VALIDATION Protocol No.

: xxxxx Reference: In-house

PROTOCOL Department : Quality control Page 1 of 9

TITLE: DULOXETINE HCl ASSAY BY POTENTIOMETRY

I Objective:

To provide a procedure for the validation of the method used in the analysis of
Duloxetine HCl assay by potentiometry on dried basis.

II Scope:
This protocol applies to the In-house method (Assay by potentiometry on dried basis),
used for carrying out the analysis of Duloxetine HCl.
III Responsibility:
It is the responsibility of Quality control department to carry out the validation study
for the method as per the procedure mentioned in the protocol.
IV Method details

Apparatus : Potentiometer.
Reagents : 0.1 N Perchloric acid solution.
Glacial acetic acid.
Procedure for Assay :
 Weigh accurately about 0.300 g of test sample and transfer to the 100mL beaker.
 Add 50 mL of glacial acetic acid and dissolve the contents.
 Feed the parameters to the system (Autotitrator) like Normality of 0.1N Perchloric acid,
factor of Duloxetine HCl, weight of the test sample taken for sample preparations
as per operational procedure of the Autotitrator.
 Titrate the contents of the beaker against 0.1N Perchloric acid and determine
end point potentiometrically (TV).

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 VALIDATION Protocol No. :xxxxxxxxxx Reference: In-house
PROTOCOL Department : Quality control Page 2 of 9

TITLE: DULOXETINE HCl ASSAY BY POTENTIOMETRY

 Perform the blank in the same manner by omitting the sample being examined.
 The titrant volume consumed for blank is (B)
 Calculate the assay % with the following formula.
Sample preparation –I
TV = Volume of the Titrant consumed for sample.
Assay, % w/w =
(TV-B) x Normality of 0.1N Perchloric acid x 0.3319 x 100
Weight of the test sample taken in grams

% Assay on dried basis, w/w = % Assay of sample preparation -I x 100

(100 – %LOD)
Sample preparation –II
Assay, % w/w =
(TV-B) x Normality of 0.1N Perchloric acid x 0.3319 x 100
Weight of the test sample taken in grams

% Assay on dried basis, w/w = % Assay of sample preparation -II x 100

(100 – %LOD)
 The % assay on dried basis values obtained for sample preparation - I and sample

preparation – II should comply with the specification limit individually.

 If the % assay on dried basis values are with in the specified limit for both the
sample preparations – I and II , calculate the variation for % assay on dried basis

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 VALIDATION Protocol No. :xxxxxxxxxx Reference: In-house
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TITLE: DULOXETINE HCl ASSAY BY POTENTIOMETRY

by the following formula.

Difference in % assay on dried basis x 100 = Variation in %

Highest % assay on dried basis
 The % assay on dried basis results are acceptable only if the variation is less than 1.0.

 If the acceptance criteria for variation is met, then report the mean % assay on dried

basis, w/w as the final result.

 Calculate the mean % assay on dried basis using the following formula.
Mean % = % assay on dried basis of sample prep’n - I + % assay on dried basis of sample prep’n – II
2
 Report the mean assay value as the result of Assay by potentiometry on dried basis.
Specification: Assay by potentiometry on dried basis should be not less than
98.0% and not more than 102.0% w/w.

V. Experimental design:
The following parameters should be considered for the Validation:
1. Specificity
2. Precision
3. Linearity and Range
4. Intermediate Precision
Note : Accuracy is not required, since the methodology is the spiked samples.

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TITLE: DULOXETINE HCl ASSAY BY POTENTIOMETRY

Linearity and Precision gives the inference for accuracy, simply estimating assay of
standard(true value)
Comparing the result of true value and the value found gives accuracy.

1) Specificity
Blank:
 Take 50 mL of glacial acetic acid in a 100 mL beaker.
 Titrate the contents of the beaker against 0.1N Perchloric acid and determine end point
potentiometrically (TV).
Acceptance criteria: The volume of 0.1N Perchloric acid solution consumed
for blank should not be more than 0.3mL.
2) Precision:

 Take 50mL of glacial acetic acid in a 100 mL beaker.

