Development and validation of the stability indicating RP-UHPLC method for the determination of the chemical purity...

A simple, rapid and accurate ultra-high performance liquid chromatographic (UHPLC) method with a UV detection for the determination of the chemical purity and assay of bimatoprost (BT-1) was developed. The chromatographic separation was achieved with the use of an Acquity BEH C8, 150 × 2.1 mm, 1.7 μm reversed phase analytical column. The mobile phase consisted of 0.01% H₃PO₄: acetonitrile (initial conditions 80 : 20, v/v) was passed through the column at the flow rate of 0.7 mL min^-1. The separation was carried out in the gradient elution mode. The presented method allows to separate ten potential impurities of BT-1. The full validation according to the ICH Q2 (R1) guidelines was carried out for five of the potential impurities while limit tests were performed for four BT-1 related substances. The performed validation tests proved the suitability of the method for its intended purposes. An additional LC/MS method was utilized for the identification of the unknown impurities in bimatoprost as well as the degradation impurities generated during the forced degradation studies.