (intermediate) precision is additionally determined. The interlaboratory precision (reproducibility) of a method is evaluated when it is supposed to be included in a draft general pharmacopoeial monograph, pharmacopoeial monograph, or in the regulatory documentation for pharmacopoeial reference materials.

7.1 Repeatability (convergence)

The repeatability of an analytical procedure is evaluated by independent results obtained under the same regulated conditions in the same laboratory (the same performer, the same equipment, the same set of reagents) within a short period of time.

7.2 Intralaboratory (Intermediate) Precision

The intralaboratory (intermediate) precision of the method being validated is evaluated under the same laboratory conditions (different days, different performers, different equipment, etc.).

7.3 Interlaboratory precision (reproducibility)

Interlaboratory precision (reproducibility) of a validated method is assessed when testing in different laboratories.

1. STABILITY

The stability of a validated technique is the ability to maintain the characteristics found for it under optimal (nominal) conditions, given in the table, with probable small deviations from these analysis conditions.

Method robustness should not be determined in relation to easily controlled assay conditions. This drastically reduces the need for a special stability study.

Stability should only be studied if the method being validated is based on the use of particularly environmentally sensitive methods of analysis, such as various types of chromatography and functional analysis. If necessary, the assessment of the stability of the methodology is carried out at the stage of its development. If the low stability of the technique is likely, its suitability is checked without fail directly in the process of practical use.

Analytical System Validation

Validation of the suitability of an analytical system is a verification of the fulfillment of the basic requirements for it. The system whose suitability is being tested is a collection of specific instruments, reagents, standards, and samples under analysis. The requirements for such a system are usually specified in the general monograph for the corresponding analytical method. Thus, testing the suitability of the analytical system becomes a procedure included in the method being validated.

Presentation of validation results

The validation protocol for the analytical method should contain:

– its complete description, sufficient for reproduction and reflecting all the conditions necessary for performing the analysis;

– evaluated characteristics;

- all primary results that were included in the statistical data processing;

– results of statistical processing of data obtained experimentally during the development or verification of a validated methodology;

- illustrative materials, such as copies of chromatograms obtained by high performance liquid chromatography or gas chromatography; electrophoregrams, electronic and infrared spectra; photographs or drawings of chromatograms obtained by thin-layer or paper chromatography; drawings of titration curves, calibration graphs;

– a conclusion on the suitability of the method being validated for inclusion in the regulatory document.

Validation materials for individual analytical methods should be presented in the form of a combined validation report.

BOARD

SOLUTION

In accordance with Article 30 of the Treaty on the Eurasian Economic Union of May 29, 2014 and paragraph 2 of Article 3 of the Agreement on Common Principles and Rules for the Circulation of Medicines within the Eurasian Economic Union of December 23, 2014, the Board of the Eurasian Economic Commission

decided:

1. Approve the attached Guidelines for the Validation of Analytical Methods for Testing Medicinal Products.

2. This Decision comes into force after 6 months from the date of its official publication.

Chairman of the Board
Eurasian Economic Commission
T. Sargsyan

Guidance on the Validation of Analytical Methods for Testing Medicinal Products

APPROVED
Board decision
Eurasian Economic Commission
dated July 17, 2018 N 113

I. General provisions

1. This Guide defines the rules for the validation of analytical methods for testing medicinal products, as well as a list of characteristics to be assessed during the validation of these methods and included in registration dossiers submitted to the authorized bodies of the Member States of the Eurasian Economic Union (hereinafter, respectively, the Member States, Union).

2. The purpose of the validation of an analytical method for testing medicinal products is to document its suitability for the intended purpose.

