reviewing and approving the results to ensure consistency and reliability in API testing.

5. Procedure

5.1 Sample Preparation

1. Weigh an appropriate amount of tablet sample (usually 10 tablets) to obtain a representative quantity for testing.
2. Crush the tablets to a fine powder using a mortar and pestle or mechanical grinder to ensure uniformity.
3. Dissolve the powder in an appropriate solvent (e.g., methanol or acetonitrile) based on the solubility of the API, and filter the solution using a 0.45 µm filter.
4. If necessary, dilute the solution to bring the concentration within the optimal range for HPLC analysis.

5.2 HPLC System Setup

1. Ensure that the HPLC system is properly calibrated and the appropriate column is installed for API separation.
2. Set up the mobile phase, flow rate, and other conditions (e.g., temperature, detection wavelength) based on the method validation specifications.
3. Prime the system to ensure consistent flow and eliminate air bubbles in the pump or lines.

5.3 Retention Time Validation

1. Inject a known standard of the API into the HPLC system to establish a baseline retention time under the selected conditions.
2. Inject the prepared tablet sample and record the retention time for the API peak in the chromatogram.
3. Compare the retention time of the tablet sample with that of the standard. The retention time should match within an acceptable range (typically ±5% of the standard’s retention time).
4. Perform the test in triplicate to ensure reproducibility of the results.

5.4 Data Analysis and Calculation

1. Calculate the average retention time for the API from the triplicate injections.
2. Ensure that the average retention time falls within the specified range of the standard’s retention time. Any significant deviations must be investigated.
3. Document all observations and results, including chromatograms, retention time data, and any deviations from the expected results.

5.5 Acceptance Criteria

1. The retention time of the API should be consistent with the standard within the allowable limits (±5%).
2. If the retention time falls outside the acceptable range, initiate an investigation to identify potential issues, such as incorrect mobile phase composition, column problems, or sample contamination.
3. Corrective actions should be taken as necessary, and re-testing should be performed once the issue is resolved.

5.6 Documentation and Record-Keeping

1. Record all validation results, including chromatograms, retention time calculations, and observations in the batch record (Annexure-2).
2. Ensure that all records are signed, dated, and stored according to the company’s record retention policy for future audits and inspections.
3. Maintain raw data, including instrument logs, standard preparation details, and deviation reports, for regulatory compliance and future reference.

5.7 Post-Test Actions

1. Clean the HPLC system and associated equipment to remove any residual sample and prevent cross-contamination between tests.
2. Dispose of used samples, solvents, and reagents according to the company’s waste disposal procedures.
3. Ensure that the HPLC system is calibrated and maintained according to the manufacturer’s guidelines and company SOPs.

6. Abbreviations

• SOP: Standard Operating Procedure
• GMP: Good Manufacturing Practice
• QC: Quality Control
• QA: Quality Assurance
• API: Active Pharmaceutical Ingredient
• HPLC: High-Performance Liquid Chromatography

7. Documents

1. Batch Record (Annexure-2)
2. Deviation Report (Annexure-1)

8. References

• USP <621> – Chromatography
• 21 CFR Part 211 – Current Good Manufacturing Practice for Finished Pharmaceuticals (US FDA)
• European Pharmacopoeia (EP) – Specifications for Chromatography and Method Validation

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Deviation Report

Deviation Date	Batch Number	Deviation Description	Corrective Action	Responsible Person
15/12/2025	Batch 001	Retention time variation beyond acceptable limit	Re-calibrated HPLC system and re-tested	Jane Smith

Annexure-2: Batch Record

Batch Number	Sample Weight (g)	API Retention Time (min)	Result
Batch 001	100 g	8.5 min	Pass

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
01/01/2024	1.0	Initial Version	New SOP Creation	QA Head
01/02/2025	2.0	Updated retention time limits	Improved precision and accuracy	QA Head