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- updated specifications for Column Inner Diameter, Column Temperature, Oven Temperature)
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

None.

5. References

A Laboratory Guide to Method Validation, (Eurachem).

6. Procedure

6.1 Method Selection

1. Selection of Methods
   
   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. 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.

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
   
   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 Assocaition 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

ORA-LAB1 Microbiological Controls for Sample Analysis
International Conference on Harmonization (ICH) Topic Q2A Code of Federal Regulations. Title 40, Part 136, Appendix B .

10. Attachments

• Attachment A:  Modification Criteria (PDF , 30 kb)
• Attachment B:   Example Validation Form (PDF , 24 kb)

11. Appendix

• Appendix 1:  ORA Validation and Verification Guidance for Human Drug Analytical Methods (PDF , 46 kb)

12. Document History

Approving Official's signature: _________________________________ Date: _____________