Bioanalytical Method 
Validation 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Veterinary Medicine (CVM) 
 
 
May 2018 
Biopharmaceutics 
Bioanalytical Method Validation 
05/24/18   
Bioanalytical Method 
Validation 
Guidance for Industry 
 
 
 
 
Additional copies are available from: 
Office of Communications, Division of Drug Information  
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
and/or 
Policy and Regulations Staff, HFV-6 
Center for Veterinary Medicine 
Food and Drug Administration 
7500 Standish Place, Rockville, MD  20855 
http://www.fda.gov/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/default.htm 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Veterinary Medicine (CVM) 
 
 
May 2018 
Biopharmaceutics 
 
 
Bioanalytical Method Validation 
05/24/18  Contains Nonbinding Recommendations 
TABLE OF CONTENTS 
 
 
 
I.  INTRODUCTION ...................................................................................................... 1 
II.  BACKGROUND ........................................................................................................ 2 
III.  BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION ................. 4 
A.  Guiding Principles ........................................................................................................ 4 
B.  Bioanalytical Parameters of CCs and LBAs .................................................................... 5 
1. Reference Standards and Critical Reagents ........................................................................ 5 
2. Calibration Curve........................................................................................................... 6 
3. Quality Control Samples.................................................................................................. 7 
4. Selectivity and Specificity................................................................................................. 7 
5. Sensitivity...................................................................................................................... 8 
6. Accuracy, Precision, and Recovery ................................................................................... 8 
7. Stability ........................................................................................................................ 9 
8. Dilution Effects .............................................................................................................10 
9. Partial and Cross Validations..........................................................................................11 
C.  Validated Methods:  Expectations of In-Study Analysis and Reporting ............................12 
IV.  INCURRED SAMPLE REANALYSIS .................................................................... 14 
V.  ADDITIONAL ISSUES............................................................................................ 15 
A.  Endogenous Compounds ..............................................................................................15 
B.  Biomarkers .................................................................................................................15 
C.  Diagnostic Kits ............................................................................................................16 
D.  Bridging Data From Multiple Bioanalytical Technologies ...............................................17 
E.  Dried Blood Spots ........................................................................................................18 
VI.  DOCUMENTATION ............................................................................................... 18 
A.  Summary Information..................................................................................................18 
B.  Documentation for Method Validation and Bioanalytical Reports ...................................19 
VII.  APPENDIX............................................................................................................... 20 
Table 1.  Recommendations and Acceptance Criteria for Bioanalytical Method Validation and 
In-Study Conduct. ...............................................................................................................20 
Table 2.  Documentation and Reporting ................................................................................28 
Table 3.  Example of an Overall Summary Table for a Method Validation Report* or a Clinical 
Study Report.......................................................................................................................33 
Table 4.  Example of Summary Analytical Runs for a Bioanalytical Study Report....................36 
VIII.  GLOSSARY ............................................................................................................. 37 
 
Bioanalytical Method Validation 
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Bioanalytical Method Validation 
Guidance for Industry1 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page.   
 
 
 
 
I.  INTRODUCTION   
 
This guidance helps sponsors of investigational  new drug applications  (INDs) or applicants of 
new drug applications  (NDAs), abbreviated new drug applications  (ANDAs), biologic  license 
applications  (BLAs), and supplements validate bioanalytical  methods used in human clinical 
pharmacology,  bioavailability  (BA), and bioequivalence  (BE) studies that require 
pharmacokinetic,  toxicokinetic,  or biomarker concentration evaluation.2  This guidance can also 
inform the development of bioanalytical  methods used for nonclinical  studies that require 
toxicokinetic  or biomarker concentration data.  For studies related to the veterinary drug 
approval process such as investigational  new animal drug applications  (INADs), new animal 
drug applications  (NADAs), and abbreviated new animal drug applications  (ANADAs), this 
guidance may apply to blood and urine BA, BE, and pharmacokinetic  studies. 
 
The information  in this guidance applies to bioanalytical  procedures such as chromatographic 
assays (CCs) and ligand binding  assays (LBAs) that quantitatively  determine the levels of drugs,  
their metabolites,   therapeutic proteins, and  biomarkers in biological  matrices such as blood, 
serum, plasma, urine, and tissue such as skin.  
 
This final guidance incorporates public  comments to the revised draft published  in 2013 and 
provides recommendations  for the development,  validation,  and in-study use of bioanalytical 
methods.  The recommendations  can be modified  with justification,  depending  on the specific 
type of bioanalytical  method.  This guidance reflects advances in science and technology related 
to validating  bioanalytical  methods.   
 
In general, FDAs guidance documents do not establish legally  enforceable responsibilities.   
Instead, guidances describe the Agencys current thinking  on a topic and should be viewed only 
                                              
1 This guidance has been prepared by the Office of Clinical Pharmacology in the Center for Drug Evaluation and 
Research and the Center for Veterinary Medicine at the Food and Drug Administration.  
 
2 This guidance applies to both sponsors and applicants.  The use of the word sponsor applies to both sponsors and 
applicants and hence, INDs, NDAs, BLAs, and ANDAs. 
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as recommendations,  unless specific regulatory or statutory requirements are cited.  The use of 
the word should in Agency guidances means that something  is suggested or recommended, but 
not required.  
 
 
II.  BACKGROUND 
 
The 2001 guidance for industry on Bioanalytical Method Validation was originally  based on the 
deliberations  of two workshops described in publications  entitled:    
 
  Analytical Methods Validation: Bioavailability, Bioequivalence, and Pharmacokinetic 
Studies3  
 
 
  Bioanalytical Methods Validation: A Revisit With a Decade of Progress4   
 
Additional  workshops, summarized in the following  publications,  have informed subsequent 
revisions (e.g., the 2013 draft guidance for industry entitled Bioanalytical Method Validation5):    
 
  Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for 
Chromatographic and Ligand Binding Assays6   
 
  The AAPS/FDA Workshop on Incurred Sample Reanalysis7 
 
  The AAPS Workshop on Crystal City V  Quantitative Bioanalytical Method Validation 
and Implementation: 2013 Revised FDA Guidance8 
                                              
3 Shah, VP, KK Midha, S Dighe, IJ McGilveray, JP Skelly, A Yacobi, T Layloff, CT Viswanathan, CE Cook, RD 
McDowell, KA Pittman, S Spector, 1992, Analytical Methods Validation:  Bioavailability, Bioequivalence, and 
Pharmacokinetic Studies, Pharm Res, 9:588-592. 
 
4 Shah, VP, KK Midha, JW Findlay, HM Hill, JD Hulse, IJ McGilveray, G McKay, KJ Miller, RN Patnaik, ML 
Powell, A Tonelli, CT Viswanathan, A Yacobi, 2000, Bioanalytical Methods Validation:  A Revisit With a Decade 
of Progress, Pharm Res, 17:1551-1557.  
 
5 When final, this guidance will represent the FDAs current thinking on this topic.  For the most recent version of a 
guidance, check the FDA Drugs guidance Web page at 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
 
6 Viswanathan, CT, B Surendra, B Booth, AJ DeStefano, MJ Rose, J Sailstad, VP Shah, JP Skelly, PG Swann, R 
Weiner, 2007, Quantitative Bioanalytical Methods Validation and Implementation:  Best Practices for 
Chromatographic and Ligand Binding Assays, Pharm Res, 24:1962-1973.  
 
7 Fast, DM, M Kelley, CT Viswanathan, J OShaughnessy, SP King, A Chaudhary, R Weiner, AJ DeStefano, D 
Tang, 2009, Workshop Report and Follow-Up  AAPS Workshop on Current Topics in GLP Bioanalysis:  Assay 
Reproducibility for Incurred Samples  Implications of Crystal City Recommendations, AAPS J, 11:238-241. 
 
8 Booth, B, ME Arnold, B DeSilva, L Amaravadi, S Dudal, E Fluhler, B Gorovits, SH Haidar, J Kadavil, S Lowes, 
R Nicholson, M Rock, M Skelly, L Stevenson, S Subramaniam, R Weiner, E Woolf, 2015, Workshop Report:  
 
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Validated analytical methods for the quantitative  evaluation of analytes (i.e., drugs, including 
biologic  products, and their metabolites) and biomarkers in a given biological  matrix (e.g. blood, 
plasma, serum, or urine) are critical for the successful conduct of nonclinical,  biopharmaceutics, 
and clinical  pharmacology  studies.  These validated methods provide critical data to support the 
safety and effectiveness of drugs and biologic  products.  Validating  the analytical method 
ensures that the data are reliable by addressing certain key questions, including: 
 
  Does the method measure the intended analyte?  For example, does anything interfere 
with the measurement, and is the method specific or selective for the analyte? 
 
  What is the variability  associated with these measurements?  For example, what are the 
accuracy and precision of the method? 
 
  What is the range in measurements that provide reliable data?  For example, what is the 
sensitivity  of the method (e.g., what is the lower limit  of quantitation  (LLOQ) of the 
method, and what is the upper limit  of quantitation the method (ULOQ)?) 
 
  How do sample collection,  handling,  and storage affect the reliability  of the data from the 
bioanalytical  method?  For example, what steps need to be followed while collecting 
samples?  Do the samples need to be frozen during shipping?   What temperatures are 
required to store the samples, and how long can  the samples be stored? 
 
When changes are made to a validated method, the sponsor should conduct additional  validation 
(i.e., partial or cross validation). 
 
The fit-for-purpose (FFP) concept states that the level of validation  should be appropriate for the 
intended purpose of the study.  The key questions listed above should be evaluated relative to the 
stage of drug development.  Pivotal studies submitted in an NDA, BLA, or ANDA that require 
regulatory decision making for approval, safety or labeling,  such as BE or pharmacokinetic 
studies, should include bioanalytical  methods that are fully validated.  Exploratory  methods that 
would not be used to support regulatory decision making (e.g., candidate selection) may not 
require such stringent validation.   This FFP concept applies to drugs, their metabolites,  and 
biomarkers. 
 
The analytical laboratory conducting toxicology  studies for regulatory submissions  should 
adhere to 21 CFR 58, Good Laboratory Practices (GLPs).9 The bioanalytical  method for human 
  
BA, BE, and pharmacokinetic studies must meet the criteria specified in 21 CFR 320 
Bioequivalence  and Bioavailability  Requirements (i.e., 21 CFR 320.29).  
 
