64 
 
Guidance for Industry 
 
V :
ALIDATION OF ANALYTICAL PROCEDURES  
M  
ETHODOLOGY
 
 FINAL GUIDANCE
   
(October 21, 2010, this guidance document was revised to update contact information, correct broken 
internet links and minor formatting changes) 
 
This guidance document provides recommendations on how to consider various validation 
characteristics for each analytical procedure included as part of registration applications. 
for approval of veterinary medicinal products submitted to the European Union, Japan 
and the United States. The implementation date is set for October 1999. 
 
This guidance represents current thinking and does not create or confer any rights for or on any 
person and does not operate to bind FDA or the public.  An alternative approach may be used if 
such approach satisfies the requirements of the applicable statute, regulation, or both.  
 
Submit comments on this guidance at any time.  Submit written comments to the Division of 
Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Room 
1061, Rockville, MD 20852.  Comments may also be submitted electronically on the Internet at 
http://www.regulations.gov.   
 
 For questions regarding this document, contact Rebecca Owen, Center for Veterinary Medicine, 
(HFV-140), Food and Drug Administration, 7500 Standish Place, Rockville, MD 20855, 240-
276-9842, E-mail: Rebecca.Owen@fda.hhs.gov. 
 
Additional copies of this guidance document may be requested from the Communications Staff, 
HFV-12, Center for Veterinary Medicine, U.S. Food and Drug Administration, 7519 Standish 
Place, Rockville, MD  20855, and may be viewed on the Internet at 
http://www.fda.gov/AnimalVeterinary/default.htm.  
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
JULY 1999 
     
VICH GL2 (VALIDATION METHODOLOGY) 
October 1998 
For implementation Step 7 
 
 
 
 
 
 
V A P : 
  ALIDATION OF  NALYTICAL  ROCEDURES
M  
ETHODOLOGY
 
 
 
 
 
 
Recommended for Implementation  
at Step 7 of the VICH Process 
on 22 October 1998 
by the VICH Steering Committee 
 
 
 
 
 
 
 
 
 
 
 
 
 
THIS GUIDANCE HAS BEEN DEVELOPED BY THE APPROPRIATE VICH EXPERT WORKING GROUP ON THE 
BASIS OF THE ICH GUIDELINES ON THE SAME SUBJECT AND HAS BEEN SUBJECT TO CONSULTATION BY THE 
PARTIES, IN ACCORDANCE WITH THE VICH PROCESS.  AT STEP 7 OF THE PROCESS THE FINAL DRAFT IS 
RECOMMENDED FOR ADOPTION TO THE REGULATORY BODIES OF THE EUROPEAN UNION, JAPAN AND USA. 
Pa ge 2   
  VALIDATION OF ANALYTICAL PROCEDURES: 
METHODOLOGY 
 
TABLE OF CONTENTS 
 
INTRODUCTION..........................................................................................................................4 
1.Specificity.............................................................................................................................4 
1.1.  Identification...................................................................................................5 
1.2.  Assay and Impurity Test(s).............................................................................5 
2.LINEARITY.........................................................................................................................6 
3.RANGE.................................................................................................................................7 
4.ACCURACY........................................................................................................................8 
4.1.  Assay...............................................................................................................8 
4.2.  Impurities (Quantitation)................................................................................8 
4.3.  Recommended Data........................................................................................9 
5.PRECISION..........................................................................................................................9 
5.1.  Repeatability...................................................................................................9 
5.2.  Intermediate Precision....................................................................................9 
5.3.  Reproducibility.............................................................................................10 
5.4.  Recommended Data......................................................................................10 
6.DETECTION LIMIT..........................................................................................................10 
6.1.  Based on Visual Evaluation..........................................................................10 
  6.2. Based on Signal-to-Noise..............................................................................10 
6.3  Based on the Standard Deviation of the Response and the Slope....................11 
6.4  Recommended Data..........................................................................................11 
7.QUANTITATION LIMIT..................................................................................................12 
7.1.  Based on Visual Evaluation..........................................................................12 
7.2.  Based on Signal-to-Noise Approach.............................................................12 
7.3.  Based on the Standard Deviation of the Response and the Slope................12 
7.4  Recommended Data..........................................................................................13 
8 .ROBUSTNESS..................................................................................................................13 
9. SYSTEM SUITABILITY TESTING................................................................... 14 
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VALIDATION OF ANALYTICAL PROCEDURES: 
METHODOLOGY 
INTRODUCTION 
 
