ASSAY METHOD VALIDATION PROTOCOL OF Ofloxacin and Ornidazole Tablets

ASSAY METHOD VALIDATION PROTOCOL OF Ofloxacin & Ornidazole Tablets

Superseded Protocol No.	Nil
Effective Date

Table of contents :

Sr. No.	Subject
	Protocol Approval
	Objective
	Scope
	Responsibility of validation team
	Product profile
	Methodology
	Revision History

1. Protocol Approval:

Prepared By:

Functional Area	Name	Signature	Date
Officer / Executive Quality Control

Reviewed By:

Functional Area	Name	Signature	Date
Manager QA
Head QC

Approved By:

Functional Area	Name	Signature	Date
Head QA

Authorized By:

Functional Area	Name	Signature	Date
Head Quality

• Objective:

The objective of this validation is to validate the assay method of Ofloxacin & Ornidazole Tablets by considering the following parameters:

• Precision
• System Precision
• Method Precision
• Intermediate Precision (Ruggedness)
• Specificity
• Forced Degradation
• Linearity
• Accuracy
• Range
• Stability in Analytical Solutions
• Filter Paper Selection Study
• Robustness  

Following parameters will be seen during system suitability:

Sr. No.	Parameters	Limit
1.	Tailing Factor of the Ofloxacin & Ornidazole.	Not more than 2.0
2.	% RSD for peak area of Ofloxacin & Ornidazole from 6 replicate injections of standard solution.	Not more than 2.0
3.	Resolution between Ofloxacin & Ornidazole.	Not Less than 2.0
4.	Theoretical plates of the Ofloxacin & Ornidazole.	Not Less than 2000

When the above parameter meets the method, the actual experiment shall be started.

• Scope :

This protocol is based on SOP and applicable for the validation of assay method of Ofloxacin & Ornidazole in Ofloxacin & Ornidazole tablet.

• Responsibility of Validation Team:

Departments	Responsibilities
QC	Preparation & Review of Protocol.
Analysis of samples and recording of data.
Compilation and checking of data
Preparation of Summary Report.
To impart training of protocol to concerned department/persons.
QA	Review and approval of protocol.
Co-ordination with QC to carryout Validation.
Review of data and summary report.
Head Quality	Authorization of protocol.

• Product Profile:

Category (% API Content)
Reason for Validation
Active Ingredient	Ofloxacin & Ornidazole
Strength	Each Film Coated tablet contains: Ofloxacin IP 200 mg Ornidazole IP 500 mg
Methodology	Non-Pharmacopeial
Method Reference	IH
Specification Limits	90 % -110 %

• Methodology:
  • Precision:
    • System Precision:

Test Procedure:

Prepare standard solution as per test method and inject for 6 times.

Documentation:

Record the retention time, peak area, and peak height on the datasheet. Calculate the mean, standard deviation, and % RSD of area.

Acceptance criteria

% RSD of area shall be not more than 2.

• Method Precision:

Test Procedure:

Prepare six samples and analyze as per finished product testing procedure.

Documentation:

Record the % Assay of Ofloxacin & Ornidazole in Ofloxacin & Ornidazole Tablet on the datasheet. Calculate the mean, standard deviation, and % RSD of area.

Acceptance criteria

% RSD of area shall be not more than 2.

• Intermediate Precision (Ruggedness):

Test Procedure:

Prepare six samples and analyse as per finished product testing procedure by different analyst by using different systems on different day. Evaluate the system suitability criteria.

Documentation:

Calculate the mean, standard deviation, and % RSD of area for the operators and instruments and record on data sheet.

Acceptance criteria

% RSD of area shall be not more than 2.

• Specificity:

Test Procedure:

Prepare and inject blank (diluents), standard, sample & placebo.

Documentation:

Print chromatograms.

Acceptance criteria

The excipient compounds must not interfere with the analysis of the targeted analyte. No peak shall be eluted at the retention time of Ofloxacin & Ornidazole in blank and placebo.

• Forced Degradation:

Test Procedure:

Stress the sample at the following conditions and evaluate the peak purity.

• Degradation by Hydrochloric Acid
• Degradation by Sodium Hydroxide
• Degradation by hydrogen peroxide
• Degradation by thermal
• Degradation by water hydrolysis

Documentation:

Print chromatograms.

Acceptance criteria

Peak purity shall be passed. The difference between treated and untreated sample shall be not more than 10 %.

• Linearity:

Test Procedure:

Prepare the standard solutions at six concentrations, typically 25, 50, 75, 100, 150, and 200% of target concentration following finished product testing procedure. Three individually prepared replicates at each concentration will be analyzed.

Documentation:

Record results on a datasheet. Calculate the mean, standard deviation, and Relative Standard Deviation (RSD) for each concentration. Plot concentration (x-axis) versus mean response (y-axis) for each concentration. Calculate the correlation coefficient (r). Record these calculations on the datasheet.

Acceptance criteria

The correlation coefficient (r) shall be not less than 0.99.

• Accuracy:

Test Procedure:

Prepare sample solution by spiking the Ofloxacin & Ornidazole (Drug) with placebo at about 50%, 100% and 150% of test concentration and analyze as per finished product testing procedure.

Documentation:

For each sample, record the theoretical value, assay value, and percent recovery on test data sheet. Calculate the mean, standard deviation, RSD, and percent recovery for all samples. Record results on the datasheet.

Acceptance criteria

The mean recovery at each level shall be within 50% to 150 % of the theoretical value and the % RSD not more than 2.

• Range:

Test Procedure:

The data obtained during the linearity and accuracy studies will be used to assess the range of the method. The precision data used for this assessment is the precision of the three replicate samples analyzed at each level in the accuracy studies.

Documentation:

Record the range on the datasheet.

Acceptance criteria

The acceptable range shall be defined as the concentration interval over which linearity and accuracy are obtained as per the above criteria.

• Stability in Analytical Solutions;

Test Procedure:

Prepare standard and one sample solution as per finished product testing procedure and inject into HPLC at initial and different time interval 12 and 24 hours and determine the % assay.

Documentation:

Calculate the % assay.

Acceptance criteria

The difference in the results shall be not more than 2 %.

• Filter Paper Selection Study :

Test Procedure:

Prepare sample solution in triplicate as per the finished product testing procedure and filter test with 0.45m PVDF filter and 0.45m Nylon filter and centrifuge a portion of filter.

Documentation:

Calculate the % assay of centrifuged and filtered samples.

Acceptance criteria

The difference in the results shall be not more than 2%.

• Robustness:

Test Procedure:

Prepare the sample solution in triplicate and analyze under the following conditions as per finished product testing procedure.

• By changing the flow rate by ±10%
• By changing the temperature by ±5°C
• By changing the organic content by ±10%
• By changing the pH of solvent in mobile phase by ±0.2 units

System suitability shall be done in each condition.

 Documentation:

Calculate the % assay. Calculate the % RSD for % assay for individual above experiment with method precision results. 

Acceptance criteria

Overall % RSD shall be not more than 2.

• Revision History:

Revision No.	Details of changes	Reason
00	Nil	New Document