Characterization Of Degradation Products Research Articles

LC/Q-TOF-MS-based structural characterization of enasidenib degradation products and establishment of a stability-indicating assay method: Insights into chemical stability.

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. 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 1mL/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). 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. 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.

Mechanical properties, in vitro biodegradable behavior, biocompatibility and osteogenic ability of additively manufactured Zn-0.8Li-0.1Mg alloy scaffolds

Alloying and structural design provide flexibility to modulate performance of biodegradable porous implants manufactured by laser powder bed fusion (L-PBF). Herein, bulk Zn-0.8Li-0.1Mg was first fabricated to indicate the influence of the ternary alloy system on strengthening effect. Porous scaffolds with different porosities, including 60 % (P60), 70 % (P70) and 80 % (P80), were designed and fabricated to study the influence of porosity on mechanical properties, in vitro degradation behavior, biocompatibility and osteogenic ability. Pure Zn (Zn-P70) scaffolds with a porosity of 70 % were utilized for the comparison. The results showed Zn-0.8Li-0.1Mg bulks had an ultimate tensile strength of 460.78 ± 5.79 MPa, which was more than 3 times that of pure Zn ones and was the highest value ever reported for Zn alloys fabricated by L-PBF. The compressive strength (CS) and elastic modulus (E) of scaffolds decreased with increasing porosities. The CS of P70 scaffolds was 24.59 MPa, more than 2 times that of Zn-P70. The weight loss of scaffolds during in vitro immersion increased with increasing porosities. Compared with Zn-P70, a lower weight loss, better biocompatibility and improved osteogenic ability were observed for P70 scaffolds. P70 scaffolds also exhibited the best biocompatibility and osteogenic ability among all the used porosities. Influence mechanism of alloying elements and structural porosities on mechanical behaviors, in vitro biodegradation behavior, biocompatibility and osteogenic ability of scaffolds were discussed using finite element analysis and the characterization of degradation products. The results indicated that the proper design of alloying and porosity made Zn-0.8Li-0.1Mg scaffolds promising for biodegradable applications.

Characterization of Degradation Products and Drug–Excipient Interaction Products of Erdafitinib by LC–Q-TOF-MS/MS and NMR

Characterization of Degradation Products and Drug–Excipient Interaction Products of Erdafitinib by LC–Q-TOF-MS/MS and NMR

Development of a stability-indicating HPLC method for Lasmiditan and its process related impurities with characterization of degradation products by LCMS/MS

Development of a stability-indicating HPLC method for Lasmiditan and its process related impurities with characterization of degradation products by LCMS/MS

Green adherent degradation kinetics study of Nirmatrelvir, an oral anti-COVID-19: characterization of degradation products using LC–MS with insilico toxicity profile

The SARS-CoV-2 virus sets up a global catastrophe, and countries all around the world made significant efforts to halt the spread. Nirmatrelvir (NMV) was lately approved by the FDA as a safe and well-tolerated oral direct-acting antiviral medication for SARS-CoV-2 virus infection. Therefore, a fast completely validated stability indicating method was established-for the first time- for NMV determination. The study used NaOH, HCl, neutral, H2O2, and sunlight to test NMV stability under various stress conditions followed by kinetics degradation investigation and derivation of Arrhenius plot. The analysis was performed using Agilent Zorbax Eclipse-C18 column (5 µm, 4.6 × 250 mm) with a mobile phase consisting of acetonitrile: 50 mM ammonium acetate, pH = 5 (50:50, v/v, respectively) at a flow rate of 1.0 mL/min with 5 min run time. Diode array detector (DAD) was set at 225 nm to quantify NMV at the concentration range of 5–500 µg/mL with LOD and LOQ of 0.6 and 2 µg/mL, respectively. Method’s greenness was assessed using different metrics including Analytical Eco-Scale, Greenness Assessment Procedure Index, GAPI, and Analytical Greenness, AGREE. A thorough study of stress stability revealed that NMV was more susceptible to alkaline hydrolysis compared with acid hydrolysis. In contrast, it was found that NMV remained stable when subjected to oxidative, neutral, and sun-induced degradation conditions. Moreover, acid and alkali-induced hydrolysis were found to follow pseudo first order kinetics. Consequently, the half lifetime of the studied degradation conditions at room temperature were calculated using the Arrhenius plot. The mechanism of the degradation pathways under stress circumstances was proposed using LC–MS-UV. Toxicities of the proposed degradation products were assessed using ProTox-II, along with the parent medication NMV, and were shown to be hardly hazardous.

