GUIDELINES ON ANALYTICAL METHOD OF VALIDATION

Introduction
Validation of analytical procedure is the process for proving that an analytical procedure is
suitable for its intended purpose. Results obtained from method validation study can be used
to judge the quality, reliability and consistency of analytical results. Several articles have been
published on the requirements of validation for analytical methods [1,2]. Green gave a practical
guide for analytical method validation with a set of requirements for a method [3]. For the
pharmaceutical industry, guidelines from the FDA [4−6] and US pharmacopoeia (USP) [7]
provide a framework for performing validation study. Unfortunately, some of the definitions
vary between the different organizations. To achieve harmonization for pharmaceutical
applications, International Conference on Harmonization (ICH) was organized, and
representatives from the pharmaceutical industry and regulatory agencies from the United
States, Europe and Japan defined validation characteristics, requirements and methodology for
analytical methods validation. For pharmaceutical analyses, an ICH guideline (Q2 (R1): Text
on Validation of Analytical procedures and Methodology [8]) was issued for performing
validation study. In this guideline, analytical procedures are classified into four categories.
These four types of analytical procedures are: 1) identification tests, 2) quantitative tests for
impurities, 3) limit tests for the control of impurities, 4) quantitative tests of the active moiety
in bulk active pharmaceutical ingredient, formulated product, or other selected components in
the formulated product. The assessment of validation characteristics should be based on the
intended use of the method, and the level of stringency is proportional to the criticality of the
analytical procedure in measurement. The ICH also recognizes that it is not always necessary
to evaluate every validation characteristics. For identification test, only the validation
characteristic of “specificity” should be established. Table 1 shows the two types of the
analytical method for chromatographic analysis, such as assay method for measurement of the
active moiety and impurity method for determination of target compounds at trace level, and
validation characteristics to be investigated. For assay method, evaluation of detection limit
(DL) and quantitation limit (QL) is not essential, because the target compound to be measured
exists at high level. For quantitative analysis, a determination of DL is not necessary. There are
no official guidelines on the sequence of validation experiments,
Protocol on analytical validation
The protocol on the validation study should include the following points in the validation study:
1) the purpose and scope of the analytical method, 2) the type of analytical method and
validation characteristics, 3) acceptance criteria for each validation characteristics.
Consideration on the following points will be useful to prepare the protocol. ・What type of
the samples will be measured by the analytical method? Will the samples be whole blood,
serum, plasma, purified protein, chemicals? Are there interfering substances contained in the
samples, if so, should they be detected or quantified? ・What is the expected concentration
range? ・What level of specificity, detection limit or quantitation limit, linearity, accuracy and
precision is required? The purpose of answering the questions described above is to determine
how best to meet the objective of the validation for analytical procedure.

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Following are the method of validation
Sr Method
no.
1 Specificity
2 Linearity and Range
3 Precision
4 Accuracy
5 Limit of detection
6 Robustness
7 Range

1)Specificity
One of the significant features of HPLC is its ability to generate signals free from interference.
Specificity refers to the ability of the analytical method to differentiate and quantify the analyte
in complex mixtures. An investigation of specificity is to be conducted during the
determination of impurities and validation of identification tests.
An ICH guideline defines specificity as ability to assess unequivocally the analyte in the
presence of other compounds that may be likely to be present.
A Typically these might be impurities, degradants, matrix, etc. The definition has the following
implications
Identification test: Identification tests should be able to differentiate compounds of closely
related structure which are expected to be present i.e., to assure identity of an analyte.
Purity test: To ensure that the analytical procedure performed allows an accurate statement of
content of the impurity of an analyte i.e. related substances, residual solvents content, heavy
metals,
Assay: To arrive at an accurate result, this permits a correct report on the potency or content of
analyte in a sample.
2)Linearity and Range
The linearity of a method is a measure of how well a calibration plot of response vs.
concentration approximates a straight line. Linearity can be assessed by performing single
measurements at several analyte concentrations. The data is then processed using a linear least-
squares regression. The resulting plot slope, intercept and correlation coefficient provide the
desired information on lineari ty.
3)Precision
The precision of an analytical procedure represents the nearness of agreement between series
of measurements got from multiple sampling of the same homogenous sample under the
similar analytical conditions and it is divided into 3 categories.
• Repeatability: precision under same operating conditions, same analyst over a short period
of time.
• Intermediate precision: method is tested on multiple days, instruments, analysts etc.
• Reproducibility: inter-laboratory studies. The ICH guidelines suggest that repeatability

