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    Handout METHOD VERIFICATION AND VALIDATIONS

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    Handout METHOD VERIFICATION AND VALIDATIONS

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    25 views33 pages

    Handout METHOD VERIFICATION AND VALIDATIONS

    Uploaded by

    maslinda alias

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    METHOD VERIFICATION

    AND VALIDATIONS

    BY
    PUAN SALMA ARIFIN
    MS ISO/IEC 17025:2017
    requirements
     Verification and validation of methods
    are stated in the clause 7: Process
    Requirements
     Specifically mentioned in clause 7.2
    Selection, Verification and Validation of
    methods
    Method Verification
    requirements:
    The laboratory shall verify that it can properly
    perform methods before introducing them by
    ensuring that it can achieve the required
    performance.

    It is applicable to all methods or measurements


    applied in the laboratory including equipment and
    test kits.
    What is Method
    Verification?
    Verification:
    the process of establishing the truth, accuracy
    or validity of something.

    Method verification:
    validity of test method, valid to use.
    METHOD VERIFICATION

    How to verify the test


    method.
    > use suitable reference cultures
    > conduct the verification as per test method
    procedure.
    ➢ Calculate the recovery of reference cultures
    used(for quantitative method) or
    ➢ Absent or present of reference cultures by
    confirmation or identification test.
    METHOD VERIFICATION
    Report of method verification:
    An official report need to be
    established containing:
    >Title
    >Date of conducted and
    completed
    >Name and signed of person
    conducted the test
    > Name and signed of person
    verified the test
    >procedure of verification
    ➢ Results
    ➢ Discussion and conclusion
    Example of official report of method
    verification
     Title : Verification of Total Aerobic Plate Count in food (AOAC
    method)
     Date conducted: 12/4/2023
     Date completed : 20/4/2023
     Conducted by: Ali
     Reviewed by: Kumar
     verified by : Ah Seng

    Scope: Apply to all food


    Purpose : To verify the method used and to ensure the method is
    valid to use.
    Procedure : According to AOAC method
    Calculation:
    Results : raw and test report
    Discussion :
    Conclusion
    METHOD VALIDATION
    MS ISO/IEC 17025:2017-Stated in clause 7.2.2
    *Validation is the confirmation by examination and the
    provision of objective evidence that the particular
    requirements for a specific intended use are fulfilled.

    *The laboratory shall validate non-standard methods,


    laboratory-develop methods and standard methods use
    outside their intended scope or otherwise modified.

    *The validation shall be as extensive as is necessary to


    meet the needs of the given application or field of
    application
    METHOD VALIDATION
    MS ISO/IEC 17025:2017-Stated in clause
    7.2.2

     It is applicable to non-standard methods, laboratory


    designed/develop methods, standard methods used
    outside their intended scope, modification of
    standard methods(in house based on standard
    methods)
    NON-STANDARD METHOD
    A method that is not a published method and may or
    may not be developed by the laboratory.

    LABORATORY DESIGNED/DEVELOP METHOD


    A method that is developed by the laboratory.

    STANDARD METHOD USED OUTSIDE OF INTENDED SCOPE


    A method is essentially the same with non-standard
    method.

    MODIFICATION OF STANDARD METHOD


    A method that have been modified from standard
    method (In house method based on standard
    method).
    SCOPE OF METHOD
    VALIDATION
     Validation covers both quantitative and qualitative
    methods

     Microbial counts : Plating technique (cfu) and Most


    Probable Number (MPN) technic.
    METHOD VALIDATION

     Characteristic of the quantitative method


     i) Accuracy – comparing results obtained from
    validated method with standard method and
    microbial detected is correctly identified as per test
    method required.
     ii) Recovery – is a fraction of microbial count in sample
    matrix
     Iii) Linearity – apply to MPN method.
     iv) Limits of detection – plating method (0-100 cfu/g),
    MPN method (0.03 cfu/g or less)
    METHOD VALIDATION

     v) Limits of quantitation and determination


    – lowest microbial count to be determined
    (200-300cfu/g of food sample)
    - Limits of determination – 100 cfu/g(plating
    technique) and 0.01g (MPN technic).
    METHOD VALIDATION

     vi) Precision
    Repeatability and reproducibility
    Intra-laboratory
    > Duplicate sample with 5 replicates per lot of sample
    and per microbial level
    > microbial level of spike (control, low, medium, high)
    > calculate the standard deviation by logarithm the
    results.
    METHOD VALIDATION

     Precision continue…
     Inter-laboratory
    > minimum 2 laboratories participate
    > calculate the standard deviation, Sr values
    > compare the values obtained between lab
    False positive and false negative
    sample that gives interfering substances( affect this
    results) has to be removed.
    METHOD VALIDATION

     Sensitivity
    Same as recovery and accuracy

    Ruggedness Testing
    >small changes in operating conditions and
    environment, example : incubation temperature.
    METHOD VALIDATION

     Sample Matrixes
    > ideally, 5 matrixes for each type of food category.