                                                                                                                                                    
Crystal City V  Quantitative Bioanalytical Method Validation and Implementation:  The 2013 Revised FDA 
Guidance, AAPS J, 17:277-288.  
9 For the Center for Veterinary Medicine, all BE studies are subject to Good Laboratory Practices. 
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The following  sections discuss the development,  validation,  and in-study use of bioanalytical 
methods and how best to document validation  methods and results.  Refer to the Glossary for the 
definitions  of assay parameters and analytical terms used in this guidance. 
 
 
III.  BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION     
 
A.  Guiding Principles  
 
The purpose of bioanalytical  method development is to define the design, operating conditions, 
limitations,  and suitability  of the method for its intended purpose and to ensure that the method is 
optimized  for validation.    
 
Before the development of a bioanalytical  method, the sponsor should understand the analyte of 
interest (e.g., determine the  physicochemical  properties of the drug, in vitro and in vivo 
metabolism,  and protein binding)  and consider aspects of any prior analytical methods that may 
be applicable.   
 
The elements and acceptance criteria of method development and validation  are summarized in 
Table 1.  Table 2 describes how the sponsor should document the development and validation  of 
the bioanalytical  assay and where it should be stored or submitted. 
 
Method development  involves optimizing  the procedures and conditions  involved  with extracting 
and detecting the analyte.  Method development includes the optimization  of the following 
bioanalytical  parameters (which are discussed in greater detail in section III.B) to ensure that the 
method is suitable for validation:    
 
  Reference standards  
  Critical reagents 
  Calibration  curve 
  Quality control samples (QCs) 
  Selectivity  and specificity 
  Sensitivity 
  Accuracy 
  Precision 
  Recovery 
  Stability  of the analyte in the matrix  
 
Bioanalytical  method development  does not require extensive record keeping or notation.  
However, the sponsor should record the changes to procedures as well as any issues and their 
resolutions  during development  of the bioanalytical  method to provide a rationale for any 
changes during the development of the method. 
 
Bioanalytical  method validation  proves that the optimized  method is suited to the analysis of the 
study samples.  The sponsor should:    
 
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  Conduct a full validation  of any new bioanalytical  method for the analysis of a new drug 
entity, its metabolite(s), or biomarkers.   
 
  Conduct a full validation  for any revisions to an existing validated method that adds a 
metabolite or an additional  analyte.  
 
  Establish a detailed, written description  (e.g., protocol, study plan, and/or standard 
operating procedure (SOP)) for the bioanalytical  method before initiating  validation.   The 
description should identify  procedures that control critical parameters in the method (e.g., 
environmental,  matrix, procedural variables) from the time of collection  of the samples to 
the time of analysis to minimize  their effects on the measurement of the analyte in the 
matrix. 
 
  Document and report (in the method validation  report) all experiments used to make 
claims or draw conclusions about the validity  of the method. 
 
  Validate the measurement of each analyte in the biological  matrix.  The specific 
recommendations and acceptance criteria for each bioanalytical  parameter are listed in 
Table 1. 
 
B.  Bioanalytical Parameters of CCs and LBAs 
 
The bioanalytical  parameters applicable to CCs and LBAs are discussed below.  Issues unique to 
either CCs or LBAs are specifically  identified.   
 
1.  Reference Standards and Critical Reagents 
 
The sponsor should appropriately  characterize and document (e.g. determine the identity,  purity, 
and stability)  all reference standards and critical reagents, such as antibodies,  labeled analytes, 
and matrices and store them under defined conditions. 
 
a.  Reference standards 
 
The purity of reference standards used to prepare calibrators and QCs can affect the study data.  
Therefore, the sponsor should use authenticated analytical reference standards with known 
identities  and purities to prepare solutions  of known concentrations.  The reference standard 
should be identical to the analyte; however, when this scenario is not possible,  the sponsor can 
use an established chemical form (e.g., free base, free acid, or salt) of known purity.   
 
The sponsor should provide the certificates of analyses (CoA), including  the source, lot number, 
and expiration  date (with the exception of United States Pharmacopeia (USP) standards) for 
commercially  available reference standards.  For internally  or externally generated reference 
standards that do not have a CoA, the sponsor should provide evidence of the standards identity 
and purity in addition  to the source and the lot number.  When using expired reference standards, 
the sponsor should provide an updated CoA or re-establish the identity  and purity of the 
standard.  If the reference standard expires, the sponsor should not make stock solutions  with this 
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lot of standard unless the standards purity is re-established.  For internal standards (ISs), the 
sponsor does not have to provide a CoA or evidence of purity if it demonstrates that the IS is 
suitable for the specific use (e.g., lack of interference with an analyte). 
 
b.  Critical reagents 
 
The sponsor should appropriately characterize and document (i.e., determine the identity,  purity 
and stability)  the critical reagents, including   but not limited  to  any reference standards, 
antibodies,  labeled analytes, and matrices.   
 
Assay validation  is important when there are changes to the critical reagents, such as lot-to-lot 
changes or switches to another reagent.  For example, if there are changes to the labeled analytes, 
detector reagents, or antibodies,  the sponsor should: 
 
  Evaluate binding  and re-optimize  assays 
 
  Verify performance with a standard curve and QCs 
 
  Evaluate cross-reactivities 
 
2.  Calibration Curve 
 
During method development,  the sponsor should choose the quantitation  range of the assay and 
the concentrations of the calibration standards on the basis of the concentration range expected in 
a particular study.  For LBAs, in addition  to the calibration  standards, anchor points outside the 
range of quantification  can facilitate the fitting  of the curve.  Anchor points should not be used as 
part of the acceptance criteria for the run.  For most LBAs, calibration (standard) curves are 
inherently  nonlinear,  and in general, more calibration  standards are needed to define the fit over 
the calibration  curve range for LBAs than for CCs.  In addition,  the response-error relationship 
for LBA standard curves is a variable function of the mean response (i.e., heteroscadisticity).  
 
The sponsor should use the simplest model that adequately describes the concentration-response 
relationship,  as well as an appropriate weighting  scheme and regression equation.  For LBAs, the 
concentration-response relationship  is most often fitted to a four- or five-parameter logistic 
model, although other models can be assessed. 
 
When the method is validated,  the calibration  curve should be continuous  and reproducible.   The 
sponsor should prepare the calibration  standards in the same biological  matrix as the samples in 
the intended study.  Study samples may contain more than one analyte.  The sponsor should 
generate a calibration  curve for each analyte in the sample.  When surrogate matrices are 
necessary, the sponsor should justify and validate the calibration  curves.  
 
The requirements for the calibration  curve, including  the LLOQ, ULOQ, as well as the 
acceptance criteria are listed in Table 1. 
 
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3.  Quality Control Samples 
 
Quality controls are used to assess the precision and accuracy of an assay and the stability  of the 
samples.  Sponsors should prepare QCs in the same matrix as the study samples to be assayed 
with the validated method.  Freshly prepared QCs are recommended for precision and accuracy 
analyses during method development,  as stability  data are generally not available at this time.  
 
During method validation,  QCs evaluate the performance of a method and the stability  of an 
analyte.  Performance QCs are included  in validation  runs to determine the precision and 
accuracy of the method (see section III.B).  Stability  QCs evaluate the stability  of an analyte 
under various stress conditions  (Refer to section III.B for the selection of QC concentrations). 
 
The sponsor should prepare any calibration  standards and QCs from separate stock solutions.  
However, if the sponsor can demonstrate the precision and accuracy in one validation  run using 
calibrators and QCs prepared from separate stock solutions,  then the sponsor can use calibrators 
and QCs prepared from the same stock solution in subsequent runs.  The sponsor should make up 
calibrators and QCs in lots of blank matrix that is free of interference or matrix effects. 
 
4.  Selectivity and Specificity  
 
During method development,  the sponsor should verify that the substance being measured is the 
intended analyte to minimize  or avoid interference.  Selectivity  of the method is routinely 
demonstrated by analyzing  blank samples of the appropriate biological  matrix (e.g., plasma) 
from multiple  sources.  Depending on the intended use of the assay, the impact of hemolyzed 
samples, lipemic  samples, or samples from special populations  can be included  in the selectivity 
assessment.  When using liquid  chromatography/mass  spectrometry (LC/MS) methods, the 
sponsor or applicant should determine the effects of the matrix on ion suppression, ion 
enhancement, or extraction efficiency.  Internal standards should be assessed to avoid 
interference with the analyte.  Potential interfering  substances in a biological  matrix include 
endogenous matrix components such as metabolites,  decomposition  products  and from the 
actual study  concomitant medications and other xenobiotics.   If a stabilizer  or enzyme inhibitor 
is used during sample collection,  the sponsor should evaluate the potential for interference on the 
quantitation  of the analyte.  Sponsors should make a scientific judgment  about the need to assess 
these (and any other) potential interferences during method development.   
 
During validation,  the sponsor should confirm that the assay is free of potential interfering 
substances including  endogenous matrix components, metabolites, anticipated concomitant 
medications,  etc.  If the study sample contains more than one analyte and the analytes are 
intended to be quantified by different methods, the sponsor should test each method for 
interference from the other analyte. 
 
The sponsor should analyze blank samples of the appropriate biological  matrix (e.g. plasma) 
from at least six (for CCs) or ten (for LBAs) individual  sources.  The sponsor should ensure that 
there are no matrix effects throughout  the application  of the method.  Refer to Table 1 for 
details of selectivity  and specificity requirements and acceptance criteria. 
 
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For LBAs, it is important to investigate any interference originating  from structurally or 
physiologically  similar  analytes (i.e., exogenous interference) or matrix effects (i.e., endogenous 
interference).  Investigating exogenous interference involves  determining  the cross-reactivity of 
molecules that could potentially  interfere with the binding  interaction,  including  molecules 
structurally related to the drug, any metabolites, concomitant medications (and their significant 
metabolites),  or endogenous matrix components.  The sponsor should evaluate each factor 
individually  and in combination  with the analyte of interest to determine its ability  to cause 
interference.  Matrix effects evaluation involves  comparing calibration  curves in multiple  sources 
of the biological  matrix against a calibration curve in the matrix for parallelism  (serial dilution  of 
incurred samples) and nonspecific binding.   The sponsor should eliminate  or minimize  any 
significant  interference.  If such attempts are unsuccessful, the sponsor could consider the 
development of an orthogonal  method to eliminate  or minimize  the interference.   
 
