(T1330-09-57) Simultaneous Determination of Naphazoline Hydrochloride and Pheniramine Maleate Along with Their Related Compounds by High Performance Liquid Chromatography

Purpose: This single HPLC method can replace several separate methods for the analysis of assays and organic impurities of naphazoline hydrochloride and pheniramine maleate active pharmaceutical ingredients (APIs).

Methods: The analysis was performed using an Alliance iS HPLC System equipped with a Tunable UV detector. The detection was carried out using a TUV detector with dual wavelength capabilities at 260 nm and 280 nm. A 5 µm XSelect CSH C18 column with dimensions of 4.6×150 mm was used for separation. The column temperature was maintained at 40 ºC. The sample temperature was kept at 5 ºC. An injection volume of 8 µL was used, and the flow rate was set at 2.0 mL min^-1. The mobile phase consisted of two components. Mobile Phase A consisted of 0.05% (v/v) triethyl amine and 0.05% (v/v) phosphoric acid in water, without pH adjustment. Mobile Phase B consisted of 0.05% (v/v) phosphoric acid in Acetonitrile. The gradient profile employed a 6-minute initial hold at 5% organic (Mobile Phase B) and 95% aqueous (Mobile Phase A), followed by a linear gradient from 5% to 95% organic over 7 minutes.

Results: System Suitability and Precision: To verify the functionality of the chromatographic system, it was important to perform System Suitability Testing (SST). SST is a standard procedure used to verify the efficiency and repeatability of a chromatographic system to ensure its suitability for a specific analysis. To demonstrate this, the system underwent 12 replicate injections of the SST working standard (500/40 µg/mL of pheniramine maleate/naphazoline HCl), and the results, presented in Table 2, showed that the relative standard deviation (%RSD) for the peak areas of naphazoline and pheniramine was less than 0.1 for 12 consecutive injections. The %RSD for the retention time for these two peaks was 0.02 for pheniramine and 0.14 for naphazoline. These findings indicate that the developed method and the system offer outstanding repeatability of retention times and peak areas. Linearity of APIs: Assessing the linearity of a chromatographic method is crucial for accurate and reliable quantification of analytes across a broad range of concentrations. A calibration curve is typically established by testing the same analyte at various concentrations to determine its concentration in unknown samples. In this study, linearity was assessed by preparing five mixtures \ at concentrations ranging from 80% to 120% of the target concentration of 500/40 µg/mL of pheniramine maleate/naphazoline HCl. Each solution was then injected in duplicate into the chromatographic system, and the response area was recorded. The resulting linear calibration curves were constructed by plotting peak area against concentration, and regression equations were computed, indicating a strong correlation coefficient (R²) greater than 0.997. Related compounds: The ability of an analytical method to separate related compounds from API peaks is of critical importance to ensure that the drug is safe, effective, and stable. To assess the developed method’s capability in separating the active ingredients from their related compounds, it was interesting to run the method on a standard Resolution Mixture that contains naphazoline hydrochloride/ pheniramine maleate and their related compounds. The obtained results demonstrated that the method effectively separated all the compounds in the mixture, with a minimum USP resolution of 2.4. Intra-day and inter-day Precisions: Precision refers to the level of agreement among individual results when the procedure is repeatedly applied to several samples taken from the same homogenous sample. Intra-day precision of the method was evaluated by performing 12 replicate injections of the system suitability mixture as previously demonstrated in the system suitability section. For inter-day precision, the same samples were analyzed in two different days (12 replicate injections on day one and additional five replicate injections on the second day). Results revealed that the % RSD for the peak area and the retention time of Pheniramine over the 17 replicate injections was 0.15 and 0.58 respectively. Similar results were also observed for Naphazoline of %RSD values of peak area and retention time of 0.03 and 0.59 respectively. The results indicate that the method has good precision for detection of targeted compounds and %RSD below 0.6% confirming that the method is sufficiently precise.

Conclusion: A single LC method, specific for analysis of active ingredients and their related compounds was developed to combine three USP monographs for naphazoline HCl and pheniramine maleate ophthalmic and nasal solutions.

References: [1] A. Uncini, G. De Nicola, A. Di Muzio, G. Rancitelli, L. Colangelo, D. Gambi, P.E. Gallenga, Topical naphazoline in treatment of myopathic ptosis, Acta Neurol Scand 87(4) (1993) 322-4.
[2] L. Quan, H. He, Treatment with olopatadine and naphazoline hydrochloride reduces allergic conjunctivitis in mice through alterations in inflammation, NGF and VEGF, Mol Med Rep 13(4) (2016) 3319-3325.
[3] USP Monograph, Naphazoline Hydrochloride Nasal Solution, USP40-NF35, The United States Pharmacopeia Convention, official December 2017.
[4] USP Monograph, Naphazoline Hydrochloride Ophthalmic Solution, USP40-NF35, The United States Pharmacopeia Convention, official December 2017.
[5] USP Monograph, Naphazoline Hydrochloride and Pheniramine Maleate Ophthalmic Solution, USP40-NF35, The United States Pharmacopeia.