On approval of guidelines for the validation of analytical methods for testing medicinal products. Limit of quantitation Validation of quantitation methods in aqueous solutions

24.11.202227.05.2017

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION

GENERAL PHARMACOPOEIAN ARTICLE

Validation of analytical methods OFS.1.1.0012.15

Introduced for the first time

Validation of an analytical method is experimental evidence that the method is suitable for solving the intended problems.

This general pharmacopoeial 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 for quantitative determination are subject to validation, including methods for determining impurities and methods for determining the content limit. Authentication methods are validated when it is necessary to confirm their specificity.

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

specificity;
detection limit;
quantitation limit;
analytical area (range);
linearity;
correctness (trueness);
precision;
robustness.

Table 1 - Characteristics of methods determined during validation

Name characteristics	Main types of techniques
	Authenticity test	Foreign matter		quantitation
	Authenticity test	Quantitative Methods	Content limit	Main active ingredient, standardized components	Active ingredient in the “Dissolution” test
Specificity **)	Yes	Yes	Yes	Yes	Yes
Detection limit	No	No	Yes	No	No
Limit of Quantitation	No	Yes	No	No	No
Analytical area	No	Yes	No	Yes	Yes
Linearity	No	Yes	No	Yes	Yes
Right	No	Yes	*	Yes	Yes
Precision : – repeatability (convergence) – intermediate (in-laboratory) precision
Sustainability	No	*	*	*	*

*) can be defined if necessary;

**) the lack of specificity of one analytical technique can be compensated by the use of another analytical technique.

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

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

Specificity

Specificity is the ability of an analytical technique to unambiguously assess the analyte in the presence of accompanying components.

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

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

1.1 For authenticity test methods

The validated method (or set of methods) must provide reliable information about the presence of a given active substance in a substance or dosage form if it contains the components specified in the recipe, which is subject to experimental confirmation.

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

1.2 For quantitation and impurity testing procedures

The quantitation and impurity testing method being validated is subject to the same approach: its specificity for the analyte must be assessed, i.e., it must be experimentally verified that the presence of concomitant components does not unduly influence the analytical result.

It is possible 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 using the validated and another, obviously specific method. The results of relevant experiments must be statistically processed.

The lack of test specificity may be compensated for by other additional test(s).

When validating methods, if appropriate, drug samples may be used that have been exposed to extreme conditions (light, temperature, humidity) or chemically modified by any suitable method to accumulate impurities.

For chromatographic techniques, the resolution between the two most closely eluting substances at appropriate concentrations is shown.

DETECTION LIMIT

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

The detection limit 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 visual assessment of the analysis result

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

2.2 For methods with instrumental assessment of the analysis result

2.2.1 By signal-to-noise ratio

This approach is applicable to methods for which baseline noise is observed. The magnitudes of the signals obtained for the control experiment and for samples with low concentrations of the analyte are compared. Set the minimum amount (concentration) of the analyte in the sample at which the ratio of the analytical signal to the noise level is 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 graph

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

PO = 3.3 · S/b,

Where S

b– sensitivity coefficient, which is the ratio of the analytical signal to the value being determined (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 quantities (concentrations) of the analyte that are close to the found value of the detection limit.

In general, if there is evidence that a procedure is suitable for reliably detecting a substance at concentrations both above and below its specification limits, it is not necessary to determine the actual detection limit for that procedure.

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 procedure with the required accuracy and within-laboratory (intermediate) precision.

The limit of quantitation is a necessary validation characteristic of methods used to assess small quantities (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 visual assessment of the analysis result

Test samples with various known quantities (concentrations) of the analyte and set the minimum value at which the analysis result can be obtained visually with the required accuracy and intra-laboratory (intermediate) precision.

3.2 For methods with instrumental assessment of the analysis result

3.2.1 By 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 Based on the standard deviation of the signal and the slope of the calibration graph

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

PKO = 10 · S/b,

Where S is the standard deviation of the analytical signal;

b– 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 quantitation, if necessary, can be confirmed by direct experiment at quantities (concentrations) of the substance being determined that are close to the found value of the limit of quantitation.

