Science & Research

Volume II - Methods, Method Verification and Validation ORA-LAB.5.4.5

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Orientation and Training
Food and Drug Administration



Section 2 - Microbiology



Sections Included in this Document and Change History

  1. Purpose
  2. Scope
  3. Responsibilities
  4. Background
  5. References
  6. Procedure
  7. Definitions
  8. Records
  9. Supporting Documents
  10. Attachments (Attachment A-revised)
  11. Appendices
  12. Document History

1. Purpose

This document establishes the guidelines for method selection and the procedures for verification of standard method performance, as well as the validation of non-standard methods.

2. Scope

ORA laboratories verify standard method performance and validate non-standard methods introduced into the laboratory.

3. Responsibilities

  1. Directors:
    • ensures implementation of method verification and validation procedures.
  2. Supervisors:
    • implements method verification and validation procedures in respective division.
  3. Staff:
    • adheres to written protocol for method performance verification, validation or modification.

4. Background


5. References

A Laboratory Guide to Method Validation, (Eurachem).

6. Procedure

6.1 Method Selection

  1. Selection of Methods
    1. An integral part of the laboratory quality system is the use of standard methods. Standard methods are used, whenever possible, or unless otherwise specified by the Compliance Program or the customer.
    2. Non-standard methods are used in cases where a standard method does not exist and the customer has agreed to its use. A clear expression of quality objectives and testing parameters or criteria are made when a non-standard method is employed.

6.2 Standard Method Verification

  1. Standard or FDA official methods need verification to ensure that the laboratory is capable of performing the analysis. The laboratory’s quality control program as described in ORA-LAB.5.9 addresses this need. Verification of an analytical procedure is the demonstration that a laboratory is capable of replicating with an acceptable level of performance a standard method. Verification under conditions of use is demonstrated by meeting system suitability specifications established for the method, as well as a demonstration of accuracy and precision or other method parameters for the type of method. Method performance is accomplished by using performance characteristics such as:
    • blanks in chemistry, or un-inoculated media in microbiology, to assess contamination;
    • laboratory control samples - spiked samples for chemistry or positive culture controls for microbiology, to assess accuracy;
    • precision based on the analysis of duplicates;
    • calibration check standards analyzed periodically in the analytical batch for quantitative analyses; and
    • monitoring quality control samples, usually through the use of control charts.
  2. Verification of Standard Method Performance is defined for two situations, (1) for verifying method performance with each analytical batch (EAB) and (2) the first use of a standard method within the laboratory.
    1. Verifying method performance with each analytical batch (EAB) as a continuing check includes the following quality controls:
      1. General Chemistry
        All of these are required, even if not specified in the method. If an equivalent control is included in the method, it should be performed as specified. End the batch or run sequence with a QC sample, i.e. CCV.
        1. Meet system suitability requirements
          System suitability is intended to determine whether the ‘system’ including instruments, analysts, etc. is capable of performing a particular process, test, or assay.
        2. Analyze a blank, i.e. reagent, matrix to assess contamination from the laboratory environment and demonstrate low system background. The blank must be less than the determined MDL.
        3. Analyze matrix spike and/or reagent spike (match matrix and analyte) to assess and demonstrate accuracy.
        4. The accuracy expressed as percent recovery must be 80-120% unless otherwise specified, i.e. by in-house statistical analysis.
        5. Analyze duplicate matrix/reagent spike or sample duplicate to assess and demonstrate precision.
        6. The precision expressed as relative percent difference (RPD) must be < 20% unless otherwise specified, i.e. in-house statistical analysis. In procedures, where multiple determinations or subs are analyzed, one can be chosen as the duplicate quality control sample.
        7. Analyze calibration check standards/samples. Each Initial Calibration Verification (ICV) and Continuing Calibration Verification (CCV) must have a percent recovery of 90-110% unless otherwise specified, i.e. by in-house statistical analysis.
      2. Drug chemistry
        Meet the above requirements. Refer to appendix 1. Drugs must meet USP system suitability requirements when running a USP method. Requirements are stated in USP monographs.
      3. Microbiology
        1. Prepare and analyze a positive culture control to assess and demonstrate accuracy.
          Accuracy must be ≥ 95%.
          A positive culture control must exhibit positive growth or exhibit expected characteristics to assure the system is working. For example, turbidity in a tube filled with enrichment broth showing growth or a physical (phenotypic) change to the bacterial culture showing a positive test result.
        2. Include all other controls as required by ORA-Lab.1 (Microbiological Controls for Sample Analysis).
      4. Other scientific disciplines (e.g. radionuclides, microscopy, metrology, etc.). The following performance parameters should be included, if applicable:
        1. Blanks to assess contamination.
        2. Laboratory control samples to assess accuracy.
        3. Mechanism to assess precision;
        4. If applicable, calibration check standards analyzed periodically in the analytical batch for quantitative analyses; and
        5. Monitoring quality control samples, usually through the use of control charts.
    2. First use of standard method, an initial demonstration of acceptable laboratory capability to perform the method shall include:
      (NOTE: Can be run concurrently with samples. End batch or run sequence with a QC sample, i.e. CCV).
      1. General Chemistry
        1. Meet method system suitability requirements, if applicable.
        2. Meet all requirements in section 6.2.B.1.a. General Chemistry.
        3. Investigate the method detection limit, MDL.
          If a MDL is stated in the method, verify with one prepared sample at the stated concentration. The result should be within ±20% of the known value.

