is to improve resolution, reduce run time, and enhance overall method sensitivity and efficiency.

2. Scope

This procedure applies to the Analytical Method Development (AMD) department and covers all development and optimization activities using UPLC technology for both single-component and multi-component pharmaceutical formulations, including assay, impurity profiling, and stability testing.

3. Responsibilities

• Analytical Chemist: Executes UPLC method trials, adjusts instrument parameters, documents findings, and prepares optimization reports.
• Reviewer: Reviews method optimization logs and chromatographic data for peak shape, resolution, and compliance.
• QA Officer: Verifies procedural compliance and supports audit readiness through documentation review.
• Head – AMD: Authorizes the optimized UPLC method for validation or transfer to quality control.

4. Accountability

The Head of AMD is accountable for ensuring that all UPLC methods developed and optimized are scientifically justified, robust, and suitable for routine and regulatory use.

5. Procedure

5.1 Pre-Development Planning

1. Review API properties: solubility, pKa, UV absorbance, molecular weight.
2. Evaluate formulation matrix and potential interfering excipients.
3. Select UV detection wavelength using PDA scan; prioritize λmax values.
4. Document findings in Annexure-1: UPLC Method Planning Sheet.

5.2 Column and Mobile Phase Selection

1. Select sub-2 µm particle size UPLC columns (e.g., BEH C18 50×2.1 mm, 1.7 µm).
2. Choose mobile phases: aqueous buffer (A) and organic solvent (B) such as acetonitrile or methanol.
3. Optimize pH using low ionic strength buffers (e.g., ammonium formate or phosphate, 10–20 mM).
4. Filter and degas mobile phases; document in Annexure-2: Mobile Phase Preparation Record.

5.3 Gradient or Isocratic Flow Optimization

1. For complex mixtures, use fast gradients:
   • Start: 5% B → End: 95% B in 2–5 min
   • Re-equilibration: 2–3 min
2. For single components or known APIs, use isocratic flow for higher reproducibility.
3. Optimize flow rate: 0.3–0.6 mL/min based on backpressure and resolution.
4. Adjust injection volume (1–5 µL) for sharp peak shape and minimal fronting.
5. Record method adjustments in Annexure-3: Flow Program Optimization Sheet.

5.4 Sample and Standard Preparation

1. Prepare standard API solution using certified reference standards.
2. Prepare placebo or matrix-matched blank to evaluate specificity.
3. Filter solutions using 0.22 µm or 0.45 µm membrane filters.
4. Document details in Annexure-4: Sample Preparation Log.

5.5 Chromatographic Evaluation

1. Evaluate:
   • Retention time: ideal 0.5–4 min
   • Resolution between peaks ≥ 2.0
   • Plate count ≥ 5000
   • Tailing factor ≤ 1.5
2. Compare baseline noise and drift across injections.
3. Perform replicate injections (n=5) and calculate %RSD.
4. Log evaluation in Annexure-5: Chromatographic Evaluation Sheet.

5.6 System Suitability and Method Robustness

1. Run system suitability solution at the start of sequence.
2. Test small deliberate changes:
   • ±0.05 mL/min in flow rate
   • ±2°C column temperature
   • ±2% in mobile phase ratio
3. Document robustness trials in Annexure-6: Method Robustness Record.

5.7 Finalization of UPLC Method

1. Confirm method parameters:
   • Column type and dimensions
   • Mobile phase composition
   • Gradient or isocratic program
   • Detection wavelength
   • Run time and flow rate
2. Confirm system suitability meets pre-defined criteria.
3. Summarize finalized method in Annexure-7: Final UPLC Method Summary.

6. Abbreviations

• UPLC: Ultra Performance Liquid Chromatography
• API: Active Pharmaceutical Ingredient
• PDA: Photodiode Array
• RSD: Relative Standard Deviation
• SOP: Standard Operating Procedure

7. Documents

1. UPLC Method Planning Sheet – Annexure-1
2. Mobile Phase Preparation Record – Annexure-2
3. Flow Program Optimization Sheet – Annexure-3
4. Sample Preparation Log – Annexure-4
5. Chromatographic Evaluation Sheet – Annexure-5
6. Method Robustness Record – Annexure-6
7. Final UPLC Method Summary – Annexure-7

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP <621> – Chromatography
• FDA Guidance on Analytical Procedures and Methods Validation
• Waters UPLC System Manuals

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: UPLC Method Planning Sheet

API	λmax	Matrix Complexity	Method Type	Analyst
XYZ	245 nm	Moderate	Gradient	Sunita Reddy

Annexure-2: Mobile Phase Preparation Record

Mobile Phase A	Mobile Phase B	pH	Prepared On	Checked By
10 mM Ammonium Formate	Acetonitrile	3.8	18/05/2025	Rajesh Kumar

Annexure-3: Flow Program Optimization Sheet

Time (min)	%A	%B	Flow Rate
0.0	95	5	0.3 mL/min
2.0	50	50	0.3 mL/min
4.0	5	95	0.3 mL/min
6.0	95	5	0.3 mL/min

Annexure-4: Sample Preparation Log

Sample Code	Concentration	Diluent	Filtered	Prepared By
XYZ-STD	100 µg/mL	Mobile Phase	Yes (0.22 µm)	Sunita Reddy

Annexure-5: Chromatographic Evaluation Sheet

Retention Time	Plate Count	Tailing	Resolution	RSD (%)
2.56	9800	1.15	3.2	0.65

Annexure-6: Method Robustness Record

Parameter	Variation	Impact	Status
Flow Rate	±0.05 mL/min	Acceptable variation in retention	Pass

Annexure-7: Final UPLC Method Summary

Column	Mobile Phase	Program	λ	Flow	Injection Volume	Run Time
BEH C18, 50×2.1 mm, 1.7 µm	A: Buffer, B: ACN	Gradient 5–95% B in 5 min	245 nm	0.3 mL/min	2 µL	6 min

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included robustness evaluation and final summary format	Annual SOP Review