Hydrolytic Degradation Products Research Articles

Stability Indicating High‐performance Liquid Chromatography Method for Quantification of Amoxapine and Characterization of Forced Degradation Products Employing Quadrupole‐Time of Flight Mass Spectrometry

ABSTRACTAmoxapine, a tricyclic antidepressant, has been widely used for long‐term treatment of depression along with other psychiatric disorders involving neurosis, agitation, and anxiety. The drug may undergo degradation during manufacturing, transportation, and storage. To ensure drug safety, both identification and characterization of the degradation products (DPs) should be considered. Accordingly, a sensitive and selective stability‐indicating assay method was developed for amoxapine using high‐performance liquid chromatography (HPLC). The amoxapine drug was subjected to acidic hydrolysis, basic hydrolysis, and oxidation separately. The degradation susceptibility of the amoxapine was found to be in the order of Acid hydrolysis > Oxidation > Base hydrolysis. The developed method was validated as per the International Council for Harmonization Q2(R2) guideline. Further, LC‐electrospray ionization‐quadrupole‐time of flight‐tandem mass spectrometry was employed for the identification and characterization of the DPs formed under stress conditions. The study revealed that under accelerated stress conditions, three hydrolytic DPs and one oxidative DP were formed. Three of the four DPs were found to be novel and never reported before. In addition, a plausible mechanism representing the formation of all DPs has also been established.

Identification and characterization of alkaline hydrolytic degradation products of ivacaftor by UPLC-PDA and UPLC-ESI-QTOF-MS techniques.

The study of inherent stability characteristics of drugs has been advocated to be important by various regulatory agencies like the ICH, USFDA, and others. The current work was envisaged to investigate the forced degradation profile of the drug ivacaftor under ICH-prescribed stress conditions, identification of its potential degradants, and postulation of the degradation routes for their generation. The stress degradation studies were performed on the drug as per the ICH guideline Q1A(R2). A UPLC-photodiode array-based chromatographic method was developed to satisfactorily resolve the drug from its degradation products, validated in accordance with various ICH prescribed parameters, and assessed for its BAGI practicality index. The degradation products were identified and characterized by UPLC-ESI-QTOF-MS studies. The drug was found to significantly degrade under conditions of alkaline hydrolytic stress and it was found to be stable under all other stressor environments including acid/neutral hydrolytic, photolytic, thermal, and oxidative stress. Four alkaline hydrolytic degradation products (I-IV) were revealed by UPLC-QTOF-MS studies which were well-resolved from the drug over a C18 UPLC column by the developed UPLC-PDA method. The detection wavelength was selected as 310 nm. Characterization of the four degradation products (I-IV) was carried out by their mass spectral data and their respective degradation routes were elucidated. A UPLC-PDA method was developed and validated for ivacaftor and its practicality BAGI index was computed. Four alkaline hydrolytic degradation products of ivacaftor were revealed through UPLC-ESI-QTOF-MS studies and corresponding degradation routes were elucidated.

Degradation of high concentrations of commercial polylactide packaging on food waste composting in pilot-scale test

The increasing use of synthetic biodegradable polymers, such as aliphatic polyesters, has led to a greater need to understand their behavior in an end-of-life scenario as food packaging materials. The aim of this work was to investigate the effect on composting of high to 10 wt% concentration of commercial polylactide packaging in food waste during a 98-day pilot-scale test. Members of the genera Bacillus, Geobacillus, Caldibacillus, Compostibacillus, Novibacillus, Planifilum and Aeribacillus accounted for 77 % of the bacterial community at the initial stage. Significant fragmentation of the polylactide packaging was observed after 14 days, and the appearance of low-molecular weight (approximately 5.4 kDa) hydrolytic degradation products led to an increase in biodiversity and a prolongation of the thermophilic stage by 12 days. The results obtained show the possibility of efficient disposal of food waste with high concentration of polylactide packaging under industrial composting conditions.

Development and validation of stability indicating assay method for mitapivat: Identification of novel hydrolytic, photolytic, and oxidative forced degradation products employing quadrupole-time of flight mass spectrometry.

Mitapivat is a novel, first-in-class orally active pyruvate kinase activator approved by the US Food and Drug Administration in 2022 for the treatment of hemolytic anemia. There is no literature available regarding the identification of degradation impurities of mitapivat. The present study deals with the degradation behavior of mitapivat under various stress conditions such as hydrolytic, photolytic, thermal, and oxidative stress. The multivariate analysis found that the independent variables, that is, molarity, temperature, and time, are interacting with each other to affect the degradation of mitapivat. A specific, accurate, and precise high-performance liquid chromatographic (HPLC) method was developed to separate mitapivat from its degradation products. The separation was achieved on the C-18 column (250mm × 4.6mm × 5µm) using the combination of 0.1% formic acid buffer and acetonitrile in gradient elution profile. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Q2(R2) guideline. LC-electrospray ionization-Quadrupole-time of flight was employed to identify degradation products. A total of seven novel degradation products of mitapivat were identified based on tandem mass spectrometry and accurate mass measurement. In-silico toxicity of mitapivat and its degradation products was qualitatively evaluated by the DEREK toxicity prediction tool.