          If a MDL is not provided in the method, run three preparations at a concentration approximately one-third to one-half of the lowest calibrator of a standard curve or at a concentration of at least 20% below target level for a one point calibration.

          The relative standard deviation, RSD, will be 20% if the concentration chosen is at or above the true MDL.

        4. Determine linear calibration range or target level.

          As a general rule for a curve, the mid-point is set at the target level with the lowest calibrator at one-half this concentration and the highest calibrator at twice this concentration. The coefficient of determination (r2) for a calibration curve must be ≥ 0.995. For a one point calibration, the range is the target level, i.e. 100%.

        5. Verify method linearity at a level above the highest calibrator or above the target level.

          For a standard curve, analyze a spike at approximately 1.5 to 3 times the highest calibrator. For a one point calibration, analyze a spike at concentration at least 20% higher than the target level.

          Calculate percent recovery. The result should be within ±20% of the known value for acceptable method performance.

        6. If available, prepare and analyze a Quality Control Sample/Reference Sample.

          The concentration must be within ±10% of stated or known value. This check is intended as an independent check of technique, methodology and standards.

      2. Drug chemistry
        Meet the above requirements. Refer to appendix 1. Drugs must meet USP system suitability requirements when running a USP method. Requirements are stated in USP monographs.
      3. Qualitative Chemistry Methods
        1. Meet system suitability requirements, if applicable.
        2. Establish a detection limit, i.e. LOD. The detection limit is the lowest level of analyte that gives a positive response (e.g. the lowest standard visible on a TLC plate).
        3. Analyze a blank. The result must be negative.
        4. Analyze a positive control, i.e. standard.
        5. Analyze a matrix spike. The matrix or sample should be spiked at the expected analyte concentration and the result must be positive.
        6. Analyze a duplicate matrix spike or sample duplicate. Result must be positive.
        7. Analyze a quality control sample or reference material, if available. The results must be positive.
      4. Microbiology
        1. Meet requirements in section 6.2.B.1.c. Microbiology.
        2. Include all other controls as required by ORA-Lab.1 (Microbiological Controls for Sample Analysis).
        3. Sample duplicates are not required as precision is calculated by the number of false negatives or false positives. Precision must be ≤ 5%.
      5. Other Scientific Disciplines
        Laboratories will address, at their local level, method verification of scientific disciplines not discussed in this procedure. At a minimum, scientific disciplines must meet the requirements to demonstrate lack of contamination, accuracy, precision, detection limits, quantitation limits, and linearity as applicable.

6.3 Method Validation

  1. Validation of Method Performance
    1. Non-standard and laboratory-developed methods need method validation. This activity is planned and assigned to qualified personnel. The method’s performance characteristics are based on the intended use of the method. For example, if the method will be used for qualitative analysis, there is no need to test and validate the method’s linearity over the full dynamic range of the equipment.
    2. Typical validation characteristics which should be considered are:
      • accuracy,
      • precision,
      • specificity,
      • detection limit,
      • limit of quantitation,
      • linearity,
      • range, and
      • ruggedness and robustness.
      See Section 7 for definitions of these characteristics.
  2. Validation Tools
    The following tools can be used to demonstrate the ability to meet method specifications of performance:
    1. Blanks: Use of various types of blanks enables assessment of how much is attributable to the analyte and how much is attributable to other causes.
    2. Reference materials and certified reference materials: Use of known materials can be used to assess the accuracy of the method, as well as obtaining information on interferences.
    3. Fortified (spiked) materials and solutions: Recovery determinations can be made from fortification or spiking with a known amount of analyte.
    4. Incurred materials: These are materials in which the analyte of interest may be essentially alien, but has been introduced to the bulk at some point prior to the material being samples.
    5. Measurement standards: These are substances used for calibration or identification purposes. When placed periodically in an analytical batch, checks can be made that the response of the analytical process to the analyte is stable.
    6. Replication: Replicate analysis provides a means of checking for changes in precision in an analytical process which could adversely affect the results.
    7. Statistics: Statistical techniques are employed to evaluate accuracy, precision, linear range, limits of detection and quantification, and measurement uncertainty.
  3. Validation Protocol Guidance