If there is data on the ability of a technique to reliably determine the analyte at concentrations above and below the norm of its content established in the specification, determining the real value of the limit of quantitation for such a technique, as a rule, is not required.

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 component being determined in the object of analysis (its quantity, concentration, activity, etc.). Within this range, the results obtained using the method being validated must 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:

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

– methods for assessing dosage uniformity 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;

– purity test methods must be applicable in the range from the “Limit of Quantitation” or “Limit of Detection” to 120% of the permissible content of the impurity being determined.

The analytical scope of the technique can be established from the range of experimental data that satisfies the linear model.

LINEARITY

Linearity of a 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 range of the technique.

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

y = b · x + a,

X- the amount or concentration of the analyte;

y– response magnitude;

b– angular coefficient;

a– free member (OFS “Statistical processing of chemical experiment results”).

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

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

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

RIGHT

The correctness of a technique is characterized by the deviation of the average result of determinations made using it from the value accepted as true.

The validated method is considered correct if the values accepted as true lie within the confidence intervals of the corresponding average test results obtained experimentally using this method.

To assess the correctness of quantitation methods, the following approaches are applicable:

a) analysis using a validated methodology of standard samples or model mixtures with a known content (concentration) of the substance being determined;

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

c) consideration of the results of studying the linearity of the validated method: if the free term in the equation given in Section 5 is not statistically significantly different from zero, then the use of such a method gives results free from systematic error.

For approaches “a” and “b”, it is possible to present the obtained data in the form of an equation of linear dependence (regression) between the experimentally found and true values. For this equation, hypotheses are tested about the equality of the tangent of the angle of inclination to unity b and about 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 the validated methodology gives correct, i.e., free from systematic error, results.

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 scattering is the value of the standard deviation of the result of an individual determination, obtained for a sample of a sufficiently large size.

Precision is assessed for any quantitative determination method based on the results of at least three determinations for each of the three levels of determined values (lower, middle and upper) lying within the analytical scope of the method. Repeatability can also be assessed for any quantitation procedure based on the results of at least six determinations for samples with close to nominal analyte content. In many cases, precision can be assessed based on the results of processing experimental data using the least squares method, as indicated in the General Pharmacopoeia Monograph “Statistical processing of chemical experiment results.”

Precision should be studied on homogeneous samples and can be assessed in three ways:

– as repeatability (convergence);

– as intra-laboratory (intermediate) precision;

– as interlaboratory precision (reproducibility).

The results of assessing the analytical technique for each of the precision options are usually characterized by the corresponding value of the standard deviation of the result of a separate determination.

Usually, when developing an original method, the repeatability (convergence) of the results obtained using it is determined. If it is necessary to include the developed method in the regulatory documentation, its intra-laboratory (intermediate) precision is additionally determined. The interlaboratory precision (reproducibility) of a method is assessed upon its intended inclusion in a draft general pharmacopoeial monograph, a pharmacopoeial monograph, or in the regulatory documentation for pharmacopoeial reference materials.

7.1 Repeatability (convergence)

The repeatability of an analytical technique is assessed 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 validated method is assessed under the operating conditions of one laboratory (different days, different performers, different equipment, etc.).

7.3 Interlaboratory precision (reproducibility)

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

STABILITY

The stability of a validated method 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.

The robustness of a procedure should not be determined in relation to easily controlled analytical conditions. This dramatically reduces the need for dedicated sustainability studies.

Stability should only be studied when the procedure being validated is based on the use of particularly sensitive analytical methods, such as various types of chromatography and functional analysis. If necessary, the stability of the methodology is assessed at the stage of its development. If the stability of a method is likely to be low, its suitability must be checked directly during practical use.

Testing the suitability of the analytical system

Validation of the suitability of an analytical system is a check 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 to be analyzed. The requirements for such a system are usually specified in the general pharmacopoeial monograph for the corresponding analytical method. Thus, testing the suitability of the analytical system becomes a procedure included in the procedure being validated.

Presentation of validation results

The analytical procedure validation protocol should contain:

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

– characteristics being assessed;

– all primary results that were included in statistical data processing;

– results of statistical processing of data obtained experimentally during the development or testing of the validated method;

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

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

It is advisable to document the validation materials for individual analytical methods in the form of a combined validation report.

