Guidance for Industry

Bioanalytical Method Validation

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Veterinary Medicine (CVM)

May 2001

BP
Guidance for Industry

Bioanalytical Method Validation

Additional copies are available from:

Drug Information Branch (HFD-210)

Center for Drug Evaluation and Research (CDER)

5600 Fishers Lane, Rockville, MD 20857 (Tel) 301-827-4573

Internet at http://www.fda.gov/cder/guidance/index.htm

or

Communications Staff (HFV-12)

Center for Veterinary Medicine (CVM)

7500 Standish Place, Rockville, MD 20855 (Tel) 301–594-1755

Internet at http://www.fda.gov/cvm

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Veterinary Medicine (CVM)

May 2001

BP
Table of Contents

I. INTRODUCTION..................................................................................................................................................................1

II. BACKGROUND....................................................................................................................................................................1

A. FULL VALIDATION .............................................................................................................................................................. 2

B. PARTIAL VALIDATION ........................................................................................................................................................ 2

C. CROSS-VALIDATION ........................................................................................................................................................... 3

III. REFERENCE STANDARD.............................................................................................................................................4

IV. METHOD DEVELOPMENT: CHEMICAL ASSAY........................................................................................................4

A. SELECTIVITY....................................................................................................................................................................... 4

B. ACCURACY, PRECISION, AND RECOVERY......................................................................................................................... 5

C. CALIBRATION/STANDARD CURVE...................................................................................................................................... 5

D. STABILITY........................................................................................................................................................................... 6

E. PRINCIPLES OF BIOANALYTICAL METHOD VALIDATION AND ESTABLISHMENT ............................................................ 8

F. SPECIFIC RECOMMENDATIONS FOR METHOD VALIDATION........................................................................................... 10

V. METHOD DEVELOPMENT: MICROBIOLOGICAL AND LIGAND-BINDING ASSAYS ..................................11

A. SELECTIVITY ISSUES ........................................................................................................................................................ 11

B. QUANTIFICATION ISSUES ................................................................................................................................................. 12

VI. APPLICATION OF VALIDATED METHOD TO ROUTINE DRUG ANALYSIS....................................................13

ACCEPTANCE CRITERIA FOR THE RUN...................................................................................................................................... 15

VII. DOCUMENTATION......................................................................................................................................................16

A. SUMMARY INFORMATION................................................................................................................................................. 16

B. DOCUMENTATION FOR METHOD ESTABLISHMENT ....................................................................................................... 17

C. APPLICATION TO ROUTINE DRUG ANALYSIS ................................................................................................................. 17

D. OTHER INFORMATION...................................................................................................................................................... 19

GLOSSARY....................................................................................................................................................................................20
GUIDANCE FOR INDUSTRY1

Bioanalytical Method Validation

I. INTRODUCTION

This guidance provides assistance to sponsors of investigational new drug applications (INDs), new

drug applications (NDAs), abbreviated new drug applications (ANDAs), and supplements in

developing bioanalytical method validation information used in human clinical pharmacology,

bioavailability (BA), and bioequivalence (BE) studies requiring pharmacokinetic (PK) evaluation. This

guidance also applies to bioanalytical methods used for non-human pharmacology/toxicology studies

and preclinical studies. For studies related to the veterinary drug approval process, this guidance

applies only to blood and urine BA, BE, and PK studies. 

The information in this guidance generally applies to bioanalytical procedures such as gas

chromatography (GC), high-pressure liquid chromatography (LC), combined GC and LC mass

spectrometric (MS) procedures such as LC-MS, LC-MS-MS, GC-MS, and GC-MS-MS performed

for the quantitative determination of drugs and/or metabolites in biological matrices

such as blood, serum, plasma, or urine. This guidance also applies to other bioanalytical methods, such

as immunological and microbiological procedures, and to other biological matrices, such as tissue and

skin samples.

This guidance provides general recommendations for bioanalytical method validation. The

recommendations can be adjusted or modified depending on the specific type of analytical method used.

II. BACKGROUND

 

1

This guidance has been prepared by the Biopharmaceutics Coordinating Committee in the Center for Drug Evaluation and

Research (CDER) in cooperation with the Center for Veterinary Medicine (CVM) at the Food and Drug Administration. 

This guidance represents the Food and Drug Administration's current thinking on this topic. It

does not create or confer any rights for or on any person and does not operate to bind FDA or the

public. An alternative approach may be used if such approach satisfies the requirements of the

applicable statutes and regulations.
2

This guidance has been developed based on the deliberations of two workshops: (1) Analytical

Methods Validation: Bioavailability, Bioequivalence, and Pharmacokinetic Studies (held on December

3B5, 19902 ) and (2) Bioanalytical Methods Validation C A Revisit With a Decade of Progress (held

on January 12B14, 20003).