    NOTE: Please refer to FDA/Office of Foods, Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods and of Chemical Methods (when approved) for validation of analytical food methods for Agency-wide implementation in a regulatory capacity.

    Laboratories document their protocol, the performance characteristics measured, and acceptance limits for the validation of non-standard and laboratory developed methods. The extent of validation will depend on constraints imposed such as time, cost, amount of sample or standard, future use of method, or type of information (quantitative, qualitative, screening). Due to these constraints, not all characteristics may be applicable. The following gives guidelines for determination of performance characteristics:

    1. Chemistry
      1. Perform system suitability requirements: injection repeatability, peak resolution, relative retention for liquid chromatography analyses.
      2. Quantitative measurements: Determine detection limit, either method detection limit (MDL) according to 40 CFR, Part 136, Appendix B or limit of detection (LOD). LOD is determined by analyzing sample blanks, calculating the standard deviation, and expressed as the mean plus 3 standard deviations. For qualitative measurements, determine the concentration threshold below which specificity becomes unreliable.
      3. Quantitative measurements: Determine linear calibration range if a standard curve is to be used or determine the target calibration standard and linearity if only a one calibration point is to be used.
      4. Quantitative measurements: Prepare and analyze spiked blanks, solvent or matrix samples of known concentration utilizing at least three different concentration levels: low, middle, and high. These samples are carried through the complete sample preparation procedure. Matrix effects can also be assessed with these samples. Accuracy (percent recovery) and precision (relative standard deviation or relative percent recovery) are calculated from the results.
      5. Analyze blanks (reagent, solvent and matrix).
      6. Evaluate interferences: spectral, physical, chemical or memory by analyzing a sample containing various suspected interferences in the presence of the measure:
        • Spectral interference may be observed when an overlap of a spectral line from another element or background contribution occurs.
        • Physical interference may occur from effects associated with sample transport processes on instruments.
        • Chemical interferences are characterized by compound formation, ionization or vaporization effects.
        • Memory interference occurs from contribution of signal from previous sample to sample being tested.
      7. Prepare the laboratory procedure (LP). Infrequently used or non-routine methods do not need an LP until they become routine.
    2. Microbiology
      1. Meet method system suitability requirements, if applicable. The suitability of the method is checked and confirmed by comparing with requirements typical for the intended use of the method. For example, a filtration method for a non-filterable food, a five day test where three days are needed, a 1 gram test where 100 grams are needed, surface tests for Colony Forming Units (CFU)/square area where CFU/gram is needed.
      2. Include un-inoculated medium control to assess contamination from the laboratory. This control is considered a blank and is to exhibit no growth.
      3. Prepare and analyze positive and negative culture controls. A negative control is atypical, negative or no growth and the positive control is positive or shows microbial growth.
      4. A spike positive culture control is prepared and analyzed. Unless otherwise specified, it is recommended that a 25 gram sample be spiked with an inoculum of 30 cells or less. This assesses the matrix effects as well as the sensitivity of the method.
      5. Evaluate interferences. This assesses the selectivity and specificity of the method.
      6. Prepare the laboratory procedure (LP). Infrequently used or non-routine methods do not need a LP until they become routine.
  4. Validation of Method Modifications
    1. In cases where the testing procedure is modified from the standard or existing testing procedure and protocol, it is demonstrated that the modifications do not adversely affect the precision and accuracy of the data obtained.
    2. See Attachment A for general guidelines for allowable modifications to a method before a modification protocol is needed.
    3. In order to implement the modification, the standard or existing method is first performed. Each major modification is then verified against the original method.
    4. Additional statistics employed to verify performance specifications are:
      1. The t test for significance of difference between the two sample means to determine degree of accuracy. The t-Stat value is to be less than or equal to the t-critical value.
      2. The F test for significance of difference between the two sample variances to determine degree of precision. The F value is to be less than or equal to the F-critical value.
  5. Documentation
    The results from the validation study are submitted for approval as designated by the laboratory. Statistical techniques are employed to evaluate the method performance and determine its use. See Attachment B for an example of a form to record validation results.