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

Detection limit C min , P – the lowest content at which this method can detect the presence of a component with a given confidence probability.

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 Chebyshev’s inequality have proven that the detection limit can be determined quantitatively using the expression

Where s background is the standard deviation of the analytical background signal; S - 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 straight-line calibration graphs, this is the tangent of the angle of inclination:

(attention: don't confuse sensitivity factorS with standard deviations!)

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

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

The analyst is often interested in the lower limit of determined concentrations With n or content m n component determined using 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, measuring the optical densities of solutions colored as a result of the interaction of the Fe 3+ ion with sulfosalicylic acid. To construct the calibration dependence, the optical densities of solutions with increasing (specified) 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 iron detection limit.

Solution

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

Calculated values for constructing a calibration graph

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

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

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

Test task No. 6

Determine the detection limit of iron in water.

Initial data : the optical density values of the background (reference solution) when constructing the calibration graph for the determination of iron were 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 detection limit of iron in mg/cm 3 using the sensitivity coefficients S calculated based on the data obtained to construct 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 analyte in an analyte sample that can be quantified with an acceptable level of precision and accuracy, as demonstrated by laboratory collaborative testing or other suitable method validation..."

Source:

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

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

Official terminology. Akademik.ru. 2012.

See what “Limit of Quantification” is in other dictionaries:

limit of quantitation- 3.7 limit of quantification (LOQ): A tenfold increase in the standard deviation of the sample mass. Note The LOQ value is used as a threshold value, above which the mass... ...

repeatability limit- 3.7 repeatability limit: The absolute difference between the results of the maximum and minimum values from the specified number of measurements performed under repeatability conditions according to GOST R ISO 5725 1. Source ... Dictionary-reference book of terms of normative and technical documentation

reproducibility limit- 2.9 reproducibility limit: The 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 limit (convergence)- 3.11 repeatability limit: A value that, with a confidence probability 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

Intralaboratory precision limit- 3.11 Limit of intra-laboratory precision: The absolute discrepancy allowed for the accepted probability P between two analytical results obtained under conditions of intra-laboratory precision. Source … Dictionary-reference book of terms of normative and technical documentation

reproducibility limit R- 2.19.2 reproducibility limit R: The absolute value of the difference between two test results under reproducibility conditions (see 2.19.1) with a confidence level of 95%. 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 analytical results- Terminology MI 2881 2004: Recommendation. GSI. Methods of quantitative chemical analysis. Procedures for checking the acceptability of analysis results: 3.17 critical difference: The absolute difference allowed for an accepted probability of 95% between ... ... Dictionary-reference book of terms of normative and technical documentation

GOST R 50779.11-2000: Statistical methods. Statistical quality management. Terms and Definitions- Terminology GOST R 50779.11 2000: Statistical methods. Statistical quality management. Terms and definitions original document: 3.4.3 (upper and lower) control 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 basic statistics. Terms and Definitions- Terminology GOST R 50779.10 2000: Statistical methods. Probability and basic statistics. Terms and definitions original document: 2.3. (general) population The set of all units considered. 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. Assessment methods: 3.12 intra-laboratory precision: Precision ... Dictionary-reference book of terms of normative and technical documentation

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 framework of 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 Validation of Analytical Methods for Drug Testing

APPROVED
By decision of the Board
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 referred to as the member states, respectively). Union).

2. The purpose of validating an analytical procedure for testing medicinal products is documented confirmation of its suitability for its intended purpose.

II. Definitions

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

"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, standard samples, reagents, use of equipment, construction of a calibration curve, calculation formulas used, etc.);

“reproducibility” is a property characterizing precision in interlaboratory tests;

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

“linearity” is a directly proportional dependence of the analytical signal on the concentration (amount) of the analyte in the sample within the range of application (analytical area) of the technique;

“recovery” (recovery) - the ratio between the obtained average and the true (reference) values, taking into account the corresponding 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 the same reagents, etc.) for a short period of time;

“correctness” (accuracy, trueness) - the closeness between the accepted true (reference) value and the resulting value, which is expressed by the openability value;

“quantitation limit” - the smallest amount of a substance in a sample that can be quantified with appropriate precision and accuracy;

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

“precision” (precision) - an expression of the closeness (degree of scatter) of results (values) between a series of measurements carried out on multiple samples taken from the same homogeneous sample, under the conditions prescribed by the method;

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

“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;

“robustness” is the ability of an analytical technique to be resistant to the influence of small specified changes in test conditions, which indicates its reliability under normal (standard) use.