Selective and sensitive analytical methods for the quantitative evaluation of drugs and their metabolites

(analytes) are critical for the successful conduct of preclinical and/or biopharmaceutics and clinical

pharmacology studies. Bioanalytical method validation includes all of the procedures that demonstrate

that a particular method used for quantitative measurement of analytes in a given biological matrix, such

as blood, plasma, serum, or urine, is reliable and reproducible for the intended use. The fundamental

parameters for this validation include (1) accuracy, (2) precision, (3) selectivity, (4) sensitivity, (5)

reproducibility, and (6) stability. Validation involves documenting, through the use of specific laboratory

investigations, that the performance characteristics of the method are suitable and reliable for the

intended analytical applications. The acceptability of analytical data corresponds directly to the criteria

used to validate the method.

Published methods of analysis are often modified to suit the requirements of the laboratory performing

the assay. These modifications should be validated to ensure suitable performance of the analytical

method. When changes are made to a previously validated method, the analyst should exercise

judgment as to how much additional validation is needed. During the course of a typical drug

development program, a defined bioanalytical method undergoes many modifications. The evolutionary

changes to support specific studies and different levels of validation demonstrate the validity of an

assay’s performance. Different types and levels of validation are defined and characterized as follows:

A. Full Validation

• Full validation is important when developing and implementing a bioanalytical method for

the first time.

• Full validation is important for a new drug entity.

• A full validation of the revised assay is important if metabolites are added to an existing

assay for quantification.

B. Partial Validation

Partial validations are modifications of already validated bioanalytical methods. Partial validation

can range from as little as one intra-assay accuracy and precision determination to a nearly full

 

2

 Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 1992; 9:588-592.

3

 Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 2000; 17:in press.
3

validation. Typical bioanalytical method changes that fall into this category include, but are not

limited to:

• Bioanalytical method transfers between laboratories or analysts

• Change in analytical methodology (e.g., change in detection systems)

• Change in anticoagulant in harvesting biological fluid

• Change in matrix within species (e.g., human plasma to human urine)

• Change in sample processing procedures

• Change in species within matrix (e.g., rat plasma to mouse plasma)

• Change in relevant concentration range

• Changes in instruments and/or software platforms

• Limited sample volume (e.g., pediatric study)

• Rare matrices

• Selectivity demonstration of an analyte in the presence of concomitant medications

• Selectivity demonstration of an analyte in the presence of specific metabolites

C. Cross-Validation

Cross-validation is a comparison of validation parameters when two or more bioanalytical methods

are used to generate data within the same study or across different studies. An example of crossvalidation would be a situation where an original validated bioanalytical method serves as the

reference and the revised bioanalytical method is the comparator. The comparisons should be

done both ways.

When sample analyses within a single study are conducted at more than one site or more than one

laboratory, cross-validation with spiked matrix standards and subject samples should be conducted

at each site or laboratory to establish interlaboratory reliability. Cross-validation should also be

considered when data generated using different analytical techniques (e.g., LC-MS-MS vs.

ELISA4) in different studies are included in a regulatory submission.

All modifications should be assessed to determine the recommended degree of validation. The

analytical laboratory conducting pharmacology/toxicology and other preclinical studies for regulatory

submissions should adhere to FDA=s Good Laboratory Practices (GLPs)5 (21 CFR part 58) and to

sound principles of quality assurance throughout the testing process. The bioanalytical method for

human BA, BE, PK, and drug interaction studies must meet the criteria in 21 CFR 320.29. The

analytical laboratory should have a written set of standard operating procedures (SOPs) to ensure a

complete system of quality control and assurance. The SOPs should cover all aspects of analysis

from the time the sample is collected and reaches the laboratory until the results of the analysis are

reported. The SOPs also should include record keeping, security and chain of sample custody

 

4

 Enzyme linked immune sorbent assay

5

 For the Center for Veterinary Medicine, all bioequivalence studies are subject to Good Laboratory Practices.
4

(accountability systems that ensure integrity of test articles), sample preparation, and analytical tools

such as methods, reagents, equipment, instrumentation, and procedures for quality control and

verification of results.

The process by which a specific bioanalytical method is developed, validated, and used in routine

sample analysis can be divided into (1) reference standard preparation, (2) bioanalytical method

development and establishment of assay procedure, and (3) application of validated bioanalytical

method to routine drug analysis and acceptance criteria for the analytical run and/or batch. These

three processes are described in the following sections of this guidance.

III. REFERENCE STANDARD

Analysis of drugs and their metabolites in a biological matrix is carried out using samples spiked with

calibration (reference) standards and using quality control (QC) samples. The purity of the reference

standard used to prepare spiked samples can affect study data. For this reason, an authenticated

analytical reference standard of known identity and purity should be used to prepare solutions of known

concentrations. If possible, the reference standard should be identical to the analyte. When this is not

possible, an established chemical form (free base or acid, salt or ester) of known purity can be used. 

Three types of reference standards are usually used: (1) certified reference standards (e.g., USP

compendial standards); (2) commercially supplied reference standards obtained from a reputable

commercial source; and/or (3) other materials of documented purity custom-synthesized by an analytical

laboratory or other noncommercial establishment. The source and lot number, expiration date,

certificates of analyses when available, and/or internally or externally generated evidence of identity and

purity should be furnished for each reference standard.

