Efficient Method Development for the Analysis of Naphazoline Hydrochloride, Pheniramine Maleate, and Associated Related Substances Using a Systematic Screening Protocol

Significance of the Topic

Naphazoline hydrochloride and pheniramine maleate are key ophthalmic APIs used to relieve eye inflammation and allergic conjunctivitis. Reliable simultaneous analysis of these compounds and their related impurities is critical for product safety and regulatory compliance. Conventional methods often address APIs or impurities separately and lack comprehensive impurity profiling. A single, robust UHPLC method with high resolution and reproducibility improves laboratory throughput and ensures accurate quality control of multi-component ophthalmic formulations.

Objectives and Study Overview

This study aimed to develop and optimize an ultra-high performance liquid chromatography (UHPLC) method capable of separating naphazoline HCl, pheniramine maleate, and all associated related substances in one run. A predefined systematic screening protocol guided scouting, screening, and optimization steps to identify ideal pH, column chemistry, and organic modifiers. The final method was required to achieve United States Pharmacopeia (USP) resolution ≥ 1.5 between all peaks, peak tailing ≤ 1.5, and retention factor ≥ 2.0, while maintaining mass spectrometry compatibility.

Methodology and Instrumentation Used

Sample Preparation:

• Individual stock solutions in methanol (4.0 mg/mL), diluted to 0.5 mg/mL for APIs and 40 µg/mL for impurities in 80:20 water:methanol.
• Over-the-counter ophthalmic solution diluted to 500 µg/mL pheniramine and 40 µg/mL naphazoline.

Chromatographic System:

• Arc Premier UHPLC System with automated column manager and solvent select valve.
• Columns screened: Premier CSH C18, Premier CSH Phenyl Hexyl, Premier HSS T3, BEH C18 AX, XBridge BEH C18 (all 4.6 × 100 mm, 2.5 µm).
• Mobile phases: A – 1 % formic acid in water; B – 1 % ammonium hydroxide in water; organic modifiers acetonitrile and methanol.
• Gradient scouting: 5–90 % organic over 10 min at variable slopes and times.

Detection and Data Handling:

• PDA detection 210–400 nm (derived at 260 nm) and ACQUITY QDa mass detector (m/z 50–250, ±0.8 kV capillary).
• Empower 3 CDS with Sample Set Generator for automated method creation, ApexTrack integration for peak detection, MS peak tracking, and custom scoring reports.

Main Results and Discussion

Rapid Scouting identified low pH (formic acid) conditions as providing superior retention and resolution for all analytes. Automated MS peak tracking in Empower revealed co-eluting masses and maleate salt peaks separate from free-base pheniramine. In the screening phase, Premier CSH C18 with methanol yielded the highest number of peaks meeting resolution and tailing criteria. Optimization extended column length to 150 mm, fine-tuned gradient time, pH (0.1 % formic acid), flow rate (1.1 mL/min), and temperature (42 °C). The final method achieved USP resolution ≥ 1.5, tailing ≤ 1.5, and retention factor ≥ 2.0 for all compounds. System suitability testing (six replicates) demonstrated excellent repeatability: peak area RSD ≤ 0.58 %, retention time RSD ≤ 0.05 %. Analysis of the commercial ophthalmic solution confirmed method specificity, with UV and MS spectral purity verification showing homogenous peaks free of excipient interference.

Benefits and Practical Applications of the Method

• Efficient method development through a systematic, automated screening protocol.
• Simultaneous quantitation of APIs and related substances in multi-component formulations.
• Compatibility with UV and mass detection for unambiguous peak identification and purity confirmation.
• Reduced development time and robust transferability across laboratories.

Future Trends and Potential Applications

Integration of advanced data analytics and machine learning into systematic screening protocols can further accelerate method development and optimization. Emerging column chemistries and higher-throughput UHPLC systems will support even more complex multi-component analyses. Expanding this approach to biopharmaceuticals and complex drug products can enhance regulatory compliance and quality assurance across the pharmaceutical industry.

Conclusion

A single, robust UHPLC method was established for naphazoline HCl, pheniramine maleate, and their related impurities using a systematic screening protocol. The Arc Premier System, ACQUITY QDa, PDA detector, and Empower 3 software enabled automated column and solvent screening, peak tracking, and purity confirmation. The resulting method meets USP resolution criteria, delivers high reproducibility, and streamlines routine QC of ophthalmic formulations.

References

1. Hong P., McConville P. A Complete Solution to Perform a Systematic Screening Protocol for LC Method Development. Waters White Paper, 720005268, 2018.
2. WebMD. Naphazoline-pheniramine ophthalmic eye details. 2023.
3. United States Pharmacopeia. Naphazoline Hydrochloride and Pheniramine Maleate Ophthalmic Solution, USP40-NF35.
4. United States Pharmacopeia. Naphazoline Hydrochloride Ophthalmic Solution, USP40-NF35, December 2017.
5. United States Pharmacopeia. Naphazoline Hydrochloride Nasal Solution, USP40-NF35, December 2017.
6. European Pharmacopeia. Monograph Naphazoline Hydrochloride, 10.0, January 2009.
7. European Pharmacopeia. Monograph Pheniramine Maleate, 10.0, January 2009.
8. Maziarz M., Rainville P.D. Automated Creation of Chromatographic Methods with Empower Sample Set Generator. Waters Application Brief, 720007775, 2022.