II. Definitions

3. For the purposes of this Guide, terms are used that mean the following:

"analytical procedure" (analytical procedure) - a methodology for testing medicinal products, which includes a detailed description of the sequence of actions necessary to perform an analytical test (including a description of the preparation of test samples, reference materials, reagents, use of equipment, construction of a calibration curve, calculation formulas used, etc.);

"reproducibility" - a property characterizing the precision in interlaboratory tests;

"range of application (analytical area)" (range) - the interval between the highest and lowest concentrations (amount) of the analyte in the sample (including these concentrations), for which the analytical method is shown to have an acceptable level of precision, accuracy and linearity;

"linearity" (linearity) - directly proportional dependence of the analytical signal on the concentration (quantity) of the analyte in the sample within the range of application (analytical area) of the technique;

"discovery (recovery)" (recovery) - the ratio between the obtained average and true (reference) values, taking into account the appropriate confidence intervals;

"repeatability (intra-assay precision)" - the precision of a method when repeated tests are performed under the same operating conditions (for example, by the same analyst or group of analysts, on the same equipment, with the same and the same reagents, etc.) for a short period of time;

"correctness" (accuracy, trueness) - the proximity between the accepted true (reference) value and the received value, which is expressed by the openness value;

"limit of quantitative determination" (quantitation limit) - the smallest amount of a substance in a sample that can be quantitatively determined with appropriate precision and accuracy;

"detection limit" - the smallest amount of an analyte in a sample that can be detected, but not necessarily accurately quantified;

"precision" (precision) - an expression of the closeness (degree of spread) of the results (values) between series of measurements carried out on a plurality of samples taken from the same homogeneous sample, under the conditions prescribed by the procedure;

"intermediate (intralaboratory) precision" (intermediate precision) - the effect of variations within the laboratory (different days, different analysts, different equipment, different series (batches) of reagents, etc.) on the test results of identical samples taken from the same series;

"specificity" (specificity) - the ability of an analytical technique to unambiguously evaluate the substance being determined, regardless of other substances (impurities, degradation products, excipients, matrix (medium), etc.) present in the test sample;

"stability (robustness)" (robustness) - the ability of an analytical procedure to be resistant to the influence of small specified changes in test conditions, which indicates its reliability in normal (standard) use.

III. Types of analytical methods to be validated

4. This Guide covers validation approaches for the 4 most common types of analytical methods:

a) identification tests (authenticity);

b) tests to determine the quantitative content of impurities (quantitative tests for impurities content);

c) tests to determine the limit of impurities in the sample (limit tests for the control impurities);

d) quantitative tests (for content or activity) (quantitative tests of the active moiety) to determine the active part of the molecule of the active substance in the test sample.

5. All analytical methods used for quality control of medicinal products must be validated. This Guide does not cover the validation of analytical methods for types of tests not included in paragraph 4 of this Guide (for example, tests for dissolution or determination of particle size (dispersity) of a pharmaceutical substance, etc.).

6. Tests for identification (authenticity) usually consist of comparing the properties (eg spectral characteristics, chromatographic behavior, reactivity, etc.) of the test and reference samples.

7. Tests to determine the quantitative content of impurities and tests to determine the limit of the content of impurities in the sample are aimed at the correct description of the characteristics of the purity of the sample. The requirements for the validation of methods for the quantitative determination of impurities are different from the requirements for the validation of methods for determining the limiting content of impurities in a sample.

8. Methods of quantitative testing are aimed at measuring the content of the analyte in the test sample. In these Guidelines, quantification refers to the quantitative measurement of the main components of a pharmaceutical substance. Similar validation parameters apply to the assay of the active substance or other components of the medicinal product. Validation quantitation parameters may be used in other analytical procedures (eg dissolution testing).

The purpose of the analytical methods should be clearly defined, as this determines the choice of validation characteristics that should be evaluated during the validation.

9. The following typical validation characteristics of an analytical method are subject to evaluation:

a) correctness (accuracy (trueness));

b) precision (precision):

repeatability;

intermediate (intralaboratory) precision (intermediate precision);

c) specificity;

d) detection limit;

e) quantitative limit;

f) linearity;

g) application range (analytical area) (range).

10. The most important validation characteristics for the validation of various types of analytical methods are summarized in the table.