IV. METHOD DEVELOPMENT: CHEMICAL ASSAY

The method development and establishment phase defines the chemical assay. The fundamental

parameters for a bioanalytical method validation are accuracy, precision, selectivity, sensitivity,

reproducibility, and stability. Measurements for each analyte in the biological matrix should be

validated. In addition, the stability of the analyte in spiked samples should be determined. Typical

method development and establishment for a bioanalytical method include determination of (1)

selectivity, (2) accuracy, precision, recovery, (3) calibration curve, and (4) stability of analyte in spiked

samples.

A. Selectivity

Selectivity is the ability of an analytical method to differentiate and quantify the analyte in the

presence of other components in the sample. For selectivity, analyses of blank samples of the

appropriate biological matrix (plasma, urine, or other matrix) should be obtained from at least
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six sources. Each blank sample should be tested for interference, and selectivity should be

ensured at the lower limit of quantification (LLOQ).

Potential interfering substances in a biological matrix include endogenous matrix components,

metabolites, decomposition products, and in the actual study, concomitant medication and other

exogenous xenobiotics. If the method is intended to quantify more than one analyte, each

analyte should be tested to ensure that there is no interference.

B. Accuracy, Precision, and Recovery

The accuracy of an analytical method describes the closeness of mean test results obtained by

the method to the true value (concentration) of the analyte. Accuracy is determined by replicate

analysis of samples containing known amounts of the analyte. Accuracy should be measured

using a minimum of five determinations per concentration. A minimum of three concentrations in

the range of expected concentrations is recommended. The mean value should be within 15%

of the actual value except at LLOQ, where it should not deviate by more than 20%. The

deviation of the mean from the true value serves as the measure of accuracy.

The precision of an analytical method describes the closeness of individual measures of an

analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous

volume of biological matrix. Precision should be measured using a minimum of five

determinations per concentration. A minimum of three concentrations in the range of expected

concentrations is recommended. The precision determined at each concentration level should

not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not

exceed 20% of the CV. Precision is further subdivided into within-run, intra-batch precision or

repeatability, which assesses precision during a single analytical run, and between-run, interbatch precision or repeatability, which measures precision with time, and may involve different

analysts, equipment, reagents, and laboratories.

The recovery of an analyte in an assay is the detector response obtained from an amount of the

analyte added to and extracted from the biological matrix, compared to the detector response

obtained for the true concentration of the pure authentic standard. Recovery pertains to the

extraction efficiency of an analytical method within the limits of variability. Recovery of the

analyte need not be 100%, but the extent of recovery of an analyte and of the internal standard

should be consistent, precise, and reproducible. Recovery experiments should be performed by

comparing the analytical results for extracted samples at three concentrations (low, medium, and

high) with unextracted standards that represent 100% recovery.

C. Calibration/Standard Curve

A calibration (standard) curve is the relationship between instrument response and known

concentrations of the analyte. A calibration curve should be generated for each analyte in the
6

sample. A sufficient number of standards should be used to adequately define the relationship

between concentration and response. A calibration curve should be prepared in the same

biological matrix as the samples in the intended study by spiking the matrix with known

concentrations of the analyte. The number of standards used in constructing a calibration curve

will be a function of the anticipated range of analytical values and the nature of the

analyte/response relationship. Concentrations of standards should be chosen on the basis of the

concentration range expected in a particular study. A calibration curve should consist of a blank

sample (matrix sample processed without internal standard), a zero sample (matrix sample

processed with internal standard), and six to eight non-zero samples covering the expected

range, including LLOQ.

1. Lower Limit of Quantification (LLOQ)

The lowest standard on the calibration curve should be accepted as the limit of

quantification if the following conditions are met:

C The analyte response at the LLOQ should be at least 5 times the response

compared to blank response.

C Analyte peak (response) should be identifiable, discrete, and reproducible with

a precision of 20% and accuracy of 80-120%.

2. Calibration Curve/Standard Curve/Concentration-Response

The simplest model that adequately describes the concentration-response relationship

should be used. Selection of weighting and use of a complex regression equation should

be justified. The following conditions should be met in developing a calibration curve:

C #20% deviation of the LLOQ from nominal concentration

C #15% deviation of standards other than LLOQ from nominal concentration

At least four out of six non-zero standards should meet the above criteria, including the

LLOQ and the calibration standard at the highest concentration. Excluding the

standards should not change the model used.

D. Stability

Drug stability in a biological fluid is a function of the storage conditions, the chemical properties

of the drug, the matrix, and the container system. The stability of an analyte in a particular

matrix and container system is relevant only to that matrix and container system and should not

be extrapolated to other matrices and container systems. Stability procedures should evaluate

the stability of the analytes during sample collection and handling, after long-term (frozen at the
7

intended storage temperature) and short-term (bench top, room temperature) storage, and after

going through freeze and thaw cycles and the analytical process. Conditions used in stability

experiments should reflect situations likely to be encountered during actual sample handling and

analysis. The procedure should also include an evaluation of analyte stability in stock solution. 

All stability determinations should use a set of samples prepared from a freshly made stock

solution of the analyte in the appropriate analyte-free, interference-free biological matrix. Stock

solutions of the analyte for stability evaluation should be prepared in an appropriate solvent at

known concentrations. 

