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) Guidance for Industry Bioanalytical Method Validation Additional copies are available from:Center for Drug Evaluation and Research (CDER)5600 Fishers Lane, Rockville, MD 20857 (Tel) 301-827-4573Internet at http://www.fda.gov/cder/guidance/index.htmCenter for Veterinary Medicine (CVM)7500 Standish Place, Rockville, MD 20855 (Tel) 301–594-1755Internet at http://www.fda.gov/cvmU.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Veterinary Medicine (CVM) Table of Contents INTRODUCTION.1 BACKGROUND.1 REFERENCE STANDARD.4 METHOD DEVELOPMENT: CHEMICAL ASSAY.4
PRINCIPLES OF BIOANALYTICAL METHOD VALIDATION AND ESTABLISHMENT . 8
SPECIFIC RECOMMENDATIONS FOR METHOD VALIDATION. 10
METHOD DEVELOPMENT: MICROBIOLOGICAL AND LIGAND-BINDING ASSAYS .11 APPLICATION OF VALIDATED METHOD TO ROUTINE DRUG ANALYSIS .13 DOCUMENTATION.16
DOCUMENTATION FOR METHOD ESTABLISHMENT . 17
APPLICATION TO ROUTINE DRUG ANALYSIS . 17
GLOSSARY.20 GUIDANCE FOR INDUSTRY1 Bioanalytical Method Validation
This guidance represents the Food and Drug Administration's current thinking on this topic. Itdoes not create or confer any rights for or on any person and does not operate to bind FDA or thepublic. An alternative approach may be used if such approach satisfies the requirements of theapplicable statutes and regulations. INTRODUCTION
This guidance provides assistance to sponsors of investigational new drug applications (INDs), newdrug applications (NDAs), abbreviated new drug applications (ANDAs), and supplements indeveloping bioanalytical method validation information used in human clinical pharmacology,bioavailability (BA), and bioequivalence (BE) studies requiring pharmacokinetic (PK) evaluation. Thisguidance also applies to bioanalytical methods used for non-human pharmacology/toxicology studiesand preclinical studies. For studies related to the veterinary drug approval process, this guidanceapplies only to blood and urine BA, BE, and PK studies.
The information in this guidance generally applies to bioanalytical procedures such as gaschromatography (GC), high-pressure liquid chromatography (LC), combined GC and LC massspectrometric (MS) procedures such as LC-MS, LC-MS-MS, GC-MS, and GC-MS-MS performedfor the quantitative determination of drugs and/or metabolites in biological matricessuch as blood, serum, plasma, or urine. This guidance also applies to other bioanalytical methods, suchas immunological and microbiological procedures, and to other biological matrices, such as tissue andskin samples.
This guidance provides general recommendations for bioanalytical method validation. Therecommendations can be adjusted or modified depending on the specific type of analytical method used. BACKGROUND
1 This guidance has been prepared by the Biopharmaceutics Coordinating Committee in the Center for Drug Evaluation andResearch (CDER) in cooperation with the Center for Veterinary Medicine (CVM) at the Food and Drug Administration.
This guidance has been developed based on the deliberations of two workshops: (1) AnalyticalMethods Validation: Bioavailability, Bioequivalence, and Pharmacokinetic Studies (held on December3B5, 19902 ) and (2) Bioanalytical Methods Validation C A Revisit With a Decade of Progress (heldon 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 clinicalpharmacology studies. Bioanalytical method validation includes all of the procedures that demonstratethat a particular method used for quantitative measurement of analytes in a given biological matrix, suchas blood, plasma, serum, or urine, is reliable and reproducible for the intended use. The fundamentalparameters 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 laboratoryinvestigations, that the performance characteristics of the method are suitable and reliable for theintended analytical applications. The acceptability of analytical data corresponds directly to the criteriaused to validate the method.