 Weigh accurately about 0.400 g of test sample and transfer to the above beaker and
dissolve the contents.
 Titrate the contents of the beaker against 0.1N Perchloric acid solution and determine

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TITLE: DULOXETINE HCl ASSAY BY POTENTIOMETRY

end point potentiometrically (TV).
 Perform blank in the same manner by omitting the sample being examined (B)
 Calculate the Assay % on dried basis and record the results in data sheet.
 Determine the Assay on dried basis in replicates (6 times) and calculate the % RSD.
Acceptance criterion:
1. The %RSD value Duloxetine HCl assay on dried basis determined for
sample in replicate (6 times) should be less than 1.0.
2. 95% confidence interval (x± 1.96 (s /√n ) of mean is between 98 to 102 %.
[Where x = Average, s = standard deviation and n is number of replicates.
1.96 = two tailed t value from t-distribution at infinity degrees of freedom]

3) Linearity and range:

Preparation of Linearity Level-1 solution: Weigh accurately about 0.240 g of
Duloxetine HCl test sample in a 100 mL beaker containing 50 mL of glacial acetic acid
sonicate to mix well.
Preparation of Linearity Level-2 solution: Weigh accurately about 0.270 g of
Duloxetine HCl test sample in a 100 mL beaker containing 50 mL of glacial acetic acid
sonicate to mix well.
Preparation of Linearity Level-3 solution: Weigh accurately about 0.300 g of
Duloxetine HCl test sample in a 100 mL beaker containing 50 mL of glacial acetic acid
sonicate to mix well.

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TITLE: DULOXETINE HCl ASSAY BY POTENTIOMETRY

Preparation of Linearity Level-4 solution: Weigh accurately about 0.330 g of
Duloxetine HCl test sample in a 100 mL beaker containing 50 mL of glacial acetic acid
sonicate to mix well.
Preparation of Linearity Level-5 solution: Weigh accurately about 0.360 g of
Duloxetine HCl test sample in a 100 mL beaker containing 50 mL of glacial acetic acid
sonicate to mix well.

Procedure: Titrate the Level-1and level-5 solutions in 5 replicates and other

levels in triplicate and note down the titer values. Obtain a linearity curve by
plotting the concentration (X-axis) against titer values of 0.1N Perchloric acid
solution (Y-axis). Determine the linearity Correlation co-efficient and %Y intercept
of the linearity curve.
Calculation
1. Linearity Correlation co-efficient: Calculate using Microsoft Excel.
2. Calculate the % Y Intercept as followed.

(Y-intercept x 100) / 100% Level Burette reading

3. Residual plot: Draw the plot using Microsoft Excel.

4. Residuals ± 2 % to 100 % of Level-3 specification Level.

5. Report the residual sum of squares.

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TITLE: DULOXETINE HCl ASSAY BY POTENTIOMETRY

Acceptance criteria
1. The linearity Correlation co-efficient should be more than 0.999.
2. The % Y intercept should be between + 2.0
3. There is no systematic trend in residuals
4. Residuals are with in  2% of the 100% concentration burette volume.
Range Lower level: Prepare the linearity level-1 in 5 replicates and determin the
Assay on dried basis content of each preparation as detailed in the method details.
Range upper level: Prepare the linearity level-5 in 5 replicates and determine the
Assay on dried basis content of each preparation as detailed in the method details.

Acceptance criteria: The % RSD of the Assay on dried basis results obtained from
5 replicates of each level should be less than 1.0.

4) Intermediate Precision:
Perform the procedure as detailed in precision study on a different day, by a different
analyst using with freshly prepared solutions using the same batch sample used in
precision study.
Calculate Assay on dried basis content and record the results in the analysis work
book.
Procedure:

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TITLE: DULOXETINE HCl ASSAY BY POTENTIOMETRY

1) Determine the Assay on dried basis in 6 replicates.
2) Calculate the % RSD of Assay on dried basis content obtained from intermediate
precision study.
3) Calculate the cumulative % RSD for the Assay on dried basis content result obtained
from precision study and Reproducibility study.
4) 95% confidence interval (x± 1.96 (s / √n ) of mean
[Where x = Avearage, s = standard deviation and n is number of replicates.
1.96 = two tailed t value from student t-distribution at infinity degrees of freedom]

Acceptance criteria:
1) The %RSD value of Assay on dried basis content determined for sample in replicates
(6 times) should be less than 1.0.
2) The cumulative % RSD for the Assay on dried basis content result obtained from
precision study and Intermediate precision study should be less than 1.0.
3) Report the 95% confidence interval of mean.

VI. Conclusion: Conclude from the results of the above experimental design, whether the
applied method is validated to carrying out the analysis (Assay by potentiometry on
dried basis) of Duloxetine HCl

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TITLE: DULOXETINE HCl ASSAY BY POTENTIOMETRY

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