GUIDELINE ON VALIDATION OF BIOANALYTICAL METHODS

INTRODUCTION (background)
Measurement of drug concentrations in biological matrices is an important aspect of medicinal product development for those products containing new active substances as well as for line extensions and generic products. Such data may be required to support new applications as well as variations to authorised drug products. The results of toxicokinetic, pharmacokinetic and bioequivalence studies are used 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 used are well characterised, fully validated and documented to a satisfactory standard in order to yield reliable results.
Acceptance criteria wider than those defined in this guideline may need to be used in special situations, such as analysis of complex matrices (e.g. solid tissues), when usual acceptance criteria cannot be met. This should be justified and prospectively defined.
SCOPE
This guideline provides requirements for the validation of bioanalytical methods.
In addition, specific aspects of the bioanalytical method itself will be addressed, e.g. the actual analysis of samples from toxicokinetic studies and clinical trials.
Furthermore, this guideline will describe when partial validation or cross validation may represent an appropriate alternative approach to the complete validation of an analytical method.
Some special techniques such as radio-labelled analysis methods using 14C labelled drugs, are not covered here, but even in such cases efforts should be made to apply to the principles of this guideline.
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 as 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 should be performed in accordance with the principles of Good Laboratory Practice (GLP). However, as human bioanalytical studies fall outside of the scope of GLP, as defined in Directive 2004/10/EC, the sites conducting the human studies are not required to be monitored as part of a national GLP compliance programme. In addition, for clinical trials in humans the principles of Good Clinical Practice (GCP) should be followed.
Furthermore, reference is made to the following EMEA guidelines:
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Note for guidance on good clinical practices (CPMP/ICH/135/95).
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Note for guidance on validation of analytical procedures: text and methodology (CPMP/ICH/381/95).
METHOD VALIDATION
A complete method validation should be performed for any analytical method whether new or based upon literature.
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, plasma, urine, saliva or tissue. Moreover, validation should be performed using the same anticoagulant as for the study samples. A full validation should be performed for each species concerned.
In some cases, it may be problematic for validation purposes to obtain an identical matrix compared to the matrix of the study samples. A suitable alternative matrix may be used, e.g. synthetically prepared cerebrospinal fluid, if justified.
The main characteristics of a bioanalytical method that are essential to ensure the acceptability of the performance and the reliability of analytical results are: selectivity, lower limit of quantitation, the response function (calibration curve performance), accuracy, precision, matrix effects, stability of the analyte(s) and any internal standard in the biological matrix and the stock and working solutions 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 these cases the principles of validation and analysis apply to all analytes of interest.
During method validation, a blank biological matrix will be spiked with the analyte of interest using solutions of reference standard. In addition, an internal standard (IS) is normally used in chromatographic methods.
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 used for the analytical validation and analysis should be obtained from an authentic and traceable source. Suitable reference standards, include certified standards such as compendial standards (EPCRS, USP, WHO), commercial available standards, or fully characterised standards prepared in-house or by an external non-commercial organisation. Suitability of the reference standard should be scientifically justified. The use of certified standards is not needed for IS, as long as the suitability for use is demonstrated, e.g. lack of interference is shown for the substance itself or any impurities thereof.
Whoever the supplier, a certificate of analysis is required to ensure quality, stability, storage conditions, expiration date, batch number and purity of the reference standards.
When MS detection is used in the bioanalytical method, a stable isotope-labelled IS is recommended to be used whenever possible. However, it is essential that the labelled standard is of the highest isotope purity and that no isotope exchange reaction occurs. The presence of any unlabelled analyte would otherwise introduce a bias in the results.
The analytical method should be able to differentiate the analyte(s) of interest and IS from endogenous components in the matrix (i.e. blood, plasma, urine) or other components in the sample. Selectivity should be proven by using at least 6 sources of the appropriate blank matrix, which are individually analysed and evaluated for interference. Absence of interfering components is accepted where the response is less than 20% of the lower limit of quantitation for the analyte.
