METHOD VALIDATION PROTOCOL

All food testing methods (chemistry and microbiology) shall be fully validated or verified
by the laboratory for “fitness of purpose” prior to implementation of the methods.
This validation or verification shall be according to fully documented procedures.
Standard published method used verbatim does not require a full validation, however, the
laboratory shall confirm that it can properly operate the standard method before
introducing the tests.
Confirmation can be achieved through method verification of specific performance
characteristics of the standard methods.
If the standard method changes, the confirmation shall be repeated.
Conditions for full validation:
a. Standard published method modified or amplified (exemption: editorial modifications)
to the extent that the modification or amplification directly affect the performance of the
test.
b. Standard published method or previously validated method applied to different matrices,
analytes, concentration range or conditions outside the scope of the published method.
c. In-house developed methods.
d. Method published in scientific journals without appropriate performance data.
e. Vendor test kits without appropriate performance data.
Conditions for verification (confirmation):
a. Standard published method used without modification.
b. Method published in scientific journals with appropriate performance data.
c. Vendor test kits with appropriate performance data.
Extent of validation and verification:
Methods shall be validated or verified using certified reference materials (where available)
or materials of known characteristics.
Validation of quantitative methods,
shall include, but not limited to, the following attributes as appropriate:
• Accuracy
• Linearity
• Range •
Reproducibility (Precision)
• Repeatability (Precision)
• Robustness
• Limit of Detection
• Limit of Quantitation
• Estimation of measurement uncertainty
• Selectivity
In addition, where applicable, the following shall be taken into consideration during
validation process:
• Matrix effect
• Interference
• Sample homogeneity
Verification of quantitative methods, shall include, but not limited to, the following as
appropriate:
• Accuracy
• Precision
• Limit of Detection
• Measurement uncertainty estimate
For validation or verification of qualitative methods the following attributes, should be
taken into consideration where applicable:
• Specificity
• Contamination
• Detection limit
• Rate of false positive and false negative
• Repeatability •

. The laboratory may include a report summarizing the validation/verification data.

Once the method is validated or verified and implemented it shall be periodically reviewed
to confirm the ongoing “fitness for purpose” of the method.
The laboratory shall define and document the frequency and the review process, preferably
in the method validation procedure.
2.0.1 Specificity and Sensitivity

Sensitivity and specificity are terms that can have different definitions
for different types or categories of analytical methods. In a general sense,
these terms are defined by the extent to which a method responds
uniquely to the specified target organism or group of organisms.
Specificity is the ability to discriminate between the target organism and
other organisms. It is mathematically expressed as:

Specificity = TNC / (TNC + TPI)

Where:

TNC = Number of samples

tested negative correctly
TPI = Number of samples
tested positive incorrectly

Specificity for microbiology methods and media is traditionally

demonstrated through the use of pure positive and negative control
cultures.

Sensitivity is the proportion of target organisms that can be detected.

It is mathematically expressed as: Sensitivity = TPC / (TPC + TNI)

Where:

TPC = Number of samples tested positive correctly

TNI = Number of samples tested negative incorrectly (Deep 2006)

Data to calculate sensitivity are typically generated by repeated testing of

serial dilutions of a “known” spike standard.

2.0.2 Precision

Precision can be defined as the closeness in agreement between

independent test results obtained under stipulated conditions (ISO ).
This term is usually expressed as the variance, standard deviation, or
coefficient of variation of a series of test results.
Precision is often expressed as the percent coefficient of variation
(%CV), where:

%CV = (standard deviation of measurements / mean) x 100

The expected precision of culture-based microbial methods is typically
derived mathematically based on the assumption that bacteria are
distributed randomly in a well-mixed sample and follow a Poisson
distribution.
Precision may be considered at four levels: within-laboratory
repeatability, within-laboratory repro- ducibility, between-laboratory
repeatability, and between-laboratory reproducibility.
The first two levels should be addressed in the primary validation of a
method over the entire concentration range of the analyte that is expected
to be relevant to its intended use.

Repeatability. Repeatability can be defined as the closeness in agreement

between results of successive measurements of the same analyte carried
out under the same conditions of measurement during a short interval of
time. Repeatability is also termed intra-assay precision.

