M10 BIOANALYTICAL 
METHOD VALIDATION 
AND STUDY SAMPLE 
ANALYSIS 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
November 2022 
ICH 
 
   
 
M10 BIOANALYTICAL 
METHOD VALIDATION 
AND STUDY SAMPLE 
ANALYSIS 
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 
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and/or 
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Email: ocod@fda.hhs.gov 
https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-guidances   
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
November 2022 
ICH 
 
  Contains Nonbinding Recommendations 
 
FOREWORD 
  
The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for 
Human Use (ICH) has the mission of achieving greater regulatory harmonization worldwide to 
ensure that safe, effective, and high-quality medicines are developed, registered, and maintained 
in the most resource-efficient manner. By harmonizing the regulatory expectations in regions 
around the world, ICH guidelines have substantially reduced duplicative clinical studies, 
prevented unnecessary animal studies, standardized safety reporting and marketing application 
submissions, and contributed to many other improvements in the quality of global drug 
development and manufacturing and the products available to patients.  
  
ICH is a consensus-driven process that involves technical experts from regulatory authorities and 
industry parties in detailed technical and science-based harmonization work that results in the 
development of ICH guidelines. The commitment to consistent adoption of these consensus-
based guidelines by regulators around the globe is critical to realizing the benefits of safe, 
effective, and high-quality medicines for patients as well as for industry. As a Founding 
Regulatory Member of ICH, the Food and Drug Administration (FDA) plays a major role in the 
development of each of the ICH guidelines, which FDA then adopts and issues as guidance to 
industry. 
 
 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
I.  INTRODUCTION (1)................................................................................................. 1 
A.  Objective (1.1) .............................................................................................................. 1 
B.  Background (1.2) .......................................................................................................... 1 
C.  Scope (1.3) .................................................................................................................... 2 
II.  GENERAL PRINCIPLES (2) .................................................................................... 3 
A.  Method Development (2.1) ............................................................................................. 3 
B.  Method Validation (2.2)................................................................................................. 3 
1.  Full Validation (2.2.1)..................................................................................................... 3 
2.  Partial Validation (2.2.2)................................................................................................. 4 
3.  Cross Validation (2.2.3) .................................................................................................. 4 
III.  CHROMATOGRAPHY (3) ....................................................................................... 4 
A.  Reference Standards (3.1) .............................................................................................. 4 
B.  Validation (3.2) ............................................................................................................. 5 
1.  Selectivity (3.2.1)............................................................................................................ 5 
2.  Specificity (3.2.2)............................................................................................................ 6 
3.  Matrix Effect (3.2.3)........................................................................................................ 7 
4.  Calibration Curve and Range (3.2.4) ................................................................................. 7 
5.  Accuracy and Precision (3.2.5) ......................................................................................... 8 
6.  Carryover (3.2.6)............................................................................................................ 9 
7.  Dilution Integrity (3.2.7).................................................................................................. 9 
8.  Stability (3.2.8) .............................................................................................................10 
9.  Reinjection Reproducibility (3.2.9) ...................................................................................12 
C.  Study Sample Analysis (3.3) ..........................................................................................13 
1.  Analytical Run (3.3.1) ....................................................................................................13 
2.  Acceptance Criteria for an Analytical Run (3.3.2)...............................................................14 
3.  Calibration Range (3.3.3) ...............................................................................................15 
4.  Reanalysis of Study Samples (3.3.4)..................................................................................15 
5.  Reinjection of Study Samples (3.3.5) .................................................................................16 
6.  Integration of Chromatograms (3.3.6) ..............................................................................17 
IV.  LIGAND BINDING ASSAYS (4) ............................................................................. 17 
A.  Key Reagents (4.1) .......................................................................................................17 
1.  Reference Standard (4.1.1)..............................................................................................17 
2.  Critical Reagents (4.1.2).................................................................................................17 
B.  Validation (4.2) ............................................................................................................18 
1.  Specificity (4.2.1)...........................................................................................................18 
2.  Selectivity (4.2.2)...........................................................................................................19 
3.  Calibration Curve and Range (4.2.3) ................................................................................19 
4.  Accuracy and Precision (4.2.4) ........................................................................................20 
5.  Carryover (4.2.5)...........................................................................................................21 
6.  Dilution Linearity and Hook Effect (4.2.6).........................................................................21 
7.  Stability (4.2.7) .............................................................................................................22 
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C.  Study Sample Analysis (4.3) ..........................................................................................23 
1.  Analytical Run (4.3.1) ....................................................................................................23 
2.  Acceptance Criteria for an Analytical Run (4.3.2)...............................................................23 
3.  Calibration Range (4.3.3) ...............................................................................................24 
4.  Reanalysis of Study Samples (4.3.4)..................................................................................25 
V.  INCURRED SAMPLE REANALYSIS (5)............................................................... 26 
VI.  PARTIAL AND CROSS VALIDATION (6) ............................................................ 27 
A.  Partial Validation (6.1) .................................................................................................27 
B.  Cross Validation (6.2)...................................................................................................28 
VII.  ADDITIONAL CONSIDERATIONS (7) ................................................................. 29 
A.  Methods for Analytes That Are Also Endogenous Molecules (7.1)....................................29 
1.  Quality Control Samples for Methods for Analytes That Are Also Endogenous Molecules (7.1.1)..31 
2.  Selectivity, Recovery, and Matrix Effects for Methods for Analytes That Are Also Endogenous 
Molecules (7.1.2) ..............................................................................................................31 
3.  Parallelism for Methods for Analytes That Are Also Endogenous Molecules (7.1.3) .................32 
4.  Accuracy and Precision for Methods for Analytes That Are Also Endogenous Molecules (7.1.4) 32 
5.  Stability for Methods for Analytes That Are Also Endogenous Molecules (7.1.5) .....................33 
B.  Parallelism (7.2) ...........................................................................................................33 
C.  Recovery (7.3)..............................................................................................................33 
D.  Minimum Required Dilution (7.4) .................................................................................34 
E.  Commercial and Diagnostic Kits (7.5)............................................................................34 
F.  New or Alternative Technologies (7.6) ...........................................................................35 
1.  Dried Matrix Methods (7.6.1) ..........................................................................................35 
VIII.  DOCUMENTATION (8) .......................................................................................... 36 
A.  Summary Information (8.1) ..........................................................................................36 
B.  Documentation for Validation and Bioanalytical Reports (8.2) ........................................37 
GLOSSARY ........................................................................................................................ 45 
 
      ii  Contains Nonbinding Recommendations 
M10 BIOANALYTICAL METHOD VALIDATION AND STUDY 
SAMPLE ANALYSIS   
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 (1)2 
 
A.  Objective (1.1) 
 
This guidance is intended to provide recommendations for the validation of bioanalytical 
methods for chemical and biological drug quantification and their application in the analysis of 
study samples. Adherence to the principles presented in this guidance will ensure the quality and 
consistency of the bioanalytical data in support of the development and market approval of both 
chemical and biological drugs.  
 
The objective of the validation of a bioanalytical method is to demonstrate that it is suitable for 
its intended purpose. You may use an alternative approach from the recommendations in this 
guidance if it satisfies the requirements of applicable statutes and regulations. Alternative 
approaches should be supported by appropriate scientific justification. Applicants are encouraged 
to consult the regulatory authority(ies) regarding significant changes in method validation 
approaches when an alternate approach is proposed or taken. This guidance replaces the ICH 
draft guidance for industry M10 Bioanalytical Method Validation, issued on June 27, 2019.3 
 
B.  Background (1.2) 
 
Concentration measurements of chemical and biological drug(s) and their metabolite(s) in 
biological matrices are an important aspect of drug development. The results of studies 
employing such methods contribute to regulatory decisions regarding the safety and efficacy of 
 
1 This guidance was developed within an Expert Working Group of the International Council for Harmonisation of 
Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and has been subject to 
consultation by the regulatory parties, in accordance with the ICH process. This document was endorsed by the 
Regulatory Members of the ICH Assembly at Step 4 of the ICH process on May 24, 2022.  
2 The numbers in parentheses reflect the organizational breakdown of the document endorsed by the Regulatory 
Members of the ICH Assembly at Step 4 of the ICH process, May 24, 2022. 
3 We update guidances periodically. For the most recent version of a guidance, check the FDA guidance web page 
at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
1  Contains Nonbinding Recommendations 
drug products. It is therefore critical that the bioanalytical methods used are well characterized, 
appropriately validated, and documented in order to ensure reliable data to support regulatory 
decisions.  
 
This guidance intends to facilitate development of drugs in accordance with the principles of the 
3Rs (Reduce, Refine, Replace) for animal studies, where valid. 
 
C.  Scope (1.3) 
 
This guidance describes the validation of bioanalytical methods and study sample analysis that 
are expected to support regulatory decisions. The guidance is applicable to the bioanalytical 
methods used to measure concentrations of chemical and biological drug(s) and their 
metabolite(s) in biological samples (e.g., blood, plasma, serum, other body fluids or tissues) 
obtained in nonclinical toxicokinetic (TK) studies conducted according to the principles of Good 
Laboratory Practice (GLP), nonclinical pharmacokinetic (PK) studies conducted as surrogates 
for clinical studies, and all phases of clinical trials, including comparative 
bioavailability/bioequivalence (BA/BE) studies, in regulatory submissions. Full method 
validation is recommended for the primary matrix intended to support regulatory submissions. 
Additional matrices should be validated as necessary.  
 
For studies that are not submitted for regulatory approval or not considered for regulatory 
decisions regarding safety, efficacy, or labeling (e.g., exploratory investigations), applicants may 
decide on the level of qualification that supports their own internal decision-making.  
 
The information in this guidance applies to the quantitative analysis by ligand binding assays 
(LBAs) and chromatographic methods such as liquid chromatography (LC) or gas 
chromatography (GC), which are typically used in combination with mass spectrometry (MS) 
detection. 
 
For studies that are subject to GLP or Good Clinical Practice (GCP) requirements, the 
bioanalysis of study samples must conform to those requirements. The bioanalysis of biomarkers 
and bioanalytical methods used for the assessment of immunogenicity are not within the scope of 
this guidance. 
 
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 
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.   
2  Contains Nonbinding Recommendations 
 
II.  GENERAL PRINCIPLES (2) 
 
A.  Method Development (2.1) 
 
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 
ready for validation. 
 
Before or during the development of a bioanalytical method, the applicant is encouraged to, if 
feasible, understand the analyte of interest (e.g., the physicochemical properties of the drug, in 
vitro and in vivo metabolism, preferential distribution between red blood cells and plasma, and 
protein binding) and consider aspects of any prior analytical methods that may be applicable. 
 
Method development involves identifying the procedures and conditions involved with 
quantifying the analyte. Method development can include the characterization of the following 
bioanalytical elements: reference standards, critical reagents, calibration curve, quality control 
samples (QCs), selectivity and specificity, sensitivity, accuracy, precision, recovery, stability of 
the analyte, and minimum required dilution (MRD). 
 
Bioanalytical method development does not require extensive record keeping or notation. Once 
the method has been developed, bioanalytical method validation proves that the method is suited 
to the analysis of the study samples.  
 
If a problem is encountered with the method during the analysis of nonclinical or clinical study 
samples that requires that the analysis be stopped, any changes to the method and the rationale 
should be documented. 
 
B.  Method Validation (2.2) 
 
1.  Full Validation (2.2.1) 
 
Bioanalytical method validation is important to ensure the acceptability of assay performance 
and the reliability of analytical results. A bioanalytical method is defined as a set of procedures 
used for measuring analyte concentrations in biological samples. A full validation of a 
bioanalytical method should be performed when establishing a bioanalytical method for the 
quantification of an analyte in clinical and in applicable nonclinical studies. Full validation 
should also be performed when implementing an analytical method that is reported in the 
literature and when a commercial kit is repurposed for bioanalytical use in drug development. 
Usually, one analyte has to be determined, but on occasion it may be appropriate to measure 
more than one analyte. This may involve two different drugs, a parent drug with its metabolites 
or the enantiomers or isomers of a drug. In these cases, the principles of validation and analysis 
apply to all analytes of interest. For chromatographic methods, a full validation should include 
the following elements, unless otherwise justified: selectivity, specificity, matrix effect, 
calibration curve (response function), range (lower limit of quantification (LLOQ) to upper limit 
3  Contains Nonbinding Recommendations 
of quantification (ULOQ)), accuracy, precision, carryover, dilution integrity, stability, and 
reinjection reproducibility.  
 
For LBAs, the following elements should be evaluated, unless otherwise justified: specificity, 
selectivity, calibration curve (response function), range (LLOQ to ULOQ), accuracy, precision, 
carryover, dilution linearity, and stability. If necessary, parallelism can be conducted when 
appropriate study samples are available.  
 
The assessments that are performed during validation should be relevant to the sample analysis 
workflow. The matrix used for bioanalytical method validation should be the same as the matrix 
of the study samples, including anticoagulants and additives. In cases in which it may be difficult 
to obtain an identical matrix to that of the study samples (e.g., rare matrices such as tissue, 
cerebrospinal fluid, bile or in cases where free drug is measured), surrogate matrices may be 
acceptable for analytical method validation.  
 
The choice of surrogate matrix should be scientifically justified. Matrix differences within 
species (e.g., age, ethnicity, gender) are generally not considered different when validating a 
method. 
 
