QbD-based stability-indicating UPLC method for the analysis of fruquintinib and its impurities with MS/MS characterization of degradation products
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A strong and selective analytical technique was also created for the simultaneous determination of fruquintinib along with its impurities through a Quality by Design (QbD) strategy. UV spectral characterization of the compounds determined 265 nm as the iso-absorptive wavelength for both fruquintinib and the impurities. Chromatographic separation was conducted on a BEH C18 column with a mobile phase consisting of 5 mM ammonium formate (pH 3.5), acetonitrile, and methanol (45:35:20, v/v) at 0.3 mL/min flow rate under isocratic conditions. Optimization using DoE indicated that mobile phase pH was the prime factor for separating closely eluting impurities. The method showed excellent specificity, linearity (R2 > 0.999), precision (%RSD < 0.74 %), accuracy (98–102 %), and sensitivity (LOD = 0.075 μg/mL). Forced degradation studies established extensive degradation under acidic hydrolysis (81.02 % assay remaining), six DPs (Degradation products) being formed. Oxidative stress resulted in moderate degradation (14.25 %) with three major DPs, whereas alkaline and reductive conditions resulted in degradation of <4 %. In peroxide degradation, DP5 (m/z 223.1976) was detected as 6,7- dimethoxyquinazolin-4-yl hydroperoxide, which with oxidative demethylation gave DP2 (m/z 195.1444). Acid hydrolysis gave DP8 (m/z 381.3506) through N-methylamide hydrolysis, which in turn converted to DP6 (m/z 353.2979) and finally to DP3 (m/z 207.1979) and DP1 (m/z 177.1443) through demethylation and ether cleavage. This QbD-based procedure provides excellent stability-indicating ability and efficiently identifies important degradation products of fruquintinib under stress conditions.
Abstract
A strong and selective analytical technique was also created for the simultaneous determination of fruquintinib along with its impurities through a Quality by Design (QbD) strategy. UV spectral characterization of the compounds determined 265 nm as the iso-absorptive wavelength for both fruquintinib and the impurities. Chromatographic separation was conducted on a BEH C18 column with a mobile phase consisting of 5 mM ammonium formate (pH 3.5), acetonitrile, and methanol (45:35:20, v/v) at 0.3 mL/min flow rate under isocratic conditions. Optimization using DoE indicated that mobile phase pH was the prime factor for separating closely eluting impurities. The method showed excellent specificity, linearity (R2 > 0.999), precision (%RSD < 0.74 %), accuracy (98–102 %), and sensitivity (LOD = 0.075 μg/mL). Forced degradation studies established extensive degradation under acidic hydrolysis (81.02 % assay remaining), six DPs (Degradation products) being formed. Oxidative stress resulted in moderate degradation (14.25 %) with three major DPs, whereas alkaline and reductive conditions resulted in degradation of <4 %. In peroxide degradation, DP5 (m/z 223.1976) was detected as 6,7- dimethoxyquinazolin-4-yl hydroperoxide, which with oxidative demethylation gave DP2 (m/z 195.1444). Acid hydrolysis gave DP8 (m/z 381.3506) through N-methylamide hydrolysis, which in turn converted to DP6 (m/z 353.2979) and finally to DP3 (m/z 207.1979) and DP1 (m/z 177.1443) through demethylation and ether cleavage. This QbD-based procedure provides excellent stability-indicating ability and efficiently identifies important degradation products of fruquintinib under stress conditions.