Development of a Reversed-Phase UPLC Method for Assay of Fipronil Including Determination of Its Related Substances in Bulk Batches of Fipronil Drug Substance.

Fipronil is a commonly used pesticide in the agricultural and animal health industries for the protection of crops and control of fleas, ticks, and chewing lice. It is difficult to obtain reproducible retention time and relative retention time (RRT) for a common hydrolytic degradation product of fipronil with the current European Pharmacopeia (EP) monograph for assay and estimation of related substances of fipronil. This situation causes misidentification, mislabeling, and/or false out-of-specification results for this hydrolytic degradation product of fipronil in bulk commercial batches during batch release and/or in the stability samples during the shelf life of a released batch. This study aimed to develop a reversed-phase ultra performance liquid chromatography (UPLC) method for assay and identification of fipronil including identification and estimation of its related substances in bulk drug substance batches of fipronil and provide consistent retention time of the hydrolytic degradation product. Fipronil and its related substances were separated by gradient elution on a Halo C18 column (50 mm × 2.1 mm id, 2.0 µm particle size) maintained at 40°C with 0.1% H3PO4 in H2O as mobile phase-A and acetonitrile-methanol (50 + 50, v/v) as mobile phase-B. Fipronil and its related substances were detected and quantified at 280 nm with a quantitation limit of 0.05% of the target (analytical) concentration. The UPLC method was able to separate all analytes of interest by gradient elution with a total run time of 7 min (approximately 40% faster than EP). In this paper, we report the development and validation of a fast, stability-indicating reversed-phase UPLC method for assay and estimation of related substances of fipronil in stability samples and bulk batches of fipronil. The new UPLC method is approximately 40% faster than the current Ph. Eur. monograph for fipronil assay and the new method provides reproducible retention of a common hydrolytic degradation product of fipronil.

The acute toxicity of Novichok's degradation products using quantitative and qualitative toxicology in silico methods.

The emergence of Novichok agents, potent organophosphorus nerve agents, has spurred the demand for advanced analytical methods and toxicity assessments as a result of their involvement in high-profile incidents. This study focuses on the degradation products of Novichok agents, particularly their potential toxic effects on biological systems. Traditional in vivo methods for toxicity evaluation face ethical and practical constraints, prompting a shift toward in silico toxicology research. In this context, we conducted a comprehensive qualitative and quantitative analysis of acute oral toxicity (AOT) for Novichok degradation products, using various in silico methods, including TEST, CATMoS, ProTox-II, ADMETlab, ACD/Labs Percepta, and QSAR Toolbox. Adopting these methodologies aligns with the 3Rs principle, emphasising Replacement, Reduction, and Refinement in the realm of toxicological studies. Qualitative assessments with STopTox and admetSAR revealed toxic profiles for all degradation products, with predicted toxicophores highlighting structural features responsible for toxicity. Quantitative predictions yielded varied estimates of acute oral toxicity, with the most toxic degradation products being EOPAA, MOPGA, MOPAA, MPGA, EOPGA, and MPAA, respectively. Structural modifications common to all examined hydrolytic degradation products involve substituting the fluorine atom with a hydroxyl group, imparting consequential effects on toxicity. The need for sophisticated analytical techniques for identifying and quantifying Novichok degradation products is underscored due to their inherent reactivity. This study represents a crucial step in unravelling the complexities of Novichok toxicity, highlighting the ongoing need for research into its degradation processes to refine analytical methodologies and fortify readiness against potential threats.

Separation and characterization of hydrolytic degradation product of deucravacitinib by validated high‐performance liquid chromatography method and liquid chromatography‐mass spectrometry

AbstractDeucravacitinib, SOTYKTU was approved recently in the year 2022 to treat psoriasis. The present work attempts to identify and characterize degradation products formed when the drug is exposed to hydrolytic, oxidative, thermal, and photolytic stress conditions as well as to study the kinetics of the drug's degradation under various stress conditions. A high‐performance liquid chromatography method was developed for the separation of deucravacitinib and its degradation product comprising mobile phase of ammonium acetate (pH 4.75) buffer as solvent A and acetonitrile as solvent B and Phenomenex Gemini, C‐18 (250 × 4.6 mm, 5µ) column as stationary phase. Injection volume, flow rate, and detection wavelength for the method were optimized as 10 µL, 1.0 mL/min, and 254 nm, respectively. Accuracy, precision, linearity, robustness, and selectivity were found to be acceptable when validated in the concentration range between 5 and 150 µg/mL of deucravacitinib. The structure of the degradation product was characterized using liquid chromatography‐tandem mass spectrometry which shows a protonated molecular ion peak at m/z 358.1805 in the electrospray ionization positive mode. In silico mutagenicity tests yielded positive results for deucravacitinib and its degradation product, triggering an alarm for mutagenicity for both structures, whereas in silico toxicity studies did not generate any structural alerts.

