Analytical Method Validation Protocol for Titration Method

TABLE OF CONTENT

1. Introduction

2. Objective

3. Method validation parameters

4. Re-validation criteria

5. Method of analysis

6. Experimental Plan and Data Evaluation

7. Deviation

8. Procedure

8.1 Specificity

8.2 Linearity and Range

8.3 Accuracy (Recovery)

8.4 Method Precision

8.5 Intermediate Precision

8.6 Solution Stability

8.7 Filter Interference

8.8 Robustness

9. Validation report

1. Introduction

The titrimetric method is used for determining the amount of Sodium Chloride in Sodium Chloride Infusion 0.9% w/v.

2. Objective

The objective of this protocol is to give in detail the procedure for validating the analytical method used for determining the amount of Sodium Chloride in Sodium Chloride Infusion 0.9% w/v.

3. Method Validation Parameters

The assay method validation will include the following parameters:

• Specificity
• Linearity
• Accuracy
• Method Precision
• Intermediate Precision
• Solution stability
• Filter Interference
• Robustness

4. Revalidation Criteria

The method will be revalidated for the following changes:

• Change in manufacturing formula
• Change in analytical procedure
• Drug substances specification change concerning the addition of impurity (ies) only.
• Any change to pharmacopeial methodology or agency requirement.
• After 10 years, validation is to be evaluated for the above points, if there is no change to the above-mentioned points, the existing validation report will be held valid.

5. Method of Analysis

Product Name: Sodium Chloride 0.9% w/v Intravenous Infusion

Title: To determine the amount of Sodium Chloride in Sodium Chloride Intravenous Infusion 0.9% w/v by Titrimetric method.

Reagents Preparation: 0.1 M Silver Nitrate

Preparation

Dissolve about 1 7 g in sufficient water to produce 1000 ml and standardize the solution as follows:

Standardization

Weigh accurately about 0.1 g of sodium chloride previously dried at 110℃ for two hours and dissolve in 5 ml of water. Add 5 ml of acetic acid, 50 ml of methyl alcohol, and three drops of eosin solution. Stir, preferably with a magnetic stirrer, and titrate with the silver nitrate solution. 

Each 0.005844 g of sodium chloride is equivalent to I ml of 0.1 M silver nitrate.

Sample Preparation:

Take 20 ml of the finished product sample in a 250 ml conical flask, add 2-3 drops of Potassium chromate solution as an indicator, and Titrate with 0.1 M Silver nitrate. The endpoint is brick red colored observed. 

Each ml of 0.1 M Silver nitrate is equivalent to 5.844 mg of Sodium Chloride. The endpoint of the sample is wine red to brick red colored.

Calculation:

Calculate the content of Sodium Chloride g/100ml by the following expression:

Limit: Assay should be 95.0% to I 05.0%

6. Experimental Plan and Data Evaluation

The analytical method validation will be executed as per the following plan:

Note:

• The experiments may be done as sequential or parallel operations.
• The sample sequence of each experiment may be done together or independently with necessary alteration of sample preparation.

7. Deviations

• Deviations from Protocol
• Deviations from Method

ALSO READ: Process Validation Protocol for Oral Rehydration Salt

8. Procedure

8.1 Specificity

Preparation of Blank Solution:

Taken 20 ml of water sample in a 250 ml conical flask, added 2-3 drops of Potassium chromate solution as indicator. Titrate with 0.1 M Silver nitrate. The endpoint is brick red colored observed. 

Each ml of 0.1 M Silver nitrate is equivalent to 5.844 mg of Sodium Chloride. End point of the sample is wine red to brick red colored.

Preparation of Placebo Solution:

Taken 20 ml of placebo sample in a 250 ml conical flask, added 2-3 drops of Potassium chromate solution as indicator. Titrate with 0.1 M Silver nitrate. The endpoint is brick-red colored observed. Each ml of 0.1 M Silver nitrate is equivalent to 5.844 mg of Sodium Chloride.

End point of the sample is wine red to brick red colored.

Preparation of Sample Solution:

Taken 20 ml of the finished product sample in a 250 ml conical flask, added 2-3 drops of Potassium chromate solution as an indicator, Titrate with 0.1 M Silver nitrate. The endpoint is brick-red colored observed. Each ml of 0.1 M Silver nitrate is equivalent to 5.844 mg of Sodium Chloride.