Characterization of potential degradation products of brexpiprazole by liquid chromatography/quadrupole-time-of-flight mass spectrometry and nuclear magnetic resonance, and prediction of their physicochemical properties by ADMET Predictor™.

Brexpiprazole (BRZ) was subjected to hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress degradation in solutions prepared in a mixture of acetonitrile-water (70:30 v/v). The oxidative study was additionally done in methanol-buffer mixture at pH3, 7 and 11. Also, compatibility of the drug with selectedexcipients was investigated in the solid state. Additionally, physicochemical and ADMET properties of BRZ and its degradation products (DPs) were predicted using ADMET Predictor™ software. It provides the conditions for quality control of BRZ and its derivatives during manufacturing, processing and storage conditions. The formed DPs were separated from the drug and among themselves on a C-18 column utilizing mobile phase composed of methanol and ammonium formate buffer (10mM, pH4.0), which was run in a gradient mode. Characterization of DPs was carried out by first establishing the mass fragmentation pathway of the drug based on its liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/Q-TOF-MS) data, followed by LC/Q-TOF-MS studies of DPs. Three DPs were isolated and, along with the drug, they were subjected to 1D (1 H, 13 C and DEPT-135) and 2D (COSY and HSQC) NMR studies for confirmation of their structures. BRZ was observed to be susceptible to hydrolytic (neutral, acid and alkali), photolytic and oxidative degradation conditions; it was stable on thermal exposure. A total of 12 DPs (BRZ-1 to BRZ-12) were formed in solution state. Mechanisms of BRZ degradation were postulated. The extent of degradation of BRZ in different stress conditions highlights that stability of BRZ in drug formulations can be improved (i) by using excipients that can impart a low-pH microenvironment, (ii) by addition of antioxidants and (iii) through protection from light.

Enzymatic degradation and metabolic pathway of acid blue 129 dye by crude laccase from newly isolated Trametes hirsuta EDN 082

Crude laccase from Trametes hirsuta EDN 082 was used in this study for the decolorization of anthraquinone and azo dyes. Characterization of degradation products from one selected dye, Acid Blue 129 (AB129), was investigated using liquid chromatography high-resolution mass spectrometry (LC-HRMS). The results showed that laccase from T. hirsuta EDN 082 (0.25 U/mL dyes) was able to decolourize AB 129, AB 113, RBBR, AB 25, RB4, and RB5 dyes with a decolorization efficiency of 88%, 73%, 71%, 72%, 42%, and 6%, respectively for 7 h. During the degradation of AB129 dye, nine metabolites were identified and characterized by electrospray ionization–liquid chromatography–mass spectrometry Quadrupole-Time of flight (ESI-LCMS-Q-TOF) analysis. Evaluation of peak area showed decreasing of the two metabolites, 1-amino-4-hydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid (D2) and 2,4,6-trimethylaniline (D4) during 7 h reaction indicated these two compounds become the centre of the degradation after cleaving the AB129 dye, and from these results, metabolic pathways were proposed. This study offers the potency of laccase from T. hirsuta EDN 082 for bioremediation of synthetic dyes in the fields.

