optimal separation, reproducibility, and system suitability for pharmaceutical substances and products.

2. Scope

This procedure applies to the Analytical Method Development (AMD) department and shall be followed for all API, excipient, and finished product analyses where chromatographic method development or optimization is required using HPLC systems.

3. Responsibilities

• Analytical Chemist: Conducts column and mobile phase screening, documents evaluations, and proposes optimized conditions.
• Method Reviewer: Verifies chromatographic responses, ensures resolution and system suitability criteria are met.
• QA Officer: Reviews documentation and ensures compliance with internal and regulatory expectations.
• Head – AMD: Approves the final selection of column and mobile phase for method validation or technology transfer.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring robust and scientifically justified chromatographic conditions are finalized before method validation.

5. Procedure

5.1 Review of API/Analyte Characteristics

1. Collect data on the physicochemical properties of the analyte:
   • pKa, logP, solubility
   • UV absorbance (λmax)
   • Stability under acidic/basic conditions
2. Document findings in Annexure-1: Analyte Characterization Sheet.

5.2 Column Selection Criteria

1. Start with a reversed-phase C18 column (150 × 4.6 mm, 5 µm) for general applications.
2. For early eluting or highly polar compounds, consider:
   • C8 columns for less retention
   • Polar-embedded or phenyl-hexyl columns for aromatic compounds
   • Amino or CN columns for sugar-type molecules or polar excipients
3. For stability-indicating methods, evaluate column performance under stress conditions (acid/base/hydrolysis).
4. Record all column trials in Annexure-2: Column Screening Log.

5.3 Mobile Phase Selection Criteria

1. Begin with water:acetonitrile or water:methanol systems in ratios of 70:30, 60:40, or 50:50.
2. Select buffer if pH-sensitive analytes are involved:
   • Common buffers: phosphate, acetate, formate (pH 3.0–7.0)
   • Buffer concentration: typically 10–20 mM
3. Ensure the buffer is UV transparent and compatible with the column and detector.
4. Document buffer composition and preparation in Annexure-3: Mobile Phase Preparation Log.

5.4 Screening Experiments

1. Run the same standard/sample across at least 3 different columns and 2 mobile phase systems.
2. Evaluate:
   • Retention time
   • Peak shape (tailing factor ≤ 2.0)
   • Resolution (Rs ≥ 2.0)
   • Theoretical plates (≥ 2000)
3. Record all observations in Annexure-4: Chromatographic Evaluation Table.

5.5 Final Selection and Justification

1. Select the column and mobile phase that provides:
   • Best peak symmetry
   • Minimum run time
   • Good resolution between API, degradants, and excipients
   • Stable baseline and reproducible system suitability
2. Justify final selection based on performance and regulatory suitability.
3. Document final method in Annexure-5: Final Selection and Justification Sheet.

6. Abbreviations

• HPLC: High-Performance Liquid Chromatography
• API: Active Pharmaceutical Ingredient
• Rs: Resolution
• PDA: Photodiode Array
• SOP: Standard Operating Procedure

7. Documents

1. Analyte Characterization Sheet – Annexure-1
2. Column Screening Log – Annexure-2
3. Mobile Phase Preparation Log – Annexure-3
4. Chromatographic Evaluation Table – Annexure-4
5. Final Selection and Justification Sheet – Annexure-5

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP General Chapter <621> – Chromatography
• FDA Guidance on Analytical Procedures 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: Analyte Characterization Sheet

API	pKa	LogP	λmax	Solubility	Analyst
Compound X	4.3	2.1	240 nm	Freely soluble in water	Sunita Reddy

Annexure-2: Column Screening Log

Column	Manufacturer	Dimensions	Performance	Selected
C18	Waters	150 × 4.6 mm	Good separation	Yes

Annexure-3: Mobile Phase Preparation Log

Aqueous Phase	Organic Phase	pH	Buffer	Prepared By
Water (Milli-Q)	Acetonitrile	3.5	10 mM phosphate	Rajesh Kumar

Annexure-4: Chromatographic Evaluation Table

Trial	Column	Mobile Phase	Retention Time	Rs	Tailing
1	C18	Water:ACN (60:40)	3.4 min	2.5	1.1

Annexure-5: Final Selection and Justification Sheet

Column	Mobile Phase	Buffer	Flow Rate	λmax	Justification
C18	Water:ACN (60:40)	10 mM phosphate	1.0 mL/min	240 nm	Provides good resolution and reproducibility

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded column evaluation criteria and included new annexure structure	Annual SOP Review