Published methods of analysis are often modified to suit the requirements of the laboratory performingthe assay. These modifications should be validated to ensure suitable performance of the analyticalmethod. When changes are made to a previously validated method, the analyst should exercisejudgment as to how much additional validation is needed. During the course of a typical drugdevelopment program, a defined bioanalytical method undergoes many modifications. The evolutionarychanges to support specific studies and different levels of validation demonstrate the validity of anassay’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 forthe 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 existingassay for quantification. B. Partial Validation
Partial validations are modifications of already validated bioanalytical methods. Partial validationcan 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.
validation. Typical bioanalytical method changes that fall into this category include, but are notlimited 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 species within matrix (e.g., rat plasma to mouse plasma)
Changes in instruments and/or software platforms
Limited sample volume (e.g., pediatric study)
Selectivity demonstration of an analyte in the presence of concomitant medications
Selectivity demonstration of an analyte in the presence of specific metabolites
Cross-validation is a comparison of validation parameters when two or more bioanalytical methodsare used to generate data within the same study or across different studies. An example of cross-validation would be a situation where an original validated bioanalytical method serves as thereference and the revised bioanalytical method is the comparator. The comparisons should bedone both ways.
When sample analyses within a single study are conducted at more than one site or more than onelaboratory, cross-validation with spiked matrix standards and subject samples should be conductedat each site or laboratory to establish interlaboratory reliability. Cross-validation should also beconsidered 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. Theanalytical laboratory conducting pharmacology/toxicology and other preclinical studies for regulatorysubmissions should adhere to FDA=s Good Laboratory Practices (GLPs)5 (21 CFR part 58) and tosound principles of quality assurance throughout the testing process. The bioanalytical method forhuman BA, BE, PK, and drug interaction studies must meet the criteria in 21 CFR 320.29. Theanalytical laboratory should have a written set of standard operating procedures (SOPs) to ensure acomplete system of quality control and assurance. The SOPs should cover all aspects of analysisfrom the time the sample is collected and reaches the laboratory until the results of the analysis arereported. The SOPs also should include record keeping, security and chain of sample custody
4 Enzyme linked immune sorbent assay5 For the Center for Veterinary Medicine, all bioequivalence studies are subject to Good Laboratory Practices.
(accountability systems that ensure integrity of test articles), sample preparation, and analytical toolssuch as methods, reagents, equipment, instrumentation, and procedures for quality control andverification of results.
The process by which a specific bioanalytical method is developed, validated, and used in routinesample analysis can be divided into (1) reference standard preparation, (2) bioanalytical methoddevelopment and establishment of assay procedure, and (3) application of validated bioanalyticalmethod to routine drug analysis and acceptance criteria for the analytical run and/or batch. Thesethree processes are described in the following sections of this guidance. REFERENCE STANDARD
Analysis of drugs and their metabolites in a biological matrix is carried out using samples spiked withcalibration (reference) standards and using quality control (QC) samples. The purity of the referencestandard used to prepare spiked samples can affect study data. For this reason, an authenticatedanalytical reference standard of known identity and purity should be used to prepare solutions of knownconcentrations. If possible, the reference standard should be identical to the analyte. When this is notpossible, 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., USPcompendial standards); (2) commercially supplied reference standards obtained from a reputablecommercial source; and/or (3) other materials of documented purity custom-synthesized by an analyticallaboratory 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 andpurity should be furnished for each reference standard. METHOD DEVELOPMENT: CHEMICAL ASSAY
The method development and establishment phase defines the chemical assay. The fundamentalparameters for a bioanalytical method validation are accuracy, precision, selectivity, sensitivity,reproducibility, and stability. Measurements for each analyte in the biological matrix should bevalidated. In addition, the stability of the analyte in spiked samples should be determined. Typicalmethod 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 spikedsamples. 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
six sources. Each blank sample should be tested for interference, and selectivity should beensured 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 otherexogenous xenobiotics. If the method is intended to quantify more than one analyte, eachanalyte 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, inter- batch 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 knownconcentrations of the analyte. A calibration curve should be generated for each analyte in the
sample. A sufficient number of standards should be used to adequately define the relationshipbetween concentration and response. A calibration curve should be prepared in the samebiological matrix as the samples in the intended study by spiking the matrix with knownconcentrations of the analyte. The number of standards used in constructing a calibration curvewill be a function of the anticipated range of analytical values and the nature of theanalyte/response relationship. Concentrations of standards should be chosen on the basis of theconcentration range expected in a particular study. A calibration curve should consist of a blanksample (matrix sample processed without internal standard), a zero sample (matrix sampleprocessed with internal standard), and six to eight non-zero samples covering the expectedrange, including LLOQ. Lower Limit of Quantification (LLOQ)
The lowest standard on the calibration curve should be accepted as the limit ofquantification if the following conditions are met:
The analyte response at the LLOQ should be at least 5 times the responsecompared to blank response.