Table. Validation characteristics for the validation of different types of analytical methods

________________
* If reproducibility is determined, determination of intermediate precision is not required.

** Insufficient specificity of one analytical method can be compensated for by using one or more additional analytical methods.

*** May be required in some cases (for example, when the limit of detection and the normalized limit of the content of the determined impurity are close).

Note. "-" - the characteristic is not evaluated, "+" - the characteristic is evaluated.


The specified list should be considered as typical when validating analytical methods. There may be exceptions that require separate justification by the manufacturer of the medicinal product. Such a characteristic of the analytical method as stability (robustness) is not listed in the table, but it should be considered at the appropriate stage in the development of the analytical method.

Re-validation (revalidation) may be necessary in the following cases (but not limited to):

change in the synthesis scheme of the pharmaceutical substance;

change in the composition of the medicinal product;

change in analytical methodology.

Re-validation is not carried out if the manufacturer provides an appropriate justification. The extent of the revalidation depends on the nature of the changes made.

IV. Methodology for the validation of analytical methods

1. General requirements for the methodology of validation of analytical methods

11. This section outlines the characteristics to be considered in the validation of analytical methods and provides some approaches and recommendations for establishing the different validation characteristics of each analytical method.

12. In some cases (for example, when proving specificity), a combination of several analytical methods can be used to ensure the quality of a pharmaceutical substance or drug product.

13. All relevant data collected during the validation and the formulas used to calculate the validation performance should be presented and analyzed.

14. Approaches other than those outlined in this Guide may be used. The choice of the validation procedure and protocol is the responsibility of the applicant. In this case, the main purpose of the validation of an analytical method is to confirm the suitability of the method for the intended purpose. Due to their complexity, approaches to analytical methods for biological and biotechnological products may differ from those described in this Guide.

15. Throughout the validation performance study, reference materials with known, documented characteristics should be used. The required degree of purity of reference materials depends on the intended use.

16. Various validation characteristics are discussed in separate subsections of this section. The structure of this section reflects the process of developing and evaluating an analytical methodology.

17. Experimental work should be planned so that relevant validation characteristics are studied simultaneously, providing reliable data on the capabilities of the analytical method (eg, specificity, linearity, range of application, trueness and precision).

2. Specificity

18. Specificity studies should be carried out during the validation of tests for identification, impurities and quantitation. Specificity validation procedures depend on the intended use of the analytical method.

19. How specificity can be confirmed depends on the objectives for which the analytical method is intended. Not in all cases it is possible to confirm that the analytical method is specific for this analyte (complete selectivity). In this case, it is recommended to use a combination of 2 or more analytical methods.

The lack of specificity of one analytical method can be compensated for by the use of one or more additional analytical methods.

20. Specificity for different types of tests means the following:

a) when testing for identification - confirmation that the method allows you to identify exactly the substance to be determined;

b) when testing for impurities, confirmation that the method correctly identifies impurities in the sample (for example, testing for related compounds, heavy metals, residual solvents, etc.);

c) in quantitative tests - confirmation that the method allows you to determine the content or activity of the substance being determined in the sample.

Identification

21. Satisfactory identification tests should be able to distinguish between structurally closely related compounds that may be present in the sample. The selectivity of an analytical procedure can be confirmed by obtaining positive results (perhaps by comparison with a known standard) for samples containing the analyte and negative results for samples not containing it.

22. To confirm the absence of false positive results, an identification test may be carried out for substances with a similar structure or substances associated with the analyte.

23. The choice of potentially interfering substances should be justified.

Quantification and testing for impurities

24. When confirming specificity for an analytical procedure using a chromatographic separation method, representative chromatograms should be provided with proper identification of individual components. Similar approaches should be used to other separation based techniques.

25. Critical separations in chromatography should be studied at the appropriate level. In the case of critical separations, the resolution value of the 2 most closely eluting components should be set.