It may also be necessary to investigate the extent of any interference caused by metabolites of the drug(s), interference from degradation products formed during sample preparation, and interference from possible co-administered medications. Co-medications normally used in the subject population studied should be taken into account.
The possibility of back-conversion of a metabolite into parent analyte during the successive steps of the analysis (including extraction procedures) should also be evaluated, when relevant (e.g. acidic metabolites to ester, unstable N-oxides or glucuronide metabolites, lactone-ring structures). Preferably, blank matrix (and/or samples spiked with analyte at a concentration not higher than 3 times the lower limit of quantitation) should be spiked with concentrations of the metabolite of interest, representing the actual highest in vivo metabolite concentrations, the sample should be processed, and the chromatogram should be evaluated for the formation of the parent analyte. The extent of back-conversion should be established and the impact on the study results discussed. 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 the analytical method is revalidated as further knowledge regarding metabolism of the active substance is gained during drug development.
Carry-over should be addressed and minimised during method development. Carry-over may not affect accuracy and precision (see section 4.1.5 and 4.1.6). During validation carry-over should be assessed by injecting blank samples after a high concentration sample or calibration standard. If it appears that carry-over is unavoidable, specific measures should be considered, tested during the validation and applied during the analysis of the study samples. This could include the injection of blank samples after samples with an expected high concentration, before the analysis of the next study sample.
Randomisation of samples should be avoided, as this may interfere with the detection and assessment of carryover problems.
The lower limit of quantitation (LLOQ) is the lowest amount of analyte in a sample which can be quantified reliably, with an acceptable accuracy and precision (see Accuracy and Precision). The LLOQ should be adapted to expected concentrations and to the aim of the study.
The response of the instrument with regard to the analyte should be known, and should be evaluated over a specified concentration range. The concentrations to be analysed (calibration standards) should be prepared in the same matrix as the matrix of the intended study samples by spiking the blank matrix with known concentrations of the analyte (and IS). There should be one calibration curve for each analyte studied in the method validation and for each analytical run.
Before carrying out the validation of the analytical method it should be known what concentration range is expected. This range should be covered by the calibration curve range, defined by the LLOQ being the lowest calibration standard and the upper limit of quantitation (ULOQ), being the highest calibration standard. The range should be justified based on scientific information.
A minimum of six calibration concentration levels should be used, excluding the blank sample (processed matrix sample without analyte and without IS) and a zero sample (processed matrix with IS).
A relationship which can simply and adequately describe the response of the instrument with regard to the analyte should be applied. The blank and zero samples should not be taken into consideration to calculate the calibration curve parameters.
The calibration curve parameters should be submitted (slope and intercept in case of linear fit). In addition, the back calculated concentrations of the calibration standards should be presented together with the calculated mean accuracy values (see definition of Accuracy below). At least 3 calibration curves should be evaluated.
The back calculated concentrations of the calibration standards should be within ±15% of the nominal value, except for the LLOQ for which it should be within ±20%. At least 75% of the calibration standards with a minimum of six, must fulfil this criterion. In case a calibration standard does not comply with these criteria, this calibration standard sample should be rejected, and the calibration curve without this calibration standard should be re-evaluated, including regression analysis.
Although it may be clear from stability data that the analyte is sufficiently stable in the matrix of interest, it is recommended that freshly prepared calibration curves are used during validation of the bioanalytical method.
The accuracy of an analytical method describes the closeness of the determined value obtained by the method to the true concentration of the analyte (expressed in percentage). Accuracy should be assessed on samples spiked with known amounts of the analyte, the quality control samples (QC samples).
During method validation accuracy should be determined by replicate analysis using a minimum of 5 determinations at a minimum of 4 concentration levels which are covering the calibration curve range: the LLOQ, within three times the LLOQ (low QC), around 50% of the calibration curve range (medium QC), and at about 75% of the upper calibration curve range (high QC).
