Determination of naphazoline HCl, pheniramine maleate and their official impurities in eye drops and biological fluid rabbit aqueous humor by a validated LC-DAD method.

Abstract

A simple RP-HPLC-DAD method was developed and validated, as per the ICH guidelines, for simultaneous determination of naphazoline HCl (NPZ) & pheniramine maleate (PHN) along with three of their official impurities. Chromatographic separation was performed on a hypersil ODS column (5 mm, 250–4.6 mm i.d.) with isocratic elution using phosphate buffer pH 6.0: acetonitrile (70 : 30, v/v) as mobile phase, at a flow rate of 1.0 mL min−1 and UV detection at 260.0 nm. The developed method was found to be linear over the concentration ranges of 5.00–45.00 μg mL−1 for NPZ and NPZ impurity B and 10.00–110.00 μg mL−1, 10–70 μg mL−1 and 10–120 μg mL−1 for PHN, and PHN impurity A and B, respectively, with correlation coefficient values <0.999 for the five cited compounds. The method was confirmed to be accurate, robust and precise with RSD >2.0%. LOD and LOQ values for the five cited compounds were calculated. Moreover, the method was also validated in rabbit aqueous humor as per the US food and drug administration (FDA) bioanalytical validation guidelines. Finally, the proposed method was applied for the analysis of the two drugs along with their impurities in dosage form and spiked aqueous humor samples.