A specific, detailed, written description of the bioanalytical method and validation procedure 
should be established a priori. This description may be in the form of a protocol, study plan, 
report, notebook, or Standard Operating Procedure (SOP). 
 
2.  Partial Validation (2.2.2) 
 
Modifications to a fully validated analytical method may be evaluated by partial validation. 
Partial validation can range from as little as one accuracy and precision determination to a nearly 
full validation (refer to section VI.A (6.1)). The items in a partial validation should be 
determined according to the extent and nature of the changes made to the method. 
 
3.  Cross Validation (2.2.3) 
 
Cross validation is required to demonstrate how the reported data are related when multiple 
bioanalytical methods and/or multiple bioanalytical laboratories are involved (refer to section 
VI.B (6.2)). 
 
 
III.  CHROMATOGRAPHY (3) 
 
A.  Reference Standards (3.1) 
 
During method validation and the analysis of study samples, a blank biological matrix is spiked 
with the analyte(s) of interest using solutions of reference standard(s) to prepare calibration 
standards and QCs. Calibration standards and QCs should be prepared from separate stock 
solutions. However, calibration standards and QCs may be prepared from the same stock 
4  Contains Nonbinding Recommendations 
solution provided the accurate preparation and stability of the stock solution should have been 
verified.  
 
A suitable internal standard (IS) should be added to all calibration standards, QCs, and study 
samples during sample processing. The absence of an IS should be justified.  
 
It is important that the reference standard is well characterized and the quality (e.g., purity, 
identity) of the reference standard and the suitability of the IS is ensured, as the quality will 
affect the outcome of the analysis and, therefore, the study data. The reference standard used 
during validation and study sample analysis should be obtained from an authentic and traceable 
source. The reference standard should be identical to the analyte. If this is not possible, an 
established form (e.g., salt or hydrate) of known quality should be used. 
 
Suitable reference standards include compendial standards, commercially available standards or 
sufficiently characterized standards prepared in-house or by an external organization. A 
certificate of analysis (CoA) or an equivalent alternative is recommended to ensure quality and to 
provide information on the purity, storage conditions, retest/expiration date and batch number of 
the reference standard.  
 
A CoA is not required for the IS as long as the suitability for use is demonstrated, e.g., a lack of 
analytical interference is shown for the substance itself or any impurities thereof.  
 
When MS detection is used, the use of the stable isotope-labeled analyte as the IS is 
recommended whenever possible. However, it is important that the labeled standard is of high 
isotope purity and that no isotope exchange reaction occurs. The presence of unlabeled analyte 
should be checked and if unlabeled analyte is detected, the potential influence should be 
evaluated during method validation. 
 
Stock and working solutions should only be prepared from reference standards that are within the 
stability period as documented in the CoA (either expiration date or the retest date). 
 
B.  Validation (3.2) 
 
1.  Selectivity (3.2.1) 
 
Selectivity is the ability of an analytical method to differentiate and measure the analyte in the 
presence of potential interfering substances in the blank biological matrix.  
 
Selectivity should be evaluated using blank samples (matrix samples processed without addition 
of an analyte or IS) obtained from at least six individual sources/lots (non-hemolyzed and non-
lipemic). Use of fewer sources may be acceptable in the case of rare matrices. Selectivity for the 
IS should also be evaluated. 
 
The evaluation of selectivity should demonstrate that no significant response attributable to 
interfering components is observed at the retention time(s) of the analyte or the IS in the blank 
samples. Responses attributable to interfering components should not be more than 20% of the 
5  Contains Nonbinding Recommendations 
analyte response at the LLOQ and not more than 5% of the IS response in the LLOQ sample for 
each matrix. 
 
For the investigation of selectivity in lipemic matrices at least one source of matrix should be 
used. To be scientifically meaningful, the matrix used for these tests should be representative as 
much as possible of the expected study samples. A naturally lipemic matrix with abnormally 
high levels of triglycerides should be obtained from donors. Although it is recommended to use 
lipemic matrix from donors, if this is difficult to obtain, matrix can be spiked with triglycerides 
even though it may not be representative of study samples. However, if the drug impacts lipid 
metabolism or if the intended patient population is hyperlipidemic, the use of spiked samples is 
discouraged. This evaluation is not necessary for nonclinical studies unless the drug impacts lipid 
metabolism or is administered in a particular animal strain that is hyperlipidemic.  
 
For the investigation of selectivity in hemolyzed matrices at least one source of matrix should be 
used. Hemolyzed matrices should be obtained by spiking matrix with hemolyzed whole blood (at 
least 2% V/V) to generate a visibly detectable hemolyzed sample.  
 
2.  Specificity (3.2.2) 
 
Specificity is the ability of a bioanalytical method to detect and differentiate the analyte from 
other substances, including its related substances (e.g., substances that are structurally similar to 
the analyte, metabolites, isomers, impurities, degradation products formed during sample 
preparation, or concomitant medications that are expected to be used in the treatment of patients 
with the intended indication). 
 
If the presence of related substances is anticipated in the biological matrix of interest, the impact 
of such substances should be evaluated during method validation, or alternatively, in the pre-
dose study samples. In the case of LC-MS based methods, to assess the impact of such 
substances, the evaluation may include comparing the molecular weight of a potential interfering 
related substance with the analyte and chromatographic separation of the related substance from 
the analyte.  
 
Responses detected and attributable to interfering components should not be more than 20% of 
the analyte response at the LLOQ and not more than 5% of the IS response in the LLOQ sample. 
 
The possibility of back-conversion of a metabolite into the parent analyte during the successive 
steps of the analysis (including extraction procedures or in the MS source) should also be 
evaluated when relevant (e.g., potentially unstable metabolites such as ester analytes to 
ester/acidic metabolites, unstable N-oxides or glucuronide metabolites, lactone-ring structures). 
It is acknowledged that this evaluation will not be possible in the early stages of drug 
development of a new chemical entity when the metabolism is not yet evaluated. However, this 
issue should be investigated, and partial validation performed, if needed. The extent of back-
6  Contains Nonbinding Recommendations 
conversion, if any, should be established and the impact on the study results should be discussed 
in the Bioanalytical Report. 
 
3.  Matrix Effect (3.2.3) 
 
A matrix effect is defined as an alteration of the analyte response due to interfering and often 
unidentified component(s) in the sample matrix. During method validation the matrix effect 
between different independent sources/lots should be evaluated. 
 
The matrix effect should be evaluated by analyzing at least three replicates of low and high QCs, 
each prepared using matrix from at least six different sources/lots. For each individual matrix 
sources/lots evaluated, the accuracy should be within 15% of the nominal concentration and the 
precision (percent coefficient of variation (%CV)) should not be greater than 15%. Use of fewer 
sources/lots may be acceptable in the case of rare matrices.  
 
The matrix effect should also be evaluated in relevant patient populations or special populations 
(e.g., hepatically impaired or renally impaired) when available. An additional evaluation of the 
matrix effect is recommended using hemolyzed or lipemic matrix samples during method 
validation on a case-by-case basis, especially when these conditions are expected to occur within 
the study.  
 
4.  Calibration Curve and Range (3.2.4) 
 
The calibration curve demonstrates the relationship between the nominal analyte concentration 
and the response of the analytical platform to the analyte. Calibration standards, prepared by 
spiking matrix with a known quantity of analyte(s), span the calibration range and comprise the 
calibration curve. Calibration standards should be prepared in the same biological matrix as the 
study samples. The calibration range is defined by the LLOQ, which is the lowest calibration 
standard, and the ULOQ, which is the highest calibration standard. There should be one 
calibration curve for each analyte studied during method validation and for each analytical run. 
 
A calibration curve should be generated with a blank sample, a zero sample (blank sample spiked 
with IS), and at least six concentration levels of calibration standards, including the LLOQ and 
the ULOQ.  
 
A simple regression model that adequately describes the concentration-response relationship 
should be used. The selection of the regression model should be directed by written procedures. 
The regression model, weighting scheme, and transformation should be determined during the 
method validation. Blank and zero samples should not be included in the determination of the 
regression equation for the calibration curve. Each calibration standard may be analyzed in 
replicate, in which case data from all acceptable replicates should be used in the regression 
analysis. 
 
The calibration curve parameters should be reported (e.g., slope and intercept in the case of a 
linear model). The back-calculated concentrations of the calibration standards should be 
presented together with the calculated mean accuracy and precision values. All acceptable curves 
7  Contains Nonbinding Recommendations 
obtained during validation, based on a minimum of three independent runs over several days, 
should be reported. The accuracy of the back-calculated concentrations of each calibration 
standard should be within 20% of the nominal concentration at the LLOQ and within 15% at 
all the other levels. At least 75% of the calibration standards with a minimum of six calibration 
standard levels should meet the above criteria.  
 
In the case that replicates are used, the criteria (within 15% or 20% for LLOQ) should also be 
fulfilled for at least 50% of the calibration standards tested per concentration level. In the case 
that a calibration standard does not comply with these criteria, this calibration standard sample 
should be rejected, and the calibration curve without this calibration standard should be 
reevaluated, including regression analysis. For accuracy and precision runs, if all replicates of the 
LLOQ or the ULOQ calibration standard in a run are rejected, then the run should be rejected, 
the possible source of the failure should be determined and the method revised, if necessary. If 
the next validation run also fails, then the method should be revised before restarting validation.  
 
The calibration curve should be prepared using freshly spiked calibration standards in at least 
one assessment. Subsequently, frozen calibration standards can be used within their defined 
period of stability. 
 
5.  Accuracy and Precision (3.2.5) 
 
a.  Preparation of quality control samples (3.2.5.1) 
 
The QCs are intended to mimic study samples and should be prepared by spiking matrix with a 
known quantity of analyte, storing them under the conditions anticipated for study samples and 
analyzing them to assess the validity of the analytical method. 
 
Calibration standards and the QCs should be prepared from separate stock solutions in order to 
avoid biased estimations which are not related to the analytical performance of the method. If 
calibration standards and the QCs may be prepared from the same stock solution, the accuracy 
and stability of the stock solution should be verified. A single source of blank matrix may be 
used, which should be free of interference or matrix effects, as described in section III.B.3 
(3.2.3). 
 
During method validation the QCs for accuracy and precision runs should be prepared at a 
minimum of 4 concentration levels within the calibration curve range: the LLOQ, within 3 times 
of the LLOQ (low QC), around 30% to 50% of the calibration curve range (medium QC) and at 
least 75% of the ULOQ (high QC).  
 
For non-accuracy and precision validation runs, low, medium, and high QCs may be analyzed in 
duplicate. These QCs, along with the calibration standards, will provide the basis for the 
acceptance or rejection of the run.  
 
b.  Evaluation of accuracy and precision (3.2.5.2) 
 
8  Contains Nonbinding Recommendations 
Accuracy and precision should be determined by analyzing the QCs within each run (within-run) 
and in different runs (between-run). Accuracy and precision should be evaluated using the same 
runs and data. 
 
Within-run accuracy and precision should be evaluated by analyzing at least five replicates at 
each QC concentration level in each analytical run. Between-run accuracy and precision should 
be evaluated by analyzing each QC concentration level in at least three analytical runs over at 
least two days. To enable the evaluation of any trends over time within one run, it is 
recommended to demonstrate accuracy and precision of the QCs over at least one of the runs in a 
size equivalent to a prospective analytical run of study samples. Reported method validation data 
and the determination of accuracy and precision should include all results obtained, including 
individual QCs outside of the acceptance criteria, except those cases where errors are obvious 
and documented. Within-run accuracy and precision data should be reported for each run. If the 
within-run accuracy or precision criteria are not met in all runs, an overall estimate of within-run 
accuracy and precision for each QC level should be calculated. Between-run (intermediate) 
precision and accuracy should be calculated by combining the data from all runs.  
 
The calibration curves for these assessments should be prepared using freshly spiked calibration 
standards in at least one run. If freshly spiked calibration standards are not used in the other runs, 
stability of the frozen calibration standards should be demonstrated. 
 
The accuracy at each concentration level should be within 15% of the nominal concentration, 
except at the LLOQ, where it should be within 20%. The precision (%CV) of the 
concentrations determined at each level should not exceed 15%, except at the LLOQ, where it 
should not exceed 20%. For non-accuracy and precision validation runs, at least 2/3 of the total 
QCs and at least 50% at each concentration level should be within 15% of the nominal values. 
 
6.  Carryover (3.2.6) 
 
Carryover is an alteration of a measured concentration due to residual analyte from a preceding 
sample that remains in the analytical instrument.  
 
Carryover should be assessed and minimized during method development. During validation 
carryover should be assessed by analyzing blank samples after the calibration standard at the 
ULOQ. Carryover in the blank samples following the highest calibration standard should not be 
greater than 20% of the analyte response at the LLOQ and 5% of the response for the IS. If it 
appears that carryover is unavoidable, study samples should not be randomized. Specific 
measures should be considered, validated, and applied during the analysis of the study samples, 
so that carryover does not affect accuracy and precision. This could include the injection of blank 
sample(s) after samples with an expected high concentration, before the next study sample. 
 
7.  Dilution Integrity (3.2.7) 
 
Dilution integrity is the assessment of the sample dilution procedure, when required, to confirm 
that it does not impact the accuracy and precision of the measured concentration of the analyte. 
9  Contains Nonbinding Recommendations 
The same matrix from the same species used for preparation of the QCs should be used for 
dilution. 
 