The QC samples are analysed against the calibration curve, and the obtained concentrations are compared with the nominal value. The accuracy should be reported as percent of the nominal value. Accuracy should be evaluated for the values of the QC samples obtained within a single run (the within run accuracy) and in different runs (the between-run accuracy). The latter will support the accuracy over time.
To enable evaluation of any trends over time within one run, it is recommended to demonstrate accuracy of QC samples over at least one of the runs with a size equivalent to a prospective analytical run.
For the validation of the within-run accuracy, there should be a minimum of five samples per concentration level at LLOQ, low, medium and high QC samples in a single run. The mean accuracy value should be within 15% of the nominal values for the QC samples, except for the LLOQ which should be within 20% of the nominal value.
For the validation of the between-run accuracy at least five determinations per concentration per run at LLOQ, low, medium and high QC samples from three runs analysed on at least two different days should be evaluated. The mean accuracy value should be within 15% of the nominal values for the QC samples, except for the LLOQ which should be within 20% of the nominal value.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 should additionally be reported.
Stability
Evaluation of the stability should be carried out to ensure that every step taken during sample preparation and sample analysis, as well as the storage conditions used do not affect the concentration of the analyte. Any deviation from the initial concentration that does occur must be within acceptable limits.
Stability should be ensured for every step in the analytical method, meaning that the conditions applied to the stability tests, such as sample matrix, materials storage and analytical conditions should be similar to those used for the actual study samples. Stability cannot be proven by literature data.
Stability of the analyte and IS in the studied matrix is evaluated using at least triplicates samples of the low and high QC samples which are analysed immediately after preparation and after the applied storage conditions that are to be evaluated. The QC samples are analysed against a calibration curve, obtained from freshly prepared calibration standards, and the obtained concentrations are compared to the nominal concentrations. The deviation should be within ±15%.
Stability of the stock and working solutions should be tested with an appropriate dilution, taking into consideration the linearity and measuring range of the detector.
Stability studies should investigate different storage conditions over time periods that equal or exceed those applied to the actual study samples.
Normally, as an example, the following stability tests should be evaluated:
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stock solution and working solution stability,
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freeze and thaw stability of the analyte in the matrix from freezer storage conditions to room temperature,
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stability of the analyte in matrix stored in the refrigerator, if applicable,
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bench top stability of the analyte in matrix at room temperature,
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long term stability of the analyte in matrix stored in the freezer at the same storage temperature as the study samples,
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bench top stability of the processed sample at room temperature or under the storage conditions to be used during the study (dry extract or in the injection phase), if applicable,
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on-instrument/ autosampler stability of the processed sample at injector or autosampler temperature.
Regarding the freeze and thaw stability: The QC samples are stored and frozen in the freezer at the intended temperature and thereafter thawed at room temperature. After thawing, samples are refrozen again applying the same conditions. At each cycle, samples should be frozen for at least 12 hoursbefore they are thawed. The number of cycles in the freeze-thaw stability should equal or exceed that of the freeze/thaw cycles of study samples.
Regarding long term stability of the analyte in matrix stored in the freezer: The QC samples should be stored in the freezer under the same storage conditions and at least for the same duration as the study samples. For the evaluation of the long term stability it is not acceptable to use study samples, as the nominal concentration is unknown, and can therefore not be used as reference. It is recommended that evaluation of long term stability is carried out before the start of the actual study.
Sufficient attention should be paid to the stability of the analyte in the sampled matrix directly after blood sampling of subjects and further preparation before storage, to ensure that the obtained concentrations by the analytical method reflect the concentrations of the analyte in the subject at the moment of sampling..
In situation where minor changes are made to an analytical method that has already been validated, a full validation may not be necessary, depending onto the nature of the applied changes. Changes for which a partial validation may be needed include transfer of the bioanalytical method to another laboratory, change in equipment, calibration concentration range, storage conditions etc. All modifications should be reported and the scope of revalidation or partial validation justified.
In most cases, provision of additional accuracy and precision data or relevant additional stability data on the modified issue may be sufficient.
