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21 July 2011
EMEA/CHMP/EWP/192217/2009 Committee for Medicinal Products for Human U (CHMP)
Guideline on bioanalytical method validation
Draft agreed by the Efficacy Working Party
September 2009 Adoption by CHMP for relea for consultation 19 November 2009 End of consultation (deadline for comments)
31 May 2010 Agreed by Pharmacokinetics Working Party (PKWP) June 2011 Adoption by CHMP
21 July 2011 Date for coming into effect
1 February 2012
Comments should be provided using this template . The completed comments form should be nt to PKWPcretariat@ema.europa.eu .
Keywords CHMP, EMEA, Guideline, validation, bioanalytical method, analys
Guideline on bioanalytical method validation
Table of contents
1. Introduction (background) (3)
2. Scope (3)
3. Legal basis (3)
4. Method validation (4)
4.1. Full validation of an analytical method (4)
4.1.1. Selectivity (5)
4.1.2. Carry-over (5)
4.1.3. Lower limit of quantification (6)
4.1.4. Calibration curve (6)
4.1.5. Accuracy (7)
4.1.6. Precision (7)
4.1.7. Dilution integrity (8)
4.1.8. Matrix effect (8)
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4.1.9. Stability (9)
4.2. Partial validation (10)
4.3. Cross validation (10)
5. Analysis of study samples (10)
5.1. Analytical run (11)
5.2. Acceptance criteria of an analytical run (11)
5.3. Calibration range (12)
5.4. Reanalysis of study samples (12)
5.5. Integration (13)
6. Incurred samples reanalysis (13)
7. Ligand binding assays (14)
7.1.1. Full validation (14)
7.2. Partial validation and cross-validation (17)
7.3. Analysis of study samples (17)
7.3.1. Analytical run (17)
7.3.2. .Acceptance criteria for study sample analysis (17)
7.3.3. Incurred samples reanalysis (18)
8. Reports (18)
楼开头的成语8.1. Validation report (18)
8.2. Analytical report (19)
发音方法Definitions (20)
分数混合运算题Executive summary
This guideline defines key elements necessary for the validation of bioanalytical methods. The guideline focus on the validation of the bioanalytical methods generating quantitative concentration data ud for pharmacokinetic and toxicokinetic parameter determinations. Guidance and criteria are given on the application of the validated methods in the routine analysis of study samples from animal and human studies.
1. Introduction (background)
Measurement of drug concentrations in biological matrices (such as rum, plasma, blood, urine, and saliva) is an important aspect of medicinal product development. Such data may be required to support applications for new actives substances and generics as well as variations to authorid drug products. The results of animal toxicokinetic studies and of clinical trials, including bioequivalence studies are ud to make critical decisions supporting the safety and efficacy of a medicinal drug substance or product. It is therefore paramount that the applied bioanalytical methods ud are well characterid, fully validated and documented to a satisfactory standard in order to yield reliable results.
Acceptance criteria wider than tho defined in this guideline may be ud in special situations. This should be prospectively defined bad on the intended u of the method.
2. Scope
This guideline provides recommendations for the validation of bioanalytical methods applied to measure drug concentrations in biological matrices obtained in animal toxicokinetic studies and all phas of clinical trials. As ligand binding assays differ substantially from chromatographic analytical methods, parate validation recommendations for ligand binding assays are provided.
In addition, specific aspects for the analysis of study samples will be addresd.
Furthermore, this guideline will describe when partial validation or cross validation should be carried out in addition to the full validation of an analytical method.
Methods ud for determining quantitative concentrations of biomarkers ud in asssing pharmacodynamic endpoints are out of the scope of this guideline.
3. Legal basis
This guideline has to be read in conjunction with the introduction and general principles (4) and Part I and II of the Annex I to Directive 2001/83 as amended. It applies to Marketing Authorisation Applications for human medicinal products submitted in accordance with the Directive 2001/83/EC a
s amended, and Regulation (EC) No. 726/2004, in which the analysis of drug concentrations in a biological matrix is part of the application.
The validation of bioanalytical methods and the analysis of study samples for clinical trials in humans should be performed following the principles of Good Clinical Practice (GCP). Further guidance that will help clinical laboratories develop and maintain quality systems which will comply with relevant European Union Directives, national regulations and associated guidance documents can be found in the “Reflection Paper for Laboratories That Perform The Analysis Or Evaluation Of Clinical Trial Samples.” (EMA/INS/GCP/532137/2010).
Non-clinical (pharmaco-toxicological) studies submitted in a marketing authorisation application shall be carried out in conformity with the provisions related to Good Laboratory Practice, Directive
2004/10/EC on the harmonisation of laws, regulations and administrative provisions relating to the application of the principles of good laboratory practice and the verification of their applications for tests on chemical substances and Directive 2004/9/EC on the inspection and verification of good laboratory practice (GLP). Normally, the validation of bioanalytical methods ud in non-clinical pharmacotoxicological studies that are carried out in conformity with the provisions related to Good L
aboratory Practice should be performed following the Principles of Good Laboratory Practice. Aspects of method validation not performed according to GLP should be clearly identified and their potential impact on the validation status of the method indicated. Methods ud in pre-clinical studies not required to be performed to GLP should be fit for purpo but not necessarily developed in a GLP facility.
