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# Method Validation and Verification Protocols for Test Methods

## 1. Method Validation and Verification Protocols for Test Methods

## 2. What is it ?

• Method validation & verification provides objective evidencethat a test method is fit for purpose,

i.e. that the particular requirements for a specific intended

use are fulfilled.

• The term ‘method’ includes kits, individual reagents,

instruments, platforms and software.

• Method Validation : in-house and modified standard methods

• Method Verification : standard methods

## 3. When it is required ?

• Method Validation : in-house and modified standard methods• Method Verification : standard methods

Method

Fully validated standard

methods

Standard methods –

modifications

Standard methods – outside

their intended scope

Laboratory developed and nonstandard methods

Requirement

Verification

Validation

Validation

Validation

## 4. Why it is necessary ?

• A test method must be shown to be fit forpurpose by validation and verification for the

customers to gain confidence in the test results

## 5. Verification

• Standard validated methods - AOAC, ASTM, ISO, etc• Peer accepted methods published in scientific literature

• Commercial test kits

Laboratory needs to verify that analysts using their

equipment in their laboratory environment obtain the

same outcomes as defined in the validation data

## 6. Verification

• Method performance demonstrated by– blanks or un-inoculated media - to assess contamination;

– laboratory control samples - to assess accuracy;

– duplicates - to assess precision

– calibration check standards - for quantitative analyses

– monitoring quality control samples, and

– participation in a PT testing program

## 7. Some examples

Methodusing the same type of chromatographic

column from a different manufacturer

a slight change in a non-critical

incubation temperature

use of a different non-selective growth

medium,

differences in details of sample dilutions

as a consequence of expected counts

Requirement

Verification

Verification

Verification

Verification

## 8. Some examples

Methoduse of a different extraction solvent; use

of HPLC instead of GLC

differences in the formulation of the

selective/differential medium (e.g.

addition of an alternative antibiotic)

different antibiotic concentration to the

base medium

a change to a critical incubation

temperature or time (e.g. 3 days rather

than 5 days incubation)

different confirmation procedure (e.g. use

of an alternative suite of biochemical tests

other than those specified)

Requirement

Validation

Validation

Validation

Validation

Validation

## 9. Key parameters for verification

TestsFor quantitative results

For trace analyses

For qualitative methods

For diagnostic methods

Parameters

measurement of bias and

measurement of precision minimum requirements

limit of detection (LOD) and

limit of quantification (LOQ)

correlation studies with validated

methods or comparisons with

known outcomes

sensitivity and selectivity

(specificity)

## 10. Validation

• Non-standard and in-house-developed methods• Scope and validation criteria to be defined and

documented

Tools to demonstrate the method performance

– Blanks

– Certified Reference Material (CRMs)

– Fortified materials

– Replication

– Statistical analysis

## 11. Types of Validation

Comparative ValidationPrimary Validation

• To demonstrate equivalent

performance between two

methods (validated and

revised analytical method)

• an exploratory process to

establish operational

limits and performance

characteristics for

alternative or new method

## 12. Validation

Two steps1. to specify what you intend to identify or measure

2. to determine selected performance parameters

## 13. Validation Parameters

1.2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

Linearity range

Measuring interval

Matrix effects

Selectivity

Sensitivity

Accuracy .

Precision

Repeatability

Reproducibility

Trueness

Limit of detection (LOD) and limit of quantitation (LOQ)

Ruggedness

Measurement Uncertainty.

## 14. Analytical Performance Characteristics Procedure

• Before validation, design, maintain, calibrate and validate theanalytical system (protocol, conc. range and specified material)

• Train all the personnel who perform the validation testing

• Get approval of method validation protocol from CA before

execution.

1. Specificity

Test procedure: Investigate by injecting of the extracted

sample to demonstrate the absence of interference with the

elution of analyte

Documentation : Print chromatograms.

Acceptance criteria : The excipient compounds must not

interfere with the analysis of the targeted analyte.

## 15.

2. Linearity• Test procedure :

• Prepare standard solutions at six concentrations, typically 25,

50, 75, 100, 150, and 200% of target conc.

• Analyze three individually prepared replicates at each

concentration.

• Use same method of standard preparation and number of

injections as in the protocol

Documentation:

Record results on a datasheet.

Calculate the mean, standard deviation, and RSD for each conc.

Plot concentration (x-axis) versus mean response (y-axis) for

each conc.

• Calculate the simple regression or weighted regression equation

& correlation coefficient and record.

## 16.

2. Linearity• Acceptance criteria :

• The correlation coefficient for six conc. levels will be ≥ 0.999 for

the range of 80 to 120% of the target conc.

• The y-intercept must ≤ 2% of the target conc. response.