Development and Validation of a Stability-indicating Method for Neostigmine Bromide: Separation, Identification and Characterization of Degradation Product using LC-MS and ESI-Q-TOF-MS/MS

Abstract: Objectives: Forced degradation study of the drug product and drug substance is very much important in drug development and drug discovery to establish the intrinsic stability and understand its behaviors towards different stress conditions. In the present work, stress testing of neostigmine bromide (NBr) was carried out as per ICH guidelines to identify and characterize the degradation product (DP) formed. Materials and Methods: According to ICH guidelines, drug substance was subjected to various degradation conditions. Waters BEH (Ethylene Bridge Hybrid) C-18 column (1.7 m, 100 mm 2.1 mm) having the composition of mobile phase Eluent A: 0.01M KH2PO4 in water pH-2.5 with orthophosphoric acid and Eluent B: Acetonitrile utilizing 220 nm wavelengths provided the best separation of DP and drug component. Throughout the analysis, the injection volume (5 μL) and flow rate (0.3 mLmin -1) were kept constant. Results: The limits of detection (LOD) and quantitation (LOQ) were set at 25 ngmL-1 and 50 ngmL-1, respectively. The method showed excellent linearity over a concentration range of 25-250 ngmL-1 with a regression coefficient (R2) value of 0.9999. The results showed that significant degradation was observed in base and oxidative degradation conditions whereas found in neutral, acidic, photolytic and thermal degradation conditions. Conclusion: The DP was identified and characterized using a sophisticated HRMS/MS/TOF approach for accurate mass measurement using the ESI positive mode of ionization. In the present study, the establishment of the degradation pathway of drug substance and fragmentation pathway of DP-I were explained which was never reported in any literature. Keywords: Neostigmine bromide, RP-UPLC, Stability-indicating, Stress study, Validation.

Identification and characterization of novel hydrolytic degradation products of netarsudil by LC-Q-TOF-MS/MS: In silico toxicity prediction

A forced degradation study on novel glaucoma drug netarsudil was performed as per ICH and WHO guidelines. All the degradation products were separated from the drug on a C-18 column utilizing ammonium acetate buffer (10 mM, pH 4.90) as a mobile phase-A and acetonitrile: methanol (70:30) v/v as mobile phase-B, by employing gradient elution mode. The injection volume was 10 µL with a 0.8 mL/min flow rate. The detection wavelength was 245 nm and the study was executed at a constant column temperature of 40 °C. Initially, the mass fragmentation pathway of the drug was postulated followed by characterization of degradation products from LC-MS-TOF/MS data. This was followed by LC-MS/TOF studies on the degradation products. Significant degradation was witnessed in alkaline and acidic conditions whereas no signification decline in the drug was observed in other decomposition environments. A total of four degradation products were formed and characterized with the aid of high-resolution mass spectrometry. The mechanistic pathway of formation of all DPs from netarsudil is established. In addition, comparative toxicity properties of the drug and degradation products were established using the online web server Pro Tox II.

Inherent stability testing of empagliflozin in the presence of metformin HCl by HPTLC and characterization of degradation products of empagliflozin by LC–ESI–QTOF–MS/MS

Inherent stability testing of empagliflozin in the presence of metformin HCl by HPTLC and characterization of degradation products of empagliflozin by LC–ESI–QTOF–MS/MS

Identification, Isolation and Characterization of Degradation Products (DPs) in Osimertinib Mesylate (OSM) Tablets by UHPLC-IMS-QTOF-MS, and NMR: Evaluation of Genotoxicity for OSM and DPs

