Printed by: Date: 11.10.

2014 GMP MANUAL © Maas & Peither AG

14.F Validation of analytical methods
Up06 Dr. Josef Künzle, Dr. Ralph Gomez

Here you will find answers to the following questions:

● What methods have to be validated?
● What parameters have to be validated?
● How is this to be documented?
● When should a revalidation be carried out?

14.F.1 Principles
The analytical methods used for batch testing must be validated in order to ensure that the results obtained are “correct and precise”. As illustrated in
Chapter 15.C.3 Testing procedures and test protocol, a series of points are to be described. The last and most important point is the “Validation” part.
Validation is defined in accordance with USP <1225> (Sucker, 1983)1.
Figure 14.F-1 Validation in accordance with H. Sucker
Validation of an analytical method is the process by which it is established, by laboratory studies that the performance characteristics of the method
meet the requirements for the intended analytical applications.

Four groups of techniques are distinguished in terms of their field of use:

● Identity test
● Quantitative determination of impurities
● Limit tests of impurities
● Assay determination

A further group includes the testing of special parameters (performance characteristics), e.g. testing the dissolution rate (DR) or the content uniformity
(CU).
Depending on the application group, the following parameters must be validated:
● Accuracy,
● Precision,
● Specificity/selectivity,
● LOD = Limit of Detection,
● LOQ = Limit of Quantitation and Linearity, (linear) range – and Robustness
Figure 14.F-1 summarizes the groups of techniques and the corresponding parameters to be validated.
Figure 14.F-2 Overview of technique validation
Parameter Identity Impurities Assay Other
quantitative limit
EC WH US EC WH US US EC WH US EC WH US

Precision R R R R R R R

Accuracy R R T R R R R T

LOD R R R T

LOQ R R T

Selectivity R R R R R R R R R R T

Linearity R R R R R R T

Range R R R R R R R T

Robustness R R R R R R R R

R = Required, T = Required depending on the test

EC Analytical Validation, Note for Guidance, III, 844/87, FINAL, August 1989 (Europe)
WH Validation of analytical procedures used in the examination of pharmaceutical materials,
WHO Technical Report Series 823, 1992
US USP <1225>

14.F.2 Definitions of the parameters

The parameters to be validated are listed below with their definitions. In addition, the method for validating the corresponding parameters is also specified.
14.F.2.1 Precision
Precision expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same
homogeneous sample under prescribed conditions. It is expressed as the variance, standard deviation or coefficient of variation (relative standard
deviation) and may be performed at three levels: repeatability, intermediate precision and reproducibility. The following are distinguished:
Repeatability n determinations are executed by a person quickly one after the other, under conditions as similar as possible. At least 6 100%
determinations are required (note: “100%” corresponds to the concentration of the test solution if the content of the analyte to be investigated holds
exactly 100% of the declaration), or at total of at least 9 determinations with 3 different concentrations (e.g. 3 determinations each at 80%, 100%,
120%).
Intermediate precision Variation of the determinations within a lab if executed by several persons, on different days using different equipment.
Reproducibility: Reproducibility from ring trials with the participation of several labs (cf. Determination of content of standards of Ph. Eur., for more
information: see Chapter 14.C Standards and reference substances).
The standard deviation, the relative standard deviation (coefficient of variation) and the confidence area must be specified.
14.F.2.2 Accuracy
Accuracy expresses the closeness of agreement between the value, which is accepted either as a conventional or an accepted reference value and the
value found. It can be determined with a known accuracy from a second technique, or through determination of the recovery rate. Here, different, known
quantities of analyte (API) are added to the sample to be investigated and the recovery is determined (spiked placebo method). The accuracy can also
be derived from the regression lines of the linearity. If zero is included in the confidence area of the axis section, the technique is considered to be
accurate, i.e. not tainted with a systematic error.
Validation should ensure that the results obtained are correct and precise. For clarification, please refer to the model of the target (see Figure 14.F-3).
Figure 14.F-3 Precision (P) versus accuracy (A)