This document is complementary to the parent document which presents a discussion of the 
characteristics  that  should  be  considered  during  the  validation  of  analytical  procedures.  Its 
purpose is to provide some guidance and recommendations on how to consider the various 
validation  characteristics  for  each  analytical  procedure  included  as  part  of  a  registration 
application for approval of veterinary medicinal products submitted to the European Union, 
Japan, and the United States.  In some cases (for example, demonstration of specificity), the 
overall capabilities of a number of analytical procedures in combination may be investigated in 
order to ensure the quality of the drug substance or drug product. In addition, the document 
provides an indication of the data which should be presented in a registration application. 
 
All  relevant  data  collected  during  validation  and  formulae  used  for  calculating  validation 
characteristics should be submitted and discussed as appropriate. 
 
Approaches other than those set forth in this guideline may be applicable and acceptable. It is the 
responsibility of the applicant to choose the validation procedure and protocol most suitable for 
their product. However it is important to remember that the main objective of validation of an 
analytical procedure is to demonstrate that the procedure is suitable for its intended purpose.  
Due to their complex nature, analytical procedures for biological and biotechnological products 
in some cases may be approached differently than in this document. 
 
Well-characterized reference materials, with documented purity, should be used throughout the 
validation study. The degree of purity necessary depends on the intended use. 
 
In accordance with the parent document, and for the sake of clarity, this document considers the 
various validation characteristics in distinct sections. The arrangement of these sections reflects 
the process by which an analytical procedure may be developed and evaluated. 
 
In practice, it is usually possible to design the experimental work such that the appropriate 
validation  characteristics  can  be  considered  simultaneously  to  provide  a  sound,  overall 
knowledge of the capabilities of the analytical procedure, for instance: specificity, linearity, 
range, accuracy and precision. 
 
1.  SPECIFICITY 
 
An investigation of specificity should be conducted during the validation of identification 
tests, the determination of impurities and the assay. The procedures used to demonstrate 
specificity will depend on the intended objective of the analytical procedure.  
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It  is  not  always  possible  to  demonstrate  that  an  analytical  procedure  is  specific  for  a 
particular analyte (complete discrimination). In this case a combination of two or more 
analytical procedures is recommended to achieve the necessary level of discrimination. 
 
  1.1.  Identification  
   
Suitable identification tests should be able to discriminate between compounds of 
closely related structures which are likely to be present. The discrimination of a 
procedure may be confirmed by obtaining positive results (perhaps by comparison 
with a known reference material) from samples containing the analyte, coupled with 
negative results from samples which do not contain the analyte. In addition, the 
identification  test  may  be  applied  to  materials  structurally  similar  to  or  closely 
related to the analyte to confirm that a positive response is not obtained. The choice 
of  such  potentially  interfering  materials  should  be  based  on  sound  scientific 
judgement with a consideration of the interferences that could occur.  
 
  1.2.  Assay and Impurity Test(s) 
   
For chromatographic procedures, representative chromatograms should be used to 
demonstrate specificity and individual components should be appropriately labelled. 
 Similar considerations should be given to other separation techniques.  
 
Critical separations in chromatography should be investigated at an appropriate level. 
For critical separations, specificity can be demonstrated by the resolution of the two 
components which elute closest to each other. 
 
In cases where a non-specific assay is used, other supporting analytical procedures 
should be used to demonstrate overall specificity. For example, where a titration is 
adopted to assay the drug substance for release, the combination of the assay and a 
suitable test for impurities can be used. 
 
The approach is similar for both assay and impurity tests: 
 
1.2.1  Impurities are available 
 
For the assay, this should involve demonstration of the discrimination of the analyte 
in the presence of impurities and/or excipients; practically, this can be done by 
spiking pure substances (drug substance or drug product) with appropriate levels of 
impurities and/or excipients and demonstrating that the assay result is unaffected by 
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  the presence of these materials (by comparison with the assay result obtained on 
unspiked samples). 
 
For  the  impurity  test,  the  discrimination  may  be  established  by  spiking  drug 
substance or drug product with appropriate levels of impurities and demonstrating 
the separation of these impurities individually and/or from other components in the 
sample matrix. 
 