26. When using a non-specific quantification method, additional analytical methods should be used and the specificity of the entire set of methods should be confirmed. For example, if the quantitative determination is carried out by the titrimetric method during the release of the pharmaceutical substance, it can be supplemented by an appropriate test for impurities.

27. The approach is similar for both quantification and testing for impurities.

Presence of impurity samples

28. In the presence of impurity samples, the determination of the specificity of the analytical procedure is as follows:

a) when quantifying, it is necessary to confirm the selectivity of the determination of a substance in the presence of impurities and (or) other components of the sample. In practice, this is done by adding impurities and (or) excipients to the sample (pharmaceutical substance or medicinal product) in the appropriate amount and if there is evidence of the absence of their influence on the result of the quantitative determination of the active substance;

b) when testing for impurities, specificity can be established by adding certain amounts of impurities to the pharmaceutical substance or medicinal product and if there is evidence of the separation of these impurities from each other and (or) from other components of the sample.

No samples of impurities

29. If reference materials for impurities or degradation products are not available, specificity can be confirmed by comparing the test results of samples containing impurities or degradation products with the results of another validated method (for example, a pharmacopoeial or other validated analytical (independent) method). Where appropriate, impurity reference standards should include samples stored under specified stress conditions (light, heat, humidity, acid (base) hydrolysis and oxidation).

30. In the case of a quantitative determination, 2 results must be compared.

31. In the case of impurity tests, impurity profiles should be compared.

32. To prove that the peak of the analyte corresponds to only one component, it is advisable to conduct studies on the purity of the peaks (for example, the use of diode array detection, mass spectrometry).

3. Linearity

33. A linear relationship must be evaluated over the full range of application of the analytical technique. It can be confirmed directly on the pharmaceutical substance (by diluting the main standard solution) and (or) on separate samples of artificial (model) mixtures of drug components using the proposed method. The latter aspect can be studied in the course of determining the range of application (analytical area) of the methodology.

34. Linearity is assessed visually by plotting the analytical signal as a function of the concentration or amount of the analyte. If there is a clear linear relationship, the results obtained must be processed by suitable statistical methods (for example, by calculating the regression line using the least squares method). Mathematical transformation of test results may be required to obtain linearity between quantitation results and sample concentrations prior to regression analysis. The results of the analysis of the regression line can be used for a mathematical assessment of the degree of linearity.

35. If there is no linearity, the test data should be subjected to a mathematical transformation prior to regression analysis.

36. To confirm linearity, the correlation coefficient or coefficient of determination, the constant term of the linear regression, the tangent of the slope of the regression line and the residual sum of squared deviations must be determined and presented, and a graph with all experimental data is attached.

37. If linearity is not observed under any type of mathematical transformation (for example, when validating immunoassay methods), the analytical signal must be described using the appropriate function of the concentration (amount) of the analyte in the sample.

V. Application Range (Analytical Area)

39. The range of application of an analytical technique depends on its purpose and is determined in the study of linearity. Within the range of application, the technique should provide the required linearity, correctness and precision.

40. The following ranges of application (analytical areas) of analytical methods should be considered as the minimum allowable:

a) for the quantitative determination of the active substance in a pharmaceutical substance or medicinal product - from a concentration (content) of 80 percent to a concentration (content) of 120 percent of the nominal concentration (content);

b) for dosage uniformity - from a concentration (content) of 70 percent to a concentration (content) of 130 percent, unless a wider range is justified for the medicinal product depending on the dosage form (for example, metered-dose inhalers);

c) for the dissolution test, ±20 percent (absolute) of the rated application range. For example, if the specifications for a modified release product cover a range from 20 percent in the first hour to 90 percent of the claimed content in 24 hours, the validated range of use should be 0 to 110 percent of the claimed content;

d) for the determination of impurities - from the limit of detection of an impurity to 120% of the value specified in the specification;

e) for impurities that are extremely potent or have a toxic or unexpected pharmacological effect, the limit of detection and the limit of quantitation should be commensurate with the level at which these impurities are to be controlled. In order to validate impurity testing methods used during development, it may be necessary to set the analytical area near the expected (possible) limit;

e) if quantitation and purity are being studied simultaneously with the same test and only a 100% standard is used, the relationship should be linear over the entire range of application of the analytical method from the reporting threshold for the impurity (in accordance with the rules for studying impurities in medicinal products and setting requirements to them in the specifications approved by the Eurasian Economic Commission) up to 120 percent content specified in the specification for quantitative determination.