• A plot of response factor vs conc. must show all values within

2.5% of the target level response factor.

• The coefficient for active ingredients should be ≥ 0.997, for

impurities 0.98 and for biologics 0.95

## 17.

3. Range• Test procedure :

• Use the data obtained during linearity and accuracy studies to

assess the range of the method.

• We can use the precision data for this assessment, if precision

of the three replicate samples is analyzed at each level in the

accuracy studies.

• Documentation : Record the range on the datasheet.

• Acceptance criteria

Acceptable range (- defined as the conc. interval over which

linearity and accuracy are obtained)

It yields a precision of ≤ 3% RSD.

## 18.

4. Accuracy• Test procedure

• Prepare spiked samples at three conc. over the range of 50 to

150% of the target conc.

• Analyze three individually prepared replicates at each conc..

• When it is impossible or difficult to prepare known sample,

use a low concentration of a known standard.

• Documentation :

• For each sample, report the theoretical value, assay value, and

percent recovery.

• Calculate the mean, standard deviation, RSD, and percent

recovery for all samples.

• Record results on the datasheet.

## 19.

4. Accuracy• Acceptance criteria

• The mean recovery will be within 90 to 110% of the

theoretical value for non-regulated products.

• For the U.S. pharmaceutical industry, 100 ± 2% is typical for

an assay of an active ingredient in a drug product over the

range of 80 to 120% of the target concentration.

• Lower percent recoveries may be acceptable based on the

needs of the methods.

• Health Canada states that the required accuracy is a bias of ≤

2% for dosage forms and ≤ 1% for drug substance.

## 20.

5. Precision - Repeatability• Test procedure:

• Prepare one sample solution containing the target level of analyte

• Make ten replicates from this sample solution

• Documentation:

• Record retention time, peak area, & peak height on datasheet.

• Calculate the mean, standard deviation, and RSD.

• Acceptance criteria:

• FDA states - typical RSD should be 1% for drug substances and

drug products, ± 2% for bulk drugs and finished products.

• HC states - RSD should be 1% for drug substances and 2% for drug

products. For minor components, it should be ± 5% but may reach

10% at the LOQ.

## 21.

6. Intermediate Precision• Test procedure:

• Demonstrate Intermediate precision (within-laboratory

variation) by two analysts, using two HPLC systems on

different days and evaluate the relative percent purity data

across the two HPLC systems at three conc. levels (50%,

100%, 150%) covering range of 80 to 120%.

• Documentation:

• Record the relative % purity (% area) of each conc. on the

datasheet.

• Calculate the mean, standard deviation, and RSD for operators

and instruments.

• Acceptance criteria:

• The results obtained by two operators using two instruments

on different days should have a statistical RSD ≤ 2%.

## 22.

7. Limit of Detection• Test procedure

• Determine the lowest concentration of the standard solution

by sequentially diluting the sample.

• Make six replicates from this sample solution.

• Documentation

• Print the chromatogram and record the lowest detectable

concentration and RSD on the datasheet.

• Acceptance criteria

• The International Conference on Harmonization (ICH)

references a signal-to-noise ratio of 3:1.2

• Health Canada recommends a signal-to-noise ratio of 3:1.

• Some analysts calculate the standard deviation of signal (or

response) of a number of blank samples and then multiply

this number by 2 to estimate the signal at LOD

## 23.

8. Limit of Quantitation• Test procedure

• Determine the lowest concentration at which an analyte in the

sample matrix can be measured with the accuracy & precision.

• This value may be the lowest concentration in standard curve.

• Make six replicates from this solution.

• Documentation

• Print the chromatogram and record the lowest quantified

concentration and RSD on the datasheet.

• Provide data that demonstrates the accuracy and precision

required in the acceptance criteria.

## 24.

8. Limit of Quantitation• Acceptance criteria:

• The limit of quantitation for chromatographic methods is

described as the conc. that gives a signal-to-noise ratio of 10:1.2

• Quantitation limit is the best estimate of a low conc. that gives

an RSD of approx. 10% for a minimum of six replicate

determinations.

## 25.

9. System Suitability• Test procedure

• Perform system suitability tests on both HPLC systems to

determine the accuracy and precision of the system by injecting

six injections of a solution containing analyte at 100% of test

conc..

• Determine plate count, tailing factors, resolution, &

reproducibility (% RSD of retention time, peak area, & height)

• Documentation:

• Print the chromatogram and record the data on the datasheet

## 26.

9. System Suitability• Acceptance criteria:

• Retention factor (k): the peak of interest be well resolved from

other peaks and the void volume; generally k should be ≥2.0.