Dilution QCs should be prepared with analyte concentrations in matrix that are greater than the 
ULOQ and then diluted with blank matrix. At least five replicates per dilution factor should be 
tested in one run to determine if concentrations are accurately and precisely measured within the 
calibration range. The dilution factor(s) and concentrations applied during study sample analysis 
should be within the range of the dilution factors and concentrations evaluated during validation. 
The mean accuracy of the dilution QCs should be within 15% of the nominal concentration and 
the precision (%CV) should not exceed 15%. 
 
In the cases of rare matrices, use of a surrogate matrix for dilution may be acceptable. It should 
be demonstrated that this does not affect precision and accuracy.  
 
8.  Stability (3.2.8) 
 
Stability evaluations should be carried out to ensure that every step taken during sample 
preparation, processing, and analysis as well as the storage conditions used do not affect the 
concentration of the analyte.  
 
The storage and analytical conditions applied to the stability tests, such as the sample storage 
times and temperatures, sample matrix, anticoagulant and container materials, should reflect 
those used for the study samples. Reference to data published in the literature is not considered 
sufficient. Validation of storage periods should be performed on QCs that have been stored for a 
time that is equal to or longer than the study sample storage periods. 
 
Stability of the analyte in the matrix is evaluated using low and high concentration QCs. Aliquots 
of the low and high QCs are analyzed at time zero and after the applied storage conditions that 
are to be evaluated. One bulk QC should be prepared at each concentration level. For each 
concentration tested, the bulk sample should be divided into a minimum of three aliquots that 
will be stored, stressed, and analyzed.  
 
The QCs should be analyzed against a calibration curve, obtained from freshly spiked calibration 
standards in a run with its corresponding freshly spiked QCs or QCs for which stability has been 
proven. The mean concentration at each QC level should be within 15% of the nominal 
concentration. If the concentrations of the study samples are consistently higher than the ULOQ 
of the calibration range, the concentration of the high QC should be adjusted to reflect these 
higher concentrations. It is recognized that this may not be possible in nonclinical studies due to 
solubility limitations. 
 
For fixed dose combination products and specifically labeled drug regimens, the freeze-thaw, 
bench top, and long-term stability tests of an analyte in matrix should be conducted with the 
matrix spiked with all of the dosed compounds.  
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The following stability tests should be evaluated: 
 
(1) Stability of the Analyte in Matrix  
 
Freeze-Thaw Stability in Matrix  
 
To assess the impact of repeatedly removing samples from frozen storage, the stability of the 
analyte should be assessed after multiple cycles of freezing and thawing. Low and high QCs 
should be thawed and analyzed according to the same procedures as the study samples. QCs 
should be kept frozen for at least 12 hours between the thawing cycles. QCs for freeze-thaw 
stability should be assessed using freshly prepared calibration standards and QCs, or QCs for 
which stability has been proven. The number of freeze-thaw cycles validated should equal or 
exceed that of the freeze-thaw cycles undergone by the study samples, but a minimum of 
three cycles should be conducted. 
 
Bench Top (Short-Term) Stability in Matrix  
 
Bench top matrix stability experiments should be designed and conducted to cover the 
laboratory handling conditions for the study samples. 
 
Low and high QCs should be thawed in the same manner as the study samples and kept on 
the bench top at the same temperature and for at least the same duration as the study samples. 
 
The total time on the bench top should be concurrent; additive exposure to bench top 
conditions should not be used (i.e., time from each freeze-thaw evaluation should not be 
added up). 
 
Long-Term Stability in Matrix  
 
The long-term stability of the analyte in matrix stored in the freezer should be established. 
Low and high QCs should be stored in the freezer under the same storage conditions and at 
least for the same duration as the study samples.  
 
For chemical drugs, the stability at one temperature (e.g., -20C) to can be extrapolated to 
lower temperatures (e.g., -70/-80C). 
 
For biological drugs, a bracketing approach can be applied, e.g., in the case that the stability 
has been demonstrated at -70/-80C and at -20C, then it is not necessary to investigate the 
stability at temperatures in between those two points at which study samples will be stored.  
 
(2) Stability of the Analyte in Processed Samples  
 
The stability of processed samples, including the time until completion of analysis (in the 
autosampler/instrument), should be determined. For example: 
 
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  Stability of the processed sample under the storage conditions to be used during the 
analysis of study samples (dry extract or in the injection phase)  
 
  On-instrument/autosampler stability of the processed sample at injector or autosampler 
temperature  
 
The total time that a processed sample is stored should be concurrent (i.e., autosampler and 
other storage times should not be added together). 
 
(3) Stability of the Analyte and IS in Stock and Working Solutions 
 
The stability of the stock and working solutions of the analyte and IS should be determined 
under the storage conditions used during the analysis of study samples by using the lowest 
and the highest concentrations of these solutions. They should be assessed using the response 
of the detector. Stability of the stock and working solutions should be tested with an 
appropriate dilution, taking into consideration the linearity and measuring range of the 
detector. If the stability varies with concentration, then the stability of all concentrations of 
the stock and working solutions should be assessed. If no isotopic exchange occurs for the 
stable isotopically labeled IS under the same storage conditions as the analyte for which the 
stability is demonstrated, then no additional stability determinations for the IS are necessary. 
If the reference standard expires, or it is past the retest date, the stability of the stock 
solutions made previously with this lot of reference standard are defined by the expiration or 
retest date established for the stock solution. Stock and working solutions should not be made 
from reference standards solely for extending the expiration date for the use of the reference 
standard.  
 
In addition, the following test should be performed, if applicable: 
 
(4) Stability of the Analyte in Whole Blood  
 
Sufficient attention should be paid to the stability of the analyte in the sampled matrix 
(blood) directly after collection from subjects and prior to preparation for storage to ensure 
that the concentrations obtained by the analytical method reflect the concentrations of the 
analyte in the subjects blood at the time of sample collection.  
 
If the matrix used is plasma, the stability of the analyte in blood should be evaluated during 
method development (e.g., using an exploratory method in blood) or during method 
validation. The results should be provided in the Validation Report.  
 
9.  Reinjection Reproducibility (3.2.9) 
 
Reproducibility of the method is assessed by replicate measurements of the QCs and is usually 
included in the assessment of precision and accuracy. However, if samples could be reinjected 
(e.g., in the case of instrument interruptions or other reasons such as equipment failure), 
reinjection reproducibility should be evaluated to establish the viability of the processed samples 
and to support their storage prior to reinjection. 
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Reinjection reproducibility is assessed by reinjecting a run that is comprised of calibration 
standards and a minimum of five replicates of the low, middle, and high QCs after storage. The 
precision and accuracy of the reinjected QCs establish the viability of the processed samples.  
 
The results should be included in the Validation Report or provided in the Bioanalytical Report 
of the study where it was conducted. 
 
C.  Study Sample Analysis (3.3) 
 
The analysis of study samples can be carried out after validation has been completed, however, it 
is understood that some parameters may be completed at a later stage (e.g., long-term stability). 
By the time the data are submitted to a regulatory authority, the bioanalytical method validation 
should have been completed. The study samples, QCs, and calibration standards should be 
processed in accordance with the validated analytical method. If system suitability is assessed, a 
predefined specific study plan, protocol, or SOP should be used. System suitability, including 
apparatus conditioning and instrument performance, should be determined using samples that are 
independent of the calibration standards and QCs for the run. Subject samples should not be used 
for system suitability. The IS responses of the study samples should be monitored to determine 
whether there is systemic IS variability. Refer to Table 1 for recommendations regarding 
documentation. 
 
1.  Analytical Run (3.3.1) 
 
An analytical run should consist of a blank sample (processed matrix sample without analyte and 
without IS), a zero sample (processed matrix with IS), calibration standards at a minimum of six 
concentration levels, at least three levels of QCs (low, medium, and high) in duplicate (or at least 
5% of the number of study samples, whichever is higher) and the study samples to be analyzed. 
The QCs should be interspersed in the run in such a way that the accuracy and precision of the 
whole run is ensured. Study samples should always be bracketed by QCs.  
 
The calibration standards and QCs should be spiked independently using separately prepared 
stock solutions unless the accuracy and stability of the stock solutions have been verified. All 
samples (calibration standards, QCs, and study samples) should be processed and extracted as 
one single batch of samples in the order in which they are intended to be analyzed. Analyzing 
samples that were processed as several separate batches in a single analytical run is discouraged. 
If such an approach cannot be avoided, for instance due to bench top stability limitations, each 
batch of samples should include low, medium, and high QCs. 
 
For comparative BA/BE studies, it is advisable to analyze all samples of one subject together in 
one analytical run to reduce variability.  
 
The impact of any carryover that occurs during study sample analysis should be assessed and 
reported (refer to section III.B.6 (3.2.6)). If carryover is detected, its impact on the measured 
concentrations should be mitigated (e.g., non-randomization of study samples, injection of blank 
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samples after samples with an expected high concentration) or the validity of the reported 
concentrations should be justified in the Bioanalytical Report.  
 
2.  Acceptance Criteria for an Analytical Run (3.3.2) 
 
Criteria for the acceptance or rejection of an analytical run should be defined in the protocol, in 
the study plan, or in an SOP. In the case that a run contains multiple batches, acceptance criteria 
should be applied to the whole run and to the individual batches. It is possible for the run to meet 
acceptance criteria, even if a batch within that run is rejected for failing to meet the batch 
acceptance criteria. Calibration standards in a failed batch should not be used to support the 
acceptance of other batches within the analytical run. 
 
The back-calculated concentrations of the calibration standards should be within 15% of the 
nominal value, except for the LLOQ for which it should be within 20%. At least 75% of the 
calibration standard concentrations, which should include a minimum of six concentration levels, 
should fulfill these criteria. If more than six calibration standard levels are used and one of the 
calibration standards does not meet the criteria, this calibration standard should be rejected and 
the calibration curve without this calibration standard should be reevaluated, and a new 
regression analysis performed. 
 
If the rejected calibration standard is the LLOQ, the new lower limit for this analytical run 
should be the next lowest acceptable calibration standard of the calibration curve. This new 
lower limit calibration standard should retain its original acceptance criteria (i.e., 15%). If the 
highest calibration standard is rejected, the ULOQ for this analytical run should be the next 
acceptable highest calibration standard of the calibration curve. The revised calibration range 
should cover at least three QC concentration levels (low, medium, and high). Study samples 
outside of the revised range should be reanalyzed. If replicate calibration standards are used and 
only one of the LLOQ or ULOQ standards fails, the calibration range is unchanged.  
 
At least 2/3 of the total QCs and at least 50% at each concentration level should be within 15% 
of the nominal values. If these criteria are not fulfilled the analytical run should be rejected. A 
new analytical batch should be prepared for all study samples within the failed analytical run for 
subsequent analysis. In the cases where the failure is due to an assignable technical cause, 
samples may be reinjected. 
 
Analytical runs containing samples that are diluted and reanalyzed should include dilution QCs 
to verify the accuracy and precision of the dilution method during study sample analysis. The 
concentration of the dilution QCs should exceed that of the study samples being diluted (or of the 
ULOQ) and they should be diluted using the same dilution factor. If multiple dilution factors are 
used in one analytical run, then dilution QCs need only be diluted by the highest and lowest 
dilution factors. The within-run acceptance criteria of the dilution QC(s) will only affect the 
acceptance of the diluted study samples and not the outcome of the analytical run.  
 
When several analytes are assayed simultaneously, there should be one calibration curve for each 
analyte studied. If an analytical run is acceptable for one analyte but has to be rejected for 
another analyte, the data for the accepted analyte should be used. The determination of the 
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rejected analyte requires reextraction and analysis only for the analyte that is reanalyzed. Only 
data for this reanalyzed analyte needs to be reported. 
 
The back-calculated concentrations of the calibration standards and QCs of passed and accepted 
runs should be reported. The overall (between-run) accuracy and precision of the QCs of all 
accepted runs should be calculated at each concentration level and reported in the analytical 
report (refer to section VIII. (8) Documentation and Table 1). If the overall mean accuracy 
and/or precision fails the 15% criterion, an investigation to determine the cause of the deviation 
should be conducted. In the case of comparative BA/BE studies, it may result in the rejection of 
the data. 
 
3.  Calibration Range (3.3.3) 
 
If a narrow range of analyte concentrations of the study samples is known or anticipated before 
the start of study sample analysis, it is recommended to either narrow the calibration curve range, 
adapt the concentrations of the QCs, or add new QCs at different concentration levels as 
appropriate, to adequately reflect the concentrations of the study samples. 
 
At the intended therapeutic dose(s), if an unanticipated clustering of study samples at one end of 
the calibration curve is encountered after the start of sample analysis, the analysis should be 
stopped and either the standard calibration range narrowed (i.e., partial validation), existing QC 
concentrations revised, or QCs at additional concentrations added to the original curve within the 
observed range before continuing with study sample analysis. It is not necessary to reanalyze 
samples analyzed before optimizing the calibration curve range or QC concentrations. 
 
The same applies if a large number of the analyte concentrations of the study samples are above 
the ULOQ. The calibration curve range should be changed, if possible, and QC(s) added, or their 
concentrations modified. If it is not possible to change the calibration curve range or the number 
of samples with a concentration above the ULOQ is not large, samples should be diluted 
according to the validated dilution method. 
 
At least two QC levels should fall within the range of concentrations measured in study samples. 
If the calibration curve range is changed, the bioanalytical method should be revalidated (partial 
validation) to verify the response function and to ensure accuracy and precision. 
 
