is transferred from one injection to the next, thereby maintaining the integrity and reproducibility of results.

2. Scope

This SOP applies to all HPLC methods developed in the Analytical Method Development (AMD) laboratory, particularly for drug products and substances that require high sensitivity or involve potent compounds.

3. Responsibilities

• Analytical Chemist: Executes the carryover test, maintains records, and implements rinse strategies.
• Method Reviewer: Verifies test results and approves the adequacy of rinsing techniques.
• QA Officer: Ensures compliance with regulatory expectations and GMP documentation.
• Head – AMD: Reviews and authorizes the method for validation and routine analysis.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring that all validated HPLC methods are free from unacceptable levels of carryover that could impact analytical accuracy and precision.

5. Procedure

5.1 Preparation for Carryover Study

1. Identify target analyte and prepare high concentration standard (e.g., 150–200% of specification).
2. Prepare blank solution using same diluent or mobile phase as sample preparation.
3. Record all preparation details in Annexure-1: Sample and Blank Preparation Log.

5.2 System Setup and Initial Conditions

1. Use validated chromatographic conditions for the method under development (refer Annexure-2).
2. Ensure autosampler wash settings are configured for maximum rinse (needle wash, needle seat, and plunger wash if applicable).
3. Flush system with mobile phase and perform baseline stabilization before injecting.

5.3 Injection Sequence

1. Inject the following sequence:
   1. Blank
   2. High concentration standard
   3. Blank 1
   4. Blank 2
2. Evaluate peaks at retention time of analyte in Blank 1 and Blank 2.
3. Record chromatograms and peak areas in Annexure-3: Carryover Chromatographic Log.

5.4 Acceptance Criteria

1. Peak area of analyte in Blank 1 and Blank 2 should be:
   • <20% of LLOQ (Lower Limit of Quantification), OR
   • <0.002% relative to high concentration standard peak area
2. If above limit, repeat with improved rinsing (e.g., using strong wash solvents like ACN, IPA, or DMSO).
3. Repeat injection sequence until carryover is eliminated or within acceptable limits.

5.5 Mitigation Strategies

1. Evaluate alternative rinse solvents based on solubility and polarity.
2. Use extended needle wash times or dual wash solvents if needed.
3. Incorporate autosampler programming (wash between injections).
4. Document all mitigations in Annexure-4: Carryover Mitigation Log.

5.6 Reporting

1. Summarize observations and final decision on method suitability in Annexure-5: Carryover Study Report.
2. Attach chromatograms of high standard and blanks.
3. Sign-off by reviewer and Head – AMD.

6. Abbreviations

• HPLC: High-Performance Liquid Chromatography
• LOD: Limit of Detection
• LLOQ: Lower Limit of Quantification
• SOP: Standard Operating Procedure

7. Documents

1. Sample and Blank Preparation Log – Annexure-1
2. Chromatographic Method Parameters – Annexure-2
3. Carryover Chromatographic Log – Annexure-3
4. Carryover Mitigation Log – Annexure-4
5. Carryover Study Report – Annexure-5

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP <621> – Chromatography
• FDA Guidance – Analytical Procedures and Methods 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: Sample and Blank Preparation Log

ID	Type	Concentration	Diluent	Prepared By
STD-H	High Conc. Std	200 µg/mL	50:50 Water:ACN	Sunita Reddy
BLK-1	Blank	NA	50:50 Water:ACN	Rajesh Kumar

Annexure-2: Chromatographic Method Parameters

Column	Mobile Phase	Flow Rate	Detection	Run Time
C18, 150×4.6 mm	Buffer:ACN (60:40)	1.0 mL/min	UV 254 nm	10 min

Annexure-3: Carryover Chromatographic Log

Injection	Peak Area	% Carryover	Result
BLK-1	132	0.001%	Complies
BLK-2	112	0.0009%	Complies

Annexure-4: Carryover Mitigation Log

Solvent	Wash Duration	Change Implemented	Effectiveness
IPA	5 sec	Increased wash volume	Effective

Annexure-5: Carryover Study Report

Analyte	Result	Decision	Reviewed By
Compound X	Carryover ≤ 0.002%	Acceptable	QA Reviewer

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included dual blank injections and mitigation annexure	Regulatory update