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Bioanalytical Method Validation: Align with FDA to ICH guidelines

Method validation is a process that is used to confirm whether the analytical procedure used for drug analysis is suitable for its intended use. Analytical method validation is the key to judging the quality, consistency, and reliability of sample analysis data. For most healthcare regulators, including the FDA, method validation is a mandatory step in preclinical and clinical studies for pharmacokinetic and toxicokinetic evaluation to fulfill specific performance criteria.

Bioanalytical Method validation takes place in the following cases:

  • Once a method has been developed, but before it has been introduced into routine use.
  • When the method that was developed gets changed to the extent that’s outside the original scope of the method.
  • Whenever there is a change in conditions for which the method has already been validated.
  • If samples are analyzed in more than one laboratory, cross-validation is needed to ensure consistency.

When it comes to test method validation, it’s important to follow standard guidelines like FDA bioanalytical method validation guidance for industry. If you have your own protocols, that’s great, if not, you need to develop a method validation plan. A good method validation process will test several parameters such as calibration range, linearity, and accuracy and precision, robustness, specificity, and process stability of the analyte. Good Documentation Practice (GDP) should be in place to ensure that a full description of the method is captured in enough details to be followed by an independent analyst later.

Bioanalytical Method Validation: Parameters and Acceptance Criteria

Reference Standard(s) and Internal Standard (IS) Purity

Reference standards used in the preparation of calibration and Quality Control Samples must have a Certificate of Analysis (CoA) indicating the purity and expiration date. A CoA is not required for the internal standard, but the material must be checked for impurities that may interfere with the analyte(s).

Matrix

Whenever possible, the method validation should be done in the matrices for which the assay is intended to be used. If unavailable, then evidence must be provided to demonstrate that a substitute is acceptable.

System Suitability Sample (SST)

  • A system suitability sample (SST) must be developed during method validation. In general, the system suitability sample is a combination of all the expected analytes in the assay prepared in a suitable solvent system.
  • Analyte(s) must be at a concentration that corresponds to the low end of the calibration curve but high enough to give a robust and reproducible signal.
  • The strength and the composition of the SST solution must be documented along with a representative chromatogram generated using the SST.
  • The representative chromatogram should be used as the baseline for checking if an analytical system is suitable for conducting the validated assay. The parameters to be used for system evaluation are retention time and response.

Maximum Solvent Concentration of Standards

  • When preparing standards and QCs in a matrix such as plasma, urine, etc., the maximum amount solvent, from a stock standard(s) containing the analyte(s), should not exceed a certain percentage of the total volume.
  • This can be calculated using the equation below:

% Stock Standard = spiking amount (ml) / [matrix amount (ml) + spiking amount (ml) *100

Calibration Curves: Requirements

  • Calibration standards should be prepared in the matrix same as the study samples.
  • Calibration curve should consist of a minimum of six concentration levels analyzed in singlet or duplicate.
  • One matrix blank (Control) and one zero sample (i.e., matrix blank with IS, Control IS) should be included in each validation run.

Calibration Curves: Acceptance Criteria

  • The back-calculated concentrations of the individual standards must be within accepted deviation from the nominal value.
  • Deletion of calibrators should be done one at a time in a sequential manner beginning with the most deviant calibrator.
  • At least 3/4th of the original calibration standards are mandatory to meet the accuracy requirements.
  • The quantitation range of the study samples should not be affected by these changes. At least one standard calibrator at the LLOQ and ULQ must be within specification.
  • If the quantitation range is modified, three quality control levels within the adjusted calibration range should fulfill the accuracy requirements.

Quality Control Samples

  • Quality control samples should be prepared at a minimum of four concentration levels (QC_LLOQ, QC_Low, QC_Mid, and QC_High).
  • An additional QC level QC_Dil must be included in at least one of the runs to test for dilution integrity.
  • Six replicates of each QC concentration levels should be analyzed in each validation run.
  • Acceptance criteria for QC: The individual back-calculated concentration of each QC sample for the three in-range QC levels must be within accepted deviations from nominal.

Intra-day Accuracy and Precision

  • Accuracy and precision should be calculated using the mean concentration results of the QC samples at each level. Precision should be based on the percent coefficient of variation (%CV) observed by the mean at each quality control concentration level.
  • Dixon test for the outliers: Dixon test is applied to the percent accuracy determined for the QCs based on the theoretical and measured concentrations of the QCs from the standard curve.
  • Outliers may be omitted from the precision and accuracy calculation after performing the Dixon test.

Inter-day Accuracy and Precision

The inter-day accuracy and precision should be calculated using the data from three one-day validation runs. Precision is based on the percent coefficient of variation (%CV) observed by the mean of the QC samples at each QC concentration level.

Specificity

The developed assay must be specific for the analyte(s) and internal standard in the blank matrix. At least six independent matrix blanks must be analyzed to show that no interferences are arising from different lots of plasma.

