Scope

This SOP applies to the Analytical Method Development (AMD) team for all sterile injectable products under development or undergoing method validation, including small molecules and biologics.

3. Responsibilities

• Analytical Scientist: Develops and optimizes assay method, documents validation parameters.
• QC Analyst: Supports method transfer and routine testing post-validation.
• QA Executive: Reviews method protocol, ensures documentation compliance, and approves method validation report.

4. Accountability

The Head of Analytical Method Development is accountable for the design, execution, and regulatory compliance of assay method development for injectable dosage forms.

5. Procedure

5.1 Pre-Method Considerations

1. Review physicochemical properties of the API (e.g., pKa, solubility, UV absorbance, stability).
2. Evaluate formulation components (e.g., buffer, stabilizers, preservatives) that may interfere with detection.
3. Choose detection technique based on API characteristics:
   • HPLC with UV/RI/DAD/PDA/ELSD detectors
   • UV-Vis Spectrophotometry
   • Ion Chromatography (for ionic compounds)

5.2 Sample Preparation

1. Withdraw 1 mL from injectable vial under aseptic conditions.
2. Transfer to a 10 mL volumetric flask, dilute with diluent (water or suitable solvent).
3. Filter using 0.22 µm syringe filter and inject or analyze directly.

5.3 Standard Preparation

1. Weigh accurately a known quantity of API reference standard.
2. Dissolve and dilute in suitable solvent to obtain standard concentration in the range of sample.
3. Store under recommended conditions; document storage and stability.

5.4 Method Development and Optimization

1. Select appropriate chromatographic conditions (if using HPLC):
   • Column: C18, 250 mm × 4.6 mm, 5 µm or equivalent
   • Mobile phase: Buffer/organic solvent combination (e.g., phosphate buffer:acetonitrile)
   • Flow rate: 1.0 mL/min
   • Wavelength: As per UV λmax of API (e.g., 210–280 nm)
   • Injection volume: 20 µL
2. Optimize retention time, resolution, and peak shape.
3. Record chromatograms and validate against system suitability parameters.

5.5 Acceptance Criteria

• System suitability: % RSD of 5 replicate injections ≤ 2%
• Theoretical plates ≥ 2000
• Tailing factor ≤ 2.0
• Assay result within 90–110% of label claim

5.6 Method Validation Parameters

1. Specificity: No interference from excipients or degradants
2. Linearity: r² ≥ 0.999 across 80–120% range
3. Accuracy: Recovery between 98–102%
4. Precision: Repeatability and intermediate precision RSD ≤ 2%
5. Robustness: Validate under slight changes in flow, temperature, pH
6. LOD/LOQ: Determine based on signal-to-noise ratio (3:1 and 10:1)

6. Abbreviations

• API: Active Pharmaceutical Ingredient
• HPLC: High Performance Liquid Chromatography
• LOD: Limit of Detection
• LOQ: Limit of Quantification
• QA: Quality Assurance
• AMD: Analytical Method Development

7. Documents

1. Method Development Worksheet – Annexure-1
2. Chromatograms/Spectra Set – Annexure-2
3. Validation Summary Report – Annexure-3

8. References

• ICH Q2(R1): Validation of Analytical Procedures
• USP <621>: Chromatography
• FDA Analytical Procedures and Methods Validation Guidance (July 2015)

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Rohit Sharma	Meena Joshi	Sunita Reddy
Designation	Analytical Scientist	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Method Development Worksheet

API	Mobile Phase	Column	Wavelength (nm)	Flow Rate	Retention Time
Ceftriaxone	Phosphate Buffer:Acetonitrile (60:40)	C18	254	1.0 mL/min	5.2 min

Annexure-2: Chromatograms/Spectra Set

Attached HPLC chromatograms of standard and test sample, overlay spectra confirming retention time match and peak purity.

Annexure-3: Validation Summary Report

Assay method for ceftriaxone injection was validated for specificity, accuracy, and precision. Method meets ICH and USP criteria and is suitable for routine use in QC and stability programs.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Added new validation parameters and robustness section	Annual Review	Sunita Reddy
01/05/2022	1.0	Initial Release	New SOP	QA Head