is essential where isocratic elution fails to achieve required resolution within an acceptable run time.

2. Scope

This procedure applies to all HPLC-based analytical method development activities involving multi-component APIs, impurity profiling, degradation product separation, and chromatographically challenging formulations carried out in the Analytical Method Development (AMD) department.

3. Responsibilities

• Analytical Chemist: Responsible for method scouting, gradient optimization, data acquisition, documentation, and reporting.
• Reviewer: Reviews chromatographic data, confirms peak resolution and retention reproducibility, and checks gradient program logic.
• QA Officer: Ensures the developed method complies with regulatory expectations and ICH guidelines.
• Head – AMD: Reviews and approves gradient method for further transfer or regulatory submission.

4. Accountability

The Head of AMD is accountable for ensuring method suitability, robustness, and adherence to industry and regulatory standards for gradient-based HPLC methods.

5. Procedure

5.1 Pre-Development Considerations

1. Review existing monographs, research literature, and degradation profiles.
2. Analyze the complexity of the target mixture (e.g., number of analytes, expected impurities).
3. Record API profiles and existing impurity data in Annexure-1: Gradient Feasibility Assessment Log.

5.2 Column and Mobile Phase Selection

1. Choose a C18 column for general applications unless specific retention issues require alternatives (e.g., phenyl, CN).
2. Mobile phases typically involve aqueous buffer (A) and organic solvent (B) such as acetonitrile or methanol.
3. Select buffer based on API pKa and solubility (phosphate, acetate, or formate); pH range 2.5 to 7.5 is preferred.
4. Document choices in Annexure-2: Mobile Phase Composition Record.

5.3 Gradient Program Design

1. Start with generic linear gradient: 5% B to 95% B over 30 minutes at 1.0 mL/min.
2. Assess elution behavior of all components:
   • Retention window
   • Peak shape and symmetry
   • Baseline resolution
3. Refine gradient slope and hold times based on initial results:
   • Use shallow gradient in regions of closely eluting peaks
   • Add isocratic segments to improve separation
4. Optimize stepwise gradients if tailing or overlap persists.
5. Record gradient table and rationale in Annexure-3: Gradient Program Sheet.

5.4 Sample and Standard Preparation

1. Use API standard and spiked placebo (formulation excipients + known impurities).
2. Filter through 0.45 µm membrane filter.
3. Record solution prep in Annexure-4: Standard and Sample Prep Log.

5.5 System Suitability

1. Inject system suitability mixture (API + known impurities).
2. Check for:
   • Resolution ≥ 2.0 between critical peaks
   • Tailing ≤ 2.0
   • RSD of area ≤ 2.0% for replicate injections
3. Document in Annexure-5: System Suitability Record.

5.6 Method Validation

1. Specificity: Ensure no co-elution from excipients or impurities.
2. Linearity: Evaluate from 50% to 150% concentration range (R² ≥ 0.999).
3. Accuracy: Perform recovery at 80%, 100%, 120% with recovery 98–102%.
4. Precision: RSD ≤ 2.0% (repeatability and intermediate precision).
5. LOD/LOQ: Validate via S/N or slope method.
6. Robustness: Vary gradient steps, flow rate, pH, and temperature.
7. Summarize results in Annexure-6: Gradient Method Validation Summary.

6. Abbreviations

• HPLC: High Performance Liquid Chromatography
• API: Active Pharmaceutical Ingredient
• PDA: Photodiode Array
• RSD: Relative Standard Deviation
• LOD: Limit of Detection
• LOQ: Limit of Quantification
• SOP: Standard Operating Procedure

7. Documents

1. Gradient Feasibility Assessment Log – Annexure-1
2. Mobile Phase Composition Record – Annexure-2
3. Gradient Program Sheet – Annexure-3
4. Standard and Sample Prep Log – Annexure-4
5. System Suitability Record – Annexure-5
6. Gradient Method Validation Summary – Annexure-6

8. References

• USP General Chapter <621> – Chromatography
• ICH Q2(R1) – Validation of Analytical Procedures
• FDA Guidance for Industry – Analytical Procedures and Methods Validation
• Pharmacopoeia of India (IP)

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Gradient Feasibility Assessment Log

Product	Complexity	Number of Components	Gradient Need Justification	Analyst
XYZ Combo Tablet	High	6	Co-eluting impurities	Sunita Reddy

Annexure-2: Mobile Phase Composition Record

Mobile Phase A	Mobile Phase B	pH	Filtered	Date Prepared
10 mM phosphate buffer	Acetonitrile	3.5	Yes	18/05/2025

Annexure-3: Gradient Program Sheet

Time (min)	%A	%B	Flow Rate (mL/min)
0	95	5	1.0
10	60	40	1.0
20	30	70	1.0
25	5	95	1.0
30	95	5	1.0

Annexure-4: Standard and Sample Prep Log

Sample	Concentration	Diluent	Filtered	Prepared By
API + Impurities	100 µg/mL	Mobile Phase	0.45 µm	Rajesh Kumar

Annexure-5: System Suitability Record

Injection	Retention Time	Peak Area	Resolution	Plate Count
1	12.1	145632	3.2	8500

Annexure-6: Gradient Method Validation Summary

Parameter	Acceptance Criteria	Observed Result	Status
Accuracy	98–102%	99.4%	Pass
Precision	RSD ≤ 2.0%	0.91%	Pass
Linearity	R² ≥ 0.999	0.9991	Pass
Robustness	No significant change	Compliant	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Refined gradient slope and added system suitability parameters	Annual Review