III. Types of analytical methods to be validated

4. This Guide discusses approaches to validation of the 4 most common types of analytical methods:

a) tests for identification (authenticity);

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

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

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

5. All analytical methods used to control the quality 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 these Guidelines (for example, dissolution tests or determination of particle size (dispersity) of a pharmaceutical substance, etc.).

6. Identification (authenticity) tests usually consist of comparing the properties (for example, spectral characteristics, chromatographic behavior, chemical activity, etc.) of the test sample and the standard sample.

7. Tests to determine the quantitative content of impurities and tests to determine the limiting content of impurities in a sample are aimed at correctly describing the purity of the sample. The requirements for the validation of methods for the quantitative determination of impurities differ from the requirements for the validation of methods for determining the limiting content of impurities in a sample.

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

The purpose of analytical procedures must be clearly defined, since this determines the choice of validation characteristics that must be assessed during validation.

9. The following typical validation characteristics of an analytical procedure are subject to assessment:

a) accuracy (trueness);

b) precision:

repeatability;

intermediate (intralaboratory) precision;

c) specificity;

d) detection limit;

e) quantitation limit;

f) linearity;

g) range of application (analytical area).

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

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


Validation		Type of analytical procedure
characteristic		tests for identification	impurity tests		quantitative tests
		(authenticity)	quantitative content	limit content	dissolution (measurement only), content (activity)
Right
Precision
	repeatability
	intermediate precision
Specificity**
Detection limit
Limit of Quantitation
Linearity
Range of application

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

** The lack of specificity of one analytical technique can be compensated by the use of one or more additional analytical techniques.

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

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

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

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

changing the synthesis scheme of a pharmaceutical substance;

change in the composition of the medicinal product;

change in analytical methodology.

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

IV. Methodology for validation of analytical methods

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

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

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

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

14. It is permissible to use approaches other than those set out in these Guidelines. The selection of the validation procedure and protocol is the responsibility of the applicant. In this case, the main goal of validating an analytical method is to confirm the suitability of the method for its 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 study, reference materials with known, documented characteristics should be used. The required degree of purity of standard samples depends on the intended purpose.

16. Various validation characteristics are discussed in separate subsections of this section. The structure of this section reflects the progress of the analytical methodology development and evaluation process.

17. Experimental work should be designed so that relevant validation characteristics are studied simultaneously, obtaining reliable data on the capabilities of the analytical procedure (eg, specificity, linearity, range of application, accuracy and precision).

2. Specificity

18. Specificity studies should be performed during validation of identification, impurity and quantitation tests. Procedures for confirming specificity depend on the intended purpose of the analytical procedure.

19. The method of confirming specificity depends on the tasks for which the analytical technique is intended to solve. It is not always possible to confirm that an analytical procedure is specific for a given analyte (complete selectivity). In this case, it is recommended to use a combination of 2 or more analytical techniques.

The lack of specificity of one analytical technique can be compensated by the use of one or more additional analytical techniques.

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

a) when testing for identification - confirmation that the method allows the identification of the substance being determined;

b) when testing for impurities, confirmation that the procedure can correctly identify impurities in the sample (for example, testing for related compounds, heavy metals, residual solvent content, etc.);

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

Identification

21. A satisfactory identification test must be capable of distinguishing between structurally closely related compounds that may be present in the sample. The selectivity of an analytical procedure can be demonstrated by obtaining positive results (perhaps by comparison with a known reference 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 can be carried out for substances with a similar structure or substances accompanying the substance being determined.

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

Quantification and testing for impurities

24. When demonstrating specificity for an analytical procedure using a chromatographic separation method, representative chromatograms should be provided, with individual components properly identified. Similar approaches should be taken 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 closest eluting components should be set.