1. Freeze and Thaw Stability

Analyte stability should be determined after three freeze and thaw cycles. At least three

aliquots at each of the low and high concentrations should be stored at the intended

storage temperature for 24 hours and thawed unassisted at room temperature. When

completely thawed, the samples should be refrozen for 12 to 24 hours under the same

conditions. The freeze–thaw cycle should be repeated two more times, then analyzed

on the third cycle. If an analyte is unstable at the intended storage temperature, the

stability sample should be frozen at -700C during the three freeze and thaw cycles.

2. Short-Term Temperature Stability

Three aliquots of each of the low and high concentrations should be thawed at room

temperature and kept at this temperature from 4 to 24 hours (based on the expected

duration that samples will be maintained at room temperature in the intended study) and

analyzed.

3. Long-Term Stability

The storage time in a long-term stability evaluation should exceed the time between the

date of first sample collection and the date of last sample analysis. Long-term stability

should be determined by storing at least three aliquots of each of the low and high

concentrations under the same conditions as the study samples. The volume of samples

should be sufficient for analysis on three separate occasions. The concentrations of all

the stability samples should be compared to the mean of back-calculated values for the

standards at the appropriate concentrations from the first day of long-term stability

testing.

4. Stock Solution Stability

The stability of stock solutions of drug and the internal standard should be evaluated at

room temperature for at least 6 hours. If the stock solutions are refrigerated or frozen
8

for the relevant period, the stability should be documented. After completion of the

desired storage time, the stability should be tested by comparing the instrument

response with that of freshly prepared solutions.

5. Post-Preparative Stability

The stability of processed samples, including the resident time in the autosampler, should

be determined. The stability of the drug and the internal standard should be assessed

over the anticipated run time for the batch size in validation samples by determining

concentrations on the basis of original calibration standards.

Although the traditional approach of comparing analytical results for stored samples with

those for freshly prepared samples has been referred to in this guidance, other statistical

approaches based on confidence limits for evaluation of an analyte=s stability in a

biological matrix can be used. SOPs should clearly describe the statistical method and

rules used. Additional validation may include investigation of samples from dosed

subjects.

E. Principles of Bioanalytical Method Validation and Establishment

• The fundamental parameters to ensure the acceptability of the performance of a

bioanalytical method validation are accuracy, precision, selectivity, sensitivity,

reproducibility, and stability.

• A specific, detailed description of the bioanalytical method should be written. This can be in

the form of a protocol, study plan, report, and/or SOP.

• Each step in the method should be investigated to determine the extent to which

environmental, matrix, material, or procedural variables can affect the estimation of analyte

in the matrix from the time of collection of the material up to and including the time of

analysis. 

• It may be important to consider the variability of the matrix due to the physiological nature

of the sample. In the case of LC-MS-MS-based procedures, appropriate steps should be

taken to ensure the lack of matrix effects throughout the application of the method,

especially if the nature of the matrix changes from the matrix used during method validation.

• A bioanalytical method should be validated for the intended use or application. All

experiments used to make claims or draw conclusions about the validity of the method

should be presented in a report (method validation report).
9

• Whenever possible, the same biological matrix as the matrix in the intended samples should

be used for validation purposes. (For tissues of limited availability, such as bone marrow,

physiologically appropriate proxy matrices can be substituted.)

• The stability of the analyte (drug and/or metabolite) in the matrix during the collection

process and the sample storage period should be assessed, preferably prior to sample

analysis.

• For compounds with potentially labile metabolites, the stability of analyte in matrix from

dosed subjects (or species) should be confirmed.

• The accuracy, precision, reproducibility, response function, and selectivity of the method for

endogenous substances, metabolites, and known degradation products should be

established for the biological matrix. For selectivity, there should be evidence that the

substance being quantified is the intended analyte.

• The concentration range over which the analyte will be determined should be defined in the

bioanalytical method, based on evaluation of actual standard samples over the range,

including their statistical variation. This defines the standard curve.

• A sufficient number of standards should be used to adequately define the relationship

between concentration and response. The relationship between response and concentration

should be demonstrated to be continuous and reproducible. The number of standards used

should be a function of the dynamic range and nature of the concentration-response

relationship. In many cases, six to eight concentrations (excluding blank values) can define

the standard curve. More standard concentrations may be recommended for nonlinear than

for linear relationships.

• The ability to dilute samples originally above the upper limit of the standard curve should be

demonstrated by accuracy and precision parameters in the validation.

• In consideration of high throughput analyses, including but not limited to multiplexing,

multicolumn, and parallel systems, sufficient QC samples should be used to ensure control

of the assay. The number of QC samples to ensure proper control of the assay should be

determined based on the run size. The placement of QC samples should be judiciously

considered in the run. 

• For a bioanalytical method to be considered valid, specific acceptance criteria should be set

in advance and achieved for accuracy and precision for the validation of QC samples over

the range of the standards.
10

F. Specific Recommendations for Method Validation

• The matrix-based standard curve should consist of a minimum of six standard points,

excluding blanks, using single or replicate samples. The standard curve should cover the

entire range of expected concentrations.

