Standard Operating Procedure for Spray Content Assay Using HPLC in Analytical Method Development
| Department | Analytical Method Development |
|---|---|
| SOP No. | SOP/AMD/246/2025 |
| Supersedes | SOP/AMD/246/2022 |
| Page No. | Page 1 of 14 |
| Issue Date | 21/05/2025 |
| Effective Date | 23/05/2025 |
| Review Date | 21/05/2026 |
1. Purpose
To establish a validated HPLC-based analytical method for quantifying the content of the active pharmaceutical ingredient (API) in individual spray actuations from nasal
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
- HPLC system with UV detector
- Autosampler, analytical balance (0.1 mg), vortex mixer, sonicator
- Volumetric flasks, micropipettes, spray sampling apparatus (e.g., DUSA)
5.2 Chromatographic Conditions (Example)
- Column: C18, 150 mm × 4.6 mm, 5 µm
- Mobile Phase: 60:40 (v/v) Methanol:Water with 0.1% TFA
- Flow Rate: 1.0 mL/min
- Detection Wavelength: 220 nm
- Injection Volume: 20 µL
5.3 Standard Preparation
- Weigh accurately about 25 mg of API reference standard into a 50 mL volumetric flask.
- Dissolve in diluent (same as mobile phase) and make up the volume.
- Filter through 0.45 µm membrane filter before injection.
5.4 Sample Preparation
- Prime the spray device (3 actuations to waste).
- Actuate 3 sprays into a 50 mL volumetric flask (or 1 spray per flask for unit dose analysis).
- Add diluent, vortex to dissolve content, and sonicate if required.
- Make up to volume and filter through 0.45 µm membrane.
5.5 System Suitability
- Inject standard solution five times.
- Ensure % RSD of peak areas ≤ 2.0% and theoretical plates ≥ 2000.
5.6 Assay Calculation
- Calculate API content per spray using:
Spray Content (mg) = (Sample Area / Standard Area) × Standard Concentration × Dilution Factor - Report average content for triplicate sprays and % of label claim.
5.7 Acceptance Criteria
- Individual spray content: 85–115% of labeled claim.
- RSD for three replicates: ≤ 6.0%.
5.8 Method Validation Requirements
- 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
- Spray Assay Data Log – Annexure-1
- System Suitability Record – Annexure-2
- 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 |