Carryover between samples can occur in analytical methods.  The sponsor should eliminate any 
carryover during method development.  If carryover cannot be eliminated,  the sponsor should 
assess the impact of any carryover during method validation on the accuracy of the study sample 
concentrations.   
 
5.  Sensitivity 
 
The LLOQ defines the method sensitivity  and should be determined during method 
development.  The method should be developed and validated such that it will be able to meet 
the requirements necessary for the intended study samples.  The LLOQ evaluation can be done 
separately or as part of the precision and accuracy assessment for the calibration  range.  The 
specific recommendations  to validate sensitivity  are listed in Table 1. 
 
 6.  Accuracy, Precision, and Recovery 
 
Evaluating the accuracy and precision across the quantitation  range during method development 
is essential to determine whether the method is ready for validation  and involves  analyzing 
replicate QCs at multiple  concentrations across the assay range.  Specifically,  the sponsor should 
evaluate the performance at the LLOQ, low, mid and high QCs (and the ULOQ for LBAs) to 
determine if the method is suitable to analyze study samples.  
 
Method validation  experiments for estimating  accuracy and precision should include a minimum 
of three (for CCs) and six (for LBAs) independent runs (i.e., accuracy and precision (A & P) 
runs; see Table 1) conducted over several days.  Each A & P run should include a calibration 
curve and multiple  QC concentrations that are analyzed in replicates.  The sponsor should 
determine the accuracy and precision of the method based on the performance of the QC in the A 
& P runs.  The specific validation  requirements for accuracy and precision and A & P runs are 
listed in Table 1.  The sponsor should use freshly prepared calibrators and QCs in all A & P runs.  
Use of freshly prepared QCs in all A & P runs is preferred; however, if this is not possible,  the 
sponsor should use freshly prepared QCs in one or more A & P runs.  
 
The sponsor should optimize  the recovery of the analyte to ensure that the extraction is efficient 
and reproducible.   Recovery need not be 100 percent, but the extent of the recovery of an analyte 
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and of the ISs should be consistent and reproducible.   The sponsor should perform recovery 
experiments by comparing the analytical results of extracted samples with corresponding  extracts 
of blanks spiked with the analyte post-extraction (i.e., to represent 100 percent recovery).  
Recovery evaluation is not necessary for LBAs unless sample extraction is involved.   Recovery 
experiments should be performed as described in Table 1.  
 
7.  Stability 
 
During method development, the sponsor should determine the chemical stability  of the analyte 
in a given matrix, including  the effects of sample collection,  handling,  and storage of the analyte.  
The sponsor should assess autosampler, benchtop, processed or extracted samples, freeze-thaw, 
stock solution,  and long-term stability  of the analyte.  The sponsor should assess the stability  in 
the same matrix as that intended for in-study samples; however, when the matrix is rare, the 
sponsor can explore the use of suitable surrogate matrices.    
 
For drugs administered as fixed combinations,  or part of a specific drug regimen, the stability  of 
the analyte should be assessed in the presence of the other drug.  The sponsor should also 
consider the stability  of the analyte in the presence of other co-medications that are known to be 
regularly administered to patients for the indication  of the drug under development. 
 
Depending on the analyte as well as the sample collection  and assay conditions,  evaluating  the 
stability  of the analyte in whole blood during method development can be useful.  For example, a 
drug can be unstable in whole blood or adsorb to cellular components during collection.   
 
During validation,  stability  evaluations should cover the expected sample conditions  before 
receipt at the analytical site (e.g., at the clinical  site, during shipment, and at all other secondary 
sites) as well as during receipt and analysis at the analytical site.  Validation  of drug stability  in a 
biological  fluid is a function of the storage conditions,  the physicochemical  properties of the 
drug, the matrix, and the container system.  The stability  of an analyte in a particular matrix and 
container system is relevant only to that matrix and container system and should not be 
extrapolated to other matrices and container systems. 
 
If the storage conditions  changed or the sample analysis occurred outside of the validated storage 
condition , the stability  should be re-established under these new conditions.   Stability  testing of 
the analyte in whole blood should be revalidated if necessary (e.g., if the analytes are unstable 
during blood collection).  The specific recommendations and acceptance criteria for stability  are 
listed in Table 1. 
 
Matrix-related stability  experiments should compare stability  QCs against freshly prepared 
calibration  curves and freshly prepared QCs.  Although the use of freshly prepared calibrators 
and QCs is the preferred approach, in some cases, (e.g., for macromolecules),  it may be 
necessary to freeze them overnight.  In such cases, the sponsor should provide valid justification 
and demonstrate the freeze-thaw stability. 
 
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All stability  determinations  (see list below) should use a set of samples prepared from a freshly 
made stock solution  of the analyte in the appropriate analyte-free, interference-free biological 
matrix. 
 
  Autosampler stability:  The sponsor should demonstrate the stability  of extracts in the 
autosampler only if the autosampler storage conditions  are different or not covered by 
extract (processed sample) stability.   
 
  Bench-top stability:  The sponsor should determine the stability  of samples under the 
laboratory handling  conditions  that are expected for the study samples (e.g., the stability 
of samples maintained  at room temperature or stored in an ice bucket). 
 
  Extract (or processed sample) stability:  The sponsor should assess the stability  of 
processed samples, including  the residence time in the autosampler against freshly 
prepared calibrators.  
 
  Freeze-thaw stability:  The sponsor should assess the stability  of the sample after a 
minimum  of three freeze-thaw cycles.  QC samples should be thawed and analyzed 
according to the same procedures as the study samples.  QC samples should be frozen for 
at least 12 hours between cycles.  Freeze-thaw stability  QCs should be compared to 
freshly prepared calibration  curves and QCs. 
 
  Long-term stability:  The sponsor should determine the long-term stability  of the sample 
over a period of time equal to or exceeding the time between the date of first sample 
collection  and the date of last sample analysis.  The storage temperatures studied should 
be the same as those used to store study samples.  Long-term stability  QCs should be 
compared to freshly prepared calibration  curves and QCs. Determination  of stability  at 
minus 20oC would cover stability  at colder temperatures. 
 
  Stock solution stability:  Stock solutions  should not be made from reference materials 
that are about to expire unless the purity of the analyte in the stock solutions  is re-
established.  When the stock solution  exists in a different state (e.g., solution  versus solid) 
or in a different buffer composition  (which is generally the case for macromolecules) 
from the certified reference standard, the sponsor should generate stability  data on stock 
solutions  to justify the duration of stock solution  storage stability. 
 
8.  Dilution Effects 
 
If the method measures diluted  samples, the integrity  of the dilution  should be monitored  during 
validation  by diluting  QC samples above the ULOQ with like matrix to bring to within 
quantitation  range, and the accuracy and precision of these diluted QCs should be demonstrated.  
Dilutions  used during the validation  should mimic the expected dilutions  in the study.  The 
prozone effect should be demonstrated in LBAs.  Refer to the specific recommendations and 
acceptance criteria in Table 1. 
 
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9.  Partial and Cross Validations 
 
The following  section defines other types of methods validation. 
 
a.  Partial validation 
 
Partial validations  evaluate modifications  of already validated bioanalytical  methods.  Partial 
validation  can range from as little as one intra-assay accuracy and precision determination  to a 
nearly full validation.   Raw data on partial validations  should be retained at the analytical site for 
inspection when requested.  Typical bioanalytical  method modifications  or changes that fall into 
this category include, but are not limited  to, the following: 
 
  Bioanalytical  method transfers between laboratories 
 
  Changes in analytical methodology  (e.g., a change in detection systems) 
  
  Changes in sample processing procedures 
  
  Changes in sample volume (e.g., the smaller volume of pediatric samples)  
 
  Changes in instruments and/or software platforms 
 
  Extensions of the assay range 
 
  Changes in the anticoagulant (but not changes in the counter-ion) in harvesting biological 
fluids (e.g., heparin to EDTA) 
 
  Changes in the matrix within species (e.g., switching from human plasma to human 
blood) or changes to the species within the matrix (e.g., switching from rat plasma to 
mouse plasma) 
 
  Changes to the matrices (e.g., cerebrospinal fluid) 
 
  Demonstrating the selectivity of an analyte in the presence of concomitant medications 
 
  Changes in LBA critical reagents (e.g., lot-to-lot  changes, changes in reagents)   
 
b.  Cross validation 
 
Cross validation  is a comparison of validation  parameters of two or more bioanalytical  methods 
or techniques that are used to generate data within the same study or across different studies.  
Also, cross validation  is necessary when sample analyses within a single study are conducted at 
more than one site or more than one laboratory.  In such cases, cross validation  with shared 
matrix QCs and nonpooled  subject samples should be conducted at each site or laboratory to 
establish interlaboratory  reliability.   Pooled incurred samples can be used when insufficient 
volume exists.  An SOP or validation  plan should define the criteria a priori. 
Bioanalytical Method Validation  11 
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C.  Validated Methods:  Expectations of In-Study Analysis and Reporting 
 
This section describes the expectations for the use of a validated bioanalytical  method for routine 
drug analysis.  The specific recommendations  and acceptance criteria are listed in Table 1. 
 
  If system suitability  is assessed, a specific SOP should be used.  System suitability, 
including  apparatus conditioning  and instrument performance, should be determined 
using samples that are independent of the current study calibrators, QCs, and study 
samples.  Records of system suitability  should be maintained and available for audits. 
 
  Calibration  curves and QCs should be included in all analytical runs (see Table 1 for 
details).  The QCs should cover the expected study sample concentration range. 
 
  Typically,  the same curve fitting,  weighting,  and goodness-of-fit determined during 
validation  should be used for the calibration  curve within the study.  Changes in the 
response-function relationship  between the validation  and study sample analyses indicate 
potential problems.  A SOP should be developed a priori to address such issues. 
 