End point of the sample is wine red to brick red colored.

Sample Sequence for Specificity

Sample Name	Number of Titration	Type of Testing
Blank Solution	1	Specificity
Placebo Sample	1	Specificity
Sample Solution	1	Specificity

Acceptance Criteria:

Blank & Placebo sample burette reading should not be more than 0.05% of the sample burette reading to convert wine red color to brick red color.

8.2 Linearity and Range

A series of Standard solutions of Sodium Chloride will be prepared over a range of 50% to 120% of working level concentration by weighing standard of Sodium Chloride.

Linearity Level	Amount of Chloride Standard taken (mg)	Dilute to 100 ml
50%	450	100 ml
60%	540	100 ml
80%	720	100 ml
100%	900	100 ml
120%	1080	100 ml

Sample Sequence for Linearity

Sample Name	Number of Titration	Type of Testing
Linearity 50%	1	Level 1
Linearity 60%	1	Level 2
Linearity 80%	1	Level 3
Linearity 100%	1	Level 4
Linearity 120%	1	Level 5

Acceptance Criteria:

From the linearity graph obtained, calculate the correlation coefficient and Y-intercept.

The value obtained for the correlation coefficient should be not less than 0.99.

ALSO READ: SOP for Analytical Method Verification

8.3 Accuracy (Recovery):

The accuracy will be carried out at three different levels: 80%, 100% and 120% of the working level concentration by adding a known amount of the drug into the placebo as given in the Table below:

Further, the analysis of these solutions was carried out according to the assay procedure.

Accuracy Level	Amount of Chloride added (mg)	Dilute to 100 ml
80%	720	100 ml
100%	900	100 ml
120%	1080	100 ml

Sample Sequence for Accuracy

Sample Name	Number of Titration	Type of Testing
80% - Preparation 1	1	Accuracy Level 1
80% - Preparation 2	1
80% - Preparation 3	1
100% - Preparation 1	1	Accuracy Level 2
100% - Preparation 2	1
100% - Preparation 3	1
120% - Preparation 1	1	Accuracy Level 3
120% - Preparation 2	1
120% - Preparation 3	1

Acceptance Criteria:

The recovery obtained for each respective level should be between 98% to 102.0% and % RSD should be not more than 2.0

8.4 Method Precision

Six individual assay preparations will be prepared and analyzed. Taken 20 ml of the finished product sample in a 250 ml conical flask, added 2-3 drops of Potassium chromate solution as an indicator, Titrate with 0 . 1 M Silver nitrate. The endpoint is brick red colored observed. 

Each ml of 0.1 M Silver nitrate is equivalent to 5.844 mg of Sodium Chloride. The endpoint of the sample is wine red to brick red colored.

Sample Sequence for Method Precision

Sample Name	Number of Titration	Type of Testing
Precision SPL 1	1	Method Precision
Precision SPL 2	1
Precision SPL 3	1
Precision SPL 4	1
Precision SPL 5	1
Precision SPL 6	1

Acceptance Criteria:

The % Assay content of Sodium Chloride of these six assay preparations should be within 98.0 to 102.0% and the % RSD for the same should not be more than 2.0

8.5 Intermediate Precision

Intermediate precision is precision within laboratories variation, such as different days, analyst, equipment etc.

Taken 20 ml of the finished product sample in a 250 ml conical flask, added 2-3 drops of Potassium chromate solution as an indicator, Titrate with 0.1 M Silver nitrate. The endpoint is brick red colored observed. Each ml of 0.1 M Silver nitrate is equivalent to 5.844 mg of Sodium Chloride. The end point of sample is wine red to brick red colored.

Sample Sequence for Intermediate Precision

Sample Name	Number of Titration	Type of Testing
Inter. Precision SPL 1	1	Intermediate Precision
Inter. Precision SPL 2	1
Inter. Precision SPL 3	1
Inter. Precision SPL 4	1
Inter. Precision SPL 5	1
Inter. Precision SPL 6	1

ALSO READ: SOP for Transport Validation

Acceptance Criteria:

The % Assay content of Sodium Chloride of these six assay preparations should be within 98.0% to 102.0% and the % RSD for the same should not be more than 2.0

The % Assay of these twelve assay preparations (six of method precision and six of intermediate precision) should be within 98.0% to 102.0% and % RSD for the same should not be more than 2.0

8.6 Solution Stability:

The stability of the sample solutions Sodium Chloride Infusion of contain Sodium Chloride will determine at the time periods representative for storage (About 48 hrs with intermediate time points, further it may be studied till 72 hours). Test sample solution stability will be by assessing the test sample preparation as per the method at the initial time point or 0 hr then at other intervals and compare against freshly assessed standard. % Difference of reading within the mean of each interval should be within ± 2.0 %.