26. When using a nonspecific quantitation method, additional analytical techniques should be used and the specificity of the entire set of techniques should be confirmed. For example, if the quantitative determination is carried out using a titrimetric method when releasing a pharmaceutical substance, it can be supplemented with an appropriate test for impurities.

27. The approach is similar for both quantitation and impurity testing.

Availability of impurity samples

28. If samples of impurities are available, the determination of the specificity of an analytical procedure is as follows:

a) during quantitative determination, it is necessary to confirm the selectivity of the determination of the substance in the presence of impurities and (or) other components of the sample. In practice, this is done by adding impurities and (or) excipients in an appropriate amount to the sample (pharmaceutical substance or drug product) and if there is evidence that they do not influence the result of the quantitative determination of the active substance;

b) in impurity testing, specificity can be established by adding impurities to the pharmaceutical substance or drug product in specified quantities and providing evidence of the separation of these impurities from each other and (or) from other components of the sample.

No impurity samples

29. If standard samples of 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 procedure (for example, a pharmacopoeial or other validated analytical (independent) procedure). Where appropriate, impurity reference standards should include samples subjected to storage under specified stress conditions (light, heat, humidity, acid (base) hydrolysis and oxidation).

30. In the case of quantitative determination, it is necessary to compare 2 results.

31. In the case of impurity testing, impurity profiles must be compared.

32. To prove that the peak of the analyte is determined by 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. The linear relationship must be assessed over the entire 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 individual samples of artificial (model) mixtures of drug components using the proposed method. The latter aspect can be studied during the determination of the range of application (analytical area) of the technique.

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 using suitable statistical methods (for example, by calculating a regression line using the least squares method). To obtain linearity between assay results and sample concentrations, mathematical transformations of test results may be required prior to regression analysis. The results of regression line analysis can be used to mathematically estimate the degree of linearity.

35. In the absence of linearity, test data should be subjected to mathematical transformation before performing regression analysis.

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

37. If linearity is not observed with any type of mathematical transformation (for example, during the validation of immunoanalytical methods), the analytical signal must be described using an appropriate function of the concentration (amount) of the analyte in the sample.

V. Range of application (analytical area)

39. The range of application of an analytical technique depends on its purpose and is determined by studying linearity. Within the range of application, the procedure must provide the required linearity, accuracy and precision.

40. The following ranges of application (analytical areas) of analytical methods should be considered as minimally acceptable:

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 uniformity of dosage - 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 dissolution testing - ±20 percent (absolute) of the nominal application range. For example, if the specifications for a modified-release drug cover the range from 20 percent in the first hour to 90 percent of the declared content in 24 hours, the validated application range should be from 0 to 110 percent of the declared content;

d) for determining impurities - from the impurity detection limit to 120 percent value specified in the specification;

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

f) If assay and purity are studied simultaneously in a single test and only the 100% standard is used, the relationship should be linear over the entire range of application of the analytical procedure from the reporting threshold for the impurity (in accordance with the rules for the study of impurities in medicinal products and the establishment of requirements to them in specifications approved by the Eurasian Economic Commission) up to 120 percent content specified in the specification for quantitative determination.

VI. Right

41. Accuracy must be established for the entire range of application of the analytical procedure.

1. Quantitative determination of the active pharmaceutical substance

Pharmaceutical substance

42. Several methods for assessing correctness can be used:

application of an analytical procedure to an analyte of known purity (for example, to a standard material);

comparison of analytical results obtained using a validated analytical procedure and results obtained using a known procedure and/or an independent procedure.

A conclusion about accuracy can be made after establishing precision, linearity, and specificity.

medicinal product

43. Several methods for assessing correctness can be used:

application of analytical techniques to artificial (model) mixtures of drug components, to which a pre-known amount of the 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 accuracy of which is known, and (or) an independent method.

A conclusion about accuracy can be made after determining precision, linearity and specificity.

2. Quantitative determination of impurities

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

45. In the absence of samples of identifiable impurities and (or) degradation products, comparison of results with results obtained using an independent technique is acceptable. The use of an analytical signal of the active substance is allowed.

46. A specific method of expressing the content of individual impurities or their sum should be specified (for example, as a percentage by weight or as a percentage of peak area, but in all cases relative to the main analyte).