7. Definitions

Accuracy – Accuracy is the nearness of a result or the mean of a set of measurements to the true value.

Analytical batch – An analytical batch consists of samples which are analyzed together with the same method sequence and the same lots of reagents and with the manipulations common to each sample within the same time period or in continuous sequential time periods.

Detection limit – A detection limit is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. It is often called the limit of detection (LOD) which is the lowest concentration level that can be determined statistically different from a blank at a specified level of confidence. It is determined from the analysis of sample blanks. Method detection limit (MDL) is the minimum concentration of a substance than can be measured and reported with 99% confidence that the analyte concentration is greater than zero. It is determined from analysis of a sample in a given matrix containing the analyte.

Limit of quantitation (LOQ) – This is the level above which quantitative results may be determined with acceptable accuracy and precision.

Linearity – Linearity is the ability of the method to elicit results that are directly proportional to analyte concentration within a given range.

Non-standard method – This refers to a method that is not taken from authoritative and validated sources. This includes methods from scientific journals and unpublished laboratory-developed methods.

Precision – Precision is the agreement between a set of replicate measurements without assumption of knowledge of the true value. Repeatability expresses the precision under the same operating conditions over a short period of time. Intermediate precision expresses within-laboratory variations, such as different days, different analysts, and different equipment. Reproducibility expresses the precision between laboratories.

Range – A range is the interval between the upper and lower concentration of analyte in sample for which it has been demonstrated that the analytical procedure has an acceptable level of accuracy, precision, and linearity.

Ruggedness or robustness – Ruggedness is a measure of an analytical procedure’s capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Specificity – Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.

Standard method – This is a method that is traceable to a recognized, validated method. FDA “official” methods are those in compendia specified in the Food Drug & Cosmetic Act and prescribed in the Code of Federal Regulations. Additionally, methods in Applications and Petitions that have official status are included under the umbrella of standard methods. Official methods include those in the United States Pharmacopeia, National Formulary, Homeopathic Pharmacopeia of the United States, Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC) International or any supplement of any of them, American Public Health Association (APHA) Compendium of Methods for the Microbiological Examination of Foods. Official methods also include methods that are found in a FDA Compliance Program, the Pesticide Analytical Manual (PAM), the Food Additives Analytical Manual, the Food Chemicals Codex, FDA Bacteriological Analytical Manual, FDA Macroanalytical Procedures Manual (MPM), and ORA Laboratory Information Bulletins (LIBs). In addition, methods in approved Abbreviated New Drug Applications (ANDA), New Drug Applications (NDA), New Animal Drug Applications (NADA), Food Additive Petitions (FAP) and Pesticide Petitions (PP) are considered standard methods.

Validation, method – A method validation is the process of establishing the performance characteristics and limitations of a method and the identification of the influences which may change these characteristics and to what extent. (Eurachem Guide)

Verification – A verification is the confirmation by examination and provision of objective evidence that specified requirements have been fulfilled. (ISO 8402:1994)

8. Records

Validation data and statistics

9. Supporting Documents

  1. ORA-LAB1 Microbiological Controls for Sample Analysis
  2. International Conference on Harmonization (ICH) Topic Q2A
  3. Code of Federal Regulations. Title 40, Part 136, Appendix B
  4. FDA/Office of Foods, Guidelines for the Validation of Analytical Methods located at:

10. Attachments

  • Attachment A: Modification Criteria (PDF PDF Document, 32 kb)
  • Attachment B: Validation Form Example (PDF PDF Document, 25 kb)
  • Attachment C: Request Form Example (PDF PDF Document, 16 kb)

11. Appendix

  • Appendix 1: ORA Validation and Verification Guidance for Human Drug Analytical Methods (PDF PDF Document, 36 kb)

12. Document History

(I, R, C)
Location of Change History Name & Title
Author Approving Official
Appendix 1 added as section 11 and inserted
In Document
In Document
In Document
In Document
In Document

Approving Official's signature: _________________________________ Date: _____________

Page Last Updated: 07/14/2015
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