Suppose the within laboratory precision is Sr and the

between laboratory precision is SL. Then the precision SR

(including within and between) among laboratories is

 expressed as

SR = Sr 2 +SL 2

Refer to SOP CTL-P-049 ( Training, Competence of Microbiological Staff

&calculation of count

Reproducibility. Reproducibility can be defined as the closeness of the

agreement between the results of measurements of the same analyte
carried out under variable conditions of measurement. For deter-
mination of within-laboratory reproducibility, some of the variable
conditions that should be considered include different time intervals
between analyses, analysts, lots or preparations of reagents, instruments,
and different water matrices.

Refer to SOP CTL-P-049 ( Training, Competence of Microbiological Staff

&calculation of count

2.0.3 Accuracy and Bias

Accuracy can be defined as the closeness of the agreement between a
test result and the accepted reference value, whereas bias can be defined
as the difference between the expectation of the test results and a known
or accepted reference value. The term “accuracy,” when applied to a set
of test results, has been more comprehensively defined as a combination
of random components (related to random error) and a common bias
component (related to total systematic error) associated with the method
(ISO 1994a). As with precision, this bias component should be first
characterized at the within-laboratory level as a primary attribute of
most methods.
By the above definition, the determination of bias in the analysis of a
material by a new method requires the knowledge of either the true value
for the analyte in the sample or the assignment of an accepted reference
value. In some cases, these values may be known or assigned through the
use of
1- fortified or spiked samples, 2-certified reference materials, 3-analysis
by another presumably unbiased method, or internal controls.

When analyzing fortified or spiked samples, bias is often expressed by

 the terms recovery or percent recovery (i.e., test result, divided by the
expected [assigned] value for the added spike material, multiplied by
100).

2.0.4 Limit of Detection (LOD)

LOD can be defined as the minimum amount of analyte that can be

reliably detected (i.e., distinguishable from known and characterized
background with a given level of confidence).
The LOD measurements establish a baseline detection value under
optimal conditions. Culture-based methods are typically expected to be
capable of detecting single target organisms in large samples.
Where no organisms are detected, the result is reported as <1 organism
per sample volume or mass.

2.0.5 Linearity

Linearity is defined as the ability of the analytical procedure to obtain

test results within a given range, which are directly proportional to the
concentration of analytes (microorganisms or nucleic acid) in the
sample.
2- Robustness: 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. Eurachem Guide ( The Fitness for Purpose of Analytical
Methods. A laboratory guide to method validation and related topics.
slight variations in the test conditions.
In the context of microbiology two of the most important parameters are
incubation time and incubation temperature. Others include slight
differences in the concentration of media prepared, age of the media
used and sample holding time. For any method a degree of judgement is
required to identify those experimental parameters which could influence
the results.
Uncertainty of Measurement: Parameter associated with the result of a
measurement that characterizes the dispersion of the values that could
reasonably be attributed to the measurand (Eurachem Guide ).
The Fitness for Purpose of Analytical Methods. A laboratory guide to method
validation and related topics. Copyright LGC (Teddington) Ltd 8
For standard curve development, the simplest model (e.g., linearity) that
adequately describes the concentration-response relationship should be
used.
The calibration curve is also used to establish the
limit of quantification of the method (i.e., the lowest amount of analyte
that can be quantitatively determined with suitable precision and
accuracy) and the quantitative range of the method (i.e., the interval
between and including the upper concentration and limit of detection of
the analyte over which the concentration- response model provides
suitable precision and accuracy).
During this process, the statistical dispersion of the standard
measurements by the instrument or detector (instrument precision) is
combined with the model assumptions to generate a calibration
equation.

A statistic such as R-squared combined with a

confidence interval can be used to provide a measurement of reliability
or uncertainty for an estimate from an “unknown” sample based on the
calibration curve model.

Refrence
Method Validation ofU.S. Environmental Protection Agency (EPA)-1
Microbiological Methods of Analysis
REVISION: December 21, 2016
FDA-2
AOAC-3
Iso 17025/2017-4
Eurachem Guide ( The Fitness for Purpose of Analytical Methods. A -5
.laboratory guide to method validation and related topics