Matrix Effect on Accuracy and Precision (6 independent lots)

The ability of the assay to accurately and reproducibly quantitate the analyte(s) in different lots of the matrix is assessed by spiking six independent lots to a concentration equivalent to the QC_Low concentration.

Matrix Suppression

The degree to which the signal is suppressed by the extracted components of the matrix is assessed by injecting solutions of the analyte prepared in reconstituted blank matrix extracts and comparing the signal to the analyte prepared in the mobile phase or reconstitution solution. Values are reported as percent suppression. In some instances, signal enhancement is observed.

Extraction Recovery of Analyte(s) and Internal Standards

  • Recovery of the analyte(s) is calculated using the in-range quality control samples at three different concentration levels. The study compares the responses of the analyte(s) and IS from the validation QCs to the responses in non-extracted samples prepared in mobile phase/reconstitution solvent.
  • The percent recovery of the analyte is calculated at each concentration level by dividing the mean response of the analyte (minimum of three determinations) in the matrix sample by the mean response of the analyte at that concentration in the non-extracted samples.
  • The percent recovery of the internal standard is calculated by dividing the overall mean response of the internal standard in the matrix samples by the overall mean response of the internal standard in the non-extracted samples.
  • Recovery studies for stable-labeled internal standards are not required, since, by definition, it is chemically like the analyte.

Carryover

  • During method validation runs, a double-blank sample should be injected after the highest calibration standard to determine if the equipment is cross-contaminating the sample during loading.
  • Peak response for the analyte(s) due to carryover must not be more than a specified % of the response of the lowest calibration standard (LLOQ). For internal standard again, the response must not be more than a determined % of the average internal standard response.

Limit of Quantitation

The lower limit of quantitation (LLOQ) indicates the lowest concentration of study samples that can be quantitated with acceptable accuracy. The LLOQ is the lowest calibration standard used to generate a calibration curve.

Stability of Analyte(s)

Stability testing should include process stability, as well as short term and long-term stability of analytes in solution and in the matrix.

Lipemic and Hemolytic Plasma Samples

High and low concentrations of QCs should be used to prepare 6 replicates with lipemic plasma (in mg/dL of lipids). Similarly, 6 replicate samples with hemolytic plasma (based on mg/dL of heme) should be analyzed.

Batch Run Size

Batch run size depends on a variety of factors. Consideration during LC-MS analysis includes autosampler stability, run time, column stability, MS stability, etc. Here, the batch size is determined by repeat injection of blanks with QCs at regular intervals during a validation run.

Bioanalytical Method Validation Services by NorthEast BioLab

NorthEast BioLab test method validation services are extensive and adapt to the different phases of your drug development journey. Our method validation services include but are not limited to:

Full Validation of Newly Developed Methods

We are adept at validation of newly developed methods. This entails completion of full three-day validation as per regulatory guidance (FDA, ICH) on bioanalytical method validation. All experiments for the complete validation are performed, such as determination of method specificity, matrix effect and matrix suppression, extraction recovery, and carryover, etc. Analyte stability in the biological matrix is determined during short-term bench top, autosampler, and freeze-thaw stages.

Partial Validation

Partial validations evaluate previously validated methods with recommended modifications. The validations can be as small as one intra-assay precision determination or as large as a full validation. Raw data on partial validation is sometimes requested during inspections and is retained at the analytical site.

Cross-Validation

Cross-validation compares validation parameters for different bioanalytical techniques either within the same study or across various studies. Cross-validation is mandatory for regulated sample analyses conducted at two or more analytical sites.

Previously Validated Methods

We also offer clients methods for non-proprietary compounds that have already been validated. Of course, we ensure that the approach we recommend and utilize is appropriate for their drug development stage. This helps to accelerate the drug development timeline and optimize client resources.

Re-validation of the Method

In some cases, a re-validation of the method may become necessary. This includes situations where new compounds are analyzed, the sample matrix changes, or the method itself needs update based on regulatory guidelines.

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Achieve Bioanalytical Method Validation expectations by FDA and ICH on time and under budget

Given that drug development is an enormously resource-intensive process, finding the right Contract Research Organization can help you optimize costs and save time. At NorthEast BioLab, we have over 15 years of experience in analytical method development, validation, and transfer. We make sure that we accomplish our shared goals while pursuing an accelerated timeline and cost optimization.

For method validation, we offer a whole range of stringency levels so that the client can choose what they need. We generate an extremely comprehensive validation report that details the method being validated, as well as its outcomes. We also ensure that we report on the protocol used, calculations made, procedures followed, and equipment used.

We understand that collaboration lies at the heart of this process and ensure proper communication between our clients and our front-line lab analysts and managers. We also believe in integrity and transparency and keep our clients in the loop about all research developments. We bring together our core strengths, operational excellence, regulatory expertise, and scientific experience to make sure that your drug development process- from drug discovery to clinical trials- flows seamlessly.

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