ocular administration.

2. Scope

This SOP applies to Analytical Method Development (AMD) personnel responsible for determining osmolarity of eye drop formulations during formulation development, stability studies, and routine quality control analysis.

3. Responsibilities

• Analytical Scientist: Conducts the osmolarity test, documents results, and maintains instrument calibration records.
• QC Analyst: Assists in sample preparation and verification of test standards.
• QA Executive: Reviews osmolarity test results and approves data sheets.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring accuracy, repeatability, and compliance of the osmolarity test method with regulatory and pharmacopeial standards.

5. Procedure

5.1 Principle

Osmolarity is determined by measuring the freezing point depression of a solution using a cryoscopic osmometer. The degree of freezing point depression correlates with the number of solute particles, expressed in milliosmoles per kilogram (mOsm/kg).

5.2 Equipment and Materials

1. Freezing Point Depression Osmometer (e.g., Advanced Instruments, Model 3320)
2. Osmolarity calibration standards (100, 290, and 1000 mOsm/kg)
3. Disposable sample tubes or cups
4. Micropipettes (50–100 µL range)
5. Ophthalmic formulation sample

5.3 Instrument Calibration

1. Calibrate the osmometer daily using 100 and 290 mOsm/kg standards.
2. Run 3 replicates for each standard and verify that values are within ±2% of nominal value.
3. Document calibration results in the calibration log (Annexure-2).

5.4 Sample Testing Procedure

1. Allow sample to reach room temperature (20–25°C).
2. Mix the sample gently to ensure homogeneity.
3. Using a micropipette, transfer 50 µL of the ophthalmic sample into the sample tube.
4. Insert the tube into the osmometer and initiate the test.
5. Record the result displayed in mOsm/kg.
6. Repeat for triplicate readings per sample and calculate the average.

5.5 Acceptance Criteria

1. Osmolarity range for ophthalmic solutions: 270–330 mOsm/kg.
2. Results must be consistent within ±5 mOsm/kg across replicates.

5.6 Method Validation Parameters

1. Repeatability: % RSD ≤ 2.0% across triplicates.
2. Linearity: Verify linearity across 100–1000 mOsm/kg.
3. Accuracy: Spike placebo with known concentrations and assess recovery.

5.7 Documentation

1. Record osmolarity values, instrument ID, analyst initials, and test conditions.
2. Attach raw printouts or screenshots from osmometer for each batch tested.

6. Abbreviations

• mOsm/kg: Milliosmoles per kilogram
• QA: Quality Assurance
• AMD: Analytical Method Development
• RSD: Relative Standard Deviation

7. Documents

1. Osmolarity Test Log – Annexure-1
2. Osmometer Calibration Log – Annexure-2
3. Osmolarity Validation Report – Annexure-3

8. References

• USP <785>: Osmolality and Osmolarity
• EP 2.2.35: Osmolality
• ICH Q6A: Specifications – Test Procedures and Acceptance Criteria

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Sachin Bhate	Riya Kapoor	Sunita Reddy
Designation	Analytical Chemist	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Osmolarity Test Log

Batch No.	Sample Name	Replicate 1	Replicate 2	Replicate 3	Average (mOsm/kg)	Status
OPH-250-01	Lubricant Eye Drops	292	295	294	293.7	Pass

Annexure-2: Osmometer Calibration Log

Date	Standard (mOsm/kg)	Measured Value	Deviation (%)	Status
20/05/2025	290	288.6	-0.5	Pass

Annexure-3: Osmolarity Validation Report

The osmolarity testing method using freezing point depression osmometer was validated for ophthalmic formulations. Precision was 1.3% RSD, recovery ranged from 98–102%, and linearity was established across 100–1000 mOsm/kg. Method approved for routine and stability testing.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Expanded validation criteria and updated references	Annual SOP Review	Sunita Reddy
01/03/2022	1.0	Initial Release	New SOP	QA Head