Development and validation of an RP-HPLC method for the quantification of impurities of icatibant injection in finished formulations-identification and characterization of its impurities via LC-HRMS.

Abstract

In the present study, an efficient reverse-phase high-performance liquid chromatography method was developed for the quantification of impurities of icatibant (IBT) injection in finished formulations. The stationary phase employed is Kromasil 100 C18 with dimension of (150 × 4.6) mm, 3.5 µm. A 0.05 M KH₂PO₄ (pH 2.5) and methanol in the ratio of (90:10%v/v) was used as eluent A and 0.05 M KH₂PO₄ (pH 2.5) and methanol in the ratio of (70:30%v/v) was used as eluent B pumped through 1.0 mL/min flow rate using photodiode array detector at 210 nm. The IBT injection was analyzed under various degradation conditions, including acid, base, peroxide, photolytic, and thermal, were evaluated using a present proposed developed method. The method validation was carried out based on International Conference on Harmonization guidelines, by evaluating several key parameters. The observed limit of detection values were 0.066, 0.165, 0.165, 0.197, 0.134, and 0.493 μg/mL, the observed limit of quantification values were 0.201, 0.501, 0.500, 0.598, 0.406, and 1.495 μg/mL for icatibant (7-10), Des-Pro-Impurity, Des-Arg-impurity, Des-D-Arg-impurity, D-Tic (O) 8-10 icatibant and icatibant (1-6), respectively. The average recovery percentages of impurities of IBT injection were found to be 87.4% to 108.2%. The established methodology demonstrated linearity across a concentration interval of 0.020 to 34.73μg/mL for IBT and its impurities. Specific unidentified "Impurity at RRT about 0.92" (D-Tic (O) 8-10 Icatibant), "Impurity at RRT about 1.15," "Impurity at RRT about 1.24," and "Impurity at RRT about 0.57" were identified and liquid chromatography-high resolution mass spectrometry technique was used for their characterization. This method was assessed and confirmed to be more reliable for quantifying impurities of IBT injections.