Analyte peak (response) should be identifiable, discrete, and reproducible witha precision of 20% and accuracy of 80-120%. Calibration Curve/Standard Curve/Concentration-Response
The simplest model that adequately describes the concentration-response relationshipshould be used. Selection of weighting and use of a complex regression equation shouldbe justified. The following conditions should be met in developing a calibration curve:
#20% deviation of the LLOQ from nominal concentration
#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 theLLOQ and the calibration standard at the highest concentration. Excluding thestandards should not change the model used. D. Stability
Drug stability in a biological fluid is a function of the storage conditions, the chemical propertiesof the drug, the matrix, and the container system. The stability of an analyte in a particularmatrix and container system is relevant only to that matrix and container system and should notbe extrapolated to other matrices and container systems. Stability procedures should evaluatethe stability of the analytes during sample collection and handling, after long-term (frozen at the
intended storage temperature) and short-term (bench top, room temperature) storage, and aftergoing through freeze and thaw cycles and the analytical process. Conditions used in stabilityexperiments should reflect situations likely to be encountered during actual sample handling andanalysis. 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 stocksolution of the analyte in the appropriate analyte-free, interference-free biological matrix. Stocksolutions of the analyte for stability evaluation should be prepared in an appropriate solvent atknown concentrations.
Analyte stability should be determined after three freeze and thaw cycles. At least threealiquots at each of the low and high concentrations should be stored at the intendedstorage temperature for 24 hours and thawed unassisted at room temperature. Whencompletely thawed, the samples should be refrozen for 12 to 24 hours under the sameconditions. The freeze–thaw cycle should be repeated two more times, then analyzedon the third cycle. If an analyte is unstable at the intended storage temperature, thestability 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 roomtemperature and kept at this temperature from 4 to 24 hours (based on the expectedduration that samples will be maintained at room temperature in the intended study) andanalyzed.
The storage time in a long-term stability evaluation should exceed the time between thedate of first sample collection and the date of last sample analysis. Long-term stabilityshould be determined by storing at least three aliquots of each of the low and highconcentrations under the same conditions as the study samples. The volume of samplesshould be sufficient for analysis on three separate occasions. The concentrations of allthe stability samples should be compared to the mean of back-calculated values for thestandards at the appropriate concentrations from the first day of long-term stabilitytesting.
The stability of stock solutions of drug and the internal standard should be evaluated atroom temperature for at least 6 hours. If the stock solutions are refrigerated or frozen
for the relevant period, the stability should be documented. After completion of thedesired storage time, the stability should be tested by comparing the instrumentresponse with that of freshly prepared solutions.
The stability of processed samples, including the resident time in the autosampler, shouldbe determined. The stability of the drug and the internal standard should be assessedover the anticipated run time for the batch size in validation samples by determiningconcentrations on the basis of original calibration standards.
Although the traditional approach of comparing analytical results for stored samples withthose for freshly prepared samples has been referred to in this guidance, other statisticalapproaches based on confidence limits for evaluation of an analyte=s stability in abiological matrix can be used. SOPs should clearly describe the statistical method andrules used. Additional validation may include investigation of samples from dosedsubjects. 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 analytein the matrix from the time of collection of the material up to and including the time ofanalysis.
• 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 betaken 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 methodshould be presented in a report (method validation report).
• 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 sampleanalysis.
• 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 beestablished for the biological matrix. For selectivity, there should be evidence that thesubstance 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 concentrationshould be demonstrated to be continuous and reproducible. The number of standards usedshould be a function of the dynamic range and nature of the concentration-responserelationship. In many cases, six to eight concentrations (excluding blank values) can definethe standard curve. More standard concentrations may be recommended for nonlinear thanfor 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 controlof the assay. The number of QC samples to ensure proper control of the assay should bedetermined based on the run size. The placement of QC samples should be judiciouslyconsidered 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 overthe range of the standards. 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 theentire 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 statisticaltests 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 fivesamples independent of standards and determining the coefficient of variation and/orappropriate confidence interval. The LLOQ should serve as the lowest concentration onthe standard curve and should not be confused with the limit of detection and/or the low QCsample. The highest standard will define the upper limit of quantification (ULOQ) of ananalytical 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 itshould not deviate by more than ±20%. The precision around the mean value should notexceed 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 equallyacceptable.
