amount of active ingredient within acceptable pharmacopeial limits.

2. Scope

This SOP applies to the Analytical Method Development (AMD) department and covers the HPLC method development activities related to content uniformity analysis for tablets, capsules, and similar dosage forms intended for oral administration.

3. Responsibilities

• Analytical Chemist: Executes method development trials, prepares dosage unit samples, and analyzes HPLC data for uniformity.
• Reviewer: Validates method suitability based on accuracy, linearity, and precision for individual unit analysis.
• QA Officer: Ensures documentation compliance and adherence to ICH and pharmacopeial standards.
• Head – AMD: Approves the method for validation and routine content uniformity testing.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring all HPLC methods developed for content uniformity are scientifically sound and meet regulatory and compendial requirements.

5. Procedure

5.1 Pre-Method Development Planning

1. Review monographs in USP, BP, IP, or existing validated methods.
2. Assess formulation matrix and potential excipient interferences.
3. Define acceptance criteria:
   • 85% to 115% of label claim for individual units (as per USP <905>)
4. Document plan in Annexure-1: Method Planning Sheet.

5.2 Chromatographic System Selection

1. Column: C18, 150 mm × 4.6 mm, 5 µm preferred.
2. Mobile phase: water with buffer (10–20 mM phosphate or acetate) and organic solvent (ACN or MeOH).
3. Isocratic or gradient mode depending on matrix complexity and analyte polarity.
4. Detection wavelength: λmax of API (from UV or PDA scan).
5. Flow rate: 1.0 mL/min; Injection volume: 10–20 µL.
6. Record settings in Annexure-2: Chromatographic Parameters Log.

5.3 Sample and Standard Preparation

1. Standard Preparation:
   • Prepare working standard solution using API reference material.
   • Concentration to match expected sample level (e.g., 100 µg/mL).
2. Sample Preparation:
   • Individually transfer 10 dosage units into separate volumetric flasks.
   • Add diluent, sonicate if required, and bring to volume.
   • Filter each solution using 0.45 µm syringe filters.
3. Document sample coding and prep in Annexure-3: Sample Preparation Log.

5.4 Method Optimization and System Suitability

1. Inject blank, standard, and individual units (at least 10).
2. Check:
   • Peak symmetry (tailing factor ≤ 2.0)
   • Resolution (Rs ≥ 2.0 if multiple components present)
   • Retention time reproducibility
3. Inject standard 5 times; RSD of area ≤ 2.0%.
4. System suitability must be met before analyzing samples.
5. Record observations in Annexure-4: System Suitability and Optimization Log.

5.5 Content Uniformity Evaluation

1. Calculate individual assay content of each unit using:
   
   % Content = (Sample Area / Standard Area) × (Standard Concentration / Sample Concentration) × 100
2. Report results for each unit and calculate Mean, RSD, and Acceptance Value (AV):
   AV = |M – X̄| + k × s
   Where:
   M = Reference Value (usually target),
   X̄ = Mean,
   k = Constant (2.4 for 10 units),
   s = Standard deviation
3. Acceptance Criteria (USP <905>): AV ≤ 15.0
4. Summarize results in Annexure-5: Content Uniformity Calculation Sheet.

5.6 Method Validation Parameters

1. Linearity: 50–150% of test concentration; R² ≥ 0.999
2. Accuracy: Recovery from placebo-spiked solution at 80%, 100%, 120% levels
3. Precision: Repeatability and intermediate precision (RSD ≤ 2.0%)
4. Specificity: No interference from excipients
5. Robustness: Small deliberate changes to evaluate consistency
6. Compile validation data in Annexure-6: Method Validation Summary.

6. Abbreviations

• HPLC: High Performance Liquid Chromatography
• API: Active Pharmaceutical Ingredient
• RSD: Relative Standard Deviation
• AV: Acceptance Value
• SOP: Standard Operating Procedure

7. Documents

1. Method Planning Sheet – Annexure-1
2. Chromatographic Parameters Log – Annexure-2
3. Sample Preparation Log – Annexure-3
4. System Suitability and Optimization Log – Annexure-4
5. Content Uniformity Calculation Sheet – Annexure-5
6. Method Validation Summary – Annexure-6

8. References

• USP <905> – Uniformity of Dosage Units
• ICH Q2(R1) – Validation of Analytical Procedures
• FDA Guidance for Industry – ANDA: Content Uniformity Studies
• USP <621> – Chromatography

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Method Planning Sheet

Product	Label Claim	Matrix	Units per Batch	Reference Monograph
XYZ Tablets	50 mg	Lactose-based	10	USP

Annexure-2: Chromatographic Parameters Log

Column	Mobile Phase	Flow Rate	Detection λ	Injection Volume
C18, 150×4.6 mm	60:40 Phosphate buffer:ACN	1.0 mL/min	245 nm	20 µL

Annexure-3: Sample Preparation Log

Unit ID	Sample Code	Dilution Volume	Filtered	Prepared By
Unit 1	CU-001	100 mL	Yes	Sunita Reddy

Annexure-4: System Suitability and Optimization Log

Parameter	Observed Value	Specification	Status
RSD (n=5)	0.72%	≤ 2.0%	Pass

Annexure-5: Content Uniformity Calculation Sheet

Unit	Assay (%)
1	97.8%
2	99.1%
3	100.4%
4	98.5%
5	101.2%
6	96.9%
7	98.7%
8	99.3%
9	100.1%
10	97.5%

Annexure-6: Method Validation Summary

Parameter	Acceptance Criteria	Observed	Status
Linearity	R² ≥ 0.999	0.9994	Pass
Accuracy	98–102%	99.2%	Pass
Precision	RSD ≤ 2.0%	0.86%	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included calculation formulas, AV logic, and expanded annexures	Annual Review