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

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

VII. Precision

49. Validation of quantitation and impurity tests involves determination of precision.

50. Precision is established at 3 levels: repeatability, intermediate precision and reproducibility. Precision should be established using homogeneous, authentic samples. If it is impossible to obtain a homogeneous sample, it is permissible to determine precision using artificially prepared (model) samples or a sample solution. The precision of an analytical technique 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 procedure (3 concentrations and 3 replicates for each concentration), or at least 6 concentration determinations for samples with 100% analyte content.

IX. Intermediate (in-laboratory) precision

52. The degree to which intermediate precision is established depends on the conditions of use of the analytical technique. The applicant must establish the influence of random factors on the precision of the analytical procedure. Typical factors studied (variables) 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 experimental design.

X. Reproducibility

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

XI. Data presentation

54. For each type of precision, it is necessary to indicate the standard deviation, relative standard deviation (coefficient of variation) and confidence interval.

XII. Detection limit

55. Various approaches to determining the detection limit are possible, depending on whether the technique is instrumental or non-instrumental. Other approaches can also be used.

XIII. visual assessment

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

XIV. Estimation of the detection limit based on the signal-to-noise ratio

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

58. 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 assess 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 (LOD) can be expressed as follows:

Where:

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

b) according to the calibration curve. The resulting calibration curve, constructed for samples with an analyte content close to the detection limit, should be analyzed. The residual standard deviation of the regression line or the standard deviation of the point of intersection with the ordinate axis (standard deviation of the free term of linear regression) can be used as the standard deviation.

XVI. Data presentation

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

62. If the detection limit value is obtained by calculation or extrapolation, the estimate must be confirmed by independent testing of a sufficient number of samples containing the analyte at or near the detection limit.

XVII. Limit of Quantitation

63. The limit of quantitation is a necessary validation characteristic of methods used to determine low levels 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 may be used.

XVIII. visual assessment

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

66. The limit of quantitation is usually established by analyzing samples with known concentrations of the analyte and assessing the minimum concentration at which the analyte can be quantified with acceptable accuracy and precision.

XIX. Estimation of the limit of quantification from the signal-to-noise ratio

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

68. 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. Estimation of the limit of quantitation from the standard deviation of the signal and the slope of the calibration curve

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

Where:

s is the standard deviation of the analytical signal;

k is the tangent of the angle of inclination of the calibration curve.

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

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

b) according to the calibration curve. The resulting calibration curve, constructed for samples with an analyte content close to the limit of quantitation, should be analyzed. The residual standard deviation of the regression line or the standard deviation of the point of intersection with the ordinate axis (standard deviation of the free term of linear regression) can be used as the standard deviation.

XXI. Data presentation

71. It is necessary to indicate 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 the analyte at or near the limit of quantitation.

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

XXII. Stability (robustness)

74. The study of stability (robustness) must be carried out at the development stage; the scope of research depends on the analytical technique under consideration. It is necessary to demonstrate the reliability of the analysis with deliberate variations in the parameters (conditions) of the method.

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

76. To ensure that the validity of an analytical procedure is maintained during its use, one of the consequences of robustness studies should be the establishment of a series of system suitability parameters (eg, a resolution test).

77. Common variations of parameters are:

stability of solutions used in analytical techniques;

extraction time.

The variation parameters for liquid chromatography are:

change in pH of the mobile phase;

change in the composition of the mobile phase;

different speakers (different series and suppliers);

temperature;

velocity of the mobile phase (flow rate).

The variation parameters for gas chromatography are:

various speakers (different series and suppliers);

temperature;

carrier gas velocity.

XXIII. System Suitability Assessment

78. Assessing system suitability is an integral part of many analytical techniques. These tests are based on the concept that the equipment, electronics, analytical operations and samples analyzed constitute a complete system and must be evaluated as such. System suitability criteria must be established for a specific procedure and depend on the type of analytical procedure being validated. Further information can be obtained from the Pharmacopoeia of the Union or from the pharmacopoeias of the Member States.

Electronic document text
prepared by Kodeks JSC and verified against:
official site
Eurasian Economic Union
www.eaeunion.org, 07/20/2018