1.2.2  Impurities are not available 
 
If impurity or degradation product standards are unavailable, specificity may be 
demonstrated  by  comparing  the  test  results  of  samples  containing  impurities  or 
degradation products to a second well-characterized procedure, e.g., pharmacopoeial 
method  or  other  validated  analytical  procedure  (independent  procedure).  As 
appropriate,  this  should  include  samples  stored under relevant stress conditions: 
light, heat, humidity, acid/base hydrolysis and oxidation.  
 
-  for the assay, the two results should be compared.  
-  for the impurity tests, the impurity profiles should be compared. 
Peak purity tests may be useful to show that the analyte chromatographic peak is not 
attributable to more than one component (e.g., diode array, mass spectrometry). 
 
2.  LINEARITY 
 
A linear relationship should be evaluated across the range (see section 3) of the analytical 
procedure. It may be demonstrated directly on the drug substance (by dilution of a standard 
stock  solution)  and/or  separate  weighings  of  synthetic  mixtures  of  the  drug  product 
components,  using  the  proposed  procedure.  The  latter  aspect  can  be  studied  during 
investigation of the range. 
 
Linearity should be evaluated by visual inspection of a plot of signals as a function of 
analyte concentration or content. If there is a linear relationship, test results should be 
evaluated by appropriate statistical methods, for example, by calculation of a regression line 
by the method of least squares. In some cases, to obtain linearity between assays and sample 
concentrations, the test data may need to be subjected to a mathematical transformation prior 
to the regression analysis. Data from the regression line itself may be helpful to provide 
mathematical estimates of the degree of linearity.  
 
The correlation coefficient, y-intercept, slope of the regression line and residual sum of 
squares should be submitted. A plot of the data should be included. In addition, an analysis 
Pa ge 6   
  of the deviation of the actual data points from the regression line may also be helpful for 
evaluating linearity. 
 
Some analytical procedures, such as immunoassays, do not demonstrate linearity after any 
transformation. In this case, the analytical response should be described by an appropriate 
function of the concentration (amount) of an analyte in a sample.  
 
For the establishment of linearity, a minimum of 5 concentrations is recommended. Other 
approaches should be justified. 
 
3.  RANGE  
 
The specified range is normally derived from linearity studies and depends on the intended 
application of the procedure. It is established by confirming that the analytical procedure 
provides an acceptable degree of linearity, accuracy and precision when applied to samples 
containing amounts of analyte within or at the extremes of the specified range of the 
analytical procedure.  
 
The following minimum specified ranges should be considered: 
 
-  for the assay of a drug substance or a finished (drug) product: normally from 80 to 120 
percent of the test concentration;  
-  for  content  uniformity,  covering  a  minimum  of  70  to  130  percent  of  the  test 
concentration, unless a wider more appropriate range, based on the nature of the dosage 
form, is justified; 
-  for dissolution testing: +/-20 % over the specified range; e.g., if the specifications for a 
controlled released product cover a region from 20%, after 1 hour, up to 90%, after 24 
hours, the validated range would be 0-110% of the label claim.  
-  for the determination of an impurity: from the reporting level of an impurity1 to 120% of 
the specification;  
   for  impurities  known  to  be  unusually  potent  or  to  produce  toxic  or  unexpected 
pharmacological effects, the detection/quantitation limit should be commensurate with 
the level at which the impurities must be controlled. 
Note: for validation of impurity test procedures carried out during development, it may be 
necessary to consider the range around a suggested (probable) limit; 
-  if assay and purity are performed together as one test and only a 100% standard is used, 
linearity should cover the range from the reporting level of the impurities1 to 120% of the 
assay specification; 
                                                 
1 see chapters AReporting Impurity Content of Batches@ of the corresponding VICH-Guidelines: AImpurities 
Pa ge 7   
   
4.  ACCURACY 
 
Accuracy should be established across the specified range of the analytical procedure.  
 
  4.1.  Assay 
  4.1.1  Drug Substance 
 
Several methods of determining accuracy are available: 
 
a) application  of  an  analytical  procedure  to  an  analyte  of  known  purity  (e.g., 
reference material); 
b) comparison of the results of the proposed analytical procedure with those of a 
second  well-characterized  procedure,  the  accuracy  of  which  is  stated  and/or 
defined (independent procedure, see 1.2.); 
c) accuracy  may  be  inferred  once  precision,  linearity  and  specificity  have  been 
established. 
 