  Total QCs should number at least five percent of the total samples analyzed, or be at least 
six in number (low-, mid-, and high-QCs, in duplicate), whichever is greater (see Table 1 
for details).  Duplicate low-, mid-, and high-QCs should be used on all distinct processing 
batches within a run. 
 
  If the study sample concentrations are clustered in a narrow range of the standard curve, 
additional  QCs should be added to cover the sample range.  If the additional  QC 
concentrations are not bracketed by QCs validated before the study, the accuracy and 
precision of the additional  QCs should be demonstrated before continuing  with the 
analysis.  If the partial validation  is acceptable, samples that have already been analyzed 
do not require re-analysis. 
 
  The QCs should be interspersed with study samples during processing and analysis. 
 
  In each analytical  run, the lack of analyte interference at the LLOQ should be confirmed 
(see Table 1 for Selectivity  and Sensitivity).     
 
  The analytical run fails if the calibration  and/or QC acceptance criteria are not met (see 
Table 1).  
 
  QC results (including  outliers) from analytical runs that meet the acceptance criteria 
should be included in the estimation of accuracy and precision during the studys sample 
analysis.  The QC results from all analytical runs (passed and failed) should be reported, 
but QCs results from failed runs need not be included as part of the estimation of 
accuracy and precision. 
 
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  If the bioanalytical  method necessitates separation of the overall analytical run into 
distinct processing batches (e.g., groups of samples processed at distinctly  different times 
or by different analysts), each distinct batch should process duplicate QCs at all levels 
(e.g., low, middle,  high) along with the study samples.  Examples might include when the 
number of samples exceeds the capacity of a 96-well plate or when a solid phase 
extraction manifold  cannot accommodate all samples.  See Table 1 for what constitutes 
an acceptable run based on QC acceptance criteria.  A distinct batch or batches in an 
analytical run may be rejected when it fails to meet QC acceptance criteria, but the 
remaining  batches may pass provided  that the analytical run meets the overall QC 
acceptance criteria. 
 
  Study samples with concentrations listed below the LLOQ should be reported as below 
the LLOQ (BQL).  Study samples with concentrations above the ULOQ should be diluted 
and re-analyzed, or the standard curve should be extended and revalidated.  
 
  Study sample dilutions  should use the same matrix (e.g., human plasma to human 
plasma). 
 
  Assays of all study samples of an analyte in a biological  matrix should be completed 
within the time period for which stability  data are available.  If sample handling 
conditions  are changed or exceed validated stability  data, then the stability  of the sample 
should be established at the new conditions.   
 
  For CCs, the IS response should be monitored for variability.   An SOP should be 
developed a priori to address issues with IS variability.    
 
  Drift should be monitored and its impact on the accuracy of the estimated unknown 
sample concentrations, if any, should be addressed (e.g., the impact of drift on the 
accuracy of interspersed QCs). 
 
  All study samples from a subject should be analyzed in a single run, especially for studies 
designed with repeated measures from individual  subjects (e.g., crossover or sequential 
design required for BE studies).  If other approaches are taken, the sponsor or applicant  
should justify the approach and take steps to minimize  the variability  between periods. 
 
  Carryover, if any, should be monitored,  and its impact on the quantitation  of study 
samples should be addressed. 
 
  Incurred sample reanalysis (ISR) should be performed (See section IV, Table 1 and Table 
2). 
 
  An SOP or guideline  describing the reasons for a repeat analysis should be established a 
priori.  Repeat analysis is acceptable only for assignable causes (e.g., the samples are 
above the ULOQ, there are sample processing errors, there is an equipment failure, the 
chromatography is poor).  The SOP should include the acceptance criteria for re-analysis, 
and the sponsor or applicant should report final values.  The specific recommendations 
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are described in Table 1 and Table 2.  The rationale, approach, and all data for the repeat 
analysis and reporting should be clearly documented. 
 
  For study samples involving  multiple  analytes, a valid result for one analyte should not 
be rejected because of another analyte failing  the acceptance criteria. 
 
  If a unique or disproportionately  high concentration of a metabolite is discovered in 
human studies, a fully validated assay may need to be developed for the metabolite, 
depending upon its activity (refer to the FDA guidance for industry entitled Safety 
Testing of Drug Metabolites10).   
 
  An SOP or guideline  for sample data reintegration  for CCs should be established a priori.  
This SOP or guideline  should define the criteria for re-integration and how the re-
integration  will be performed.  The rationale for the re-integration should be clearly 
described and documented.  Audit trails should be maintained.  Original  and re-integrated 
data should be documented and reported. 
 
 
IV.  INCURRED SAMPLE REANALYSIS 
 
ISR is a necessary component of bioanalytical  method validation  and verifies the reliability  of 
the reported study sample analyte concentrations.  ISR is conducted by repeating the analysis of 
a subset of subject or patient samples from a given study in separate runs, preferably during the 
study, to critically  support the precision and accuracy measurements established with the QCs.  
The original  and repeat analyses should be conducted using the same bioanalytical  method 
procedures.  If a bulk frozen calibration  curve was used for the original  analysis, then it is 
acceptable to use a frozen curve for the ISR evaluation.  The calibration  curve, QCs, and study 
samples for the ISR evaluation  should be extracted or processed separately from those used in 
the original  runs.  Incurred samples should not be pooled.  ISR should be conducted in all studies 
submitted in an NDA, BLA, or ANDA that provide pivotal data for the approval or labeling  of 
the product, regardless of the matrix.  For instance, ISR is expected for all in vivo human BE 
studies in ANDAs, or all pivotal  pharmacokinetic,  pharmacodynamic,  and biomarker studies in 
NDAs or BLAs.  For nonclinical  safety studies, the performing laboratory should conduct ISR at 
least once for each method and species.  Table 1 lists the sample requirements and acceptance 
criteria for ISR.  Written SOPs should be established for the conduct of ISR and to guide an 
investigation  in the event of ISR failure to resolve the lack of reproducibility.   All aspects of ISR 
evaluations should be documented to allow reconstruction of the study, as well as guide any 
investigations  (see Table 2). 
 
The percentage difference of the results between the original  study and the repeat study is 
determined with the following  equation:  
 
                                              
10 This guidance is available on the Internet at http://www.fda.gov/Drugs/default.htm under Guidances (Drugs). 
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(Repeat  Original)  * 100 
                  Mean 
 
V.  ADDITIONAL ISSUES 
 
A.  Endogenous Compounds 
 
For analytes that are also endogenous compounds,  the accuracy of the measurement of the 
analytes poses a challenge when the assay cannot distinguish  between the therapeutic agent and 
the endogenous counterpart.  In such situations,  the following  approaches are recommended to 
validate and monitor assay performance.  Other approaches, if justified  by scientific principles, 
can also be considered. 
 
  The biological  matrix used to prepare calibration  standards should be the same as the 
study samples and free of the endogenous analyte.  To address the suitability  of using an 
analyte-free biological  matrix, the matrix should be demonstrated to have: (1) no 
measurable endogenous analyte; and (2) no matrix effect or interference when compared 
to the biological  matrix.  The use of alternate matrices (e.g., buffers, dialyzed serum) for 
the preparation of calibration  standards should be justified.  The QCs should be prepared 
by spiking  known quantities of the analyte in the same biological  matrix as the study 
samples.  The endogenous concentrations of the analyte in the biological  matrix should 
be evaluated before QC preparation (e.g., by replicate analysis).  The concentrations for 
the QCs should account for the endogenous concentrations in the biological  matrix (i.e., 
additive) and be representative of the expected study concentrations. 
 
  Parallelism should be evaluated for assays for endogenous compounds. 
 
B.  Biomarkers 
 
The recommendations in this guidance only pertain to the validation  of assays to measure in vivo 
biomarker concentrations in biological  matrices such as blood or urine.  Considerable effort also 
goes into defining  the biological  function of biomarkers, and confusion may arise regarding 
terminology  (e.g. biomarker method validation  vs biomarker qualification).    
 
Biomarkers are increasingly  used to assess the effects of new drugs and therapeutic biological 
products in patient populations.   Because of the important roles biomarkers can play in 
evaluating the safety, activity, or effectiveness of a new medical product, it is critical to ensure 
the integrity  of the data generated by assays used to measure them.  Biomarkers can be used for a 
wide variety of purposes during drug development; therefore, a FFP approach should be used 
when determining  the appropriate extent of method validation.   When biomarker data will be 
used to support a regulatory decision making, such as the pivotal determination  of safety and/or 
effectiveness or to support dosing instructions  in product labeling,  the assay should be fully 
validated. 
 
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For assays intended to support early drug development (e.g., candidate selection, go-no-go 
decisions, proof-of-concept), the sponsor should incorporate the extent of method validation  they 
deem appropriate.  
 
Method validation  for biomarker assays should address the same questions as method validation 
for drug assays.  The accuracy, precision, sensitivity,  selectivity,  parallelism,  range, 
reproducibility,  and stability  of a biomarker assay are important  characteristics that define the 
method.  The approach used for drug assays should be the starting point for validation  of 
biomarker assays, although the FDA realizes that some characteristics may not apply or that 
different considerations  may need to be addressed. 
 
C.  Diagnostic Kits 
 
Diagnostic kits are sometimes co-developed with new drug or therapeutic biological  products as 
analytical methods that are used during the development of new drugs and therapeutic biologics.   
The recommendations in this section of the guidance do not apply to commercial diagnostic  kits 
intended for point-of-care patient diagnosis  (e.g., companion diagnostic  kits), which are 
addressed in the following  CDRH guidance documents: 
 
  Principles for Codevelopment of an In Vitro Companion Diagnostic Device with a 
Therapeutic Product11 
 
  In Vitro Companion Diagnostic Devices  
 
However, when commercial diagnostic  kits are repurposed as analytical methods to measure the 
concentrations of drugs, therapeutic biologics,  or biomarkers in development,  the FDA has the 
following  recommendations: 
 
  LBA kits with various detection platforms are sometimes used to determine analyte 
concentrations in pharmacokinetic or pharmacodynamic studies when the reported results 
must exhibit sufficient precision and accuracy.  Because such kits are generally 
developed for use as clinical  diagnostic tools, their suitability  for use in such studies 
should be demonstrated.   
 