VI. Right

41. Correctness should be established for the full range of application of the analytical procedure.

1. Quantitative determination of the active pharmaceutical substance

Pharmaceutical substance

42. Several methods of assessing correctness can be used:

application of an analytical technique to an analyzed substance with a known degree of purity (for example, to a standard material);

comparison of the results of the analysis obtained using a validated analytical method, and the results obtained using a method, the correctness of which is known, and (or) an independent method.

The conclusion about the correctness can be made after establishing the precision, linearity and specificity.

medicinal product

43. Several methods of assessing correctness can be used:

application of an analytical technique to artificial (model) mixtures of components of a medicinal product, to which a previously known amount of an analyte has been added;

in the absence of samples of all components of the medicinal product, it is possible to add a previously known amount of the pharmaceutical substance to the medicinal product or compare the results obtained using another method, the correctness of which is known, and (or) an independent method.

The conclusion about the correctness can be made after determining the precision, linearity and specificity.

2. Quantification of impurities

44. Accuracy is determined on samples (pharmaceutical substance and medicinal product) to which a known amount of impurities has been added.

45. In the absence of samples of detectable impurities and (or) degradation products, it is acceptable to compare the results with the results obtained using an independent method. The use of an analytical signal of the active substance is allowed.

46. ​​It is necessary to indicate the specific way of expressing the content of individual impurities or their sum (for example, in mass percent or in percent in relation to the peak area, but in all cases in relation to the main analyte).

47. Accuracy is assessed for at least 9 determinations at 3 different concentrations covering the entire range of application (i.e. 3 concentrations and 3 replicates for each concentration). Definitions should include all steps of the methodology.

48. Accuracy is expressed by the openness value as a percentage based on the results of a quantitative determination of a substance added in a known amount to the analyzed sample, or the difference between the obtained average and true (reference) values, taking into account the appropriate confidence intervals.

VII. precision

49. Validation of tests for quantitation and impurities involves the determination of precision.

50. Precision is set at 3 levels: repeatability, intermediate precision and reproducibility. Precision should be established using uniform, authentic samples. If it is impossible to obtain a homogeneous sample, it is allowed to determine the precision using artificially prepared (model) samples or a sample solution. The precision of an analytical procedure is usually expressed in terms of the variance, standard deviation, or coefficient of variation of a series of measurements.

VIII. Repeatability

51. Repeatability is determined by performing at least 9 concentration determinations within the range of application of the analytical method (3 concentrations and 3 repetitions for each concentration), or at least 6 concentration determinations for samples with 100% content of the analyte.

IX. Intermediate (intralaboratory) precision

52. The extent to which intermediate precision is established depends on the conditions under which the analytical method is used. The applicant must establish the influence of random factors on the precision of the analytical procedure. Typical investigated (variable) factors are different days, analysts, equipment, etc. It is not necessary to study these influences separately. When studying the influence of various factors, it is preferable to use the design of the experiment.

X. Reproducibility

53. Reproducibility characterizes the precision in an interlaboratory experiment. Reproducibility should be determined in the case of standardization of an analytical method (for example, when it is included in the Pharmacopoeia of the Union or in the pharmacopoeias of the Member States). The inclusion of reproducibility data in the registration dossier is not required.

XI. Data representation

54. For each type of precision, the standard deviation, the relative standard deviation (coefficient of variation) and the confidence interval should be reported.