Sample Preparation:

Take 20 ml of the finished product sample in a 250 ml conical flask, add 2-3 drops of Potassium chromate solution as an indicator, Titrate with 0.1 M Silver nitrate. The endpoint is brick red colored observed. Each ml of 0.1 M Silver nitrate is equivalent to 5.844 mg of Sodium Chloride. End point of a sample is wine red to brick red colored.

Sample Sequence for Solution Stability

Time in Hr	Sample Name	Titration Reading	% Assay	Absolute Difference % Assay	Type of Testing
0 Hrs	Sodium Chloride Infusion Batch No.:	1	1		Solution Stability
8 Hrs		1	1
12 Hrs		1	1
24 Hrs		1	1
36 Hrs		1	1
72 Hrs		1	1

Acceptance Criteria:

Sample solution stability will be determined by comparing it to its initial value. % Difference of Assay value will be calculated and it should be within ± 2.0 %.

8.7 Filter Interference:

The possible interference from different filters used during Sample preparation will check. A titration value obtained for the solution without filtered will check against titration value of the filtered solution through Centrifuge, Whatman PVDF filters (GD/X) 0.45μm, Millipore millex HN, Nylon 0.45μ and Millipore millex HV, Hydrophilic PVDF 0.45μ. The absolute difference will be calculated and report (NMT ± 2.0).

Sample Preparation:

Take 20 ml of the finished product sample in a 250 ml conical flask, add 2-3 drops of Potassium chromate solution as an indicator, Titrate with 0.1 M Silver nitrate. The endpoint is brick red colored observed. Each ml of 0.1 M Silver nitrate is' equivalent to 5.844 mg of Sodium Chloride. The end point of a sample is wine red to brick red colored.

Sample Sequence for Filter Interference

Type of Filter paper	Sample Name	Titration Reading	% Assay	Absolute Difference	Type of Testing
Without Filter	Sodium Chloride Infusion Batch No.:	1	1		Filter Interference
Centrifuge		1	1
Whatman PVDF filters (GD/X) 0.45m		1	1
Millipore millex HN		1	1
Nylon 0.45μ		1	1
Millipore millex HV		1	1
Hydrophilic PVDF 0.45μ		1	1

Acceptance Criteria:

The sample solution without the filter will be determined by comparing it to its initial value.% Difference will be calculated and it should be within ± 2.0 %.

8.8 Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected (i. e. no significant difference between results) by small but detectable variation in method parameters and equipment's (e.g. change in volume of sample). This will study by deliberately changing the following parameters.

Volume of Sample taken (20 ml ± 5 mL target 15 mL & 25 mL)

Sample Preparation:

Take 20 ml of the finished product sample in a 250 ml conical flask, add 2-3 drops of Potassium chromate solution as an indicator, and Titrate with 0.1 M Silver nitrate. The end point is brick red colored observed. Each ml of 0.1 M Silver nitrate is equivalent to 5.844 mg of Sodium Chloride. Endpoint of sample is wine red to brick red colored.

Sample Sequence for Robustness

Changes in Parameters	Sample Name	Titration  Reading	% Assay	Absolute Difference % Assay	Type of Testing
Initial volume 15 mL	Sodium Chloride Infusion Batch No.:	1			Robustness
Initial volume 20 mL		1
Initial volume 25 mL		1

Acceptance Criteria:

Sample solution volume sample taken changes, will determine by comparing to its initial value. % Difference will calculate and it should be within± 2.0 %.

9. Validation Report

• The validation report will be prepared under the following headers:
• Introduction
• Method -- the Method that followed in the analytical method validation including the calculation formula.
• Validation data for all the stated parameters
• Conclusion
• Figures to include linearity of response of Sodium Chloride. The raw data will be submitted according to the experimentation and a summary of the references of experimentation will be provided.

ALSO READ: Checklist for Analytical Method Validation