The aim of this study is identification, isolation, structural characterization and qualification of degradation products DP-1, DP-2, DP-3 and DP-4. These impurities are major acid hydrolysis degradation products identified i n Osimertinib M esylate ( OSM) Tablets. T he O SM i s o bserved s table u nder thermal, photolytic and neutral conditions. The degradation of drug is observed under acidic, basic and oxidative conditions. Basic and oxidative degradation products already been reported, however, four novel degradation products are formed in acid hydrolysis which are not reported in the literature. Therefore, Using HPLC/PDA gradient program with waters X-bridge (250 mm × 4.6 mm, 5 µm) C18 column, the four acid hydrolysis degradants were identified and further characterized using UHPLC-IMS-QTOF-MS (ion mobility quadrupole time-of-flight m ass spectrometer) a nd N MR. T he s tructures o f D P-1, D P-2, D P-3 a nd D P-4 w ere further elucidated as hydroxy OSM Impurity, Des-acryl OSM impurity, Chloro OSM impurity and OSM dimer impurity, respectively. The structural characterization was followed by qualification of Impurities DP-1, DP-2, DP-3 and DP-4 using several different in-silico methodologies. From the results obtained, it can be concluded that no new structural alerts have been generated for DP-1 and DP-4 relative to the parent compound OSM. However, the degradation products, DP-2 and DP-3 shows structural alert positive for genotoxicity, which were both potential genotoxic substances. The current study presents separation, isolation, characterization and in-silico toxicity study for major acid degradants of OSM. The OSM generates DP-1 around 3.1%, DP-2 around 14.1%, DP-3 around 18.3% and DP-4 around 2.8% under acid hydrolysis on HPLC. The stress study was carried out in order to properly define the quality specifications for OSM drug products.

Simultaneous Quantification of Tiotropium Bromide Impurities G and H in Inhaler Capsules by LC-MS/MS: Method Development, Validation, and Characterization of Degradation Products by LC-Q/TOF

Simultaneous Quantification of Tiotropium Bromide Impurities G and H in Inhaler Capsules by LC-MS/MS: Method Development, Validation, and Characterization of Degradation Products by LC-Q/TOF

UHPLC-DAD Method Development and Validation: Degradation Kinetic, Stress Studies of Farnesol and Characterization of Degradation Products Using LC-QTOF-ESI-MS with in silico Pharmacokinetics and Toxicity Predictions.

Farnesol (FAR) is a sesquiterpene molecule with high lipophilicity that has antibacterial and other pharmacological properties along with broad nutritional values with high commercial values. Although having potential, FAR stability behavior and degradation kinetics are not available in the literature. Hence, it is very essential to develop a simple, rapid, accurate, precise, robust, cheap UHPLC-DAD method for FAR. It was also proposed to study mechanistic insights into FAR under different degradation conditions. Therefore, we hypothesized to do systematic stability studies along with degradation kinetic and accelerated stability studies. The developed method was validated. FAR was studied for stress studies, degradation kinetics and ADMET prediction of degradants. Degradation products were characterized using LC-QTOF-ESI-MS. Developed method consists of an isocratic mobile phase with a wavelength of 215nm. The percent recoveries for FAR were observed within the acceptance limit of 98-102%. The eight major degradation products were formed during stress studies. FAR follows first-order degradation kinetics. FAR and all degradants were found to have more than 75% good human oral absorption, and are non-toxic. FAR UHPLC-DAD method was developed, validated and performed stability studies to know the possible degradation pattern along with degradation kinetic studies.

Forced Degradation Studies of Drospirenone: Isolation and Characterization of Degradation Products

Abstract: Aim and Objectives: To remain safe for further processing or human consumption, study of stressed degradation for the identification of feasible degradants is required. The stability indicating high performance thin layer chromatographic method was developed with Camag HPTLC system. Materials and Methods: Silica C60F254 precoated TLC plates were used as stationary phase for separation of degradation products. The optimized mobile phase system consist of toluene: methanol: diethylamine (7:3:0.1) at 280 nm. Results: From the mass details and IR, NMR interpretation, the plausible structure of alkaline degradation product of drospirenone could be 17α (3-hydroxy propyl)-6β, 7β, 15β, 16β-dimethylene-5β-androstane-3β,5,17β triol and acidic degradation product of drospirenone could be 3-oxo-15α,16α-dihydro-3’H-cyclopropa[15,16]-17α-pregna-4,6- diene-21,17-carbolactone. Also In silico toxicity studies of the degradation products were performed to assess the toxicity profile of the products using Protox online sever. Conclusion: This analytical method can be considered as an alternative practical and inexpensive method for simple, accurate and efficient quantitative detection of drospirenone in the presence of its degraded products. Key words: Drospirenone, Characterization, Forced degradation studies, In silico toxicity study, Degradation products of drospirenone.