• Standard curve fitting is determined by applying the simplest model that adequately

describes the concentration-response relationship using appropriate weighting and statistical

tests for goodness of fit.

• LLOQ is the lowest concentration of the standard curve that can be measured with

acceptable accuracy and precision. The LLOQ should be established using at least five

samples independent of standards and determining the coefficient of variation and/or

appropriate confidence interval. The LLOQ should serve as the lowest concentration on

the standard curve and should not be confused with the limit of detection and/or the low QC

sample. The highest standard will define the upper limit of quantification (ULOQ) of an

analytical method.

• For validation of the bioanalytical method, accuracy and precision should be determined

using a minimum of five determinations per concentration level (excluding blank samples). 

The mean value should be within ±15% of the theoretical value, except at LLOQ, where it

should not deviate by more than ±20%. The precision around the mean value should not

exceed 15% of the CV, except for LLOQ, where it should not exceed 20% of the CV. 

Other methods of assessing accuracy and precision that meet these limits may be equally

acceptable.

• The accuracy and precision with which known concentrations of analyte in biological matrix

can be determined should be demonstrated. This can be accomplished by analysis of

replicate sets of analyte samples of known concentrations C QC samples C from an

equivalent biological matrix. At a minimum, three concentrations representing the entire

range of the standard curve should be studied: one within 3x the lower limit of quantification

(LLOQ) (low QC sample), one near the center (middle QC), and one near the upper

boundary of the standard curve (high QC).

• Reported method validation data and the determination of accuracy and precision should

include all outliers; however, calculations of accuracy and precision excluding values that are

statistically determined as outliers can also be reported.

• The stability of the analyte in biological matrix at intended storage temperatures should be

established. The influence of freeze-thaw cycles (a minimum of three cycles at two

concentrations in triplicate) should be studied.
11

• The stability of the analyte in matrix at ambient temperature should be evaluated over a time

period equal to the typical sample preparation, sample handling, and analytical run times. 

• Reinjection reproducibility should be evaluated to determine if an analytical run could be

reanalyzed in the case of instrument failure.

• The specificity of the assay methodology should be established using a minimum of six

independent sources of the same matrix. For hyphenated mass spectrometry-based

methods, however, testing six independent matrices for interference may not be important. 

In the case of LC-MS and LC-MS-MS-based procedures, matrix effects should be

investigated to ensure that precision, selectivity, and sensitivity will not be compromised. 

Method selectivity should be evaluated during method development and throughout method

validation and can continue throughout application of the method to actual study samples.

• Acceptance/rejection criteria for spiked, matrix-based calibration standards and validation

QC samples should be based on the nominal (theoretical) concentration of analytes. 

Specific criteria can be set up in advance and achieved for accuracy and precision over the

range of the standards, if so desired.

V. METHOD DEVELOPMENT: MICROBIOLOGICAL AND LIGAND-BINDING

ASSAYS

Many of the bioanalytical validation parameters and principles discussed above are also applicable to

microbiological and ligand-binding assays. However, these assays possess some unique characteristics

that should be considered during method validation.

A. Selectivity Issues

As with chromatographic methods, microbiological and ligand-binding assays should be shown

to be selective for the analyte. The following recommendations for dealing with two selectivity

issues should be considered:

1. Interference From Substances Physiochemically Similar to the Analyte

• Cross-reactivity of metabolites, concomitant medications, or endogenous

compounds should be evaluated individually and in combination with the analyte

of interest.

• When possible, the immunoassay should be compared with a validated reference

method (such as LC-MS) using incurred samples and predetermined criteria for

agreement of accuracy of immunoassay and reference method.
12

• The dilutional linearity to the reference standard should be assessed using study

(incurred) samples.

• Selectivity may be improved for some analytes by incorporation of separation

steps prior to immunoassay.

2. Matrix effects Unrelated to the Analyte

• The standard curve in biological fluids should be compared with standard in

buffer to detect matrix effects.

• Parallelism of diluted study samples should be evaluated with diluted standards to

detect matrix effects.

• Nonspecific binding should be determined.

B. Quantification Issues

Microbiological and imunoassay standard curves are inherently nonlinear and, in general, more

concentration points may be recommended to define the fit over the standard curve range than

for chemical assays. In addition to their nonlinear characteristics, the response-error

relationship for immunoassay standard curves is a nonconstant function of the mean response

(heteroscadisticity). For these reasons, a minimum of six non-zero calibrator concentrations,

run in duplicate, is recommended. The concentration-response relationship is most often fitted

to a 4- or 5-parameter logistic model, although others may be used with suitable validation. The

use of anchoring points in the asymptotic high- and low-concentration ends of the standard

curve may improve the overall curve fit. Generally, these anchoring points will be at

concentrations that are below the established LLOQ and above the established ULOQ. 

Whenever possible, calibrators should be prepared in the same matrix as the study samples or

in an alternate matrix of equivalent performance. Both ULOQ and LLOQ should be defined by

acceptable accuracy, precision, or confidence interval criteria based on the study requirements.

For all assays the key factor is the accuracy of the reported results. This accuracy can be

improved by the use of replicate samples. In the case where replicate samples should be

measured during the validation to improve accuracy, the same procedure should be followed as

for unknown samples.

