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Rationale: Disulfide bridges (DSB) play an important role in stabilizing threedimensional structures of biopharmaceuticals, single purified proteins, and various cyclic peptide drugs that contain disulfide in their structures. Incorrect cross-linking known as DSB scrambling results in misfolded structures that can be inactive, immunogenic, and susceptible to aggregation. Very few articles have been published on the experimental annotation of DSBs in proteins and cyclic peptide drugs. Accurate characterization of the disulfide bond is essential for understanding protein confirmation. Methods: Characterizing DSBs using mass spectrometry (MS) involves the chemical and enzymatic digestion of samples to obtain smaller peptide fragments, in both reduced and nonreduced forms. Subsequently, these samples are analyzed using MS to locate the DSB, either through interpretation or by employing various software tools. Results: The main challenge in DSB analysis methods using sample preparation is to obtain a sample solution in which nonnative DSBs are not formed due to high pH, temperature, and presence of free sulfhydryl groups. Formation of nonnative DSBs can lead to erroneous annotation of disulfide bond. Sample preparation techniques, fragmentation methods for DSB analysis, and contemporary approaches for DSB mapping using this fragmentation were discussed. Conclusions: This review presents the latest advancement in MS-based characterization; also a critical perspective is presented for further annotation of DSBs using MS, primarily for single purified proteins or peptides that are densely connected and rich in cysteine. Despite significant breakthroughs resulting from advancements in MS, the analysis of disulfide bonds is not straightforward; it necessitates expertise in sample preparation and interpretation. [ABSTRACT FROM AUTHOR]

Rationale: Enasidenib (EDB) is an orally active selective mutant isocitrate dehydrogenase‐2 enzyme inhibitor approved by the U.S. Food and Drug Administration to treat acute myeloid leukemia. It lacks a reported forced degradation study and a stability‐indicating assay method (SIAM). This study addresses this gap by establishing a degradation profile in accordance with the International Council for Harmonisation Q1A and Q1B (R2) guidelines and developing a validated SIAM for EDB. Methods: EDB was exposed to forced degradation under various conditions (hydrolytic, photolytic, oxidative, and thermal stress). Degradation samples were analyzed using high‐performance liquid chromatography on an Agilent ZORBAX Eclipse Plus C18 column with a mobile phase consisting of 0.1% formic acid in Milli‐Q water and acetonitrile at a flow rate of 1 mL/min and detection at 270 nm. Liquid chromatography‐quadrupole time‐of‐flight‐high‐resolution mass spectrometry (LC/Q‐TOF HRMS) was used for the identification and characterization of degradation products. Nitrosamine risk assessment was conducted using a modified nitrosation assay procedure (NAP) test due to the presence of a secondary amine group in the drug, which is liable to forming nitrosamine drug substance–related impurities (NDSRI). Results: The drug exhibited significant degradation under acidic, basic, photolytic, and oxidative conditions in the solution state. A total of nine degradation products (DP) were formed (DP‐I, DP‐III, and DP‐IV: acidic conditions; DP‐I and DP‐III: basic conditions; DP‐II, DP‐V, DP‐VI, and DP‐VII: oxidative stress; and DP‐VII, DP‐VIII, and DP‐IX: photolytic conditions), which were separated and identified using reversed‐phase high‐performance liquid chromatography and characterized using liquid chromatography–tandem mass spectrometry. The mechanism behind the formation of EDB degradation products has been discussed, and this study was the first to develop a degradation pathway for EDB. In addition, the possibilities of NDSRI formation for EDB were studied using a modified NAP test, which can contribute to the risk assessment of the drug. Conclusions: Forced degradation studies were conducted by establishing a SIAM for EDB. All the degradation products were characterized by mass spectral data obtained using LC/Q‐TOF‐HRMS. [ABSTRACT FROM AUTHOR]

Several regulatory organizations encourage the quantitative analysis of potentially genotoxic impurities in finished pharmaceutical preparation. For the first time, a sensitive, accurate, and reliable reversed phase‐high‐performance liquid chromatography method was developed and validated for two antihypertensive drugs, three genotoxic impurities of Amlodipine namely methyl benzene sulfonate, ethyl benzene sulfonate, and ethyl‐p‐toluene sulfonate, as well as N‐methyl O‐phenylenediamine dihydrochloride as genotoxic impurities of Telmisartan that is marketed in a single pill combination. reversed phase‐high‐performance liquid chromatography method was developed using a C18 column Agilent (Zorbax C18; 250 × 4.6 mm internal diameter, 5 μm) along with 50 mM ammonium acetate (pH 4.5) and ACN as a mobile phase in gradient mode to achieve the retention of all analytes. The new method's sensitivity allowed the quantification of namely methyl benzene sulfonate, ethyl benzene sulfonate, and N‐methyl‐O‐phenylenediamine dihydrochloride at test concentrations as low as 0.025%. Amlodipine and Telmisartan each had a test concertation of 100 and 800 μg/ml, respectively. The developed analytical method was validated for different applications, that is, for assay and related substance methods. The calibration curves for genotoxic impurities were linear in the range of the Limit of quantitation to 1.2% of the test concentration of the respective drugs. Forced degradation investigations were conducted on selected drugs, and targeted analytes were then quantified. The stability of both drugs in acid and basic hydrolysis was improved compared to when they were evaluated separately, although Amlodipine showed an inverted degradation pattern in a more recent investigation when combined with Telmisartan under oxidative degradation conditions. The validated method was successfully applied for the quantification of the genotoxic impurities of Amlodipine and Telmisartan in finished pharmaceutical preparation. [ABSTRACT FROM AUTHOR]