  Diagnostic kit validation  data provided by the manufacturer may not ensure that the kit 
method is reliable for drug development purposes.  In such situations,  the performance of 
diagnostic kits should be assessed in the facility conducting the sample analysis.  
Validation  considerations for kit assays include,  but are not limited  to, the following 
examples:  
 
                                              
11 When final, this guidance will represent the FDAs current thinking on this topic.  For the most recent version of a 
guidance, check the FDA Drugs guidance Web page at 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
 
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-  Site-specific validation  should be performed.  The specificity,  accuracy, 
precision, and stability  of the assay should be demonstrated under actual 
conditions  of use.  Modifications  from kit processing instructions  should be 
completely validated.   
 
-  Kits that use sparse calibration  standards (e.g., one- or two-point calibration 
curves) should include in-house validation  experiments to establish the 
calibration  curve with a sufficient number of standards across the calibration 
range as specified in Table 1. 
 
-  Actual QC concentrations should be known.  Concentrations of QCs 
expressed as ranges are not sufficient for quantitative applications.   In such 
cases, QCs with known concentrations should be prepared and used, 
independent of the kit-supplied  QCs. 
 
-  Standards and QCs should be prepared in the same matrix as the subject 
samples.  Kits with standards and QCs prepared in a matrix different from the 
subject samples should be justified,  and appropriate cross-validation 
experiments should be performed.  Refer to section V.A of this guidance for 
additional  discussion.    
 
-  If the analyte source (i.e., reference standard) in the kit differs from that of the 
subject samples (e.g., the sample is a protein isoform of the reference 
standard), testing should evaluate differences in assay performance of the kit 
reagents.   
 
-  If multiple  kit lots are used within a study, lot-to-lot  variability  and 
comparability  should be addressed for any critical reagents.   
 
-  Individual  batches using multiple  assay plates (e.g., 96-well ELISA plates) 
should include sufficient replicate QCs on each plate to monitor the accuracy 
of the assay.  Acceptance criteria should be established for the individual 
plates and the overall analytical run (refer to Table 1 and section III.B).     
 
D.  Bridging Data From Multiple Bioanalytical Technologies 
 
The FDA encourages the development and use of new bioanalytical  technologies.  However, the 
use of two different bioanalytical  technologies for the development of a drug may generate data 
for the same product that could be difficult  to interpret.  This outcome can occur when one 
platform generates drug concentrations that differ from another platform.  Therefore, when a new 
platform is used in the development of a drug, the data it produces should be bridged to that of 
the other method.  This is best accomplished by assessing the output of both methods with a set 
of incurred samples (a minimum  of 20 samples).  In cases where one method produces data with 
a constant bias relative to the other, concentrations can be mathematically  transformed by that 
factor to allow for appropriate study interpretation.  Sponsors are encouraged to seek feedback 
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from the appropriate FDA review division  early in drug development.  The use of two methods 
for BE studies in ANDAs is discouraged. 
 
E.  Dried Blood Spots 
 
Dried blood spot (DBS) technology  has been under development for several years.  The benefits 
of DBS include reduced blood sample volumes collected for drug analysis as well as ease of 
collection,  storage, and transportation.  Additional  validation  of this sampling approach is 
essential before using DBS in regulatory studies.  This validation  should address, at a minimum, 
the effects of the following  issues:  storage and handling  temperatures, homogeneity  of sample 
spotting,  hematocrit, stability,  carryover, and reproducibility,  including  ISR.  Correlative studies 
with traditional  sampling  should be conducted during drug development.  Sponsors are 
encouraged to seek feedback from the appropriate FDA review division  early in drug 
development. 
 
 
VI.  DOCUMENTATION 
 
General and specific SOPs and good record keeping are essential to a properly validated 
analytical method.  The data generated for bioanalytical  method development  and/or validation 
should be documented and available for data audit and inspection.  Documentation at the 
analytical site and for submission  to the FDA is described in Table 2. 
 
All relevant documentation  necessary for reconstructing the study as it was conducted and 
reported should be maintained  in a secure environment.  Relevant documentation  includes, but is 
not limited  to, source data, protocols and reports, records supporting  procedural, operational,  and 
environmental  concerns, and correspondence records between all involved  parties.   
 
Regardless of the documentation  format (i.e., paper or electronic), records should be 
contemporaneous with the event, and subsequent alterations should not obscure the original  data.  
The basis for changing or reprocessing data should be documented with sufficient detail, and the 
original  record should be maintained.   
 
A.  Summary Information 
 
Summary information  should include the following  items: 
   summary of assay methods used for each study protocol should be included.  Each 
summary should provide the protocol number, the protocol title, the assay type, the assay 
method identification  code, the bioanalytical  report code, and the effective date of the 
method. 
  For each analyte, a summary table of all the relevant method validation  reports should be 
provided,  including  partial validation  and cross validation  reports.  The table should 
include the assay method identification  code, the type of assay, the reason for the new 
method or additional  validation  (e.g., to lower the limit  of quantification),  and the dates 
of final reports.  Changes made to the method should be clearly identified. 
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  A summary table cross-referencing multiple  identification  codes should be provided 
when an assay has different codes for the assay method, the validation  reports, and the 
bioanalytical  reports. 
 
B.  Documentation for Method Validation and Bioanalytical Reports 
 
Refer to Table 2 for the FDAs recommended documentation  for method validation  and 
bioanalytical  reports.  
 
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VII.  APPENDIX 
Table 1.  Recommendations and Acceptance Criteria for Bioanalytical Method Validation and In-Study Conduct (refer to 
sections III.A and III.B for additional information). 
Validation Recommendations 
Parameters  In-Study Analysis Recommendations 
Chromatographic Assays (CCs)  Ligand Binding Assays (LBAs) 
Elements:  Elements:  Elements: 
 A blank (no analyte, no IS), a zero calibrator   A blank and at least six, non-zero   A blank, a zero, and at least six, (in duplicate 
(blank plus IS), and at least six, non-zero  calibrator levels covering the quantitation  for LBAs) non-zero calibrator levels covering 
calibrator levels covering the quantitation  range, including LLOQ per validation  the expected range, including LLOQ per 
range, including LLOQ in every run.  run.  analytical run. 
     
 All blanks and calibrators should be in the    Calibration curves are usually run in   All blanks and calibrators should be in the same 
same matrix as the study samples.  duplicate.  matrix as the study samples. 
     
 The concentration-response relationship   Additional calibrators may be used as   The in-study analysis should use the same 
should be fit with the simplest regression  anchor points.  regression model as used in validation. 
model.     
   All blanks and calibrators should be in the   
  same matrix as the study samples.    
     
Calibration     The concentration-response relationship is   
  usually fit with a four- or five-parameter   
Curve 
  logistic model.  Other models may be   
  acceptable with justification.   
     
Acceptance Criteria:  Acceptance Criteria:  Acceptance Criteria: 
 Non-zero calibrators should be  15% of   Non-zero calibrators should be  20% of   CC:  Non-zero calibrators should be  15%, 
nominal (theoretical) concentrations, except  nominal (theoretical) concentrations,  except at LLOQ where the calibrator should be 
at LLOQ where the calibrator should be   except at LLOQ and ULOQ where the   20% of nominal concentrations in each run. 
20% of the nominal concentrations in each  calibrator should be  25% of the nominal   LBA:  Non-zero calibrators should be  20%, 
validation run.   concentrations in each validation run.   except at LLOQ and ULOQ where the 
 75% and a minimum of six non-zero   75% and a minimum of six non-zero  calibrator should be  25% of nominal 
calibrator levels should meet the above  calibrator levels should meet the above  concentrations in each run. 
criteria in each validation run.   criteria in each validation run.    CC and LBA:  75% and a minimum of six 
 Anchor points should not be included in  non-zero calibrator levels should meet the 
the curve fit.  above criteria in each run.  
Only data points that fail to meet acceptance criteria may be excluded.  Exclusion should not change the model used.   
                                                                                                                                                                                              Continued                                    
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Table 1 continued.  Recommendations and Acceptance Criteria for Bioanalytical  Method Validation  and In-Study Conduct 
Validation Recommendations 
Parameters  In-Study Analysis Recommendations 
Chromatographic Assays (CCs)  Ligand Binding Assays (LBAs) 
Elements:  Elements:  Elements: 
 For A & P Runs:  Four QCs, including    For A& P Runs:  Five QCs, including    three QC levels (L, M & H) and  two 
LLOQ, low (L: defined as three times the  LLOQ, L, M, H, and ULOQ from at least  replicates per QC level in each analytical run. 
LLOQ), mid (M: defined as mid-range), and  three replicates in at least six runs   
high (H: defined as high-range) from at least     Total QCs should be 5% of unknown samples 
five replicates in at least three runs   For Other Validation Runs:  L, M, and  or  six, whichever number is greater. 
  H QCs in duplicates   
 For Other Validation Runs:  L, M, and H     If the analytical runs consist of distinct 
QCs in duplicates    processing batches, the QC acceptance criteria 
    should be applied for the whole run and for 
Quality      each distinct batch within the runs. 
Controls       
(QC)        
     
Acceptance Criteria:   Acceptance Criteria:   Acceptance Criteria: 
 Refer to A & P Runs, Other Validation   Refer to A & P Runs, Other Validation   CC:   67% of QCs should be  15% of the 
Runs, and Stability Evaluations.  Runs, and Stability Evaluations.  nominal, and  50% of QCs per level should be 
 15% of their nominal. 
 
 LBA:   67% of QCs should be  20% of the 
nominal, and  50% of QCs per level should be 
 20% of their nominal. 
 
                                                                                                                                                                                            Continued 
 
 
 
 
 
 
 
 
 
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Table 1 continued.  Recommendations and Acceptance Criteria for Bioanalytical  Method Validation  and In-Study Conduct 
Validation Recommendations 
Parameters  In-Study Analysis Recommendations 
Chromatographic Assays (CCs)  Ligand Binding Assays (LBAs) 
Elements:  Elements:  CC Acceptance Criteria:  
 Analyze blank samples of the appropriate   Investigate parallelism (for endogenous   In each analytical run, the blank and zero 
biological matrix from at least six individual  products).  calibrators should be free of interference at the 
sources.    retention times of the analyte and the internal 
   Conduct an analysis of blank samples in  standard 
  the matrix from  10 individual sources.    
   