The following recommendations apply to quantification issues:

• If separation is used prior to assay for study samples but not for standards, it is important to

establish recovery and use it in determining results. Possible approaches to assess efficiency
13

and reproducibility of recovery are (1) the use of radiolabeled tracer analyte (quantity too

small to affect the assay), (2) the advance establishment of reproducible recovery, (3) the

use of an internal standard that is not recognized by the antibody but can be measured by

another technique.

• Key reagents, such as antibody, tracer, reference standard, and matrix should be

characterized appropriately and stored under defined conditions.

• Assessments of analyte stability should be conducted in true study matrix (e.g., should not

use a matrix stripped to remove endogenous interferences).

• Acceptance criteria: At least 67% (4 out of 6) of QC samples should be within 15% of

their respective nominal value, 33% of the QC samples (not all replicates at the same

concentration) may be outside 15% of nominal value. In certain situations, wider

acceptance criteria may be justified.

• Assay reoptimization or validation may be important when there are changes in key

reagents, as follows:

Labeled analyte (tracer)

• Binding should be reoptimized.

• Performance should be verified with standard curve and QCs.

Antibody

• Key cross-reactivities should be checked.

• Tracer experiments above should be repeated.

Matrix

• Tracer experiments above should be repeated.

Method development experiments should include a minimum of six runs conducted over several days,

with at least four concentrations (LLOQ, low, medium, and high) analyzed in duplicate in each run.

VI. APPLICATION OF VALIDATED METHOD TO ROUTINE DRUG ANALYSIS

Assays of all samples of an analyte in a biological matrix should be completed within the time period for

which stability data are available. In general, biological samples can be analyzed with a single

determination without duplicate or replicate analysis if the assay method has acceptable variability as

defined by validation data. This is true for procedures where precision and accuracy variabilities

routinely fall within acceptable tolerance limits. For a difficult procedure with a labile analyte where high

precision and accuracy specifications may be difficult to achieve, duplicate or even triplicate analyses

can be performed for a better estimate of analyte.
14

A calibration curve should be generated for each analyte to assay samples in each analytical run and

should be used to calculate the concentration of the analyte in the unknown samples in the run. The

spiked samples can contain more than one analyte. An analytical run can consist of QC samples,

calibration standards, and either (1) all the processed samples to be analyzed as one batch or (2) a

batch composed of processed unknown samples of one or more volunteers in a study. The calibration

(standard) curve should cover the expected unknown sample concentration range in addition to a

calibrator sample at LLOQ. Estimation of concentration in unknown samples by extrapolation of

standard curves below LLOQ or above the highest standard is not recommended. Instead, the

standard curve should be redefined or samples with higher concentration should be diluted and

reassayed. It is preferable to analyze all study samples from a subject in a single run.

Once the analytical method has been validated for routine use, its accuracy and precision should be

monitored regularly to ensure that the method continues to perform satisfactorily. To achieve this

objective, a number of QC samples prepared separately should be analyzed with processed test

samples at intervals based on the total number of samples. The QC samples in duplicate at three

concentrations (one near the LLOQ (i.e., #3 x LLOQ), one in midrange, and one close to the high end

of the range) should be incorporated in each assay run. The number of QC samples (in multiples of

three) will depend on the total number of samples in the run. The results of the QC samples provide the

basis of accepting or rejecting the run. At least four of every six QC samples should be within "15% of

their respective nominal value. Two of the six QC samples may be outside the "15% of their respective

nominal value, but not both at the same concentration.

The following recommendations should be noted in applying a bioanalytical method to routine drug

analysis:

• A matrix-based standard curve should consist of a minimum of six standard points,

excluding blanks (either single or replicate), covering the entire range.

• Response Function: Typically, the same curve fitting, weighting, and goodness of fit

determined during prestudy validation should be used for the standard curve within the

study. Response function is determined by appropriate statistical tests based on the actual

standard points during each run in the validation. Changes in the response function

relationship between prestudy validation and routine run validation indicate potential

problems.

• The QC samples should be used to accept or reject the run. These QC samples are matrix

spiked with analyte. 

• System suitability: Based on the analyte and technique, a specific SOP (or sample) should

be identified to ensure optimum operation of the system used.
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• Any required sample dilutions should use like matrix (e.g., human to human) obviating the

need to incorporate actual within-study dilution matrix QC samples.

• Repeat Analysis: It is important to establish an SOP or guideline for repeat analysis and

acceptance criteria. This SOP or guideline should explain the reasons for repeating sample

analysis. Reasons for repeat analyses could include repeat analysis of clinical or preclinical

samples for regulatory purposes, inconsistent replicate analysis, samples outside of the assay

range, sample processing errors, equipment failure, poor chromatography, and inconsistent

pharmacokinetic data. Reassays should be done in triplicate if sample volume allows. The

rationale for the repeat analysis and the reporting of the repeat analysis should be clearly

documented. 

• Sample Data Reintegration: An SOP or guideline for sample data reintegration should be

established. This SOP or guideline should explain the reasons for reintegration and how the

reintegration is to be performed. The rationale for the reintegration should be clearly

described and documented. Original and reintegration data should be reported.