XII. Limit of detection

55. Different approaches to determining the limit of detection are possible depending on whether the technique is instrumental or non-instrumental. Other approaches may also be used.

XIII. visual assessment

56. Visual assessment can be used for both non-instrumental and instrumental methods. The limit of detection is established by analyzing samples with known concentrations of the analyte and determining its minimum content at which it is reliably detected.

XIV. Evaluation of the limit of detection in terms of signal-to-noise ratio

57. This approach is applicable only to analytical procedures for which baseline noise is observed.

58. The determination of the signal-to-noise ratio is carried out by comparing the signals obtained from samples with known low concentrations with the signals obtained from blank samples and establishing the minimum concentration at which the analyte can be reliably detected. To estimate the detection limit, a signal-to-noise ratio of 3:1 to 2:1 is considered acceptable.

XV. Estimation of the detection limit from the standard deviation of the analytical signal and the slope of the calibration curve

59. The limit of detection (LO) can be expressed as follows:

where:

60. The value of k is calculated from the calibration curve for the analyte. Estimating s can be done in several ways:

b) according to the calibration curve. It is necessary to analyze the resulting calibration curve, constructed for samples with the content of the analyte close to the detection limit. The residual standard deviation of the regression line or the standard deviation of the point of intersection with the y-axis (standard deviation of the free term of the linear regression) can be used as the standard deviation.

XVI. Data representation

61. It is necessary to specify the limit of detection and the method of its determination. If the determination of the limit of detection is based on a visual assessment or an assessment of the signal-to-noise ratio, the presentation of the relevant chromatograms is considered sufficient to justify it.

62. If the value of the limit of detection is obtained by calculation or extrapolation, the estimate must be confirmed by independent testing of a sufficient number of samples with the content of the analyte corresponding to the limit of detection or close to it.

XVII. Limit of Quantitation

63. The limit of quantitation is a necessary validation characteristic of procedures used to determine the low content of substances in a sample, in particular for the determination of impurities and/or degradation products.

64. Several approaches to determining the limit of quantitation are possible, depending on whether the technique is instrumental or non-instrumental. Other approaches are allowed.

XVIII. visual assessment

65. Visual assessment can be used for both non-instrumental and instrumental methods.

66. The limit of quantitation is usually established by analyzing samples with known concentrations of the analyte and estimating the minimum content at which the analyte is quantifiable with acceptable accuracy and precision.

XIX. Evaluation of the limit of quantification by signal-to-noise ratio

67. This approach is applicable only to measurement methods where baseline noise is observed.

68. The determination of the signal-to-noise ratio is carried out by comparing the measured signals obtained from samples with known low concentrations of the analyte, with the signals obtained from blank samples, and establishing the minimum concentration at which the analyte can be reliably quantified. The typical signal-to-noise ratio is 10:1.

XX. Evaluation of the limit of quantitation from the standard deviation of the signal and the slope of the calibration curve

69. The limit of quantification (LOQ) can be expressed as follows:

where:

s is the standard deviation of the analytical signal;

k is the tangent of the slope of the calibration curve.

70. The value of k is calculated from the calibration curve for the analyte. Estimating s can be done in several ways:

a) according to the standard deviation of a blank sample. The magnitude of the analytical signal is measured for a sufficient number of blank samples, and the standard deviation of their values ​​is calculated;

b) according to the calibration curve. It is necessary to analyze the resulting calibration curve, constructed for samples with the content of the analyte close to the limit of quantitative determination. The residual standard deviation of the regression line or the standard deviation of the point of intersection with the y-axis (standard deviation of the free term of the linear regression) can be used as the standard deviation.

XXI. Data representation

71. It is necessary to specify the limit of quantitation and the method of its determination.

72. The limit of quantitation must subsequently be confirmed by analyzing a sufficient number of samples containing analytes equal to or close to the limit of quantitation.