Identification of degradation impurity of TGR5 receptor agonist-ZY12201 by LC\u2013MS technique during force degradation study

Forced degradation study is a systemic characterization of degradation products of active pharmaceutical ingredient (API) at conditions which posses more harsh environment that accelerates degradation of API. Forced degradation and stability studies would be useful in selection of proper, packaging material and storage conditions of the API. These are also useful to demonstrate degradation pathways and degradation products of the API and further characterisation of the degradation products using mass spectrometry. TGR5 is a G protein-coupled receptor, activation of which promotes secretion of glucagon-like peptide-1 (GLP-1) and modulates insulin secretion. The potent and orally bioavailable TGR5 agonist, ZY12201, shows activation of TGR5 which increase secretion of GLP-1 and help in lowering blood glucose level in animal models. Hence it is necessary to establish and study degradation pathway and stability of API for better handling and regulatory approval. Force degradation studies of ZY12201 have shown presence of one oxidative impurity during oxidative degradation in HPLC analysis. The oxidized product is further characterized by LC–MS to elucidate structure of impurity and characterize its degradation pathway.

Ultrasound-assisted depolymerization of kappa-carrageenan and characterization of degradation product

Degradation of polysaccharides to afford low-molecular-weight oligosaccharides have been shown to produce new bioactivities that are not present in the starting material. The simplicity of ultrasonic treatment in the degradation of a polysaccharide, such as κ-carrageenan, offers practical advantage in producing degraded products with lower molecular weight that may have new interesting potential activities. This study embarked on investigating the effects in molecular weights and structural changes of κ-carrageenan under varying ultrasonic conditions. Molecular weight (MW) monitoring of ultrasonically-treated κ-carrageenan at various conditions were done by gel permeation chromatography. The product formed using the optimized condition was characterized using FTIR and NMR. The decrease in MW has been shown to be dependent on low concentration (5.0 mg mL−1), high amplitude (85%), and long treatment time (180 mins) to afford a degraded κ-carrageenan with average molecular weight (AMW) of 41,864 Da, which is a 96.33% reduction from the raw sample with initial AMW of 1,139,927 Da. Structural analysis reveals that most of the peaks of the raw κ-carrageenan was retained with minor change. 1D and 2D NMR analyses showed that the sonic process afforded a product where the sulfate group at the G4S-4 position was cleaved forming a methylene in the G4S ring. The results would be useful in the structure–activity relationship of κ-carrageenan oligosaccharides and in understanding the effect in the various potential applications of degraded κ-carrageenan.

Oxidative degradation profile studies of tavaborole by a validated stability indicating RP-UPLC method: Isolation and characterization of novel degradant using 2D-NMR and HRMS.