• 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 ofreplicate sets of analyte samples of known concentrations C QC samples C from anequivalent biological matrix. At a minimum, three concentrations representing the entirerange 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 upperboundary 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 arestatistically 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 twoconcentrations in triplicate) should be studied.
• 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-basedmethods, 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 beinvestigated to ensure that precision, selectivity, and sensitivity will not be compromised. Method selectivity should be evaluated during method development and throughout methodvalidation 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 therange of the standards, if so desired. METHOD DEVELOPMENT: MICROBIOLOGICAL AND LIGAND-BINDING ASSAYS
Many of the bioanalytical validation parameters and principles discussed above are also applicable tomicrobiological and ligand-binding assays. However, these assays possess some unique characteristicsthat should be considered during method validation. A. Selectivity Issues
As with chromatographic methods, microbiological and ligand-binding assays should be shownto be selective for the analyte. The following recommendations for dealing with two selectivityissues should be considered:
1. Interference From Substances Physiochemically Similar to the Analyte
Cross-reactivity of metabolites, concomitant medications, or endogenouscompounds should be evaluated individually and in combination with the analyteof interest.
When possible, the immunoassay should be compared with a validated referencemethod (such as LC-MS) using incurred samples and predetermined criteria foragreement of accuracy of immunoassay and reference method.
The dilutional linearity to the reference standard should be assessed using study(incurred) samples.
Selectivity may be improved for some analytes by incorporation of separationsteps prior to immunoassay. 2. Matrix effects Unrelated to the Analyte
The standard curve in biological fluids should be compared with standard inbuffer to detect matrix effects.
Parallelism of diluted study samples should be evaluated with diluted standards todetect matrix effects.
Nonspecific binding should be determined. B. Quantification Issues
Microbiological and imunoassay standard curves are inherently nonlinear and, in general, moreconcentration points may be recommended to define the fit over the standard curve range thanfor chemical assays. In addition to their nonlinear characteristics, the response-errorrelationship 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 fittedto a 4- or 5-parameter logistic model, although others may be used with suitable validation. Theuse of anchoring points in the asymptotic high- and low-concentration ends of the standardcurve may improve the overall curve fit. Generally, these anchoring points will be atconcentrations 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 orin an alternate matrix of equivalent performance. Both ULOQ and LLOQ should be defined byacceptable 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 beimproved by the use of replicate samples. In the case where replicate samples should bemeasured during the validation to improve accuracy, the same procedure should be followed asfor 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
and reproducibility of recovery are (1) the use of radiolabeled tracer analyte (quantity toosmall to affect the assay), (2) the advance establishment of reproducible recovery, (3) theuse of an internal standard that is not recognized by the antibody but can be measured byanother 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 sameconcentration) may be outside 15% of nominal value. In certain situations, wideracceptance criteria may be justified.
• Assay reoptimization or validation may be important when there are changes in key
Performance should be verified with standard curve and QCs.
Key cross-reactivities should be checked.
Tracer experiments above should be repeated.
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. 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 forwhich stability data are available. In general, biological samples can be analyzed with a singledetermination without duplicate or replicate analysis if the assay method has acceptable variability asdefined by validation data. This is true for procedures where precision and accuracy variabilitiesroutinely fall within acceptable tolerance limits. For a difficult procedure with a labile analyte where highprecision and accuracy specifications may be difficult to achieve, duplicate or even triplicate analysescan be performed for a better estimate of analyte.
A calibration curve should be generated for each analyte to assay samples in each analytical run andshould be used to calculate the concentration of the analyte in the unknown samples in the run. Thespiked 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) abatch 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 acalibrator sample at LLOQ. Estimation of concentration in unknown samples by extrapolation ofstandard curves below LLOQ or above the highest standard is not recommended. Instead, thestandard curve should be redefined or samples with higher concentration should be diluted andreassayed. 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 bemonitored regularly to ensure that the method continues to perform satisfactorily. To achieve thisobjective, a number of QC samples prepared separately should be analyzed with processed testsamples at intervals based on the total number of samples. The QC samples in duplicate at threeconcentrations (one near the LLOQ (i.e., #3 x LLOQ), one in midrange, and one close to the high endof the range) should be incorporated in each assay run. The number of QC samples (in multiples ofthree) will depend on the total number of samples in the run. The results of the QC samples provide thebasis of accepting or rejecting the run. At least four of every six QC samples should be within "15% oftheir respective nominal value. Two of the six QC samples may be outside the "15% of their respectivenominal value, but not both at the same concentration.