4.  Reanalysis of Study Samples (3.3.4) 
 
Possible reasons for reanalysis of study samples, the number of replicates, and the decision 
criteria to select the value to be reported should be predefined in the protocol, study plan, or SOP 
before the actual start of the analysis of the study samples. For study samples in which multiple 
analytes are being analyzed, a valid result for one analyte should not be rejected if the other 
analyte fails the acceptance criteria. 
 
The number of samples (and percentage of total number of samples) that have been reanalyzed 
should be reported and discussed in the Bioanalytical Report. For comparative BA/BE studies, a 
separate table should report values from rejected runs. 
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Some examples of reasons for study sample reanalysis are: 
 
  Rejection of an analytical run because the run failed the acceptance criteria with regard to 
accuracy of the calibration standards and/or the precision and accuracy of the QCs 
 
  IS response significantly different from the response for the calibration standards and 
QCs (as predefined in an SOP) 
 
  The concentration obtained is above the ULOQ 
 
  The concentration observed is below the revised LLOQ in runs where the lowest 
calibration standard has been rejected from a calibration curve, resulting in a higher 
LLOQ compared with other runs 
 
  Improper sample injection or malfunction of equipment 
 
  The diluted study sample is below the LLOQ 
 
  Identification of quantifiable analyte levels in pre-dose samples, control, or placebo 
samples 
 
  Poor chromatography (as predefined in an SOP) 
 
For comparative BA/BE studies, reanalysis of study samples for a PK reason (e.g., a sample 
concentration does not fit with the expected profile) should not be done, as it may bias the study 
result. 
 
Any reanalyzed samples should be identified in the Bioanalytical Report and the initial value, the 
reason for reanalysis, the values obtained in the reanalyzes, the final accepted value and a 
justification for the acceptance should be provided. Further, a summary table of the total number 
of samples that have been reanalyzed for each reason should be provided. In cases where the first 
analysis yields a nonreportable result, a single reanalysis is considered sufficient (e.g., 
concentration above the ULOQ or equipment malfunction). In cases where the value needs to be 
confirmed (e.g., pre-dose sample with measurable concentrations) replicate determinations are 
required if sample volume allows.  
 
The safety of trial subjects should take precedence over any other aspect of the trial. 
Consequently, there may be other circumstances when it is necessary to reanalyze specific study 
samples for the purpose of a safety investigation.  
 
5.  Reinjection of Study Samples (3.3.5) 
 
Reinjection of processed samples can be made in the case of equipment failure if reinjection 
reproducibility has been demonstrated during validation or provided in the Bioanalytical Report 
where it was conducted. Reinjection of a full analytical run or of individual calibration standards 
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or QCs simply because the calibration standards or QCs failed, without any identified analytical 
cause, should not be done. 
 
6.  Integration of Chromatograms (3.3.6) 
 
Chromatogram integration and reintegration should be described in a study plan, protocol, or 
SOP. Any deviation from the procedures described a priori should be discussed in the 
Bioanalytical Report. The list of chromatograms that required reintegration, including any 
manual integrations, and the reasons for reintegration should be included in the Bioanalytical 
Report. Original and reintegrated chromatograms and initial and repeat integration results should 
be kept for future reference and submitted in the Bioanalytical Report for comparative BA/BE 
studies. 
 
 
IV.  LIGAND BINDING ASSAYS (4) 
 
A.  Key Reagents (4.1) 
 
1.  Reference Standard (4.1.1) 
 
The reference standard should be well characterized and documented (e.g., CoA and origin). A 
biological drug has a highly complex structure and its reactivity with binding reagents for 
bioanalysis may be influenced by a change in the manufacturing process of the drug substance. It 
is recommended that the manufacturing batch of the reference standard used for the preparation 
of calibration standards and QCs is derived from the same batch of drug substance as that used 
for dosing in the nonclinical and clinical studies whenever possible. If the reference standard 
batch used for bioanalysis is changed, bioanalytical evaluation should be carried out with QCs 
from the original material and the new material prior to use to ensure that the performance 
characteristics of the method are within the acceptance criteria. 
 
2.  Critical Reagents (4.1.2) 
 
Critical reagents, including binding reagents (e.g., binding proteins, aptamers, antibodies, or 
conjugated antibodies) and those containing enzymatic moieties, have direct impact on the 
results of the assay and, therefore, their quality should be assured. Critical reagents bind the 
analyte and, upon interaction, lead to an instrument signal corresponding to the analyte 
concentration. The critical reagents should be identified and defined in the assay method. 
 
Reliable procurement of critical reagents, whether manufactured in-house or purchased 
commercially, should be considered early in method development. The data sheet for the critical 
reagent should include at a minimum identity, source, batch/lot number, purity (if applicable), 
concentration (if applicable), and stability/retest date/storage conditions (refer to Table 1). 
Additional characteristics may be warranted.  
 
A critical reagent lifecycle management procedure is necessary to ensure consistency between 
the original and new batches of critical reagents. Reagent performance should be evaluated using 
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the bioanalytical method. Minor changes to critical reagents would not be expected to influence 
the method performance, whereas major changes may significantly impact the performance. If 
the change is minor (e.g., the source of one reagent is changed), a single comparative accuracy 
and precision assessment is sufficient for characterization. If the change is major, then additional 
validation experiments are recommended. Ideally, assessment of changes should compare the 
method with the new reagents to the method with the old reagents directly. Major changes 
include, but are not limited to, change in production method of antibodies, additional blood 
collection from animals for polyclonal antibodies, and new clones or new supplier for 
monoclonal antibody production.  
 
Retest dates and validation parameters should be documented in order to support the extension or 
replacement of the critical reagent. Stability testing of the reagents should be based upon the 
performance in the bioanalytical method and upon general guidance for reagent storage 
conditions. It can be extended beyond the expiration date from the supplier. The performance 
parameters should be documented in order to support the extension or replacement of the critical 
reagent.  
 
B.  Validation (4.2) 
 
Most often, microtiter plates are used for LBAs and study samples can be analyzed using an 
assay format of one or more well(s) per sample. The assay format should be specified in the 
protocol, study plan or SOP. If method development and method validation are performed using 
one or more well(s) per sample, then study sample analysis should also be performed using one 
or more well(s) per sample, respectively. If multiple wells per sample are used, the reportable 
sample concentration should be determined either by calculating the mean of the responses from 
the replicate wells or by averaging the concentrations calculated from each response. Data 
evaluation should be performed on reportable concentrations.  
 
1.  Specificity (4.2.1) 
 
Specificity is related to the concept of cross-reactivity in LBA. It is important that the binding 
reagent specifically binds to the target analyte but does not cross-react with coexisting 
structurally related molecules (e.g., endogenous compounds, isoforms, or structurally related 
concomitant medication). Specificity is evaluated by spiking blank matrix samples with related 
molecules at the maximal concentration(s) of the structurally related molecule anticipated in 
study samples.  
 
The accuracy of the target analyte at the LLOQ and at the ULOQ should be investigated in the 
presence of related molecules at the maximal concentration(s) anticipated in study samples. The 
response of blank samples spiked with related molecules should be below the LLOQ. The 
accuracy of the target analyte in presence of related molecules should be within 25% of the 
nominal values.  
 
In the event of nonspecificity, the impact on the method should be evaluated by spiking 
increasing concentrations of interfering molecules in blank matrix and measuring the accuracy of 
the target analyte at the LLOQ and ULOQ. It is important to determine the minimum 
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concentration of the related molecule where interference occurs. Appropriate mitigation during 
sample analysis should be employed, e.g., it may be necessary to adjust the LLOQ/ULOQ 
accordingly or consider a new method. 
 
During method development and early method validation, these related molecules are 
frequently not available. Additional evaluation of specificity may be conducted after the original 
validation is completed. 
 
2.  Selectivity (4.2.2) 
 
Selectivity is the ability of the method to detect and differentiate the analyte of interest in the 
presence of nonspecific matrix components. The matrix can contain nonspecific matrix 
component such as degrading enzymes, heterophilic antibodies or rheumatoid factor which may 
interfere with the analyte of interest. 
 
Selectivity should be evaluated at the low end of an assay where problems occur in most cases, 
but it is recommended that selectivity is also evaluated at higher analyte concentrations. 
Therefore, selectivity should be evaluated using blank samples obtained from at least 10 
individual sources and by spiking the individual blank matrices at the LLOQ and at the high QC 
level. Use of fewer sources may be acceptable in the case of rare matrices. The response of the 
blank samples should be below the LLOQ in at least 80% of the individual sources.  
 
The accuracy should be within 25% at the LLOQ and within 20% at the high QC level of the 
nominal concentration in at least 80% of the individual sources evaluated.  
 
Selectivity should be evaluated in lipemic samples and hemolyzed samples (refer to section 
III.B.1 (3.2.1)). For lipemic and hemolyzed samples, tests can be evaluated once using a single 
source of matrix. Selectivity should be assessed in samples from relevant patient populations 
(e.g., renally or hepatically impaired patients, inflammatory or immune-oncology patients if 
applicable). In the case of relevant patient populations, there should be at least five individual 
patients.  
 
3.  Calibration Curve and Range (4.2.3) 
 
The calibration curve demonstrates the relationship between the nominal analyte concentration 
and the response of the analytical platform to the analyte. Calibration standards, prepared by 
spiking matrix with a known quantity of analyte, span the calibration range and comprise the 
calibration curve. Calibration standards should be prepared in the same biological matrix as the 
study samples. The calibration range is defined by the LLOQ, which is the lowest calibration 
standard, and the ULOQ, which is the highest calibration standard. There should be one 
calibration curve for each analyte studied during method validation and for each analytical run. If 
needed, the use of surrogate matrix should be scientifically justified. 
 
A calibration curve should be generated with at least six concentration levels of calibration 
standards, including LLOQ and ULOQ standards, plus a blank sample. The blank sample should 
not be included in the calculation of calibration curve parameters. Anchor point samples at 
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concentrations below the LLOQ and above the ULOQ of the calibration curve may also be used 
to improve curve fitting. The relationship between response and concentration for a calibration 
curve is most often fitted by a 4- or 5-parameter logistic model if there are data points near the 
lower and upper asymptotes. Oher models should be suitably justified. 
 
A minimum of six independent runs should be evaluated over several days considering the 
factors that may contribute to between-run variability. 
 
The accuracy and precision of back-calculated concentrations of each calibration standard should 
be within 25% of the nominal concentration at the LLOQ and ULOQ, and within 20% at all 
other levels. At least 75% of the calibration standards excluding anchor points, and a minimum 
of 6 concentration levels of calibration standards, including the LLOQ and ULOQ, should meet 
the above criteria. The anchor points do not require acceptance criteria since they are beyond the 
quantifiable range of the curve. 
 
The calibration curve should preferably be prepared using freshly spiked calibration standards. If 
freshly spiked calibration standards are not used, the frozen calibration standards can be used 
within their defined period of stability. 
 
4.  Accuracy and Precision (4.2.4) 
 
a.  Preparation of quality control samples (4.2.4.1) 
 
The QCs are intended to mimic study samples and should be prepared by spiking matrix with a 
known quantity of analyte, stored under the conditions anticipated for study samples, and 
analyzed to assess the validity of the analytical method. 
 
The dilution series for the preparation of the QCs should be completely independent from the 
dilution series for the preparation of calibration standard samples. If they are prepared from the 
same stock solution (or working stock) the accurate preparation and stability should be verified. 
The QCs should be prepared at a minimum of five concentration levels within the calibration 
curve range: The analyte should be spiked at the LLOQ, within three times of the LLOQ (low 
QC), around the geometric mean of the calibration curve range (medium QC), and at least at 
75% of the ULOQ (high QC) and at the ULOQ. 
 
For non-accuracy and precision validation runs, low, medium, and high QCs may be analyzed in 
duplicate. These QCs, along with the calibration standards, should provide the basis for the 
acceptance or rejection of the run.  
 
b.  Evaluation of accuracy and precision (4.2.4.2) 
 
Accuracy and precision should be determined by analyzing the QCs within each run (within-run) 
and in different runs (between-run). Accuracy and precision should be evaluated using the same 
runs and data. 
 
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Accuracy and precision should be determined by analyzing at least three replicates per run at 
each QC concentration level (LLOQ, low, medium, high, ULOQ) in at least six runs over 2 or 
more days. Reported method validation data and the determination of accuracy and precision 
should include all results obtained, except those cases where errors are obvious and documented. 
Within-run accuracy and precision data should be reported for each run. If the within-run 
accuracy or precision criteria are not met in all runs, an overall estimate of within-run accuracy 
and precision for each QC level should be calculated. Between-run (intermediate) precision and 
accuracy should be calculated by combining the data from all runs. 
 
The overall within-run and between-run accuracy at each concentration level should be within 
20% of the nominal values, except for the LLOQ and ULOQ, which should be within 25% of 
the nominal value. Within-run and between-run precision of the QC concentrations determined at 
each level should not exceed 20%, except at the LLOQ and ULOQ, where it should not exceed 
25%.  
 
For non-accuracy and precision validation runs, at least 2/3 of the total QCs and at least 50% at 
each concentration level should be within 20% of the nominal values. 
 
Furthermore, the total error (i.e., sum of absolute values of the errors in accuracy (%) and 
precision (%)) should be evaluated. The total error should not exceed 30% (40% at LLOQ and 
ULOQ). 
 
