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% HPO: acetonitrile (initial conditions 80 : 20, v/v) was passed through the column at the flow rate of 0.7 mL min. 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.
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