Comprehensive characterization of bremelanotide acetate and its degradants by LC-HRMS/MS and predicting epimerization...

Bremelanotide (BRM) is a cyclic peptide therapeutic whose intrinsic stability and degradation behavior have not been extensively investigated. This study aimed to develop a stability-indicating RP-HPLC method for BRM and characterize its degradation products using LC-HRMS/MS combined with computational approaches. Stress degradation studies were conducted according to International Council for Harmonization guidelines under acidic, basic, neutral hydrolytic, oxidative, thermal, and photolytic conditions. The RP-HPLC method was developed using a YMC Pack C8 column with 10 mM ammonium formate buffer (pH 3.0) and acetonitrile containing 0.1% formic acid as the mobile phase in gradient mode. The method was partially validated and showed satisfactory system suitability, precision, and accuracy. Excellent linearity was achieved over 25-150 µg mL^-1 with a coefficient of determination (r²) of 0.9993. Forced degradation studies revealed that BRM exhibited lower degradation under acidic conditions compared with basic conditions and showed significant susceptibility to oxidative conditions. Furthermore, degradation was also observed under thermal and photolytic stress conditions. A total of eight degradation products were detected and characterized by LC-HRMS/MS. The major degradation pathways involved deacetylation, peptide-bond hydrolysis, oxidation, and epimerization. Epimerized products were identified, and probable stereochemical sites were predicted using energy minimization by correlating steric energies with MS/MS fragment relative intensities. ProTox-3.0-based in silico toxicity prediction indicated that most degradation products belonged to toxicity class 5, while two hydrolytic products were classified as class 4. Overall, this study provides valuable insights into BRM stability behavior and supports peptide API/formulation development, impurity profiling, and quality control.