5.  Carryover (4.2.5) 
 
Carryover is generally not an issue for LBA analyses. However, if the analytical platform is 
prone to carryover, the potential of carryover should be investigated by placing blank samples 
after the calibration standard at the ULOQ. The response of blank samples should be below the 
LLOQ. 
 
6.  Dilution Linearity and Hook Effect (4.2.6) 
 
Due to the narrow assay range in many LBAs, study samples may require dilution in order to 
achieve analyte concentrations within the range of the assay. Dilution linearity should be 
assessed to confirm: (i) that measured concentrations are not affected by dilution within the 
calibration range and (ii) that sample concentrations above the ULOQ of a calibration curve are 
not impacted by hook effect (i.e., a signal suppression caused by high concentrations of the 
analyte), whereby yielding an erroneous result. 
 
The same matrix as that of the study sample should be used for preparation of the QCs for 
dilution. 
 
Dilution linearity should be demonstrated by generating a dilution QC, i.e., spiking the matrix 
with an analyte concentration above the ULOQ, analyzed undiluted (for hook effect) and diluting 
this sample (to at least three different dilution factors) with blank matrix to a concentration 
within the calibration range. For each dilution factor tested, at least three independently prepared 
dilution series should be performed using the number of replicates that will be used in sample 
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analysis. The absence or presence of response reduction (hook effect) should be checked in the 
dilution QCs and, if observed and unable to be eliminated with reasonable measures, steps 
should be taken to mitigate this effect during the analysis of study samples. 
 
The calculated mean concentration for each dilution should be within 20% of the nominal 
concentration after correction for dilution and the precision should not exceed 20%.  
 
The dilution factor(s) applied during study sample analysis should be within the range of dilution 
factors evaluated during validation. 
 
7.  Stability (4.2.7) 
 
Stability evaluations should be carried out to ensure that every step taken during sample 
preparation, processing, and analysis as well as the storage conditions used do not affect the 
concentration of the analyte.  
 
The storage and analytical conditions applied to the stability tests, such as the sample storage 
times and temperatures, sample matrix, anticoagulant, and container materials should reflect 
those used for the study samples. Reference to data published in the literature is not considered 
sufficient. Validation of storage periods should be performed on QCs that have been stored for a 
time that is equal to or longer than the study sample storage periods. 
 
Stability of the analyte in the studied matrix should be evaluated using low and high 
concentration QCs. Aliquots of the low and high QCs should be analyzed at time zero and after 
the applied storage conditions that are to be evaluated. One bulk QC should be prepared at each 
concentration level. For each concentration tested, the bulk sample should be divided into a 
minimum of three aliquots that will be stored, stressed, and analyzed. 
 
The QCs should be analyzed against a calibration curve, obtained from freshly spiked calibration 
standards in a run with its corresponding freshly spiked QCs or QCs for which stability has been 
proven. While the use of freshly prepared calibration standards and QCs is the preferred 
approach, it is recognized that in some cases, for macromolecules, it may be necessary to freeze 
them overnight. In such cases, valid justification should be provided, and freeze-thaw stability 
demonstrated. QCs should be kept frozen for at least 12 hours between the thawing cycles. The 
mean concentration at each QC level should be within 20% of the nominal concentration.  
 
Since sample dilution may be required for many LBA methods due to a narrow calibration range, 
the concentrations of the study samples may be consistently higher than the ULOQ of the 
calibration curve. If this is the case, the concentration of the QCs should be adjusted, considering 
the applied sample dilution, to represent the actual sample concentration range. 
 
For fixed dose combination products and specifically labeled drug regimens, the freeze-thaw, 
bench top and long-term stability tests of an analyte in matrix should be conducted with the 
matrix spiked with all of the dosed compounds, on a case-by-case basis.  
 
22  Contains Nonbinding Recommendations 
As mentioned in section III.B.8 (3.2.8), the investigation of stability should cover bench top 
(short-term) stability at room temperature or sample preparation temperature and freeze-thaw 
stability. In addition, long-term stability should be studied.  
 
For chemical drugs, the stability at one temperature (e.g., -20C) can be extrapolated to lower 
temperatures (e.g., -70/-80C). 
 
For biological drugs, a bracketing approach can be applied, e.g., in the case that the stability has 
been demonstrated at -70/-80C and at -20C, then it is not necessary to investigate the stability 
at temperatures in between those two points at which study samples will be stored.  
 
C.  Study Sample Analysis (4.3) 
 
The analysis of study samples can be carried out after validation has been completed, however, it 
is understood that some parameters may be completed at a later stage (e.g., long-term stability). 
By the time the data are submitted to a regulatory authority, the bioanalytical method validation 
should have been completed. The study samples, QCs, and calibration standards should be 
processed in accordance with the validated analytical method. Refer to Table 1 for 
recommendations regarding documentation. 
 
1.  Analytical Run (4.3.1) 
 
An analytical run should consist of a blank sample, calibration standards at a minimum of six 
concentration levels, at least three levels of QCs (low, medium, and high) applied as two sets (or 
at least 5% of the number of study samples, whichever is higher) and the study samples to be 
analyzed. The blank sample should not be included in the calculation of calibration curve 
parameters. The QCs should be placed in the run in such a way that the accuracy and precision of 
the whole run is ensured taking into account that study samples should always be bracketed by 
QCs.  
 
Most often, microtiter plates are used for LBAs. An analytical run may comprise of one or more 
plate(s). Typically, each plate contains an individual set of calibration standards and QCs. If each 
plate contains its own calibration standards and QCs, then each plate should be assessed on its 
own. However, for some platforms the sample capacity may be limited. In this case, sets of 
calibration standards may be placed on the first and the last plate, but QCs should be placed on 
every single plate. QCs should be placed at least at the beginning (before) and at the end (after) 
of the study samples of each plate. The QCs on each plate and each calibration curve should 
fulfill the acceptance criteria for an analytical run (refer to section IV.C.2 (4.3.2)). For the 
calculation of concentrations, the calibration standards should be combined to conduct one 
regression analysis. If the combined calibration curve does not pass the acceptance criteria the 
whole run fails. 
 
2.  Acceptance Criteria for an Analytical Run (4.3.2) 
 
Criteria for the acceptance or rejection of an analytical run should be defined in the protocol, in 
the study plan, or in an SOP. In the case that a run contains multiple batches, acceptance criteria 
23  Contains Nonbinding Recommendations 
should be applied to the whole run and to the individual batches. It is possible for the run to meet 
acceptance criteria, even if a batch within that run is rejected for failing to meet the batch 
acceptance criteria. Calibration standards in a failed batch should not be used to support the 
acceptance of other batches within the analytical run. 
 
The back-calculated concentrations of the calibration standards should be within 20% of the 
nominal value at each concentration level, except for the LLOQ and the ULOQ, for which it 
should be within 25%. At least 75% of the calibration standards, with a minimum of 6 
concentration levels, should fulfill this criterion. This requirement does not apply to anchor 
calibration standards. If more than six calibration standards are used and one of the calibration 
standards does not meet these criteria, this calibration standard should be rejected and the 
calibration curve without this calibration standard should be reevaluated, and a new regression 
analysis performed. 
 
If the rejected calibration standard is the LLOQ, the new lower limit for this analytical run 
should be the next lowest acceptable calibration standard of the calibration curve. If the highest 
calibration standard is rejected, the new upper limit for this analytical run should be the next 
acceptable highest calibration standard of the calibration curve. The new lower and upper limit 
calibration standard will retain their original acceptance criteria (i.e., 20%). The revised 
calibration range should cover all QCs (low, medium, and high). The study samples outside of 
the revised assay range should be reanalyzed. 
 
Each run should contain at least three levels of QCs (low, medium, and high). During study 
sample analysis, the calibration standards and QCs should mimic the analysis of the study 
sample with regard to the number of wells used per study sample. At least 2/3 of the QCs and 
50% at each concentration level should be within 20% of the nominal value at each 
concentration level. Exceptions to these criteria should be justified and predefined in the SOP or 
protocol. 
 
The overall mean accuracy and precision of the QCs of all accepted runs should be calculated at 
each concentration level and reported in the analytical report. In the case that the overall mean 
accuracy and/or precision exceeds 20%, additional investigations should be conducted to 
determine the cause(s) of this deviation. In the case of comparative BA/BE studies, it may result 
in the rejection of the data. 
 
3.  Calibration Range (4.3.3) 
 
At least two QC sample levels should fall within the range of concentrations measured in study 
samples. At the intended therapeutic dose(s), if an unanticipated clustering of study samples at 
one end of the calibration curve is encountered after the start of sample analysis, the analysis 
should be stopped and either the standard calibration range narrowed (i.e., partial validation), 
existing QC concentrations revised, or QCs at additional concentrations added to the original 
curve within the observed range before continuing with study sample analysis. It is not necessary 
to reanalyze samples analyzed before optimizing the calibration curve range or QC 
concentrations. 
 
24  Contains Nonbinding Recommendations 
4.  Reanalysis of Study Samples (4.3.4) 
 
Possible reasons for reanalysis of study samples, the number reanalyzed, and the decision criteria 
to select the value to be reported should be predefined in the protocol, study plan, or SOP, before 
the actual start of the analysis of the study samples. 
 
The number of samples (and percentage of total number of samples) that have been reanalyzed 
should be reported and discussed in the Bioanalytical Report. For comparative BA/BE studies, a 
separate table should report values from rejected runs. 
 
Some examples of reasons for study sample reanalysis are: 
 
  Rejection of an analytical run because the run failed the acceptance criteria with regard to 
accuracy of the calibration standards and/or the precision and accuracy of the QCs 
 
  The concentration obtained is above the ULOQ 
 
  The concentration obtained is below the LLOQ in runs where the lowest calibration 
standard has been rejected from a calibration curve, resulting in a higher LLOQ 
compared with other runs 
 
  Malfunction of equipment 
 
  The diluted sample is below the LLOQ 
 
  Identification of quantifiable analyte levels in pre-dose samples, control, or placebo 
samples 
 
  When samples are analyzed in more than one well and nonreportable values are obtained 
due to one replicate failing the predefined acceptance criteria (e.g., excessive variability 
between wells, one replicate being above the ULOQ or below the LLOQ) 
 
For comparative BA/BE studies, reanalysis of study samples for a PK reason (e.g., a sample 
concentration does not fit with the expected profile) should not be done, as it may bias the study 
result. 
 
The reanalyzed samples should be identified in the Bioanalytical Report and the initial value, the 
reason for reanalysis, the values obtained in the reanalysis, the final accepted value, and a 
justification for the acceptance should be provided. Further, a summary table of the total number 
of samples that have been reanalyzed due to each reason should be provided. In cases where the 
first analysis yields a nonreportable result, a single reanalysis is considered sufficient (e.g., 
concentration above the ULOQ or excessive variability between wells). The analysis of the 
samples should be based on the same number of wells per study sample as in the initial analysis. 
In cases where the value needs to be confirmed, (e.g., pre-dose sample with measurable 
concentrations) multiple determinations are recommended where sample volume allows.  
 
25  Contains Nonbinding Recommendations 
The safety of trial subjects should take precedence over any other aspect of the trial. 
Consequently, there may be other circumstances when it is necessary to reanalyze specific study 
samples for the purpose of an investigation.  
 
 
V.  INCURRED SAMPLE REANALYSIS (5)  
 
The performance of study samples may differ from that of the calibration standards and QCs 
used during method validation, which are prepared by spiking blank matrix. Differences in 
protein binding, back-conversion of known and unknown metabolites, sample inhomogeneity, 
concomitant medications, or biological components unique to the study samples may affect 
measured concentrations of the analyte in study samples. Incurred sample reanalysis (ISR) is 
intended to verify the reliability of the reported sample analyte concentrations.  
 
ISR should be performed at least in the following situations: 
 
  For nonclinical studies within the scope of this guidance, ISR should, in general, be 
performed at least once per species.  
 
  All pivotal comparative BA/BE studies. 
 
  First clinical trial in subjects. 
 
  Pivotal early patient trial(s), once per patient population. 
 
  First or pivotal trial in patients with impaired hepatic and/or renal function. 
 
ISR is conducted by repeating the analysis of a subset of samples from a given study in separate 
(i.e., different to the original) runs on different days using the same bioanalytical method. 
 
The extent of ISR depends upon the analyte and the study samples and should be based upon an 
in-depth understanding of the analytical method and analyte. However, as a minimum, if the total 
number of study samples is less than or equal to 1000, then 10% of the samples should be 
reanalyzed; if the total number of samples is greater than 1000, then 10% of the first 1000 
samples (100) plus 5% of the number of samples that exceed 1000 samples should be assessed. 
Objective criteria for choosing the subset of study samples for ISR should be predefined in the 
protocol, study plan, or an SOP. While the subjects/animals should be picked as randomly as 
possible from the dosed study population, adequate coverage of the concentration profile is 
important. Therefore, it is recommended that the samples for ISR be chosen around the 
maximum concentration (C ) and some in the elimination phase. Additionally, the samples 
max
chosen should be representative of the whole study. 
 