The current research work reports a study on the degradation profile of tavaborole, which is an oxaborole antifungal drug used to treat infections in the toenails. This work also reports the chemical stability of tavaborole in different stress conditions along with the isolation and characterization of degradation products by high-resolution mass spectrometry and two-dimensional nuclear magnetic resonance techniques. A sensitive and reproducible stability-indicating ultra-performance liquid chromatography method was developed and validated for quantification of tavaborole bulk drug in the presence of degradation products. Significant degradation was observed during oxidative stress conditions using H2 O2 . It was observed that the drug was highly unstable under oxidation stress conditions and thus degradation profiles with various oxidizing reagents were studied. One unknown impurity (DP-1) was formed during peroxide degradation, which was isolated by reverse-phase preparative chromatography. The structure of this degradant was characterized by high-resolution mass spectrometry and multidimensional nuclear magnetic resonance techniques. The structure of this novel impurity DP-1 was identified as [4-fluoro-2-(hydroxymethyl)phenol], which was not reported as a degradant in the literature. An Acquity BEH C18 , 100 × 2.1 mm, 1.7 μm column was used to achieve the desired separation within a shorter runtime of 4.0 min. The method was validated for specificity, precision, linearity and accuracy over the concentration range of 5.0-400 μg ml-1 (r2 -0.9999) and limit of quantitation 5.0 μg ml-1 . This method is compatible with LCMS analysis which enables to identify the unknown impurities formed in the process.

Stress degradation study of bortezomib: effect of co-solvent, isolation and characterization of degradation products by UHPLC-Q-TOF-MS/MS and NMR and evaluation of the toxicity of the degradation products

Bortezomib (BTZ) is a first-in-class, potent reversible inhibitor of proteasome used in the treatment of multiple myeloma, the second most common hematological cancer.

Development and validation of stability indicating HPTLC method for estimation of pirfenidone and characterization of degradation product by using mass spectroscopy

Pirfenidone is used as a novel antifibrotic agent approved for mild-to-moderate idiopathic pulmonary fibrosis. An extensive literature search revealed that, method validation by high-performance thin-layer chromatography (HPTLC) and structural determination by tandem mass spectrometry (MS/MS) method was not reported till date. Precoated silica gels plates were used as a stationary phase. Methanol: ethyl acetate: toluene (1:2:7 v/v) was delivered best separation at 315 nm (Rf 0.49±0.03) by densitometry analysis. Degradation analysis was performed as per ICH guidelines Q2 (R1). Isolation of degradation product was done by the HPTLC method and characterized by using MS/MS method. All the validation parameters were found within the range. Moreover, its possible degradation pathway was also proposed. The Proposed developed and validated HPTLC method was found to be more sensitive, simple, precise, accurate, cost-effective and robust. This method could be applied for the analysis of bulk drug and tablet formulation, degradation study. This degradation pathway of the drug will further help to identify the degradation products of Pirfenidone which may be used for the impurity profiling of the drug.

A stability indicating method development and validation for separation of process related impurities and characterization of unknown impurities of tyrosine kinase inhibitor Nilotinib by RP-HPLC, NMR spectroscopy and ESI-MS

A selective RP-HPLC method for separation and determination of potential related impurities (process related, and degradants) of Nilotinib (NTB) drug substance has been developed and validated. The separation was accomplished on YMC Triart C-18 (150 × 4.6 mm, 3.0 µm) column connected to a photodiode array (PDA) detector using 10 mM NH 4 H 2 PO 4 (pH: 3.5 ± 0.05 adjusted with diluted Ortho phosphoric acid) as a buffer and acetonitrile in a ratio of 85:15 (%v/v) respectively as mobile phase-A, and 10 mM NH 4 H 2 PO 4 buffer, acetonitrile and methanol in a ratio of 20:27.5:52.5 (%v/v/v) respectively as mobile phase B, under gradient elution. The flow rate and column oven temperature were 1.0 mL/min and 50 °C respectively. Detection was carried out at 230 nm. NTB was stressed to degrade under various stress conditions of photolysis, thermal, hydrolysis and oxidation stress as per ICH Q1A (R2). The drug was highly susceptible to acidic and alkaline hydrolytic stress conditions. The drug degraded to two degradation products (DPs), while the remains stable under thermal, photolytic, neutral hydrolytic and oxidative stress conditions. Characterization of DPs was performed by 1 D and 2 D NMR, FT-IR and LC/MS/MS. Optimized method was validated in agreement with ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, robustness and ruggedness. This optimized method can be used for quality control of both drug substance and drug product of NTB.