73. Other approaches than those listed above may be acceptable.

XXII. Stability (robustness)

74. The study of stability (robustness) should be carried out at the development stage, the scope of studies depends on the analytical method under consideration. It is necessary to show the reliability of the analysis under deliberate variations of the parameters (conditions) of the method.

75. If the results of measurements depend on changes in the conditions of application of the analytical procedure, it is necessary to strictly control compliance with such conditions or stipulate precautions during the test.

76. In order to ensure that the validity of an analytical method is maintained when used, one of the consequences of a robustness study should be to establish a series of system suitability parameters (eg a resolution test).

77. Common parameter variations are:

stability of solutions used in analytical procedures;

extraction time.

Variation parameters for liquid chromatography are:

changing the pH of the mobile phase;

change in the composition of the mobile phase;

different columns (different series and suppliers);

temperature;

mobile phase velocity (flow rate).

Variation parameters for gas chromatography are:

different columns (different series and suppliers);

temperature;

carrier gas velocity.

XXIII. System Suitability Assessment

78. System suitability assessment is an integral part of many analytical procedures. These tests are based on the concept that equipment, electronics, analytical operations and samples under analysis constitute a complete system and must be evaluated as such. System suitability criteria should be established for a particular method and depend on the type of analytical method being validated. Additional information can be obtained from the Union Pharmacopoeia or the pharmacopoeias of the Member States.



Electronic text of the document
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Eurasian Economic Union
www.eaeunion.org, 20.07.2018

Each instrumental method is characterized by a certain level of noise associated with the specifics of the measurement process. Therefore, there is always an abundance limit below which a substance cannot be reliably detected at all.

Limit of detection C min , P - the lowest content at which the presence of a component with a given confidence probability can be detected using this method.

The detection limit can also be set by the minimum analytical signal y min , which can be confidently distinguished from the signal of the control experiment - y background.

Statistical methods using the Chebyshev inequality proved that the limit of detection can be quantitatively determined using the expression

Where s background is the standard deviation of the background analytical signal; S is the sensitivity coefficient (sometimes called simply "sensitivity"), it characterizes the response of the analytical signal to the content of the component. The sensitivity coefficient is the value of the first derivative of the calibration function for a given concentration determination. For rectilinear calibration graphs, this is the tangent of the slope angle:

(Attention: don't confuse sensitivity factorS co standard deviations!)

There are other ways to calculate the limit of detection, but this equation is the most commonly used.

In quantitative chemical analysis, a range of detectable contents or concentrations is usually given. It means the range of values ​​of the determined contents (concentrations) provided for by this method and limited by the lower and upper limits of the determined concentrations.

Analytics are more often interested in the lower limit of determined concentrations With n or content m n component determined by this method. Beyond the lower limit of determined contents usually take the minimum amount or concentration that can be determined with a relative standard deviation

. .

Example

The mass concentration of iron in the solution was determined by the spectrophotometric method, by measuring the optical densities of solutions colored as a result of the interaction of the Fe 3+ ion with sulfosalicylic acid. To construct a calibration dependence, the optical densities of solutions with increasing (given) iron concentrations treated with sulfosalicylic acid were measured.

The optical densities of the reference solution (control experiment for reagents, i.e. without the addition of iron, (background) were 0.002; 0.000; 0.008; 0.006; 0.003.

Calculate limit of detection of iron.

Solution

1) As a result of calculations by the least squares method (see the example for control task No. 5), the values ​​​​for constructing a calibration graph were obtained.

Calculated values ​​for building a calibration graph

2) We calculate the sensitivity coefficient, i.e. the angular coefficient of the calibration dependence (S) according to the table.

3) Calculate background signal standard deviation, what is 0,0032 units of optical density.

4) The limit of detection will be, mg / cm 3

Control task number 6

Determine the detection limit for iron in water.

Initial data : the values ​​of the optical density of the background (reference solution) when constructing a calibration graph for the determination of iron amounted to 0.003; 0.001; 0.007; 0.005; 0.006; 0.003; 0.001; 0.005. The values ​​of optical densities corresponding to the concentrations of iron in the solution are presented in the table of control task No. 5.