 In each analytical run, the internal standard 
Acceptance Criteria:  Acceptance Criteria: 
response in the blank should not exceed 5% of 
 Blank and zero calibrators should be free of   For  80% of sources, unspiked matrix 
average internal standard response of the 
interference at the retention times of the  should be BQL, and spiked samples 
Selectivity  calibrators and QCs. 
analyte(s) and the IS.  should be  25% at LLOQ, and  20% at 
 
  H QC. 
LBA Acceptance Criteria: 
 Spiked samples should be  20% LLOQ.     The blank should be free of interference for the 
analyte. 
 
 The IS response in the blank should not   
exceed 5% of the average IS responses of   Parallelism should be conducted if not done 
the calibrators and QCs.  during validation.   
 
Elements:  Elements:  Elements: 
 The method specificity should be assessed   The method specificity should be assessed   Check as needed. 
for interference by cross-reacting molecules,  for interference by cross-reacting 
concomitant medications, bio-transformed  molecules, concomitant medications, bio-
species, etc.  transformed species, etc. 
   
Specificity 
   Potential interfering materials should be 
  added to calibration curves in buffer. 
   
Acceptance Criteria:  Acceptance Criteria: 
 See Selectivity above.   QCs should meet  20%, or 25% at the 
LLOQ and ULOQ. 
                                                                                                                                                                                                                                     Continued 
 
 
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Table 1 continued.  Recommendations and Acceptance Criteria for Bioanalytical  Method Validation  and In-Study Conduct 
Validation Recommendations 
Parameters  In-Study Analysis Recommendations 
Chromatographic Assays (CCs)  Ligand Binding Assays (LBAs) 
Elements:    Not applicable  Elements: 
  The impact of carryover on the accuracy of    Carryover, if any, should be monitored, and its 
the study sample concentrations should be  impact on the quantitation of study samples 
assessed.  should be addressed. 
Carryover     
Acceptance Criteria:  Acceptance Criteria: 
  Carryover should not exceed 20% of    Carryover should not exceed 20% of LLOQ. 
LLOQ. 
 
Elements:  Elements:   
 The lowest nonzero standard on the   The lowest nonzero standard on the   
calibration curve defines the sensitivity  calibration curve defines the sensitivity   
(LLOQ).   (LLOQ).    
     
Acceptance Criteria:  Acceptance Criteria:  Acceptance Criteria:  
 The analyte response at the LLOQ should   The accuracy should be  25% of the  In each analytical run: 
be  five times the analyte response of the  nominal concentration (from  three   The analyte response at the LLOQ should be  
zero calibrator.  replicates in at least six runs).  five times the analyte response of the zero 
    calibrator (CC). 
 The accuracy should be  20% of nominal   The precision should be  25% CV (from   
Sensitivity  concentration (from  five replicates in at   three replicates in at least six runs).   The A & P for CC should be  20% of nominal 
least three runs).    concentration. 
   The total error should be  40%.   
 The precision should be  20% CV (from    The A & P for LBA should be  25% of 
five replicates in at least three runs).   nominal concentration. 
 
 If the above criteria are not met, the next higher 
calibrator can be selected as the new LLOQ or 
the next lower point if the ULOQ fails 
(provided the resulting calibration curve meets 
acceptance criteria) and does not change the 
calibration model. 
                                                                                                                                                                                                                                   Continued 
                                                                                                                                
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Table 1 continued.  Recommendations and Acceptance Criteria for Bioanalytical  Method Validation  and In-Study Conduct 
Validation Recommendations 
Parameters  In-Study Analysis Recommendations 
Chromatographic Assays (CCs)  Ligand Binding Assays (LBAs) 
Elements:  Elements:  Elements: 
 A & P should be established with at least   A & P should be established with at least   Not applicable 
three independent A & P runs, four QC  six  independent  A & P runs, five QC   
levels per run (LLOQ, L, M, H QC), and   levels per run (LLOQ, L, M, H, ULOQ   
five replicates per QC level.  QC), and  three replicates per QC level.   
     
A & P Run Acceptance Criteria:  A & P Run Acceptance Criteria:   
 The run should meet the calibration curve   The run should meet the calibration   
acceptance criteria and include the LLOQ  acceptance criteria and include the LLOQ   
calibrator.  calibrator.   
Accuracy 
     
and   This run has no QC acceptance criteria.   This run has no QC acceptance criteria.   
Precision        
(A & P)  Accuracy:  Within-run and between runs:  Accuracy:  Within-run and between runs:   Accuracy:  Between runs:  
  15% of nominal concentrations; except    20% of nominal concentrations; except    CC:  15% of nominal concentrations  
 
 20% at LLOQ.  25% at LLOQ, ULOQ   LBA:  20% of nominal concentrations 
     
Precision:  Within-run and between runs:  Precision:  Within-run and between runs:  Precision:  Between runs: 
  15% CV, except    20% CV, except   CC:  15% CV 
 20% CV at LLOQ   25% at LLOQ, ULOQ   LBA: 20%  CV 
     
Total Error:   Total Error:  Total Error:  
 Not applicable   QCs should be 30%, except at LLOQ,   Not applicable 
  ULOQ 40%    
 
                                                                                                                                                                                               Continued 
                                  
 
 
 
 
 
 
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Table 1 continued.  Recommendations and Acceptance Criteria for Bioanalytical  Method Validation  and In-Study Conduct 
Validation Recommendations 
Parameters  In-Study Analysis Recommendations 
Chromatographic Assays (CCs)  Ligand Binding Assays (LBAs) 
Elements:  Elements:   Not applicable 
  three QC levels (L, M, H) in at least    three QC levels (L, M, H) in at least 
duplicates in each run.   duplicates in each run 
   
Run Acceptance Criteria:  Run Acceptance Criteria: 
Other 
 Meet the calibration acceptance criteria   Meet the calibration acceptance criteria 
Validation 
   
Runs 
  67% of QCs should be  15% of the    67% of QCs should be  20% of the 
nominal (theoretical) values,  50% of QCs  nominal (theoretical) values, and  50% 
per level should be  15% of their nominal  of QCs per level should be  20% of their 
concentrations  nominal  concentrations 
 
Elements:  Elements:   
  Extracted samples at L, M, and H QC    Need to be demonstrated only if 
concentrations versus extracts of blanks  extraction is involved 
Recovery 
spiked with the analyte post extraction (at 
L, M, and H) 
 
                                                                                                                                                                                                                               Continued 
 
 
 
 
 
 
 
 
 
 
 
 
 
Bioanalytical Method Validation  25 
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Table 1 continued.  Recommendations and Acceptance Criteria for Bioanalytical  Method Validation  and In-Study Conduct 
Validation Recommendations 
Parameters  In-Study Analysis Recommendations 
Chromatographic Assays (CCs)  Ligand Binding Assays (LBAs) 
Elements:  Elements:  Elements: 
  For auto-sampler, bench-top, extract,    For auto-sampler, bench-top, extract,    Update stability parameters (e.g., long-term) 
freeze-thaw, stock solution and long-term  freeze-thaw, stock solution/reagent and  as needed. 
stability, perform at least three replicates at  long-term stability, perform at least three 
L and H QC concentrations.  replicates at L and H QC concentrations. 
Stability 
   
Acceptance Criteria:  Acceptance Criteria: 
  The accuracy (% nominal) at each level    The accuracy (% nominal) at each level 
should be  15%.  should be  20%. 
 
Elements:  Elements:  Elements: 
 QCs for planned dilutions, 5 replicates per   QCs for planned dilutions    Dilution QC (if not a validated pre-study) 
dilution factor:     
   Demonstrate dilution linearity    
  Accuracy:   15% of nominal     
o
concentrations    Demonstrate lack of prozone effect, i.e.,  Acceptance Criteria:  
  increasing analyte concentration results in   Same as described under QCs above 
o  Precision:   15% CV   no change or decreased signals compared 
Dilution  to the preceding concentration 
 
 5 replicates per dilution factor: 
 
  Accuracy:   20% of nominal 
o
concentrations 
 
  Precision:   20% CV 
o
 
                                                                                                                                                                                                                            Continued 
 
 
 
Bioanalytical Method Validation  26 
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Table 1 continued.  Recommendations and Acceptance Criteria for Bioanalytical  Method Validation  and In-Study Conduct 
Validation Recommendations 
Parameters  In-Study Analysis Recommendations 
Chromatographic Assays (CCs)  Ligand Binding Assays (LBAs) 
 Not applicable   Not applicable  Elements: 
 Sample size: 
  10% reanalysis of the first 1000 samples, 
o
and 
   5% reanalysis of the remaining samples 
o
Incurred   
Sample   Sample selection:  
Reanalysis  o  Around Cmax and in the elimination 
(ISR)   phase 
 
Acceptance Criteria: 
 CC:  67% should be  20% of the mean  
 LBA:  67% should be  30% of the mean  
 
 No re-analysis of individual calibrators and    No re-analysis of individual calibrators   Re-analysis should be based on reasons 
QCs is permitted.  and QCs is permitted.  described in a pre-existing SOP 
 
 No re-analysis of calibrators and QCs 
 
Repeat 
 At least the same number of replicates for 
Analysis  repeats as originally tested 
 
 No confirmatory repeats for BE studies 
 
 
 
 
 
Bioanalytical Method Validation  27 
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Table 2.  Documentation and Reporting (refer to sections III.B and VI for additional information) 
Items  Documentation at the Analytical Site  Validation Report*  Analytical Study Report* 
 Dates, times, QCs or samples used for suitability testing   Not applicable   Not applicable 
System 
Suitability 
 Not applicable   Synopsis of method development   Not applicable 
(e.g., evolution of methods with 
multiple revisions, unique aspects)    
Synopsis 
 
 Overall summary information   
 
 Certificate of analysis (CoA) or purity,   Batch/lot number, purity, and   Batch/Lot number, purity, and 
stability/expiration data, batch number, and  expiration (see appendix VII, Table  expiration (see appendix VII, 
manufacturer  4)  Table 4) 
Reference       
 Log records of receipt, use, and storage.   If expired, purity and stability at the   If expired, purity and stability at 
Standards 
  time of use and retest dates  the time of use and retest dates  
and 
 If expired, recertified CoA, or retest of purity & 
Critical 
identity with retest dates 
Reagents   
 Internal standard CoA, purity or demonstration of 
suitability 
 
 Log records of preparation, and use   Brief description of preparation   Brief description of preparation 
 Storage location and condition   Preparation dates    Preparation dates  
Stock Solutions     Stock solution stability    Stock solution stability  
 Storage conditions   Storage conditions  
 
 Records of matrix descriptions, receipt dates, and   Description, lot number, receipt   Description, lot number, receipt 
storage  dates  dates 
     
Blank Matrix 
 Records of interference checks   Description of interference check   Description of interference check 
   
 Matrix effect results   Matrix effect results 
                                              Continued 
 
Bioanalytical Method Validation  28 
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Table 2 continued:   Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Analytical Study Report* 
 Records of preparation   Brief description of preparation   Brief description of preparation 
Calibrators   Record of storage (e.g., in/out dates, temperatures)   Preparation dates   Preparation dates 
and QCs   Storage conditions   Storage conditions  
 
SOPs for all aspects of analysis, such as:   A detailed description of the assay  Not applicable 
 Method/procedure (validation/analytical)  procedure 
 Acceptance criteria (e.g., run, calibration curve, QCs)    
 Instrumentation 
SOPs 
 Re-analysis 
 ISR 
 Record of changes to SOP (change, date, reason, etc.) 
 