Effect of di-butyl phosphate on the distribution behavior of uranyl ions in PUREX solvent

ABSTRACT Di-butyl phosphoric acid (HDBP) is one of the major hydrolytic and radiolytic degradation product of tri-n-butyl phosphate (TBP) employed for the extraction of U(VI) and Pu (IV) from the spent nuclear fuel dissolver solution. The presence of HDBP in the solvent phase limits the recovery of actinides during stripping. In view of this, the extraction and stripping behavior of uranyl nitrate in 1.1 M TBP in n-dodecane (n-DD) was studied in presence of di-butyl phosphate (HDBP). The distribution ratio (D) of uranium (VI) in HDBP/n-DD was negligible and the D value increased gradually with an increase in HDBP content in the organic phase. An alternate method was developed for estimation of HDBP in TBP/n-DD system based on uranium metal retention in organic phase. Based on the results it was found that 1 M HDBP in 1.1 M TBP/n-DD retained about 0.63 M of uranium (VI) in the organic phase during stripping. The results were further validated using irradiated solvent. FT-IR spectroscopic investigation also revealed the retention of U(VI) in 1.1 M TBP/n-DD containing HDBP even after several stripping with 0.01 M HNO3.

A solvent-free HPLC method for the simultaneous determination of Favipiravir and its hydrolytic degradation product

During COVID-19 pandemic, Favipiravir (FPV) showed a great efficacy against COVID-19 virus, it produced noticeable improvements in recovery of the patients. The aim of this study was to develop a new, green and simple method for the simultaneous determination of FPV and its acid-induced degradation product (ADP) in its pure and pharmaceutical dosage forms. This method will be key for the inevitable development of FPV solution and inhaler formulations. A green micellar RP-HPLC method was developed using an RP-VDSPHERE PUR 100 column (5 µm, 250 × 4.6 mm) and an isocratic mixed micellar mobile phase composed of 0.02 M Brij-35, 0.1 M SDS and 0.01 M potassium dihydrogen orthophosphate anhydrous and adjusted to pH 3.0 with 1.0 mL min−1 flow rate. The detection was performed at 280 nm with a run time of less than six min. Under the optimized chromatographic conditions, linear relationship has been established between peak area and concentration of FPV and its ADP in the range of 5–100 and 10–100 µg mL−1 with elution time of 3.8 and 5.7 min, respectively. The developed method was validated according to the ICH guidelines and applied successfully for determination of FPV in its pharmaceutical dosage form.

The role of aggregation and ionization in the chemical instability of Amphotericin B in aqueous methanol

Amphotericin B (AmB) is a potent antimicrobial agent used in clinical practice. Nevertheless, the mechanism of its aqueous instability remains not yet fully understood, especially the role that its aggregation state plays in this process. Therefore, the current study used an aqueous methanol media to evaluate the AmB instability as a function of pH-, organic solvent- and concentration-dependent ionization and aggregation. To reach this goal, the aggregation status and instability were determined using UV–vis spectroscopy, LC-MS and HPLC. Moreover, not only the hydrolytic degradation products were identified by UV–vis spectroscopy and LC-MS, but also, the degradation rate constants were estimated by nonlinear regression. The results indicated that monomeric AmB was the predominant species under pH conditions, wherein the substrate was cationic (pH < 4) or anionic (pH > 9). On the other hand, aggregated AmB form was the predominant species for the zwitterionic substrate (at methanol concentration < 30 %(v/v)). Anionic substrate degraded by specific base-catalyzed lactone hydrolysis. Oxidation accounted for the loss of zwitterionic substrate. Aggregated zwitterionic AmB exhibited lower stability than monomeric zwitterionic AmB under neutral pH conditions. These studies are a step forward in comprehending the degradation kinetics of AmB in aqueous medium. In fact, along with our previous research on AmB instability in oils, it leads to a better understanding of the AmB stability in complex systems with an oil–water interface, such as disperse lipid systems.

Development of stability-indicating method for separation and characterization of benidipine forced degradation products using LC-MS/MS.