Figure 14.F-3 graphically depicts the difference between Precision and Accuracy. Precision measures the degree of agreement among a series of
repetitive analyses but it does not address the agreement with the established true value. The latter is determined by Accuracy. Excellent Precision may
be obtained for a series of values, but due to a bias in the analytical method used, the average value obtained may not be in accordance with the true
value (see the target in the lower left corner of Figure 14.F-3). In such a case, the method must be revised to remove the bias.
14.F.2.3 LOD = Limit of Detection
The limit of detection is the smallest quantity of a substance in a sample (matrix) that can be detected but not necessarily quantitated as an exact value.
This can be determined in various ways. One possibility is the visual procedure, where the minimum detectable quantity is derived from samples of a
known concentration (dilution series). Often the signal/noise ratio (S/N) is used. Here, the noise of a blank is measured over a period of time and the limit
of detection is established from this with S/N = 3:1. Another approach uses the standard deviation of the calibration lines (“3σ-Procedure”, cf. Band 3 ICH
Q2B).
14.F.2.4 LOQ = Limit of Quantitation
The limit of quantitation is the smallest quantity of a substance in a sample (matrix) that can be quantified as an exact value with acceptable precision
and accuracy. This can be determined in various ways. As with the limit of detection, it can be determined visually or via the signal/noise ratio. Here, the
limit of quantitation is established with S/N = 10:1. Another approach uses the standard deviation of the calibration lines (“10σ Procedure”, cf. Band 3
ICH Q2B).
Note: The relevant chromatograms must be added to determine the limit of detection and the limit of quantitation.
14.F.2.5 Selectivity
Selectivity is the ability of the technique to detect an analyte that is free from any interference in the presence of other components, such as by-products
and degradation products, excipients (matrix) and other impurities.
During development of the technique for API characterization, forced decomposition tests are often executed. These aim to degrade the API in order to
be able to develop the technique optimally on this basis.
14.F.2.6 Linearity, Range
The aim of checking the linearity is to derive a direct proportionality between the detector signal and the concentration of a substance in the sample over
a certain range. The correlation coefficient, the slope and the intercept with the confidence level must be specified. In addition, a graphical representation
must be included.
14.F.2.7 Robustness
Robustness is a measure to establish that (deliberate) small changes in the parameters of the method do not influence the result. It is an indicator of the
reliability of a method in normal routine use. Possible influence factors are listed in Figure 14.F-4.
Figure 14.F-4 Factors that could influence robustness
Factors that could influence robustness

● Stability of the test solution (suitability for use with the autosampler)
● Completeness of the extraction

In particular in the HPLC, the following must be noted:

● pH of the mobile phase
● Composition of the mobile phase
● Columns (different column materials, batches, suppliers, dimensions)
● Temperature
● Flow rate

In particular in the GC, the following must be noted:

● Columns (different suppliers, different batches)
● Temperature
● Flow rate

In practice it has been shown that these investigations are usually very time-consuming, which can lead to the temptation to do without the
corresponding explanations. However, the benefit of routine use is quickly apparent and pays for itself many times over. It provides to other analysts
carrying out the analysis where possible problems might occur. Problems caused by methods that are not robust can lead to false analytical results,
which will require extensive OOS investigations. Above all, the identification of critical parameters and how to avoid potential problems should be
thoroughly described in the method validation report. In the current environment where methods often have to be transferred, the existence of robust
methods is a requirement to be ready for use at the new site more quickly and more reliably.

14.F.3 Documentation
Before starting to validate an analytical method, a protocol must also be created. As the validation parameters can be well structured, there are
companies that do not write project-specific validation protocols, but have created a manual for validation. This includes the principles according to which
validation should be carried out. However, currently in the United States, FDA investigators are used to seeing approved protocols for analytical validation
studies and have an expectation that individual protocols will be prepared for each study. Two important reasons for the expectation for the existence of
individual protocols are that the procedures to be carried out can be described in detail and the acceptance criteria for each measured parameter can be
specified.
The results of the validation can be given in the form of a report, which contains the validation of all methods for a product. This corresponds to the
method description (see Chapter 15.C.3 Testing procedures and test protocol).
Alternatively, it may be of use to supplement the testing procedure with the validation section. This offers the advantage of having the corresponding
documents always present with the method.

14.F.4 Revalidation
If changes are made to the method, partial or complete revalidation must be carried out. This includes, for example, the conversion of an assay
determination from UV measurement to HPLC/UV determination. If the sample preparation of a tablet is changed from extraction/filtration to Solid Phase
Extraction (SFE), this must be revalidated. Changes in equipment or modifications in manufacturing procedures are further reasons for revalidation. The
method must stay abreast of the new circumstances, for current GMP demands that the methods be state-of-the-art.
This is a very ambitious objective, where benefits and costs must be considered. For products that have been on the market for a long time, it is often
not worth the effort. However, it is clearly the responsibility of the companies to bring only faultless goods to the market. To this end, adequate analytical
methods are required. One advantage of revising methods is to curb the historical “uncontrolled growth of methods” and to establish uniform methods for
a product for the various administration forms, dosages and markets.
Summary
In principle, all analytical methods must be validated.
The parameters are based on the field of use.
A thorough validation (keyword: robustness) saves much aggravation later.
The form of documentation is not fixed.
The methods must be state-of-the-art or have to be revised to meet this requirement.

1Corresponding definitions can be found in USP (USP <1225>), the WHO-GMP Guidelines and in FDA/ICH (Sickmüller, 1995) (Throm, 1997) (ICH
Q2(R1).