    Microbial strain (analyte)


    > either spike or natural contaminants
    ➢ Recommended 10 strain but if only for particular
    method, one strain per sample matrixes is used.
    ➢ In case of mold and yeast validation, more than one
    strain has been recommended.
    METHOD VALIDATION

     Level
    ➢ microbial level of spike : unspiked, low, middle, and high
    spike
    ➢ analyzed at least 5 times per matrixes

    Interfering Microorganism
    ➢ Related to natural contaminants : natural microflora.
    ➢ If any interfere happen, sample (matrixes) shall be
    removed from list
    ➢ Use matrixes with high and low levels of incurred
    microflora.
    METHOD VALIDATION

     Acceptance Criteria
    ➢ No specific acceptance criteria
    ➢ For Quantitative method, calculate the results from
    validated method and compare with standard
    method results or
    ➢ For Qualitative method, compared the results
    obtained from validated method against to inoculum
    of microbial suspension results.
    METHOD VALIDATION
    Quantitative
     Procedure
    ➢ Prepare sample matrixes as per required for each type
    of matrixes
    ➢ Prepare inoculum for spiking level (unspike (blank),
    low(0.1ml), middle (0.5ml), high(1 ml))
    ➢ Obtained the initial count for each level of spike
    ➢ Spike the sample matrixes
    ➢ Each level = 5 times (5 replicates)
    METHOD VALIDATION
    Quantitative
     Procedure cont…
    > Proceed with the test by following the procedure of
    the method to be validated.
    ➢ Count the results obtained
    ➢ Calculate the results in terms of comparing the results
    with initial count in percentage term.
    QUALITATIVE METHOD
    VALIDATION
     Accuracy – comparing results obtained from
    validated method with standard method and
    microbial detected is correctly identified as per test
    method required.
     Recovery – use middle or high spike for qualitative
    method. Comparing by proportion of positive results.
    Homogenity of the sample is crucial to obtained
    results.
    QUALITATIVE METHOD
    VALIDATION
     Limits of detection – theoretically, one microbe is to be
    detected for particular method to be validated. Limit
    sample is 25g.
     Limits of quantitation and determination – one
    analytical particle per chosen analytical sample size
    QUALITATIVE METHOD
    VALIDATION
     Precision
    Repeatability and reproducibility
    Intra-laboratory
    > Duplicate sample with 10 replicates per lot of sample
    and per microbial level
    > microbial level of spike (control, low, medium, high)
    QUALITATIVE METHOD
    VALIDATION
     Inter laboratory reproducibility
    > minimum 2 laboratories participate
    > 10 replicates of sample matrixes with spike level of low,
    middle and high.

    Sensitivity
    >proportion of test sample that contain microbial and
    response positively to the test

    Specificity
    > Proportion of test sample that do not contain microbial
    and response negatively to the test.
    QUALITATIVE METHOD
    VALIDATION
     Sample matrixes
    > one or more different samples
    Microbial analyte
    > Ideally a variety of strains of the target microorganisms
    would be tested.
    > If possible, use isolated microbial (natural contaminant)
    for validation.
    QUALITATIVE METHOD
    VALIDATION
     State of stress (concentration) of microbial analyte
    (microbe)
    ➢ microbes may be subject in a particular matrix; dry
    inocula are used for dry foods and wet inocula are
    used for wet foods.

    Spiking levels
    > Each strain is to be used at a minimum of 3 levels per
    analytical sample (25g/ml); 1-10cfu; 10-50cfu; blank; no
    addition. Obtained the initial count of spike level.
    QUALITATIVE METHOD
    VALIDATION
     Acceptance criteria
    > depend on matrixes type
    > inoculum level must be stated from < 10 to 100%
    QUALITATIVE METHOD
    VALIDATION
     Procedure
    ➢ Prepare sample (10 replicates of different matrixes)
    ➢ Prepare inoculum of microbial and obtained initial
    count
    ➢ Spike with microbial levels (low, middle, high)
    ➢ Proceed with the test to be validated
    ➢ Obtained the results
    ➢ Compare with the results by standard method
    ➢ Results as presence or abscence
    METHOD VALIDATION
    Report of method validation:
    An official report need to be established
    containing:
    >Title
    >Date of conducted and completed
    >Name and signed of person
    conducted the test
    > Name and signed of person verified
    the test
    >procedure of verification
    ➢ Results (calculation)
    ➢ Discussion and conclusion
    EXAMPLE OF METHOD VERIFICATION

    TEST METHOD : DETECTION OF SALMONELLA IN


    FOOD SAMPLES (FDA BAM CHAPTER 5)

    PROCEDURE : AS PER TEST METHOD FDA BAM


    CHAPTER 5

    Reference culture used: Salmonella typhimurium


    Concentration range of pure culture : 10 cfu/ml,
    10-50 cfu/ml, 100 cfu/ml, blank
    Volume of pure culture concentration used: 1 ml
    Sample matrixes : soy beans.
    EXAMPLE OF METHOD VALIDATION
    TEST METHOD: Detection of Salmonella In Oil Samples
    (In House method based on FDA BAM Chapter 5)

    PROCEDURE : As per In House Method based on FDA


    BAM Chapter 5.

    Reference culture used: Salmonella typhimurium


    Concentration range of pure culture : 1-10 cfu/ml, 10-
    50 cfu/ml, 100 cfu/ml, blank
    Volume of pure culture concentration used: 1 ml
    Sample matrixes : waste oil, corn oil, engine oil,
    Repeatability : duplicate samples of 10 replicates of
    sample for each concentration of pure culture used.
    THE END

    Thank You

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