Samples should not be pooled, as pooling may limit anomalous findings. ISR samples and QCs 
should be processed and analyzed in the same manner as in the original analysis. ISR should be 
performed within the stability window of the analyte, but not on the same day as the original 
analysis.  
26  Contains Nonbinding Recommendations 
 
The percent difference between the initial concentration and the concentration measured during 
the repeat analysis should be calculated in relation to their mean value using the following 
equation:  
% difference  
  repeat valueinitial value
=   100
For chromatographic methods, the percent diffmereeannce v sahluoeuld be within 20% for at least 2/3 of 
the repeats. For LBAs, the percent difference should be within  30% for at least 2/3 of the 
repeats. 
 
If the overall ISR results fail the acceptance criteria, an investigation should be conducted and 
the causes remediated. There should be an SOP that directs how investigations are triggered and 
conducted. If an investigation does not identify the cause of the failure, the potential impact of an 
ISR failure on study validity should also be provided in the Bioanalytical Report. If ISR meets 
the acceptance criteria yet shows large or systemic differences between results for multiple 
samples, this may indicate analytical issues and it is advisable to investigate this further. 
 
Examples of trends that are of concern may include: 
 
  All ISR samples from one subject fail 
  All ISR samples from one run fail 
 
All aspects of ISR evaluations should be documented to allow reconstruction of the study and 
any investigations. Individual samples that are quite different from the original value (e.g., > 
50%, flyers) should not trigger reanalysis of the original sample and do not need to be 
investigated. ISR sample data should not replace the original study sample data. 
 
 
VI.  PARTIAL AND CROSS VALIDATION (6)  
 
A.  Partial Validation (6.1) 
 
Partial validations evaluate modifications to already fully validated bioanalytical methods. 
Partial validation can range from as little as one within-run accuracy and precision determination 
to a nearly full validation. If stability is established at one facility it does not necessarily need to 
be repeated at another facility.  
 
For chromatographic methods, typical bioanalytical method modifications or changes that fall 
into this category include, but are not limited to, the following situations: 
 
  Analytical site change using same method (i.e., bioanalytical method transfers between 
laboratories) 
 
  A change in analytical method (e.g., change in detection systems, platform) 
 
27  Contains Nonbinding Recommendations 
  A change in sample processing procedures  
 
  A change in sample volume (e.g., the smaller volume of pediatric samples)  
 
  Changes to the calibration concentration range 
 
  A change in anticoagulant (but not changes in the counter-ion) in biological fluids (e.g., 
heparin to ethylenediaminetetraacetic acid (EDTA)) 
 
  Change from one matrix within a species to another (e.g., switching from human plasma 
to serum or cerebrospinal fluid) or changes to the species within the matrix (e.g., 
switching from rat plasma to mouse plasma)  
 
  A change in storage conditions 
 
For LBAs, typical bioanalytical method modifications or changes that fall into this category 
include, but are not limited to, the following situations: 
 
  Changes in LBA critical reagents (e.g., lot-to-lot changes) 
 
  Changes in MRD 
 
  A change in storage conditions 
 
  Changes to the calibration concentration range 
 
  A change in analytical method (e.g., change in detection systems, platform) 
 
  Analytical site change using same method (i.e., bioanalytical method transfers between 
laboratories) 
 
  A change in sample preparation 
 
  A change in anticoagulant (but not changes in the counter-ion) in biological fluids (e.g., 
heparin to ethylenediaminetetraacetic acid (EDTA)) 
 
The parameters of the partial validations should meet the full validation criteria. If these criteria 
are not satisfied, additional investigation and validation is warranted. 
 
B.  Cross Validation (6.2) 
 
Cross validation is required to demonstrate how the reported data are related when multiple 
bioanalytical methods and/or multiple bioanalytical laboratories are involved. 
 
Cross validation is required under the following situations: 
 
28  Contains Nonbinding Recommendations 
  Data are obtained from different fully validated methods within a study. 
 
  Data are obtained within a study from different laboratories with the same bioanalytical 
method.  
 
  Data are obtained from different fully validated methods across studies that are going to 
be combined or compared to support special dosing regimens, or regulatory decisions 
regarding safety, efficacy, and labeling. 
 
If data are obtained from different fully validated methods, and these data are not to be combined 
across studies, cross validation is not generally required.  
 
Cross validation should be performed in advance of study samples being analyzed, if possible. 
 
Cross validation should be assessed by measuring the same set of QCs (low, medium, and high) 
at least in triplicate and study samples (if available) that span the study sample concentration 
range (n30) with both methods, or in both laboratories.  
 
Bias can be assessed by Bland-Altman plots or Deming regression. Other methods appropriate 
for assessing agreement between two methods (e.g., concordance correlation coefficient) may be 
used too. Alternatively, the concentration vs. time curves for study samples could be plotted for 
samples analyzed by each method to assess bias.  
 
The use of multiple bioanalytical methods for the measurement of the same analyte in the 
conduct of one comparative BA/BE study is strongly discouraged. 
 
 
VII.  ADDITIONAL CONSIDERATIONS (7) 
 
A.  Methods for Analytes That Are Also Endogenous Molecules (7.1) 
   
For analytes that are also endogenous molecules (e.g., replacement therapies), the accuracy of 
the measurement of the analytes poses a challenge when the method cannot distinguish between 
the therapeutic agent and the endogenous molecule. Furthermore, the endogenous levels of the 
analyte may vary because of age, gender, race, diurnal variations, illness, or as side effect of drug 
treatment. This section describes some of the approaches that may be used to assess 
concentrations of analytes that are also endogenous molecules. As a reminder, biomarkers are 
outside of the scope of this guidance. 
 
If available, biological matrix to prepare calibration standards and QCs should be the same as the 
study samples (i.e., authentic biological matrix) and it should be free of matrix effect and 
interference, as described in sections III. (3) and IV. (4). The endogenous concentration in the 
biological matrix chosen should be low enough to obtain an adequate signal-to-noise ratio (e.g., 
<20% of the LLOQ). 
 
29  Contains Nonbinding Recommendations 
In those cases where matrices without interference are not available, the following approaches 
can be used to calculate the concentration of the analyte in the study samples: (1) the surrogate 
matrix approach, (2) the surrogate analyte approach, (3) the background subtraction approach, 
and (4) the standard addition approach. 
 
(1) Surrogate Matrix Approach: 
 
The matrix for the calibration standards is substituted by a surrogate matrix. Surrogate 
matrices can vary widely in complexity from simple buffers or artificial matrices that try 
to mimic the authentic one, to stripped matrices or matrices from other species.  
 
(2) Surrogate Analyte Approach: 
 
Stable isotope labeled analytes are used as surrogate standards in mass spectrometric 
methods to construct the calibration curve for the quantification of endogenous analytes. 
In this approach, it is assumed that the physicochemical properties of the authentic and 
surrogate analytes are the same with the exception of molecular weight. However, isotope 
standards may differ in retention time and MS sensitivity, therefore, before application of 
this approach, the ratio of the MS responses (i.e., the response factor) of the labeled to 
unlabeled analyte should be close to unity and remain constant over the entire calibration 
range. If the response factor does not comply with these requirements, it should be 
incorporated into the regression equation of the calibration curve. 
 
(3) Background Subtraction Approach: 
 
The concentration of the endogenous analyte observed in a pooled/representative matrix 
is subtracted from the concentration observed in the spiked standards, subsequently the 
net differences are used to construct the calibration curve. 
 
When the background concentrations are lowered by dilution of the blank matrices before 
spiking with the standards (e.g., if a lower LLOQ is required) the composition of the 
matrices in the study samples and the calibration standards is different, which may cause 
different recoveries and matrix effects. These differences should be considered when 
validating the method.  
 
(4) Standard Addition Approach: 
 
The standard addition approach should only be applied for analytical platforms with 
linear responses. Typically, the standard addition method is used to determine the 
concentration of the endogenous analyte in the authentic matrix to be used for preparation 
of standards and QCs. However, this approach can be employed for determination of 
study samples as well. In this approach, every study sample is divided into aliquots of 
equal volume. All aliquots, but one, are separately spiked with known and varying 
amounts of the analyte standards to construct a calibration curve for either the authentic 
blank matrix or every study sample (e.g., with three to five points). The endogenous 
30  Contains Nonbinding Recommendations 
blank concentration or the study sample concentration is then determined as the negative 
x-intercept of the standard calibration curve prepared in that particular study sample. 
 
Validation of an analytical method for an analyte that is also an endogenous compound should 
include the following considerations, in addition to the validation shown in sections III. (3) and 
IV. (4). 
 
1.  Quality Control Samples for Methods for Analytes That Are Also Endogenous 
Molecules (7.1.1) 
 
The endogenous concentrations of the analyte in the biological matrix should be evaluated prior 
to QC preparation. The matrices with the lowest possible level of the interfering endogenous 
analyte should be used. The concentrations of the QCs should account for the endogenous 
concentrations in the authentic matrix and be representative of the expected study sample 
concentrations. 
 
The QCs should resemble study samples and should be prepared in the same matrix. In principle, 
all QC concentrations used for validation should be aliquots of the authentic biological matrix 
unspiked (endogenous QC with a concentration between LLOQ and low QC, if possible) and 
spiked with known amounts of the authentic analyte (low QC, middle QC, and high QC). In 
spiked samples (e.g., LLOQ, low QC), the added amount should be enough to provide 
concentrations that are threefold higher or statistically different from the endogenous 
concentration. If options such as multiple lots, alternative vendors, and matrices of special 
populations that might contain a lower concentration of the analyte continue to yield matrices in 
which the endogenous levels are so high that it is not possible to prepare the low QC in the 
authentic matrix, diluted (surrogate) matrix may be used.  
 
2.  Selectivity, Recovery, and Matrix Effects for Methods for Analytes That Are Also 
Endogenous Molecules (7.1.2) 
 
The assessment of selectivity is complicated by the absence of interference-free matrix. For 
chromatography, peak purity should be investigated as part of method validation by analyzing 
matrices obtained from several donors (at least six normal blanks, one hemolyzed blank, and one 
lipemic blank) using a discriminative detection system (e.g., tandem mass spectrometry 
(MS/MS)). Other approaches, if justified by scientific principles, may also be considered. 
 
For the Standard Addition and Background Subtraction Approaches, as the same biological 
matrix and analyte are used for study samples and calibration standards, the same recovery and 
matrix effect occurs in the study samples and the calibration standards. However, if the 
endogenous component were not completely identical (e.g., recombinant proteins) the potential 
difference in recovery should be assessed in a parallelism test. For the Surrogate Matrix and 
Surrogate Analyte Approaches, the matrix effect and the extraction recovery may differ between 
calibration standards and study samples. Matrix effect should be evaluated to ensure that it does 
not impact accuracy and precision, mainly at LLOQ level. 
 
31  Contains Nonbinding Recommendations 
  If the Surrogate Matrix Approach is used, the impact of the different matrix effect and 
recovery in both the surrogate and authentic matrix should be assessed. This should be 
investigated in an experiment using QCs spiked with analyte in the matrix, the 
endogenous matrix only and spiked analyte in the surrogate matrix alone against the 
surrogate calibration curve. 
 
  If the Surrogate Analyte Approach is used in mass spectrometry chromatographic 
methods, the impact of the different matrix effect and recovery between surrogate and 
authentic endogenous analytes should be evaluated. This should be investigated in an 
experiment using QCs spiked with analyte in the matrix, the endogenous matrix only and 
spiked surrogate analyte in the matrix against the surrogate calibration curve. 
 
  In certain cases, dilution of the QCs with surrogate matrix may be necessary (e.g., for 
background subtraction methods) where the endogenous level is high and the LLOQ 
needs to be reduced. In these cases, the recovery and matrix effect experiments should be 
repeated with authentic biological matrices with endogenous concentrations between 
LLOQ and low QC, if available. 
 
Refer to sections III. (3) and IV. (4) for the acceptance criteria for chromatography and LBAs, 
respectively. 
 
Since the composition of the biological matrix might affect method performance, matrices from 
at least six (chromatographic methods)/10 (LBA) different donors should be investigated, except 
in the Standard Addition Approach, where each sample is analyzed with its own calibration 
curve. 
 
3.  Parallelism for Methods for Analytes That Are Also Endogenous Molecules 
(7.1.3) 
 
Parallelism assures that observed changes in response per given changes in analyte 
concentrations are equivalent for the surrogate and the authentic biological matrix across the 
range of the method. Parallelism should be evaluated in the Surrogate Matrix and Surrogate 
Analyte Approaches, taking into account that parallelism is assessed differently in LBA and 
chromatographic methods.  
 
4.  Accuracy and Precision for Methods for Analytes That Are Also Endogenous 
Molecules (7.1.4) 
 
Accuracy and precision should meet criteria specified in sections III. (3) and IV. (4) for 
chromatography and LBAs, respectively. 
 
In case of using a surrogate matrix or analyte approaches, the assessment of accuracy and 
precision should be performed by analyzing the QCs against the surrogate calibration curve.  
The concentration of the endogenous molecule in the blank matrix may be determined and 
subtracted from the total concentrations observed in the spiked samples. Accuracy is 
32  Contains Nonbinding Recommendations 
recommended to be calculated using the following formula when QCs are spiked with the 
authentic analyte in the matrix containing endogenous levels of the analyte: 
 
 
 
(Measured concentration of spiked sampleendogenous concentration )
  AAAAAAAAAAAAAAAA (%)=100 
Only the precision can be determined from the anaSlpyiskiesd o cfo necaecnht ruantisopniked/endogenous QC. 
 
