or oral sprays to ensure dose uniformity and compliance with regulatory requirements.

2. Scope

This SOP is applicable to Analytical Method Development (AMD) personnel performing quantitative assay of spray content for nasal, buccal, or sublingual metered-dose spray products during formulation development, stability studies, and batch release.

3. Responsibilities

• Analytical Scientist: Prepares standard and sample solutions, runs HPLC, and calculates assay results.
• QC Analyst: Assists in spray collection, sample weighing, and documentation.
• QA Executive: Reviews method suitability and assay results for accuracy and consistency.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring method validation, data integrity, and compliance with pharmacopeial specifications for spray product assay.

5. Procedure

5.1 Apparatus and Equipment

1. HPLC system with UV detector
2. Autosampler, analytical balance (0.1 mg), vortex mixer, sonicator
3. Volumetric flasks, micropipettes, spray sampling apparatus (e.g., DUSA)

5.2 Chromatographic Conditions (Example)

1. Column: C18, 150 mm × 4.6 mm, 5 µm
2. Mobile Phase: 60:40 (v/v) Methanol:Water with 0.1% TFA
3. Flow Rate: 1.0 mL/min
4. Detection Wavelength: 220 nm
5. Injection Volume: 20 µL

5.3 Standard Preparation

1. Weigh accurately about 25 mg of API reference standard into a 50 mL volumetric flask.
2. Dissolve in diluent (same as mobile phase) and make up the volume.
3. Filter through 0.45 µm membrane filter before injection.

5.4 Sample Preparation

1. Prime the spray device (3 actuations to waste).
2. Actuate 3 sprays into a 50 mL volumetric flask (or 1 spray per flask for unit dose analysis).
3. Add diluent, vortex to dissolve content, and sonicate if required.
4. Make up to volume and filter through 0.45 µm membrane.

5.5 System Suitability

1. Inject standard solution five times.
2. Ensure % RSD of peak areas ≤ 2.0% and theoretical plates ≥ 2000.

5.6 Assay Calculation

1. Calculate API content per spray using:
   
   Spray Content (mg) = (Sample Area / Standard Area) × Standard Concentration × Dilution Factor
2. Report average content for triplicate sprays and % of label claim.

5.7 Acceptance Criteria

1. Individual spray content: 85–115% of labeled claim.
2. RSD for three replicates: ≤ 6.0%.

5.8 Method Validation Requirements

1. Specificity, Linearity (r ≥ 0.999), Accuracy (98–102%), Precision (% RSD ≤ 2%), Robustness.

6. Abbreviations

• HPLC: High Performance Liquid Chromatography
• API: Active Pharmaceutical Ingredient
• RSD: Relative Standard Deviation
• DUSA: Dose Uniformity Sampling Apparatus

7. Documents

1. Spray Assay Data Log – Annexure-1
2. System Suitability Record – Annexure-2
3. Spray Assay Validation Report – Annexure-3

8. References

• USP <601>: Aerosols, Nasal Sprays, and Inhalers
• ICH Q2(R1): Validation of Analytical Procedures
• FDA Guidance on Nasal Spray and Inhalation Products

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Deepak Nair	Vaidehi Sharma	Sunita Reddy
Designation	Analytical Chemist	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Spray Assay Data Log

Spray No.	Sample Area	Calculated Content (mg)	% Label Claim	Status
1	13456	0.102	102%	Pass
2	13289	0.101	101%	Pass
3	13110	0.099	99%	Pass

Annexure-2: System Suitability Record

Injection	Peak Area	Retention Time
1	13470	3.58
2	13465	3.59
3	13462	3.58
4	13468	3.58
5	13466	3.59

Annexure-3: Spray Assay Validation Report

The HPLC method for spray content assay was validated as per ICH Q2(R1). Linearity was demonstrated between 50–150% of target concentration (r = 0.9995). Precision (% RSD) was 1.3% and accuracy was within 98.5–101.2%. Method approved for batch release and stability testing.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Added section on system suitability and annexure formatting	Annual Review and Enhancement	Sunita Reddy
15/02/2022	1.0	Initial Release	New SOP	QA Head