Acceptance Criteria for the Run

The following acceptance criteria should be considered for accepting the analytical run:

• Standards and QC samples can be prepared from the same spiking stock solution,

provided the solution stability and accuracy have been verified. A single source of matrix

may also be used, provided selectivity has been verified.

• Standard curve samples, blanks, QCs, and study samples can be arranged as considered

appropriate within the run.

• Placement of standards and QC samples within a run should be designed to detect assay

drift over the run.

• Matrix-based standard calibration samples: 75%, or a minimum of six standards, when

back-calculated (including ULOQ) should fall within ±15%, except for LLOQ, when it

should be ±20% of the nominal value. Values falling outside these limits can be discarded,

provided they do not change the established model.

• Acceptance criteria for accuracy and precision as outlined in section IV.F, “Specific

Recommendation for Method Validation,” should be provided for both the intra-day and

intra-run experiment. 

• Quality Control Samples: Quality control samples replicated (at least once) at a minimum of

three concentrations (one within 3x of the LLOQ (low QC), one in the midrange (middle
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QC), and one approaching the high end of the range (high QC)) should be incorporated into

each run. The results of the QC samples provide the basis of accepting or rejecting the run.

 At least 67% (four out of six) of the QC samples should be within 15% of their respective

nominal (theoretical) values; 33% of the QC samples (not all replicates at the same

concentration) can be outside the ±15% of the nominal value. A confidence interval

approach yielding comparable accuracy and precision is an appropriate alternative.

The minimum number of samples (in multiples of three) should be at least 5% of the number of

unknown samples or six total QCs, whichever is greater.

• Samples involving multiple analytes should not be rejected based on the data from one

analyte failing the acceptance criteria.

• The data from rejected runs need not be documented, but the fact that a run was rejected

and the reason for failure should be recorded.

VII. DOCUMENTATION

The validity of an analytical method should be established and verified by laboratory studies, and

documentation of successful completion of such studies should be provided in the assay validation

report. General and specific SOPs and good record keeping are an essential part of a validated

analytical method. The data generated for bioanalytical method establishment and the QCs should be

documented and available for data audit and inspection. Documentation for submission to the Agency

should include (1) summary information, (2) method development and establishment, (3) bioanalytical

reports of the application of any methods to routine sample analysis, and (4) other information

applicable to method development and establishment and/or to routine sample analysis.

A. Summary Information

Summary information should include:

• Summary table of validation reports, including analytical method validation, partial

revalidation, and cross-validation reports. The table should be in chronological sequence,

and include assay method identification code, type of assay, and the reason for the new

method or additional validation (e.g., to lower the limit of quantitation).

• Summary table with a list, by protocol, of assay methods used. The protocol number,

protocol title, assay type, assay method identification code, and bioanalytic report code

should be provided.

• A summary table allowing cross-referencing of multiple identification codes should be

provided (e.g., when an assay has different codes for the assay method, validation reports,
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and bioanalytical reports, especially when the sponsor and a contract laboratory assign

different codes). 

B. Documentation for Method Establishment

Documentation for method development and establishment should include:

• An operational description of the analytical method

• Evidence of purity and identity of drug standards, metabolite standards, and internal

standards used in validation experiments

• A description of stability studies and supporting data

• A description of experiments conducted to determine accuracy, precision, recovery,

selectivity, limit of quantification, calibration curve (equations and weighting functions used, if

any), and relevant data obtained from these studies

• Documentation of intra- and inter-assay precision and accuracy

• In NDA submissions, information about cross-validation study data, if applicable

• Legible annotated chromatograms or mass spectrograms, if applicable

• Any deviations from SOPs, protocols, or GLPs (if applicable), and justifications for

deviations

C. Application to Routine Drug Analysis

Documentation of the application of validated bioanalytical methods to routine drug analysis

should include:

• Evidence of purity and identity of drug standards, metabolite standards, and internal

standards used during routine analyses

• Summary tables containing information on sample processing and storage. Tables should

include sample identification, collection dates, storage prior to shipment, information on

shipment batch, and storage prior to analysis. Information should include dates, times,

sample condition, and any deviation from protocols.

• Summary tables of analytical runs of clinical or preclinical samples. Information should

include assay run identification, date and time of analysis, assay method, analysts, start and
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stop times, duration, significant equipment and material changes, and any potential issues or

deviation from the established method.

• Equations used for back-calculation of results

• Tables of calibration curve data used in analyzing samples and calibration curve summary

data

• Summary information on intra- and inter-assay values of QC samples and data on intra- and

inter-assay accuracy and precision from calibration curves and QC samples used for

accepting the analytical run. QC graphs and trend analyses in addition to raw data and

summary statistics are encouraged.

• Data tables from analytical runs of clinical or preclinical samples. Tables should include

assay run identification, sample identification, raw data and back-calculated results,

integration codes, and/or other reporting codes.