4.1.2  Drug Product 
 
Several methods for determining accuracy are available: 
 
a) application of the analytical procedure to synthetic mixtures of the drug product 
components to which known quantities of the drug substance to be analysed have 
been added; 
b) in cases where it is impossible to obtain samples of all drug product components, it 
may be acceptable either to add known quantities of the  analyte to the drug 
product  or  to  compare  the  results  obtained  from  a  second  well-characterized 
procedure, the accuracy of which is stated and/or defined (independent procedure, 
see 1.2.). 
c) accuracy  may  be  inferred  once  precision,  linearity  and  specificity  have  been 
established. 
 
  4.2.  Impurities (Quantitation) 
                                                                                                                                                             
in New Drug Substances@ and AImpurities in New Drug Products@ 
 
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Accuracy should be assessed on samples (drug substance/drug product) spiked with 
known amounts of impurities. 
 
In  cases  where  it  is  impossible  to  obtain  samples  of  certain  impurities  and/or 
degradation products, it is considered acceptable to compare results obtained by an 
independent procedure (see 1.2.). The response factor of the drug substance can be 
used. 
 
It should be clear how the individual or total impurities are to be determined, e.g., 
weight/weight or area percent, in all cases with respect to the major analyte. 
 
  4.3.  Recommended Data 
 
Accuracy should be assessed using a minimum of 9 determinations over a minimum 
of  3  concentration  levels  covering  the  specified  range  (e.g.,  3  concentrations/3 
replicates each of the total analytical procedure). 
 
Accuracy should be reported as percent recovery by the assay of known added 
amount of analyte in the sample or as the difference between the mean and the 
accepted true value together with the confidence intervals. 
 
5.  PRECISION 
 
Validation of tests for assay and for quantitative determination of impurities includes an 
investigation of precision. 
 
  5.1.  Repeatability 
   
Repeatability should be assessed using: 
 
a) a minimum of 9 determinations covering the specified range for the procedure 
(e.g., 3 concentrations/3 replicates each) 
or 
 
b) a minimum of 6 determinations at 100% of the test concentration. 
 
  5.2.  Intermediate Precision 
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The extent to which intermediate precision should be established depends on the 
circumstances under which the procedure is intended to be used. The applicant 
should establish the effects of random events on the precision of the analytical 
procedure. Typical variations to be studied include days, analysts, equipment, etc. It 
is  not  considered  necessary  to  study  these  effects  individually.  The  use  of  an 
experimental design (matrix) is encouraged. 
 
  5.3.  Reproducibility 
Reproducibility is assessed by means of an inter-laboratory trial. Reproducibility 
should be considered in case of the standardization of an analytical procedure, for 
instance, for inclusion of procedures in pharmacopoeias. These data are not part of 
the marketing authorization dossier.  
 
  5.4.  Recommended Data 
The standard deviation, relative standard deviation (coefficient of variation) and 
confidence interval should be reported for each type of precision investigated. 
 
6.  DETECTION LIMIT 
 
Several approaches for determining the detection limit are possible, depending on whether 
the procedure is non-instrumental or instrumental. Approaches other than those listed below 
may be acceptable. 
 
  6.1.  Based on Visual Evaluation 
 
Visual evaluation may be used for non-instrumental methods but may also be used 
with instrumental methods. 
 
The  detection  limit  is  determined  by  the  analysis  of  samples  with  known 
concentrations of analyte and by establishing the minimum level at which the analyte 
can be reliably detected.  
  6.2. Based on Signal-to-Noise  
 
This approach can only be applied to analytical procedures which exhibit baseline 
noise. 
 
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  Determination  of  the  signal-to-noise  ratio  is  performed  by  comparing  measured 
signals  from samples with known low concentrations of analyte with those of blank 
samples and  establishing the minimum concentration at which the analyte can be 
reliably detected. A  signal-to-noise ratio between 3 or 2:1 is generally considered 
acceptable for estimating  the detection limit. 
 
  6.3  Based on the Standard Deviation of the Response and the Slope 
   
The detection limit (DL) may be expressed as: 
3.3  
DL = 
  S 
where  = the standard deviation of the response 
S = the slope of the calibration curve 
 
The slope S may be estimated from the calibration curve of the analyte. The estimate 
of  may be carried out in a variety of ways, for example:  
 
6.3.1  Based on the Standard Deviation of the Blank 
 
Measurement of the magnitude of analytical background response is performed by 
analyzing  an  appropriate  number  of  blank  samples  and  calculating  the  standard 
deviation of these responses.  
 