The following recommendations should be noted in applying a bioanalytical method to routine druganalysis:
• 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 thestudy. Response function is determined by appropriate statistical tests based on the actualstandard points during each run in the validation. Changes in the response functionrelationship between prestudy validation and routine run validation indicate potentialproblems.
• The QC samples should be used to accept or reject the run. These QC samples are matrix
• System suitability: Based on the analyte and technique, a specific SOP (or sample) should
be identified to ensure optimum operation of the system used.
• 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 sampleanalysis. Reasons for repeat analyses could include repeat analysis of clinical or preclinicalsamples for regulatory purposes, inconsistent replicate analysis, samples outside of the assayrange, sample processing errors, equipment failure, poor chromatography, and inconsistentpharmacokinetic data. Reassays should be done in triplicate if sample volume allows. Therationale for the repeat analysis and the reporting of the repeat analysis should be clearlydocumented.
• 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 thereintegration is to be performed. The rationale for the reintegration should be clearlydescribed 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 matrixmay also be used, provided selectivity has been verified.
• Standard curve samples, blanks, QCs, and study samples can be arranged as considered
• Placement of standards and QC samples within a run should be designed to detect assay
• 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 itshould 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 andintra-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
QC), and one approaching the high end of the range (high QC)) should be incorporated intoeach 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 respectivenominal (theoretical) values; 33% of the QC samples (not all replicates at the sameconcentration) can be outside the ±15% of the nominal value. A confidence intervalapproach 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 ofunknown 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. DOCUMENTATION
The validity of an analytical method should be established and verified by laboratory studies, anddocumentation of successful completion of such studies should be provided in the assay validationreport. General and specific SOPs and good record keeping are an essential part of a validatedanalytical method. The data generated for bioanalytical method establishment and the QCs should bedocumented and available for data audit and inspection. Documentation for submission to the Agencyshould include (1) summary information, (2) method development and establishment, (3) bioanalyticalreports of the application of any methods to routine sample analysis, and (4) other informationapplicable to method development and establishment and/or to routine sample analysis. A. Summary Information
• 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 newmethod 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 codeshould 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,
and bioanalytical reports, especially when the sponsor and a contract laboratory assigndifferent 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
• 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, ifany), 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
C. Application to Routine Drug Analysis
Documentation of the application of validated bioanalytical methods to routine drug analysisshould include:
• Evidence of purity and identity of drug standards, metabolite standards, and internal
• Summary tables containing information on sample processing and storage. Tables should
include sample identification, collection dates, storage prior to shipment, information onshipment 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
stop times, duration, significant equipment and material changes, and any potential issues ordeviation from the established method.
• Equations used for back-calculation of results
• Tables of calibration curve data used in analyzing samples and calibration curve summary
• 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 foraccepting the analytical run. QC graphs and trend analyses in addition to raw data andsummary 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 analysisof any clinical samples.
• Documentation for repeat analyses. Documentation should include the initial and repeat
analysis results, the reported result, assay run identification, the reason for the repeatanalysis, the requestor of the repeat analysis, and the manager authorizing reanalysis. Repeat analysis of a clinical or preclinical sample should be performed only under apredefined SOP.
• Documentation for reintegrated data. Documentation should include the initial and repeat
integration results, the method used for reintegration, the reported result, assay runidentification, the reason for the reintegration, the requestor of the reintegration, and themanager authorizing reintegration. Reintegration of a clinical or preclinical sample should beperformed only under a predefined SOP.
• Deviations from the analysis protocol or SOP, with reasons and justifications for the
D. Other Information
Other information applicable to both method development and establishment and/or to routinesample 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 induplicate
• Sample code designations, including clinical or preclinical sample codes and
Assignment of clinical or preclinical samples to assay batches
Sample collection, processing, and storage
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). 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. Partial validation: Modification of validated bioanalytical methods that do not necessarily call for full revalidation. Cross-validation: Comparison validation parameters of two bioanalytical methods.
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