5.  Stability for Methods for Analytes That Are Also Endogenous Molecules (7.1.5) 
 
To mimic study samples as much as possible, stability experiments should be investigated with 
the authentic analyte in the authentic biological matrix and with unspiked/endogenous QCs 
(blank matrix with endogenous molecule) as well as spiked low QC and high QCs as defined in 
section VII.A.1 (7.1.1) However, if a surrogate matrix is used for calibration standards, stability 
should also be demonstrated for the analyte in the surrogate matrix, as this could differ from 
stability in the authentic biological matrix. 
 
B.  Parallelism (7.2) 
 
Parallelism is defined as a parallel relationship between the calibration curve and serially diluted 
study samples to detect any influence of dilution on analyte measurement. Although lack of 
parallelism is a rare occurrence for bioanalytical methods for PK evaluation, parallelism of LBA 
should be evaluated on a case-by-case basis, e.g., where interference caused by a matrix 
component (e.g., presence of endogenous binding protein) is suspected during study sample 
analysis. Parallelism investigations, or the justification for its absence, should be included in the 
Bioanalytical Report. Some methods may demonstrate parallelism for one patient population but 
lack it for another population. Generally, these experiments should be conducted during the 
analysis of the study samples due to the unavailability of study samples during method 
development or validation. A study sample with a high concentration (preferably close to Cmax) 
should be diluted to at least three concentrations with blank matrix. The consistency of the back 
calculated concentrations between samples in a dilution series should not exceed 30% CV. 
However, when applying the 30% criterion, data should be carefully monitored as results that 
pass this criterion may still reveal trends of nonparallelism. In the case that the sample does not 
dilute linearly (i.e., in a nonparallel manner), a procedure for reporting a result should be defined 
a priori. 
 
C.  Recovery (7.3) 
 
For methods that employ sample extraction, the recovery (extraction efficiency) should be 
evaluated. Recovery should be reported as a percentage of the known amount of an analyte 
carried through the sample extraction and processing steps of the method. Recovery should be 
determined by comparing the analyte response in a biological sample that is spiked with the 
analyte and processed, with the response in a biological blank sample that is processed and then 
spiked with the analyte. Recovery of the analyte does not need to be 100%, but the extent of 
recovery of an analyte and of the IS (if used) should be consistent. Recovery experiments are 
recommended to be performed by comparing the analytical results for extracted samples at 
multiple concentrations, typically three concentrations (low, medium, and high). 
33  Contains Nonbinding Recommendations 
 
D.  Minimum Required Dilution (7.4) 
 
MRD is a dilution factor employed in samples that are diluted with buffer solution to reduce the 
background signal or matrix interference on the analysis using LBA. The MRD should be 
identical for all samples, including calibration standards and the QCs, and it should be 
determined during method development. If MRD is changed after establishment of the method, 
partial validation should be done. MRD should be defined in the Validation Report of the 
analytical method. 
 
E.  Commercial and Diagnostic Kits (7.5) 
 
Commercial or diagnostic kits (referred to as kits) are sometimes codeveloped with new 
chemical or biological drugs for point-of-care patient diagnosis. The recommendations in this 
section of the guidance do not apply to the development of kits that are intended for point-of-care 
patient diagnosis (e.g., companion or complimentary diagnostic kits). Refer to the appropriate 
guidance documents regarding regulatory recommendations for the development of these kits.  
 
If an applicant repurposes a kit (instead of developing a new method) or utilizes research use 
only kits to measure chemical or biological drug concentrations during the development of a 
novel drug, the applicant should assess the kit validation to ensure that it conforms to the drug 
development standards described in this guidance. 
 
Validation considerations for kit assays include, but are not limited to, the following: 
 
  If the reference standard in the kit differs from that of the study samples, testing should 
evaluate differences in assay performance of the kit reagents. The specificity, accuracy, 
precision, and stability of the kit assay should be demonstrated under actual conditions of 
use in the facility conducting the sample analysis. 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. 
 
  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. 
 
  Calibration standards and QCs should be prepared in the same matrix as the study 
samples. Kits with calibration standards and QCs prepared in a matrix different from the 
study samples should be justified and appropriate experiments should be performed.  
 
  If multiple kit assay lots are used within a study, lot-to-lot variability and comparability 
should be addressed for any critical reagents included in the kits.  
 
34  Contains Nonbinding Recommendations 
  If a kit using multiple assay plates is employed, sufficient replicate QCs should be used 
on each plate to monitor the accuracy of the assay. Acceptance criteria should be 
established for the individual plates and for the overall analytical run. 
 
F.  New or Alternative Technologies (7.6) 
 
When a new or alternative technology is used as the sole bioanalytical technology from the onset 
of drug development, cross validation with an existing technology is not required.  
 
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 those obtained with another platform. 
Therefore, when a new or alternative analytical platform is replacing a previous platform used in 
the development of a drug it is important that the potential differences are well understood. The 
data generated from the previous platform/technology should be cross validated to that of the 
new or alternative platform/technology. Seeking feedback from the regulatory authorities is 
encouraged early in drug development. The use of two methods or technologies within a 
comparative BA/BE study is strongly discouraged. 
 
The use of new technology in regulated bioanalysis should be supported by acceptance criteria 
established a priori based on method development and verified in validation.  
 
1.  Dried Matrix Methods (7.6.1) 
 
Dried matrix methods (DMM) is a sampling methodology that offers benefits such as collection 
of reduced blood sample volumes as a microsampling technique for drug analysis and ease of 
collection, storage, and transportation. In addition to the typical methodological validation for 
LC-MS or LBA, use of DMM necessitates further validation of this sampling approach before 
using DMM in studies that support a regulatory application, such as: 
 
  Hematocrit (especially for spotting of whole blood into cards). 
 
  Sample homogeneity (especially for sub-punch of the sample on the card/device). 
 
  Extraction of the sample from the dried matrix. 
 
  DMM sample collection for ISR: 
 
  Care should be taken to ensure sufficient sample volumes or numbers of replicates are 
retained for ISR.  
 
  Should be assessed by multiple punches of the sample or samples should be taken in 
duplicate.  
 
When DMM is used for clinical or nonclinical studies in addition to typical liquid approaches 
(e.g., liquid plasma samples) in the same studies, these two methods should be cross validated as 
35  Contains Nonbinding Recommendations 
described (refer to section VI.B (6.2)). For nonclinical TK studies, refer to section IV.1 (4.1) of 
the May 2018 ICH guidance for industry S3A Guidance: Note for Guidance on Toxicokinetics: 
The Assessment of Systemic Exposure in Toxicity Studies: Focus on MicosamplingQuestions 
and Answers (ICH S3AQ&A).4 Feedback from the appropriate regulatory authorities is 
encouraged in early drug development. 
 
 
VIII.  DOCUMENTATION (8) 
 
General and specific SOPs and good record keeping are essential to a properly validated 
analytical method. The data generated for bioanalytical method validation should be documented 
and available for data audit and inspection. Table 1 describes the recommended documentation 
for submission to the regulatory authorities and documentation that should be available at the 
analytical site at times of inspection. This documentation should be stored at the analytical site or 
at another secure location. In this case, the documentation should be readily available when 
requested. 
 
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 (8.1) 
 
Summary information should include the following items in sections II.F.4 (2.6.4) and II.G.1 
(2.7.1) of the Common Technical Document (CTD; or electronic CTD, eCTD)) or reports: 
 
  A summary of methods used for each study should be included. Each summary should 
provide the method title, method identification code, the assay type, the Bioanalytical 
Report code, effective date of the method, and the associated Validation Report codes. 
 
  A summary table of all the relevant Validation Reports should be provided for each 
analyte, including Partial Validation and Cross Validation Reports. The table should 
include the method identification code, the type of method, the reason for the new 
method or additional validation (e.g., to lower the limit of quantification). Changes made 
to the method should be clearly identified. 
 
 
4 The ICH S3AQ&A guidance is available on the FDA guidance web page. 
36  Contains Nonbinding Recommendations 
  A summary table cross-referencing multiple identification codes should be provided 
when a method has different codes for the method, the Validation Reports, and the 
Bioanalytical Reports. 
 
  Discussion of method changes (e.g., evolution of methods, reason(s) for revisions, unique 
aspects). 
 
  For comparative BA/BE studies, a list of regulatory site inspections including dates and 
outcomes for each analytical site if conducted over the last 3 years, and 1 year post-study 
completion.  
 
B.  Documentation for Validation and Bioanalytical Reports (8.2) 
 
Table 1 describes the recommended documentation for the Validation and Bioanalytical Reports.
37   
Table 1: Recommended Documentation and Reporting 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Chromatographic   Dates, times, and samples used for   Not applicable   Not applicable 
System Suitability  suitability testing 
Synopsis   History/evolution of methods (e.g.,   Not applicable   Not applicable 
Overview of Method  to explain revisions, unique aspects 
Evolution  with supportive data, if available) 
Reference Standards   CoA or equivalent alternative to   A copy of the CoA or equivalent   A copy of the CoA or equivalent 
  ensure quality (including purity),  alternative including batch/lot  alternative including batch/lot 
stability/expiration/retest date(s),  number, source, quality  number, source, quality (including 
batch number, and manufacturer or  (including purity), storage  purity), storage conditions, and 
source  conditions, and expiration/retest  expiration/retest date or a table with 
 Records of receipt, use, and storage  date, or table with this  this information 
conditions  information   If expired, quality and stability at the 
 If expired, recertified CoA, or retest   If expired, quality and stability  time of use and retest dates and 
at the time of use and retest dates  retested values  
of quality and identity with retest 
and retested values 
dates 
Internal Standard   IS quality or demonstration of   Name of reagent or standard   Name of reagent or standard 
suitability   Origin   Origin 
 Records of receipt, use, and storage 
conditions 
continued 
38   
Table 1, continued  
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
 Name of reagent   Name of reagent   Name of reagent 
Critical Reagents 
 Batch/Lot number   Batch/Lot number   Batch/Lot number 
 Source/Origin   Source/Origin   Source/Origin 
 Concentration, if applicable   Concentration, if applicable   Concentration, if applicable 
 Retest date (expiration date)   Retest date (expiration date)   Retest date (expiration date) 
 Storage conditions    Storage conditions    Storage conditions  
 Record of preparation and use of   Notation that solutions were   Notation that solutions were used 
Stock/Working 
stock/working solutions  used within stability period  within stability period 
Solutions 
 Storage location and condition   Stock/working solution stability   Stock/working solution stability  
   Storage conditions   Storage conditions  
 Records of matrix descriptions, lot   Description, lot number, receipt   Description, lot number, receipt 
Blank Matrix 
numbers, receipt dates, storage  dates   dates 
conditions, and source/supplier 
 Records and date of preparation   Description of preparation,   Description of preparation 
Calibration 
Standards and QCs   Record of storage temperature (e.g.,  including matrix   Preparation dates and stability period  
log of in/out dates, analyst,   Batch number, preparation dates,   Storage conditions  
temperatures, and freezer(s))  and stability period 
 Storage conditions 
(temperatures, dates, duration, 
etc.)  
continued 
39   
Table 1, continued  
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Standard Operating  Procedures for all aspects of analysis,   A detailed description of the   A list of procedures/analytical 
Procedures (SOPs;  such as:  method procedures  protocols used for the method  
Procedures)   Method/procedure     
(validation/analytical) 
 Acceptance criteria (e.g., run, 
calibration curve, QCs) 
 Instrumentation 
 Reanalysis 
 ISR 
 Record of changes to SOP (change, 
date, reason, etc.) 
Sample Tracking   Study sample receipt and condition   Not applicable  For All Studies: 
on receipt   Dates of receipt of shipments number 
 Records that indicate how samples  of samples 
were transported and received.   Sample condition on receipt  
Sample inventory and reasons for 
 Analytical site storage condition and 
missing samples 
location 
 Location of storage (e.g., freezer 
 Storage: total duration from sample 
unit) 
collection to analysis 
 Tracking logs of QCs, calibration 
 List of any deviations from planned 
standards, and study samples 
storage conditions and potential 
 Freezer logs for QCs, calibration  impact 
standards, and study samples entry 
Additionally, for Comparative BA/BE 
and exit 
Studies Also Include: 
 The subject ID 
continued 
   
40   
Table 1, continued 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Analysis   Documentation and data for system  For All Studies:  For All Studies: 
suitability checks for   Table of all runs (including failed runs), and   Table of all runs, status (accepted and 
chromatography  analysis dates  failed), reason for failure, and analysis 
 Instrument use log, including dates of   Table of calibration standard concentration  dates 
analysis for each run  and response functions results (calibration   Table of calibration standard 
 Sample extraction logs, including  curve parameters) of all accepted runs with  concentration and response function 
documentation of processing of  accuracy and precision  results (calibration curve parameters) 
calibration standards, QCs, and study   Table of within- and between- run QC  of all accepted runs with accuracy and 
samples for each run, including dates  results and calibration standards (from  precision  
of extraction  accuracy and precision runs). Values   Table of QCs results of all accepted 
 Identity of QCs and calibration  outside the acceptance criteria should be  runs with overall (between-run) 
standard lots, and study samples in  clearly marked   accuracy and precision results of the 
each run   Include total error for LBA methods.  QCs and between-run accuracy and 
 Documentation of instrument settings   Data on selectivity, specificity, dilution  precision results from accepted runs  
and maintenance  linearity, and sensitivity (LLOQ),   Table of reinjected runs with results 
 Laboratory information management  carryover, recovery. Bench top, freeze- from reinjected runs and reason(s) for 
system (LIMS)  thaw, long-term, extract, and stock solution  reinjection 
 Validation information, including  stability   QCs graphs trend analysis encouraged 
documentation and data for:   Partial/cross-validation, if applicable   Study concentration results table 
 Selectivity, specificity, sensitivity,   Append separate report for additional  Additionally, for Comparative BA/BE 
precision and accuracy, carryover,  validation, if any  Studies Also Include: 
dilution, recovery, matrix effect  Additionally, for Comparative BA/BE Studies   Instrument ID for each run in 
 Bench top, freeze-thaw, long- Also Include:  comparative BA/BE studies 
term, extract, and stock solution   Instrument ID for each run in comparative   IS response plots for each analytical 
stability  BA/BE studies  run, including failed runs  
 Cross/partial validations, if   100% of run summary table of accepted and   100% of run summary table of 
applicable  failed runs  accepted and failed runs 
continued 
   