Calculate the limit of detection of iron in mg/cm 3 according to the sensitivity coefficients S calculated on the basis of the data obtained for the construction of a calibration graph using the least squares method when performing control task No. 5;

Limit of Quantitation

"...Limit of quantitation (LOQ) (in analytical definitions): the lowest concentration or of an analyte in an analyte that can be quantified with an acceptable level of accuracy and confidence, as can be demonstrated by collaborative laboratory testing or other suitable method validation..."

Source:

"FOOD PRODUCTS. METHODS OF ANALYSIS FOR DETECTION OF GENETICALLY MODIFIED ORGANISMS AND PRODUCTS DERIVED FROM THEM. GENERAL REQUIREMENTS AND DEFINITIONS. GOST R 53214-2008 (ISO 24276:2006)"

(approved by the Order of Rostekhregulirovanie dated December 25, 2008 N 708-st)

Official terminology. Akademik.ru. 2012 .

See what the "Limit of Quantitation" is in other dictionaries:

limit of quantitation- 3.7 limit of quantification [ LOQ ] tenfold estimate of the standard deviation of the mass of a sample Note The LOQ value is used as the threshold value above which the mass ... ...

repeatability limit- 3.7 repeatability limit Dictionary-reference book of terms of normative and technical documentation

reproducibility limit- 2.9 reproducibility limit value below which, with a probability of 95 %, lies the absolute value of the difference between two test results obtained under reproducibility conditions Source … Dictionary-reference book of terms of normative and technical documentation

repeatability (convergence) limit 3.11 repeatability limit value which, with a confidence level of 95 %, is not exceeded by the absolute value of the difference between the results of two measurements (or tests) obtained under repeatability conditions ... Dictionary-reference book of terms of normative and technical documentation

Limit of intralaboratory precision- 3.11 within-laboratory precision limit: The absolute difference allowed for an assumed probability P between two analytical results obtained under within-laboratory precision conditions. Source … Dictionary-reference book of terms of normative and technical documentation

reproducibility limit R- 2.19.2 reproducibility limit R 2.19.1, 2.19.2 (Changed edition, title = Change No. 1, IUS 12 2002). ... ... Dictionary-reference book of terms of normative and technical documentation

MI 2881-2004: Recommendation. GSI. Methods of quantitative chemical analysis. Procedures for checking the acceptability of the results of the analysis- Terminology MI 2881 2004: Recommendation. GSI. Methods of quantitative chemical analysis. Procedures for checking the acceptability of the results of the analysis: 3.17 critical difference: The absolute difference allowed for the accepted probability of 95% between ... ... Dictionary-reference book of terms of normative and technical documentation

GOST R 50779.11-2000: Statistical methods. Statistical quality control. Terms and Definitions- Terminology GOST R 50779.11 2000: Statistical methods. Statistical quality control. Terms and definitions original document: 3.4.3 (upper and lower) regulation limits The limit on the control chart, above which the upper limit, ... ... Dictionary-reference book of terms of normative and technical documentation

GOST R 50779.10-2000: Statistical methods. Probability and bases of statistics. Terms and Definitions- Terminology GOST R 50779.10 2000: Statistical methods. Probability and bases of statistics. Terms and definitions original document: 2.3. (general) set The set of all considered units. Note For a random variable ... ... Dictionary-reference book of terms of normative and technical documentation

RMG 61-2003: State system for ensuring the uniformity of measurements. Indicators of accuracy, correctness, precision of methods of quantitative chemical analysis. Assessment Methods- Terminology RMG 61 2003: State system for ensuring the uniformity of measurements. Indicators of accuracy, correctness, precision of methods of quantitative chemical analysis. Evaluation methods: 3.12 within-laboratory precision: Precision … Dictionary-reference book of terms of normative and technical documentation

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