profiles, and physicochemical stability of nanocarriers.

2. Scope

This procedure applies to all nano-based pharmaceutical products including polymeric nanoparticles, lipid-based nanoparticles, nanocrystals, nanoemulsions, and nanogels being developed or tested in the AMD laboratory.

3. Responsibilities

• Analytical Scientist: Develops and executes methods for nanoparticle characterization and validates analytical parameters.
• Nanotechnology Scientist: Provides formulation data and performance targets to guide method development.
• QA Officer: Reviews validation protocols, method verification results, and ensures compliance with regulatory guidance.
• Head – AMD: Authorizes method approval and ensures consistency with project goals and regulatory expectations.

4. Accountability

The Head of Analytical Method Development is accountable for the scientific integrity, validation, and regulatory suitability of all methods applied to nanoformulations analysis in the AMD lab.

5. Procedure

5.1 Preliminary Method Strategy

1. Understand formulation specifics including:
   • Carrier matrix (lipid/polymer/nanocrystal)
   • Drug payload characteristics
   • Route of administration
2. Identify Critical Quality Attributes (CQAs):
   • Particle size and distribution
   • Zeta potential
   • Encapsulation efficiency
   • Drug release profile
   • Stability (physical and chemical)

5.2 Analytical Techniques for Nanoformulations

1. Dynamic Light Scattering (DLS): For particle size (nm) and polydispersity index (PDI)
2. Zeta Potential Measurement: For surface charge assessment
3. Transmission Electron Microscopy (TEM): For morphological analysis
4. UV-HPLC or UPLC: For drug content, entrapment efficiency, and release
5. Stability-indicating Assays: For degradation profile under stress conditions

5.3 Entrapment Efficiency Method

1. Separate free and encapsulated drug using ultrafiltration, centrifugation, or dialysis.
2. Measure free drug concentration using a validated HPLC or UV method.
3. Calculate % Encapsulation:
   • Encapsulation (%) = [(Total – Free)/Total] × 100
4. Log data in Annexure-1: Entrapment Efficiency Sheet.

5.4 Particle Size and Zeta Potential Analysis

1. Dilute sample in filtered water or appropriate buffer.
2. Use instrument SOPs to measure mean size, PDI, and zeta potential.
3. Acceptance Criteria:
   • Size: 10–200 nm (formulation dependent)
   • PDI: < 0.3
   • Zeta Potential: ±30 mV minimum for stable formulations

5.5 In-Vitro Drug Release Studies

1. Use Franz diffusion cells or dialysis bag setup with defined release media.
2. Withdraw samples at intervals and replace with fresh media.
3. Analyze each sample using validated method (HPLC/UV).
4. Calculate cumulative drug release and model kinetics using zero-order, Higuchi, or Korsmeyer-Peppas models.
5. Log profile in Annexure-2: Drug Release Log.

5.6 Method Validation

1. Perform validation as per ICH Q2(R2) covering:
   • Specificity
   • Linearity
   • Accuracy
   • Precision
   • LOD & LOQ
   • Robustness
2. Summarize data in Annexure-3: Validation Report.

5.7 Documentation and Reporting

1. Prepare Method Development Report (MDR) and Validation Report (VR) signed by the analyst and QA.
2. Include chromatograms, spectra, graphs, and raw data.
3. All records must be archived for minimum five years or as per site SOP.

6. Abbreviations

• DLS: Dynamic Light Scattering
• PDI: Polydispersity Index
• LOD: Limit of Detection
• LOQ: Limit of Quantification
• ICH: International Council for Harmonisation
• SOP: Standard Operating Procedure

7. Documents

1. Entrapment Efficiency Sheet – Annexure-1
2. Drug Release Log – Annexure-2
3. Validation Report – Annexure-3

8. References

• ICH Q2(R2) – Validation of Analytical Procedures
• EMA Reflection Paper on Nanomedicines
• USP <729> – Globule Size Distribution
• WHO TRS 1025 – Quality of Nanotechnology Products

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Entrapment Efficiency Sheet

Batch ID	Total Drug (mg)	Free Drug (mg)	Entrapment (%)	Analyst
NANO/2025/015	5.0	0.8	84.0%	Rajesh Kumar

Annexure-2: Drug Release Log

Time (hr)	Released (%)	Release Medium	Analyst
0	0.0	PBS pH 6.8	Sunita Reddy
1	15.3	PBS pH 6.8	Sunita Reddy
4	42.1	PBS pH 6.8	Sunita Reddy

Annexure-3: Validation Report

Parameter	Result	Criteria	Status
Linearity	R² = 0.9991	≥ 0.999	Pass
Accuracy	98.7%–101.5%	98%–102%	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added drug release kinetics and expanded DLS protocol	Annual SOP Review