 Study sample receipt, and condition on receipt   Storage condition and location of   Dates of receipt of shipments and 
  QCs and calibrators  contents 
 Temperature during shipment   
   Sample condition on receipt  
 Sample inventory and reasons for missing samples   
   Analytical site storage condition 
Sample 
 Location of storage  and location 
Tracking 
   
 Tracking logs of QC, calibrators, and study samples   Total duration of sample storage 
   
 Freezer logs for QC, calibrators, and study samples   Any deviations from planned 
entry and exit  storage conditions 
 
                         Continued 
   
   
   
     
   
   
   
 
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Table 2 continued:   Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Analytical Study Report* 
 Documentation and data for system suitability checks   Table of all runs (including failed   Table of all runs, status (pass and 
  runs), instrument ID, and analysis  fail), reason for failure, instrument 
 Instrument use log, including dates of analysis for each  dates  ID, and analysis dates (see 
run    appendix VII, Table 4). 
   Tables of calibrator concentration   
 Sample extraction logs, including documentation of  and response functions results of all   Table of calibrator concentration 
processing of calibrators, QCs, and study samples for  runs with accuracy and precision.  & response function results of all 
each run, including dates of extraction    runs (pass and fail) with accuracy 
    and precision 
 Identity of QC & calibrator lots, and study samples in   Tables of within- and between- run   
each run  QC results (from accuracy and   Table of QC results of all runs 
  precision runs)  (pass and fail) with accuracy and 
 Documentation of instrument settings and maintenance    precision results of the QC 
   Interference/matrix effect,  samples and between run accuracy 
 100% of run summary sheets of passed and failed runs,  sensitivity, carryover, dilution,  and precision results from 
Analysis  including calibration curve, regression, weighting  recovery  successful runs 
function, analyte and IS response, response ratio,     
integration type   Bench-top, freeze-thaw, long-term,   Table of re-injected runs with 
  extract, and stock solution stability  results from original and re-
 100% e-chromatograms of original and re-integrations    injected runs and reason(s) for 
from passed and fail runs   Stability QC storage and handling  reinjection 
  conditions (dates, duration,   
 Laboratory information management system (LIMS)  temperature, etc.)   QC graphs trend analysis 
    encouraged 
 Validation information, including documentation and   Partial/cross-validation, if applicable   
data for:     Study concentration results table 
  Selectivity, sensitivity, precision and accuracy,   Append separate report for   
o
carryover, dilution, recovery, matrix effect  additional validation, if any 
  Bench-top, freeze-thaw, long-term, and extract    
o
stability   Include total error for LBA methods 
  Cross/partial validations, if applicable 
o
                              Continued 
   
     
   
 
Bioanalytical Method Validation  30 
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Table 2 continued:   Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Analytical Study Report* 
 Electronic audit trail: original and re-integration   Representative chromatograms   Chromatograms from 20% of 
 Reason for re-integration  (original and re-integration)  serially selected subjects for BE 
 Mode of re-integration    studies in ANDAs 
 Reason for re-integration   
   Randomly selected 
chromatograms from 5% of 
studies submitted in NDAs and 
Chromatograms  BLAs 
and   
Reintegration   Original and re-integrated 
chromatograms and initial and 
repeat integration results for BE 
studies 
 
 Reason for re-integration 
 
 SOP for re-integration 
 Contemporaneous documentation of deviations/   Description of deviations   Description of deviations 
unexpected events     
   Impact on study results   Impact on study results 
Deviations from   Investigation of unexpected events     
Procedures     Description and supporting data of   Description and supporting data of 
 Impact assessment  significant investigations  significant investigations 
 
 ISR failure investigations 
 SOP for re-analysis (Refer to Analysis)   Not applicable   Table of sample IDs, reason for 
 100% of repeat data  re-assay, original and re-assay 
 Contemporaneous records of reason for repeats  values, reason for reported values, 
and run IDs and percent difference 
Repeat Analysis 
between original and re-assay 
values 
 
 Re-analysis SOP  
                                                                                                                                                                       Continued 
 
Bioanalytical Method Validation  31 
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Table 2 continued:   Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Analytical Study Report* 
 SOP for ISR   Not applicable   SOP for ISR 
   
 ISR data:  Run IDs, run summary sheets,   ISR data table (original, 
chromatograms or other electronic instrument data  reanalysis, percent difference, 
ISR 
files  percent passed) 
   
 Document ISR failure investigations, if any   ISR failure investigations, if any 
 
 Between involved parties (Sponsor, contract research   Not applicable   Not applicable 
organizations (CROs), and consultants) related to 
Communication 
study/assay 
 
*The FDA expects the sponsor to maintain data at the analytical site to support summary data submitted in Validation and Analytical Study Reports.  
Bioanalytical Method Validation  32 
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Table 3.&  Example of an Overall Summary Table for a Method Validation Report* or a Clinical Study Report (this table 
contains fictitious information, which serves illustrative purposes only) 
Items  Results  Hyperlink  Comments 
Methodology  LC/MS/MS  01-SOP-001   
 
Method Validation  MVR-001  MVR-001   
Report (MVR)   
Number 
 
Biological matrix  Human plasma  MVR-001   
 
Anticoagulant (if  EDTA  MVR-001   
applicable)   
 
XXX-YYY ng/mL  Summary tables   
001MVR-01/CCTables 
Calibration curve   
range  Report text 
001MVR-01/CCText 
 
Analyte of interest  Compound A  NA   
 
Compound A internal  NA   
Internal standard  standard 
 
Low QC (AA ng/mL):  X%  Summary tables   
Medium QC (e.g., BB  001MVR-01/APTables 
Inter-run accuracy  ng/mL): Y%   
(for each QC  High QC (e.g. CC ng/mL):  Report text 
concentration)  Z%  001MVR-01/APText 
   
 
Low QC (AA ng/mL): X%     
Inter-run precision  Medium QC (BB 
(for each QC  ng/mL): Y% 
concentration)  High QC (CC 
ng/mL): Z% 
      Continued 
       
Bioanalytical Method Validation  33 
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Table 3 continued.  Example of an Overall Summary Table for a Method Validation 
Report* or a Clinical  Study Report 
Items  Results  Hyperlink  Comments 
Dilution integrity  Dilution QC: CC ng/mL  Summary tables   
(specify dilution  (dilution factor: X)  001MVR-01/DILTables 
factors, QC  Accuracy: Y%   
concentrations, and  Precision:  Z%  Report text 
matrices that were    001MVR-01/DILText 
evaluated)   
< 20% of the lower limit of  Summary tables   
quantification (LLOQ)  001MVR-01/SELTables   
Selectivity  -list drugs tested     
Report text   
001MVR-01/SELText 
Demonstrated for X hours at  Summary tables   
Short-term or bench-
YC  001MVR-01/STSTables 
top temperature 
 
stability 
Report text 
 
001MVR-01/STSText 
Demonstrated for X days at  Summary tables   
YC   001MVR-01/LTSTables 
Long-term stability   
Report text 
001MVR-01/LTSText 
Demonstrated for Y cycles at  Summary tables   
ZC  001MVR-01/FTSTables 
Freeze-thaw stability   
Report text 
001MVR-01/FTSText 
Demonstrated for X weeks at  Summary tables   
YoC  001MVR-01/SSSTables 
Stock solution stability   
Report text 
001MVR-01/SSSText 
    Continued 
   
 
 
 
       
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Table 3 continued.  Example of an Overall Summary Table for a Method Validation 
Report* or a Clinical  Study Report 
Items  Results  Hyperlink  Comments 
Demonstrated for Y hours at  Summary tables   
ZoC  001MVR-01/PSSTables 
Processed sample   
stability  Report text 
001MVR-01/PSSText 
 
> 67% of samples acceptable  Summary tables   
001MVR-01/ISRTables 
 
ISR 
Report text 
001MVR-01/ISRText 
 
Summary tables   
001MVR-01/EXTTables 
Recovery:  extraction 
Report text 
efficiency 
001MVR-01/EXTText 
 
Summary tables   
001MVR-01/MATTables 
Matrix effects  Report text 
001MVR-01/MATText 
 
&Report Format examples are pertinent for applications to either CDER or CVM.  Summary tables should be included in Module 2 of the eCTD. 
*Failed method validation experiments should be listed, and data may be requested. 
For eCTD submissions, a hyperlink should be provided for the summary tables and report text.  
 
 
Bioanalytical Method Validation  35 
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Table 4.  Example of Summary Analytical Runs for a Bioanalytical Study Report* (this 
table contains fictitious information, which serves illustrative purposes only) 
 
Sponsors and applicants should provide a table summarizing  both the failed and accepted runs 
for each study.  
 
