Identification and structural elucidation of a new cetrorelix methylene dimer impurity in cetrorelix acetate by using LC-MS/MS

• In this study, a forced degradation test combined with LC–MS/MS high-resolution mass spectrometry was used to quickly determine the source of cetrorelix methylene dimer impurity in cetrorelix, and determined the reaction site of cetrorelix methylene dimer impurity. • The determined of impurity named cetrorelix methylene dimer, which is synthesized for the first time. • The structure of cetrorelix methylene dimer was elucidated in detail by using liquid chromatography mass spectrometry (LC–MS/MS) method. • We found that the cetrorelix was highly unstable in formaldehyde conditions. Therefore, the methods of formaldehyde fumigation and sterilization in the workshop are not suitable for the production of such products, and sufficient attention must be paid. Cetrorelix, a potent third generation of luteinizing hormone releasing hormone (LHRH) antagonist, is a synthetic decapeptide used for treatment of infertility, prostatic hypertrophy and sexual hormone-dependent tumors. The approved drug of cetrorelix (Cetrotide, Asta Medica AG, Frankfurt, Germany.), was used for prevention of premature ovulation in patients undergoing a controlled ovarian stimulation (COS), followed by oocyte pick-up and assisted reproductive techniques, and has been shown safe and effective in controlled ovarian stimulation. Nevertheless, the study of aggregation products of cetrorelix was rarely reported. A simple liquid chromatography mass spectrometry (LC–MS/MS) method was developed for separation, identification and characterization of a new cetrorelix methylene dimer impurity in cetrorelix. The chromatographic separation was achieved on an XSelect Peptide CSH ™C 18 column (150 × 4.6 mm, 3.5 μm particle size) using gradient elution with a mobile phase of ammonium formate buffer (pH 3.0, 20 mM), acetonitrile at a flow rate 1.0 mL min −1 , and an ultraviolet detection wavelength of 226 nm. The new cetrorelix methylene dimer impurity was characterized by LC–MS/MS and it characteristic fragment ions were summarized. A simple, fast and accurate method was established for the determination of the molecular weight and structure of the new cetrorelix methylene dimer impurity. In this study, the results showed that the cetrorelix was highly unstable in formaldehyde conditions. In addition, it is proposed that the impact of formaldehyde in the environment on the quality of cetrorelix acetate for Injection should be evaluated during the production process.