41   
Table 1, continued 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Chromatograms   Electronic audit trail:   For All Studies:  For All Studies: 
and Reintegration   100% e-chromatograms of original and   Representative   Chromatograms may be submitted as a 
reintegration from accepted and fail runs  chromatograms (original  supplement 
 Reason for reintegration  and reintegration)   For studies other than comparative 
 Mode of reintegration 100% of run   Reason for reintegration  BA/BE, randomly selected 
summary tables of accepted and failed   Chromatograms may be  chromatograms from 5% of samples 
submitted in application dossiers 
runs, including calibration curve,  submitted as a supplement 
regression, weighting function, analyte  Additionally, for Comparative   Reason for reintegration 
and IS response and retention time,  BA/BE Studies Also Include:   Identification and discussion of 
response ratio, integration type   100% chromatograms of  chromatograms with manual 
  original and reintegration  reintegration 
from accepted and fail runs    SOP for reintegration, as applicable 
 100% of run summary table  Additionally, for Comparative BA/BE 
of accepted and failed runs  Studies Also Include: 
   100% of chromatograms.  
 Original and reintegrated 
chromatograms and initial and repeat 
integration results 
 100% of run summary table of accepted 
and failed runs 
continued 
   
42   
Table 1, continued 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Deviations From   Contemporaneous documentation of   Description of   Description of deviations 
Procedures  deviations/unexpected events  Deviations   Impact on study results 
 Investigation of unexpected events   Impact on study results   Description and supporting 
 Impact assessment   Description and  data of significant 
supporting data of  investigations 
significant 
investigations 
Reanalysis/Repeat Analysis   Procedures for conducting reanalysis/repeat   Not applicable  For All Studies: 
analysis (define reasons for reanalysis, etc.)   Table of sample IDs, reason 
 Retain 100% of repeat/reanalyzed data  for repeat analysis, original 
and repeat analysis values, 
 Contemporaneous records of reason for 
reason for reported values, run 
repeats 
IDs  
Additionally, for Comparative 
BA/BE Studies Also Include: 
 For comparative BA/BE 
studies, values from rejected 
runs should be included in a 
separate table 
ISR   Procedure for ISR   Not applicable   ISR data table (original and 
 ISR data: Run IDs, run summary sheets,  reanalysis values and run IDs, 
chromatograms, or other electronic  percent difference, percent 
passed) 
instrument data files 
 Document ISR failure investigations, if any   ISR failure investigations, if 
any 
 
continued 
43   
Table 1, continued 
Items  Documentation at the Analytical Site  Validation Report*  Bioanalytical Report* 
Communication   Between involved parties (Applicant,   Not applicable   Not applicable 
contract research organizations, and 
consultants) related to study/method 
Audits and Inspections   Evidence of audit and inspections    Not applicable/Refer to   Not applicable/Refer to section 
section VIII.A (8.1) for  VIII.A (8.1) for summary 
summary information to  information to include in the 
include in the eCTD  eCTD 
*The applicant should maintain data at the analytical site to support summary data submitted in Validation and Bioanalytical Reports. Validation and Bioanalytical Reports 
should be submitted in the application. 
 May append or link from Validation Report. 
Submit either in Validation Report or in Bioanalytical Report. 
 
44   
GLOSSARY 
 
Accuracy: 
The degree of closeness of the measured value to the nominal or known true value under 
prescribed conditions (or as measured by a particular method). In this document, accuracy is 
expressed as percent of the nominal value:  
 
Accuracy (%) = (Measured Value/Nominal Value)  100. 
 
Analysis:  
A series of analytical procedures from sample processing/dilution to measurement on an 
analytical instrument. 
 
Analyte:  
A specific chemical moiety being measured, including an intact drug, a biomolecule or its 
derivative, or a metabolite in a biological matrix. 
 
Analytical Run (also referred to as Run): 
A complete set of analytical and study samples with appropriate number of calibration 
standards and QCs for their validation. Several runs may be completed in one day or one run 
may take several days to complete.  
 
Anchor Calibration Standards/Anchor Points:   
Spiked samples set at concentrations below the LLOQ or above the ULOQ of the calibration 
curve and analyzed to improve curve fitting in LBAs. 
 
Batch (for Bioanalysis): 
A batch is comprised of QCs and study samples, and possibly blanks, zero samples, and 
calibration standards, which are handled during a fixed period of time and by the same group 
of analysts with the same reagents under homogenous conditions. 
 
Batch (for Reference Standards and Reagents): 
A specific quantity of material produced in a process or series of processes so that it is 
expected to be homogeneous within specified limits. Also referred to as Lot. 
 
Bias: 
The tendency of a measurement process to over- or underestimate the value of a population 
parameter. 
 
Bioanalytical Method:  
Analytical method used in the quantitative determination of analytes in biological matrices. 
 
Biological Drugs: 
Drugs that are made by living organisms or cells (e.g., therapeutic proteins). 
 
Biological Matrix:  
A biological material including, but not limited to, blood, serum, plasma, and urine. 
 
Binding Reagent:  
A reagent that binds to the analyte in LBA-based bioanalytical methods. 
45   
 
Blank Sample:   
A sample of a biological matrix to which no analyte, no IS, and no additional-alternative 
matrix or buffer has been added.  
 
Calibration Curve: 
The relationship between the instrument response (e.g., peak area, height, or signal) and the 
concentration (amount) of analyte in the calibration standards within a given range. Also 
referred to as Standard Curve.  
 
Calibration Range:  
The interval between the upper and lower concentration (amounts) of analyte in the 
calibration standards (including these concentrations).  
 
Calibration Standard:  
A matrix to which a known amount of analyte has been added or spiked. Calibration standards 
are used to construct calibration curves. 
 
Carryover:  
The appearance of an analyte signal in a sample from a preceding sample.  
 
Chemical Drugs: 
Chemically synthesized drugs.  
 
Critical Reagent:  
Critical reagents for LBAs include binding reagents (e.g., antibodies, binding proteins, and 
peptides) and those containing enzymatic moieties that have a direct impact on the results of 
the assay. 
 
Cross Validation:  
Assessment of potential bias between two bioanalytical methods or the same bioanalytical 
method used in different laboratories in order to determine whether reported data are 
comparable. 
 
Dilution Integrity:  
Assessment of the sample dilution procedure to confirm that the procedure does not impact 
the measured concentration of the analyte. 
 
Dilution Linearity:  
A parameter demonstrating that the method can appropriately analyze samples at a 
concentration exceeding the ULOQ of the calibration curve without influence of prozone 
(hook) effect and that the measured concentrations are not affected by dilution within the 
calibration range in LBAs. 
 
Dilution Factor: 
The magnitude by which a sample is diluted. 
 
Full Validation: 
Establishment of all validation parameters that ensure the integrity of the method when 
applied to sample analysis. 
46   
 
Hook Effect:  
Suppression of response due to very high concentrations of a particular analyte. A hook effect 
may occur in LBAs that use a liquid-phase reaction step for incubating the binding reagents 
with the analyte. Also referred to as Prozone Effect. 
 
Incurred Sample:   
A sample obtained from study subjects or animals.  
 
Incurred Sample Reanalysis (ISR): 
Reanalysis of a portion of the incurred samples in a separate analytical run on a different day 
to determine whether the original analytical results are reproducible.  
 
Interfering Substance:  
A substance that is present in the matrix that may affect the quantification of an analyte. 
 
Internal Standard (IS):   
A structurally similar analogue or stable isotope labeled compound added to calibration 
standards, QCs, and study samples at a known and constant concentration to facilitate 
quantification of the target analyte. 
 
Ligand Binding Assay (LBA):  
A method to analyze an analyte of interest using reagents that specifically bind to the analyte. 
The analyte is detected using reagents labeled with, e.g., an enzyme, radioisotope, 
fluorophore, or chromophore. Reactions are carried out in microtiter plates, test tubes, disks, 
etc. 
 
Lower Limit of Quantification (LLOQ):  
The lowest amount of an analyte in a sample that can be quantitatively determined using a 
method with predefined precision and accuracy. 
 
Matrix Effect:  
The direct or indirect alteration or interference in response due to the presence of unintended 
analytes or other interfering substances in the sample. 
 
Minimum Required Dilution (MRD):  
The initial dilution factor by which biological samples are diluted with buffer solution for the 
analysis by LBAs. The MRD may not necessarily be the ultimate dilution but should be 
identical for all samples including calibration standards and QCs. However, samples may 
require further dilution. 
 
Nominal Concentration:  
Theoretical or expected concentration. 
 
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.  
 
 
47   
Partial Validation:  
Validation based on evaluation of selected validation parameters. Applicable to methods that 
were changed after full validation. 
 
Precision: 
The closeness of agreement (i.e., degree of scatter) among a series of measurements. Precision 
is expressed as the coefficient of variation (CV), or the relative standard deviation (RSD) 
expressed as a percentage:  
 
%CV = (Standard Deviation/Mean) x 100. 
 
Processed Sample:  
The final sample that has been subjected to various manipulations (e.g., extraction, dilution, 
concentration). 
 
Quality Control Sample (QC):  
A biological matrix spiked with a known quantity of analyte that is used to monitor the 
performance of a bioanalytical method and assess the integrity and validity of the results of 
the unknown samples analyzed in an individual batch or run.  
 
Reanalysis: 
An additional evaluation of a previously assayed sample. Also referred to as Repeat Analysis. 
 
Recovery:  
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.  
 
Reference Standard: 
A well-characterized substance of known purity and identity used to prepare calibration and 
quality control samples.  
 
Reintegration:  
Change of the original integration of a chromatographic peak 
 
Replicate:  
One of several determinations or measurements of a sample, calibration standards or QC. 
 
Reproducibility:  
The extent to which consistent results are obtained when an experiment is repeated.  
 
Response Function: 
A mathematical expression which adequately describes the relationship between instrument 
response (e.g., peak area or height ratio or signal) and the concentration (amount) of analyte 
in the calibration standards. Response function is defined within a given range. See also 
Calibration Curve. 
 
Run Summary Table: 
Tabular output of all data from individual samples, QCs, and calibration standards within the 
analytical run (e.g., for chromatography retention times, analyte and IS responses, 
48   
concentrations, and dilution factors if any; for ligand binding assays analyte responses 
concentrations, and dilution factors). 
 
Selectivity:  
Ability of an analytical method to differentiate and measure the analyte in the presence of 
interfering substances in the biological matrix (nonspecific interference).  
 
Sensitivity:  
The lowest analyte concentration that can be measured with acceptable accuracy and 
precision (i.e., LLOQ). 
 
Specificity:  
Ability of an analytical method to detect and differentiate the analyte from other substances, 
including its related substances (e.g., substances that are structurally similar to the analyte, 
metabolites, isomers, impurities, or concomitant medications). 
 
Stability: 
Measure of the intactness of 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:  
The relationship between the instrument response (e.g., peak area, height, or signal) and the 
concentration (amount) of analyte in the calibration standards within a given range. Also 
referred to as Calibration Curve.  
 
Standard Operating Procedure (SOP):  
Detailed written instructions to achieve uniformity of the performance of a specific function 
and/or process(es). 
 
Stock Solution: 
An analyte in a solvent or mixture of solvents at a known concentration, which is used to 
prepare calibration standards or QCs. 
 
Study Samples:   
Samples from animals or subjects enrolled in nonclinical or clinical studies. 
 
Surrogate Matrix:  
An alternative to a study matrix of limited availability (e.g., tissue, cerebrospinal fluid, bile) 
or where the study matrix contains an interfering endogenous counterpart. 
 
System Suitability:   
Determination of instrument performance (e.g., Signal-to-Noise ratio, peak shape, retention 
time) by analysis of a prepared, spiked sample conducted prior to the analytical run and is not 
a part of the sample analysis. 
 
Total Error:  
The sum of the absolute value of the errors in accuracy (%) and precision (%). Total error is 
reported as percent (%) error. 
 
 
49   
Upper Limit of Quantification (ULOQ):  
The highest amount of an analyte in a sample that can be quantitatively determined with 
predefined precision and accuracy. 
 
Validation:  
Demonstration that a bioanalytical method is suitable for its intended purpose. 
 
Working Solution:   
A non-matrix solution prepared by diluting the stock solution in an appropriate solvent. It is 
mainly added to matrix to prepare calibration standards and QCs. 
 
Zero Sample:   
A blank sample spiked with an IS. 
50