Clinical Study XXYY-0032456 
Batch 
number  Results  Comments 
Analytical  Dates of 
within  (Accepted  Hyperlink  (e.g. information on runs 
run *  analysis 
analytical  /Rejected)  that failed) 
run 
001-100-01  Not  MM/DD/YY  Rejected  Summary tables for  001BR-01/01Failure 
applicable  calibration curve  67% of the QCs passed; 
standards and QCs  however both QCs that 
  exceeded 15% 
001BR- were at the low QC 
01/01CALTables  concentration.  The follow-
001BR- up investigation concluded 
01/01QCTables  that the LC/MS/MS 
  instrument required a 
Report text  recalibration. 
001BR-01/01CALText   
001BR-01/01QCText 
 
Raw Data 
001BR-
01/01CALData 
001BR-01/01QCData 
001-100-02  Not  MM/DD/YY  Accepted  Summary tables for  This is the reanalysis of the 
applicable  calibration curve  samples from run  001-100-
standards and QCs  01 
001BR-
01/02CALTables 
001BR-
01/02QCTables 
 
Report text 
001BR-01/02CALText 
001BR-01/02QCText 
 
Raw Data 
001BR-
01/02CALData 
001BR-01/02QCData 
*If multiple batches are analyzed within an analytical run, each batch should be separately evaluated to determine if 
the batch meets acceptance criteria.  
For eCTD submissions, a hyperlink should be provided for the summary tables, report text, and raw data.  
 
 
 
Bioanalytical Method Validation  36 
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VIII.  GLOSSARY 
 
Accuracy:  Accuracy is the degree of closeness of the determined value to the nominal  or known 
true value under prescribed conditions.   Accuracy is also sometimes termed trueness. 
 
Analyte:  An analyte is the specific chemical moiety being measured; it can be an intact drug, a 
biomolecule  or its derivative,  a metabolite,  or a degradation product in a biologic  matrix. 
 
Analytical run:  An analytical run is a complete set of analytical and study samples with an 
appropriate number of standards and QCs for their validation.   Several runs can be completed in 
one day, or one run may take several days to complete. 
 
Autosampler stability:  Autosampler stability  is the stability of the analyte in the processed 
sample under the conditions  in the autosampler. 
 
Biological matrix:  A biological  matrix is discrete material of biological  origin  that can be 
sampled and processed in a reproducible manner.  Examples are blood, serum, plasma, urine, 
feces, cerebrospinal fluid,  saliva, sputum, and various discrete tissues. 
 
Batch:  For purposes of this guidance, a batch is a number of unknown samples from one or 
more patients in a study and QCs that are processed at one time. 
 
Between run:  Between run refers to the distinct period between or among several analytical or 
validation  runs. 
 
Bench-top stability:  Bench-top stability  is the stability  of an analyte in a matrix under 
conditions  of sample handling  during sample processing. 
 
Blank:  A blank is a sample of a biological  matrix to which no analytes have been added that is 
used to assess the selectivity  of the bioanalytical  method.  
 
Calibration curve:  The calibration  curve  also known as the standard curve  is the 
relationship  between the instrument response and the calibration  standards within the intended 
quantitation  range.    
 
Calibrators/Calibration standards:  Calibrators, or calibration  standards, refer to a biological 
matrix to which a known amount of analyte has been added.  Calibration  standards are used to 
construct calibration  curves from which the concentrations of analytes in QC samples and in-
study samples are determined. 
 
Carryover:  Carryover is the appearance of an analyte in a sample from a preceding sample.  
 
Critical reagents:  Critical reagents are requisite components of an assay, which include 
antibodies,  labeled analytes, matrices, etc.  
 
Bioanalytical Method Validation  37 
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Dilutional linearity:  Dilutional  linearity  demonstrates the accurate measurement of 
concentrations of spiked samples (i.e., QCs) exceeding the quantitation  range when serially 
diluted to within the quantitative  assay range.   
 
Extract:  An extract is a sample treated to remove impurities  or interfering substances (also 
known as a processed sample).   
 
Extract stability:  Extract stability  assesses the degradation of the processed sample relative to 
the starting material.  
 
Freeze-thaw stability:  Freeze-thaw stability  refers to the stability  of the analyte in the matrix 
upon freezing and thawing.  
 
Freshly prepared:  Freshly prepared refers to QC sample preparation (i.e., spiked) on the day of 
the experiment; not frozen before use. 
 
Full validation:  Full validation  refers to the establishment  of all validation  parameters that 
apply to sample analysis for the bioanalytical  method for each analyte. 
 
Heteroscadisticity:  Heteroscadisticity occurs when the variance of a response is not constant 
but changes with the response. 
 
Hook effect:  The hook effect occurs when increasing analyte concentrations result in no change 
or decreased signals when compared to the preceding concentration. 
 
Incurred samples:  Incurred samples are study samples or samples from subjects or patients 
who were dosed. 
 
Incurred Sample Reanalysis (ISR):  ISR is the repeated measurement of an analytes 
concentration from study samples to demonstrate reproducibility.    
 
Interference:  Interference refers to the action of sample components, including  structurally 
similar analytes, metabolites,  impurities,  degradants, or matrix components that may impact 
quantitation  of the analyte of interest.  Refer to Selectivity and Matrix effect for further 
information. 
 
Internal standard (IS):  ISs are test compounds (e.g., structurally similar analogs, stable isotope 
labeled compounds) added to both calibration  standards and samples at known and constant 
concentrations to facilitate quantification  of the target analyte(s). 
 
Long-term stability:  Long-term stability  assesses the degradation of an analyte in the matrix 
relative to the starting material after periods of frozen storage.  
 
Lower limit of quantification (LLOQ):  The LLOQ is the lowest amount of an analyte that can 
be quantitatively  determined with acceptable precision and accuracy. 
 
Bioanalytical Method Validation  38 
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Matrix effect:  The matrix effect is a direct or indirect alteration or interference in response 
because of the presence of unintended analytes (for analysis) or other interfering substances in 
the sample. 
 
Method:  A method is a comprehensive description of all procedures used in the collection, 
storage, and analysis of samples. 
 
Non-zero calibrator:  A non-zero calibrator is a calibrator to which the internal standard is 
added. 
 
Nominal concentration:  The nominal  concentration is the actual or intended concentration of 
the calibrator or quality control samples. 
 
Parallelism:  Parallelism demonstrates that the serially diluted  incurred sample response curve is 
parallel to the calibration  curve.  Parallelism is a performance characteristic that can detect 
potential matrix effects and interactions between critical reagents in an assay. 
 
Precision:  Precision is the closeness of agreement (i.e., degree of scatter) among a series of 
measurements obtained from multiple  sampling of the same homogenous  sample under the 
prescribed conditions. 
 
Processed sample:  A processed sample is the final extract (before instrumental  analysis) of a 
sample that has been subjected to various manipulations  (e.g., extraction, dilution, 
concentration). 
 
Processing batch:  A processing batch is a group of unknown samples from one or more study 
subjects, calibrators, and a set of QCs that are subjected to the analytical methodology  together.   
 
Prozone:  The prozone is an effect observed when increasing analyte concentrations result in 
either no change or decreased detector response when compared to the preceding concentration.  
(Also see the Hook effect) 
 
Quality control sample (QC):  A QC is a biological  matrix with a known quantity of analyte 
that is used to monitor the performance of a bioanalytical  method and to assess the integrity  and 
validity  of the results of study samples analyzed in an individual  run. 
 
Quantification range:  The quantification  range is the range of concentrations, including  the 
ULOQ and the LLOQ that can be reliably  and reproducibly  quantified with accuracy and 
precision with a concentration-response relationship. 
 
Recovery:  Recovery refers to the extraction efficiency of an analytical process, reported as a 
percentage of the known amount of an analyte carried through the sample extraction and 
processing steps of the method. 
 
Reintegration:  Reintegration is a reanalysis of the chromatographic  peak. 
 
Bioanalytical Method Validation  39 
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Reference standard: A reference standard is a chemical substance of known purity and identity 
which is used to prepare calibration standards and quality  controls.  Three types of reference 
standards are usually  used:  (1) certified (e.g., USP compendial  standards), (2) commercially-
supplied,  and (3) custom-synthesized. 
 
Reproducibility:  Reproducibility  is the precision between two laboratories.  It also represents 
the precision of the method under the same operating conditions  over a short period of time. 
 
Response function:  Response function is the mathematical expression that describes the 
relationship  between known sample concentrations and the response of the instrument (Also 
refer to Calibration curve). 
 
Sample:  A sample is a generic term encompassing controls, blanks, unknowns, and processed 
samples. 
 
Selectivity:  Selectivity is the extent to which the method can determine a particular compound 
in the analyzed matrices without interference from matrix components.  
 
Sensitivity:  Sensitivity  is defined as the lowest analyte concentration in the matrix that can be 
measured with acceptable accuracy and precision (i.e., LLOQ). 
 
Specificity:  Specificity  is the ability  of the method to assess, unequivocally,  the analyte in the 
presence of other components that are expected to be present (e.g., impurities,  degradation 
products, matrix components, etc.). 
 
Spiked samples:  A spiked sample is a general term that refers to calibrators (calibration 
standards) and quality controls. 
 
Stability:  Stability  is a measure of the intactness an analyte (lack of degradation) in a given 
matrix under specific storage and use conditions  relative to the starting material for given time 
intervals. 
 
Standard curve:  Refer to Calibration curve. 
 
Stock Solution:  A stock solution  refers to an analyte in a solvent or mixture of solvents at a 
known concentration, which is used to prepare calibrators or QCs. 
 
Study samples:  Study samples refer to samples from subjects or patients enrolled in a study. 
 
System suitability:  System suitability  is a determination  of instrument performance (e.g., 
sensitivity  and chromatographic retention) by analyzing  a set of reference standards before the 
analytical run. 
 
Total error:  Total error is the sum of the absolute value of the errors in accuracy (%) and 
precision (%).  Total error is reported as percent (%) error. 
 
Bioanalytical Method Validation  40 
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Unknown:  An unknown is a biological  sample that is the subject of the analysis. 
 
Upper limit of quantification (ULOQ):  The ULOQ is the highest amount of an analyte in a 
sample that can be quantitatively  determined with precision and accuracy. 
 
Within-run:  Within-run  refers to the time period during a single analytical or validation  run. 
 
Zero calibrator:  A zero calibrator is a blank sample to which the internal standard is added. 
 
 
Bioanalytical Method Validation  41 
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