Development and validation of a reversed-phase HPLC method for analysis of tetrahydrozoline hydrochloride in eye drop formulations

Abstract

A simple, precise, accurate, and stability-indicating method is developed and validated for analysis of tetrahydrozoline hydrochloride in eye drop formulations. Separation was achieved on a reversed-phase C₈ column (125 mm×4.6 mm i.d., 5 μm) using a mobile phase consisting of acetonitrile/phosphate buffer of pH 3.0 (20:80, v/v) at a flow rate of 1.0 mL/min and UV detection at 240 nm. This method is validated according to United States Pharmacopeia requirements for new methods, which include accuracy, precision, selectivity, robustness, and linearity and range. This method shows enough selectivity, accuracy, precision, and linearity and range to satisfy Federal Drug Administration/International Conference on Harmonization regulatory requirements. The current method demonstrates good linearity over the range of 0.025-0.075 mg/mL of tetrahydrozoline with r² 0.999. The average recovery of the method is 100.8% with a relative standard deviation of 0.47%. The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters and by changing analytical operators has proven that the method is robust and rugged.

Keywords: Accuracy; Linearity; Precision; Tetrahydrozoline hydrochloride; Validation.

Figure 1

Structure of tetrahydrozoline hydrochloride.

Figure 2

Chromatogram of tetrahydrozoline in eye drop formulation. Mobile phase: acetonitrile/potassium dihydrogen phosphate buffer—pH 3.0 (20:80, v/v), flow rate—1.0 mL/min, injection volume—20 μL. Column: C₈, 5 μm and 12.5 cm—length, 4.6 mm—inner diameter, UV detection: 240 nm. Peak asymmetry and theoretical plates of tetrahydrozoline peak are 0.98 and 3100, respectively.