ANALYSIS OF STUDY SAMPLES
After complete validation of the analytical method, analysis of study or subject samples may be carried out. Depending on the time period between validation and the analysis of the study samples it may be necessary to verify the performance of the method before start of the analysis of study samples.The study samples should be processed in accordance with the validated analytical method, and together with the QC samples and the calibration curve to ensure the acceptability of the analytical run.An analytical run consists of the blank sample (processed matrix sample without analyte and without IS) and a zero sample (processed matrix with IS), a set of calibration standards at a minimum of 6 concentration levels, at least 3 levels of QC samples (low, medium and high) in duplicate (or at least 5 % of the number of study samples, whichever is higher), and study samples to be analysed. All samples (calibration standards, QC, and study samples) should be processed and extracted as one single batch of samples. Analysing as a single analytical run samples prepared separately as several batches should be avoided. If such an approach cannot be avoided, for instance due to bench-top stability limitations, each batch of samples should preferably include low, medium and high QC samples. Acceptance criteria should be pre-established in a Standard Operating Procedure (SOP) or in the study plan and should be defined both globally for the full analytical run, and for each individual batch of samples.
It is advised to analyse all samples of one subject together in one analytical run to reduce the variability in outcome. Moreover, it is considered acceptable to analyse study samples of more than one subject in one analytical run. The QC samples should be divided over the run i.e. at the beginning, middle and at the end of the run. It is also acceptable to place one of each low, medium and high QC samples at the beginning of the analytical run followed by the study samples and at the end of the run again one of each low, medium and high QC samples.Criteria for acceptance or rejection of an analytical run should be defined in the analytical study protocol, in the study plan or in a SOP. The following acceptance criteria should apply:
Accuracy:
The back calculated concentrations of the calibration standards should be within ±15% of the nominal value, except for the LLOQ for which it should be within ±20%. At least 75% of the calibration standards with a minimum of six, must fulfil this criterion. If one of the calibration standards does not meet these criteria, this calibration standard sample should be rejected and the calibration curve without this calibration standard should be re-evaluated, and regression analysis performed. Criteria for decision to exclude calibration standards or not should be pre-defined in a SOP, and should be independent from the results of the QC samples.
If the rejected calibration standard is the LLOQ, it should be realised that the LLOQ for this analytical run is the next acceptable higher calibration standard concentration of the calibration curve. If the highest calibration standard is rejected, the ULOQ, for this analytical run is the concentration of the next acceptable lower calibration standard of the calibration curve. The revised calibration range must cover all QC samples (low, medium and high).
The accuracy values of the QC samples should be within ±15% of the nominal values. At least 4 out of 6 (67%) QC samples and at least 50% at each concentration level should comply with this criterion. In case these criteria are not fulfilled the analytical run should be rejected, and the study samples reanalysed.
The between-run (mean) accuracy of the QC samples should be within 15% of the nominal value.
In the case of the simultaneous determination of several analytes, if an analytical run is acceptable for one analyte but has to be rejected for another analyte, the data for the accepted analyte can be used, but the samples should be re-analysed for determination of the rejected analyte.Precision:
The between-run precision should not exceed 15%.
Ligand-binding assays:
The back calculated concentrations of the calibration standards should be within ±20% of the nominal value, except for the LLOQ and the ULOQ for which it should be within ±25%. At least 75% of the calibration standards with a minimum of six, must fulfil this criterion. This requirement does not apply to ‘anchor’ calibrators.
The accuracy values of the QC samples should be within ±20% of the nominal values. At least 4 out of 6 (67%) QC samples and at least 50% at each concentration level should apply to this criterion. Exceptions to this criterion should be justified.If a large number of the analyte concentrations of the study samples appear to be above the ULOQ, the calibration curve range should be extended, if possible. The same applies if it appears that the calibration curve range is too wide, such that most analyte concentrations fall in the lower part of the calibration curve range. The calibration curve range should then be narrowed, or an additional QC sample level should be included so that at least 2 QC sample levels fall within the range of concentrations measured in study samples. If the calibration curve range is extended, the analytical method should be revalidated to ensure accuracy and precision.