4. Method validation
4.1. Full validation of an analytical method
A full method validation should be performed for any analytical method whether new or bad upon literature.
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The main objective of method validation is to demonstrate the reliability of a particular method for the determination of an analyte concentration in a specific biological matrix, such as blood, rum, plasma, urine, or saliva. Moreover, if an anticoagulant is ud, validation should be performed using the same anticoagulant as for the study samples. Generally a full validation should be performed for each species and matrix concerned.
In some cas, it may be problematic for validation purpos to obtain an identical matrix compared to the matrix of the study samples. A suitable alternative matrix may be ud, e.g. synthetically prepared cerebrospinal fluid, if justified.
The main characteristics of a bioanalytical method that are esntial to ensure the acceptability of the performance and the reliability of analytical results are: lectivity, lower limit of quantification, the respon function and calibration range (calibration curve performance), accuracy, precision, matrix effects, stability of the analyte(s) in the biological matrix and stability of the analyte(s) and of the internal standard in the stock and working solutions and in extracts under the entire period of storage and processing conditions.
Usually one analyte or drug has to be determined, but on occasions it may be appropriate to measure more than one analyte. This may involve two different drugs, but can also involve a parent drug with its metabolites, or the enantiomers or isomers of a drug. In the cas the principles of validation and analysis apply to all analytes of interest.
Reference standards
During method validation and analysis of study samples, a blank biological matrix will be spiked with
the analyte(s) of interest using solutions of reference standard(s) to prepare calibration standards, quality control samples and stability samples. In addition, suitable internal standard(s) (IS) can be added during sample processing in chromatographic methods.
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It is important that the quality of the reference standard and IS is ensured, as the quality (purity) may affect the outcome of the analysis, and therefore the outcome of the study data. Therefore the reference standards ud during the validation and study sample analysis should be obtained from an authentic and traceable source. Suitable reference standards, include certified standards such as
compendial standards (EPCRS, USP, WHO), commercially available standards, or sufficiently characterid standards prepared in-hou or by an external non-commercial organisation. A certificate of analysis is required to ensure purity and provide information on storage conditions, expiration date and batch number of the reference standard.
The u of certified standards is not needed for IS, as long as the suitability for u is demonstrated, e.g. lack of analytical interference is shown for the substance itlf or any impurities thereof. A certificate of analysis is not required.
When mass-spectrometry (MS) detection is ud in the bioanalytical method, a stable isotope-labelle
d IS is recommended to be ud whenever possible. However, it is esntial that the labelled standard is of the highest isotope purity and that no isotope exchange reaction occurs. The prence of any unlabelled analyte should be checked and if relative amounts of unlabelled analyte are detected the potential influence has to be evaluated during method validation.
4.1.1. Selectivity
The analytical method should be able to differentiate the analyte(s) of interest and IS from endogenous components in the matrix or other components in the sample. Selectivity should be proved using at least 6 individual sources of the appropriate blank matrix, which are individually analyd and evaluated for interference. U of fewer sources is acceptable in ca of rare matrices. Normally, abnce of interfering components is accepted where the respon is less than 20% of the lower limit of quantification for the analyte and 5% for the internal standard.
It may also be necessary to investigate the extent of any interference caud by metabolites of the drug(s), interference from degradation products formed during sample preparation, and interference from possible co-administered medications. Co-medications normally ud in the subject population studied which may potentially interfere should be taken into account at the stage of method validation, or on a study specific and compound specific ba.
The possibility of back-conversion of a metabolite into parent analyte during the successive steps of the analysis (including extraction procedures or in the MS source) should also be evaluated, when relevant (i.e. potentially unstable acidic metabolites to ester, unstable N-oxides or glucuronide metabolites, lactone-ring structures). The extent of back-conversion should be established and the impact on the study results discusd. It is acknowledged that this evaluation will not be possible early during drug development of a new chemical entity when the metabolism is not yet evaluated. However, it is expected that this issue is taken into account and a partial validation is performed if relevant as further knowledge regarding metabolism of the active substance is gained during drug development.
It is recognized that in some cas it is very difficult to obtain the metabolites of interest. Alternatively, back-conversion of a metabolite can be checked by applying incurred sample reanalysis. However, in this ca potential back conversion during sample processing cannot be ruled out.
4.1.2. Carry-over
Carry-over should be addresd and minimid during method development. During validation carry-磕绊
over should be assd by injecting blank samples after a high concentration sample or calibration standard at the upper limit of quantification. Carry over in the blank sample following the high concentration standard should not be greater than 20% of the lower limit of quantification (LLOQ; e below) and 5% for the internal standard. If it appears that carry-over is unavoidable, study samples should not be randomid. Specific measures should be considered, tested during the validation and