• Complete serial chromatograms from 5B20% of subjects, with standards and QC samples

from those analytical runs. For pivotal bioequivalence studies for marketing,

chromatograms from 20% of serially selected subjects should be included. In other studies,

chromatograms from 5% of randomly selected subjects in each study should be included. 

Subjects whose chromatograms are to be submitted should be defined prior to the analysis

of any clinical samples.

• Reasons for missing samples

• Documentation for repeat analyses. Documentation should include the initial and repeat

analysis results, the reported result, assay run identification, the reason for the repeat

analysis, the requestor of the repeat analysis, and the manager authorizing reanalysis. 

Repeat analysis of a clinical or preclinical sample should be performed only under a

predefined SOP.

• Documentation for reintegrated data. Documentation should include the initial and repeat

integration results, the method used for reintegration, the reported result, assay run

identification, the reason for the reintegration, the requestor of the reintegration, and the

manager authorizing reintegration. Reintegration of a clinical or preclinical sample should be

performed only under a predefined SOP.

• Deviations from the analysis protocol or SOP, with reasons and justifications for the

deviations
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D. Other Information

Other information applicable to both method development and establishment and/or to routine

sample analysis could include: 

• Lists of abbreviations and any additional codes used, including sample condition codes,

integration codes, and reporting codes

• Reference lists and legible copies of any references

• SOPs or protocols covering the following areas:

• Calibration standard acceptance or rejection criteria

• Calibration curve acceptance or rejection criteria

• Quality control sample and assay run acceptance or rejection criteria

• Acceptance criteria for reported values when all unknown samples are assayed in

duplicate

• Sample code designations, including clinical or preclinical sample codes and

bioassay sample code

• Assignment of clinical or preclinical samples to assay batches

• Sample collection, processing, and storage

• Repeat analyses of samples

• Reintegration of samples
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GLOSSARY

Accuracy: The degree of closeness of the determined value to the nominal or known true value under

prescribed conditions. This is sometimes termed trueness. 

Analyte: A specific chemical moiety being measured, which can be intact drug, biomolecule or its

derivative, metabolite, and/or degradation product in a biologic matrix.

Analytical run (or batch): A complete set of analytical and study samples with appropriate number of

standards and QCs for their validation. Several runs (or batches) may be completed in one day, or one

run (or batch) may take several days to complete.

Biological matrix: A discrete material of biological origin that can be sampled and processed in a

reproducible manner. Examples are blood, serum, plasma, urine, feces, saliva, sputum, and various

discrete tissues.

Calibration standard: A biological matrix to which a known amount of analyte has been added or

spiked. Calibration standards are used to construct calibration curves from which the concentrations of

analytes in QCs and in unknown study samples are determined. 

Internal standard: Test compound(s) (e.g. structurally similar analog, stable labeled compound)

added to both calibration standards and samples at known and constant concentration to facilitate

quantification of the target analyte(s). 

Limit of detection (LOD): The lowest concentration of an analyte that the bioanalytical procedure

can reliably differentiate from background noise.

Lower limit of quantification (LLOQ): The lowest amount of an analyte in a sample that can be

quantitatively determined with suitable precision and accuracy.

Matrix effect: The direct or indirect alteration or interference in response due to the presence of

unintended analytes (for analysis) or other interfering substances in the sample. 

Method: A comprehensive description of all procedures used in sample analysis.

Precision: The closeness of agreement (degree of scatter) between a series of measurements

obtained from multiple sampling of the same homogenous sample under the prescribed conditions.

Processed: The final extract (prior to instrumental analysis) of a sample that has been subjected to

various manipulations (e.g., extraction, dilution, concentration).
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Quantification range: The range of concentration, including ULOQ and LLOQ, that can be reliably

and reproducibly quantified with accuracy and precision through the use of a concentration-response

relationship.

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.

Reproducibility: The precision between two laboratories. It also represents precision of the method

under the same operating conditions over a short period of time.

Sample: A generic term encompassing controls, blanks, unknowns, and processed samples, as

described below:

Blank: A sample of a biological matrix to which no analytes have been added that is used to

assess the specificity of the bioanalytical method. 

Quality control sample (QC): A spiked sample used to monitor the performance of a

bioanalytical method and to assess the integrity and validity of the results of the unknown

samples analyzed in an individual batch.

Unknown: A biological sample that is the subject of the analysis.

Selectivity: The ability of the bioanalytical method to measure and differentiate the analytes in the

presence of components that may be expected to be present. These could include metabolites,

impurities, degradants, or matrix components.

Stability: The chemical stability of an analyte in a given matrix under specific conditions for given time

intervals.

Standard curve: The relationship between the experimental response value and the analytical

concentration (also called a calibration curve).

System suitability: Determination of instrument performance (e.g., sensitivity and chromatographic

retention) by analysis of a reference standard prior to running the analytical batch. 

Upper limit of quantification (ULOQ): The highest amount of an analyte in a sample that can be

quantitatively determined with precision and accuracy.

Validation:

Full validation: Establishment of all validation parameters to apply to sample analysis for the

bioanalytical method for each analyte.
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Partial validation: Modification of validated bioanalytical methods that do not necessarily call

for full revalidation.

Cross-validation: Comparison validation parameters of two bioanalytical methods.