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should be conformed duly utilizing at least 9 determinations with specified range for the
procedure (e.g., three concentrations / three replicates each) or a minimum of 6
determinations at 100 % of the test concentration
4)Accuracy
The accuracy of a measurement is defined as the closeness of the measured value to the true
value. In a method with high accuracy, a sample (whose “true value” is known) is analyzed
and the measured value is identical to the true value. Typically, accuracy is represented and
determined by recovery studies. There are three ways to determine accuracy:
1Comparison to reference standard.
2. Recovery of the analyte spiked into blank matrix.
3. Standard addition of the analyte. It should be clear how the individual or total impurities
are to be determined.

5)Limit of detection
LOD is determined by the analysis of samples with known concentration of analyte and by
establishing that minimum level at which the analyte can reliably detected, but not
necessarily quantitated as precise value, under the stated experimental conditions. The
detection limit is generally expressed in the concentration of analyte (ppm) in the sample. A
number of approaches are recommended by the ICH for determining the detection limit of
sample, depending on instrument used for analysis, nature of analyte and suitability of the
method.
The acceptable approaches are
• Visual evaluation.
• Signal-to-noise ratio.
• Standard deviation of the response.
• Standard deviation of the slope of linearity plot.
The formula for calculating
LOD is LOD = 3.3 δ/S (7)
Where δ = standard deviation of intercepts of calibration curves.
S = the slope of linearity plot.
Limit of quantitation
Limit of quantitation is the least concentration of drug in a sample which is estimated with
appropriate precision and accuracy under the affirmed experimental conditions. Similar to
LOD, ICH recommends the following four methods for estimation of LOQ. The acceptable
approaches are
• Visual evaluation. • Signal-to-noise ratio. • Standard deviation of the response. •
Standard deviation of the slope of linearity plot.
The formula for calculating LOQ is
LOQ = 10 δ/S (8)

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Where δ = standard deviation of response. S = Mean of slopes of the calibration curves. 4.8.
Robustness
6)Robustness
is defined by the measure of the capability of an analytical method to stay unchanged by
small deliberate changes in method parameters. The variable method parameters in HPLC
technique may involves flow rate, column temperature, sample temperature, pH and mobile
phase composition.
7)Range The range of an analytical method is the interval between the upper and lower levels
(including these levels) that have been demonstrated with precision, accuracy and linearity
using the analytical method. So, the acceptable range will be defined as the concentration
interval over which linearity, accuracy and precision are acceptable.
Conclusion
If the analysts in the pharmaceutical industry obtained the doubtful testing results through the
invalid analytical procedure, they would realize that much amount of time should be required
for solving problems. This kind of trouble would be avoided, provided that the validation study
is performed properly. A well−defined validation process provides evidence that the system
and method are suitable for its intended use. Performing a throughout validation study on an
analytical procedure can be a tedious process. However, once validation studies are completed,
the analysts can be confident in the ability of the analytical procedure to provide good
quantitation. We hope that we could provide a guide to help to understand how to perform a
validation study on an analytical procedure that generates both useful and meaning data. This
report focuses on performing a validation study for pharmaceuticals by HPLC system. This
validation approach would be applied to the analytical methods using GC, HPLC, GC−MS,
LC−MS for the biological samples or environmental pollution substances.
References
[1] Inman, E. L.; Frischmann, J. K.; Jimenez, P. J.; Winkle, G. D.; Persinger, M. L.; Rutherford,
B. S. J. Chromatogr. Sci. 1987, 25, 252−256.
[2] Shah, V. P.; Midha, K. K.; Dighe, S.; McGilverray, I. J.; Skey, J. P.; Yacobi, A.; Layoff,
T.; Viswanathan, C. T.; Cook, C. E.; McDowall, R. D.; Pittman, K. A.; Spector, S. Pharma.
Res. 1992, 9, 588−592.
[3] Green, J. M. Anal. Chem. 1996, 305A−309A.
[4] U.S. Department of Health and Human Services, Food and Drug Administration, Center
for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM). Guidance
for Industry, Bioanalytical Method Validation, 2001.
[5] U.S. Department of Health and Human Services, Food and Drug Administration, Center
for Drug Evaluation and Research (CDER). Reviewer Guidance, Validation of
chromatographic methods, 1994.
NAME:RUSHIKESH SANGAMESHWAR TEKALE
ID NO:2019H1460163P

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