6.3.2  Based on the Calibration Curve 
 
A specific calibration curve should be studied using samples containing an analyte in 
the range of DL. The residual standard deviation of a regression line or the standard 
deviation of y-intercepts of regression lines may be used as the standard deviation.  
 
  6.4  Recommended Data 
The detection limit and the method used for determining the detection limit should be 
presented. If DL is determined based on visual evaluation or based on signal-to-noise 
ratio, the presentation of the relevant chromatograms is considered acceptable for 
justification. 
 
In cases where an estimated value for the detection limit is obtained by calculation or 
extrapolation,  this  estimate  may  subsequently  be  validated  by  the  independent 
analysis  of  a  suitable  number  of  samples  known  to  be  near  or  prepared  at  the 
detection limit.  
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  7. QUANTITATION LIMIT 
Several  approaches  for  determining  the  quantitation  limit  are  possible,  depending  on 
whether the procedure is non-instrumental or instrumental. Approaches other than those 
listed below may be acceptable. 
 
  7.1.  Based on Visual Evaluation 
 
Visual evaluation may be used for non-instrumental methods but may also be used 
with instrumental methods. 
 
The quantitation limit is generally determined by the analysis of samples with known 
concentrations of analyte and by establishing the minimum level at which the analyte 
can be quantified with acceptable accuracy and precision.  
 
  7.2.  Based on Signal-to-Noise Approach 
 
This approach can only be applied to analytical procedures that exhibit baseline 
noise. 
 
Determination  of  the  signal-to-noise  ratio  is  performed  by  comparing  measured 
signals from samples with known low concentrations of analyte with those of blank 
samples and by establishing the minimum concentration at which the analyte can be 
reliably quantified.  A typical signal-to-noise ratio is 10:1.  
 
 
  7.3.  Based on the Standard Deviation of the Response and the Slope 
   
The quantitation limit (QL) may be expressed as: 
10  
QL = 
  S 
where   = the standard deviation of the response 
S = the slope of the calibration curve 
 
The slope S may be estimated from the calibration curve of the analyte. The estimate 
of  may be carried out in a variety of ways for example:  
 
7.3.1  Based on Standard Deviation of the Blank 
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Measurement of the magnitude of analytical background response is performed by 
analyzing  an  appropriate  number  of  blank  samples  and  calculating  the  standard 
deviation of these responses.  
 
7.3.2  Based on the Calibration Curve 
 
A specific calibration curve should be studied using samples, containing an analyte 
in the range of QL. The residual standard deviation of a regression line or the 
standard deviation of y-intercepts of regression lines may be used as the standard 
deviation.  
 
  7.4  Recommended Data 
The quantitation limit and the method used for determining the quantitation limit 
should be presented. 
 
The limit should be subsequently validated by the analysis of a suitable number of samples 
known to be near or prepared at the quantitation limit.  
 
8.  ROBUSTNESS 
 
The  evaluation  of  robustness  should  be  considered  during  the  development  phase  and 
depends on the type of procedure under study. It should show the reliability of an analysis 
with respect to deliberate variations in method parameters. 
 
If  measurements  are  susceptible  to  variations  in  analytical  conditions,  the  analytical 
conditions should be suitably controlled or a precautionary statement should be included in 
the procedure. One consequence of the evaluation of robustness should be that a series of 
system suitability parameters (e.g., resolution test) is established to ensure that the validity 
of the analytical procedure is maintained whenever used. 
 
Examples of typical variations are: 
-  stability of analytical solutions, 
-  extraction time 
In the case of liquid chromatography, examples of typical variations are: 
-  influence of variations of pH in a mobile phase, 
-  influence of variations in mobile phase composition, 
-  different columns (different lots and/or suppliers), 
-  temperature, 
Pa ge 13   
  -  flow rate. 
In the case of gas-chromatography, examples of typical variations are: 
-  different columns (different lots and/or suppliers), 
-  temperature, 
-  flow rate. 
 
 
9.  SYSTEM SUITABILITY TESTING 
 
System suitability testing is an integral part of many analytical procedures. The tests are 
based on the concept that the equipment, electronics, analytical operations and samples to be 
analyzed constitute an integral system that can be evaluated as such. System suitability test 
parameters to be established for a particular procedure depend on the type of procedure 
being validated. See Pharmacopoeias for additional information. 
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