• Resolution (Rs): Rs should be ≥2 between the peak of interest

and the closest eluted peak (impurity, excipient, and

degradation product).

• Reproducibility: RSD for peak area, height, and retention time

will be 1% for six injections.

• Tailing factor (T): T should be 2.

• Theoretical plates (N): ≥2000

## 27.

10. Robustness• Measures the capacity of an analytical method to remain

unaffected by small but deliberate variations in method

parameters.

• Provides some indication of the reliability of an analytical

method during normal usage.

• Parameters investigated - % organic content in the mobile

phase or gradient ramp, pH of the mobile phase, buffer

concentration, temperature, and injection volume.

• Evaluate these parameters - one factor at a time or

simultaneously as part of a factorial experiment.

## 28.

10. Robustness• Compare the chromatography obtained for a sample containing

representative impurities, when using modified parameter(s),

to the chromatography obtained using the target parameters.

• Determine the effects of the following changes in

chromatographic conditions :

– methanol content in mobile phase adjusted by ± 2%,

– mobile phase pH adjusted by ± 0.1 pH units,

– Column temperature adjusted by ± 5˚C.

• If these changes are within the limits that produce acceptable

chromatography, incorporate in the method procedure.

## 29.

11. Measurement Uncertainty• Calculation of measurement uncertainty by mathematical

model according to law of propagation of uncertainty

u [y (x1. x2…..)] = √ ci2 u(xi)2

i=l,n

Where

u [y (x1. x2…..)] is a function of several independent variables x1, x2, …

ci is a sensitivity coefficient evaluated as ci = δy/ δx, the partial differential of y with respect to xi

u(xi) and u(y) are standard uncertainties i.e measurement uncertainties expressed as SD

So, u [y (x1. x2…..)] is referred as a combined standard uncertainty

## 30.

Estimation of UncertaintyUncertainty calculation for Chloramphenicol analysis

• Type A and Type B errors are the sources to calculate uncertainty.

• Type A – Due to sample (Repeatability Measurement) (URep)

• Type B – a). Due to Equipments (UEquip)

b). Due to Purity of Chemicals and CRM (UPur)

c). Due to Glassware (Ug)

• Coverage factor k = 2 at 95 % confidence level.

## 31.

Type A ErrorRepeatability

Readings Xi

0.28000

0.27300

0.30000

0.26700

0.32000

0.30700

Average X

Std.

0.2912

0.02100

USTD = Sd/√n

0.00860

Type B

i. Uncertainty due to Equipments

Equipment

Weighing

Balance

Refrigerated

Centrifuge

Vortex Mixer

Uncertainty

k

U. Equip= U/k

0.09

2

0.045

0.06

0.06

2

2

0.03

0.03

## 32.

ii. Uncertainty due to Chemicals and CRM (Upur)Chemical

Purity U.

% Conv = U

% Chem %

Chloramphenic

ol (CRM)

99.7

Acetonitrile

99.9

Carbon

Tetrachloride

99

Ethyl Acetate

99.7

k

Std Uncertainty =

U/k

0.3

0.1

0.003

0.001

2

1.732

0.0015

0.0006

1

0.3

0.01

0.003

1.732

1.732

0.0058

0.0017

iii. Due to Standard Uncertainty Glassware (Ug)

Glassware

Volumentric Flask (UVol)

Measuring Cylinder

Micro Pipette (UPip)

Micro Pipette (UPip)

Micro Pipette (UPip)

Capacity

10 ml

25 ml

1000 µl

100 µl

20 µl

Std Uncertainity

0.00200

0.00200

0.11000

0.01000

0.09500

## 33.

Calculation of Combined Standard UncertainityValue

X

Standard

Uncertainity U(X)

URep

UBal

U.RF

U. VM

Upur (CAP)

Upur (ACN)

Upur (Ethyl Acetate)

Upur (Carbon Tetrachloride)

0.2912

2.0050

6000.00

2500.00

99.70

99.90

99.70

99.00

0.0086

0.04500

0.03000

0.03000

0.0015

0.0006

0.0017

0.0058

Relative

Uncertainity

UR = U(X) / X

0.029536

0.022444

0.000005

0.000012

0.000015

0.000006

0.000017

0.000059

Volumentric Flask (UVol)

Measuring Cylinder

Micro Pipette (UPip)

Micro Pipette (UPip)

Micro Pipette (UPip)

10.00

25.00

1000.00

100.00

20.00

0.0020

0.00200

0.00200

0.11000

0.01000

0.000200

0.000080

0.000002

0.001100

0.000500

Uncertainity Sources

Combined Standard Uncertainity

√UR2 =

0.01080707

Therefore, Chloramphenicol residues in shrimp (ppb) = 0.2912 ± 0.011