The present study describes forced degradation of benidipine (BEN) as per Q1A (R2) and Q1B guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. BEN degraded under hydrolysis (neutral, acidic, and alkaline), hydrogen peroxide induced oxidation, and UV light mediated photolytic degradation. A total of 14 degradation products (DPs) were found in all degradation studies, comprising 4 hydrolytic DPs, 8 oxidative DPs, and 4 photolytic DPs. A selective stability-indicating method was developed using an XBridge BEH C18 column with gradient elution program consisting of ammonium acetate (10mM, 4.8 pH, acetic acid) and acetonitrile. The flow rate was maintained at 1ml min-1 . All DPs were separated well using the developed HPLC method and were characterized using LC-MS/MS data. As this method is effective in identifying and separating BEN and its DPs with sufficient resolution, it can be used in laboratories for quality control of drugs in daily routine analysis and stability studies.

A synergistic framework for development of green HPLC methods for determination of amlodipine besylate and candesartan cilexetil in presence of their hydrolytic degradation products in two different matrices

The world is changing so fast towards rationalization of resources consumption and employing safer practices for the surrounding environment and humans. Therefore, there is a renewed interest in integrating green analytical chemistry principles and experimental design methodologies for screening and optimizing complex analytical procedures such as HPLC. Thus, this work introduces three sustainable versatile multi-analyte HPLC methods with two types of detection techniques. Firstly, amlodipine besylate and candesartan cilexetil with three of their hydrolytic products were chromatographed on CSH-C18 (150 mm) stationary phase within short run time (about 8.9 min) through gradient elution mode, flow rate 1 mL min−1 and UV-detection at 210 nm and 254 nm. Face-centered composite design and Derringer's desirability function were implemented to optimize the chromatographic conditions to fit the goals of pharmaceutical and biological analysis via fluorometric detection. A good separation was obtained utilizing HSS-Cayno (250 mm) column and mobile phase composed of ethanol and phosphate buffer, pH 3.5, (55.6: 44.4, v/v) delivered at 1.03 mL min−1 at 245 and 446 nm as excitation and emission wavelengths, respectively, with quantification range of 1.00–400.00 ng mL−1 for amlodipine and 1.00–600.00 ng mL−1 for candesartan cilexetil. The conditions were modified for plasma analysis to a new ratio (45:55, v/v) pH 3.7 and flow rate 0.7 mL min−1. The methods' greenness profiles have been evaluated and compared with the reported ones via several comprehensive tools.

Structural characterization of novel hydrolytic and oxidative degradation products of acalabrutinib by LC-Q-TOF-MS, H/D exchange and NMR

The drug substance, acalabrutinib was subjected to hydrolytic (acid, base and neutral) and oxidative stress degradation as per ICH recommendations. Degradation products (DPs) generated from the drug substance were separated on a Shimadzu Shim-pak C-8 column utilizing a mobile phase composed of methanol: acetonitrile (90:10 v/v) and ammonium acetate buffer (10 mM, pH 3.80) in a gradient elution mode. Acalabrutinib was found to be labile under acid, basic, neutral and oxidative conditions. A total of eighteen DPs of drug substance were formed in hydrolytic (fourteen DPs) and oxidative degradation conditions (four DPs). All the DPs were characterized by comparing the LC-Q-TOF mass spectrometric fragmentation pathway of the drug substance with DPs. Further, hydrogen/deuterium (H/D) exchange studies were also carried out on the DPs to confirm the presence of labile hydrogens in their structures. Four DPs (H-12, O-2, O-3 and O-4) were isolated for chemical structural elucidation by NMR. Probable mechanisms involved in degradation of acalabrutinib were also postulated.

Identification of 2,2‐dinitropropanol, a Hydrolyzed Product of Aged Eutectic Bis(2,2‐dinitropropyl) Acetal – Bis(2,2‐dinitropropyl) Formal Mixture

AbstractEutectic bis(2,2‐dinitropropyl) acetal ‐ bis(2,2‐dinitropropyl) formal mixture, nitroplasticizer (herein called NP) has historically been produced by either the ter Meer or oxidative nitration synthesis process, wherein 2,2‐dinitropropanol (DNPOH) is produced as an intermediate step in both processes. Therefore that DNPOH, could be present in NP either as a production or hydrolysis degradation product is worth investigation. Here, we synthesized DNPOH, validated the synthesis using NMR, and identified DNPOH in aged NP using liquid chromatography tandem time of flight – quadrupole mass spectrometry (LC‐QTOF). Using these results, for the first time we positively identify that DNPOH is absent from NP after its production, but is present as a degradation product through hydrolysis from a thermo‐chemical aging profile of NP. To hydrolyze NP, prerequisite is the presence of both water and acid. Despite the presence of water in NP, DNPOH is only generated in the late stage of the aging process, when acid concentration is sufficiently high. It has been previously shown both theoretically and experimentally that a primary step of NP degradation is HONO elimination followed by decomposition, wherein nitric acid, nitrous acid, and water are produced (NP→NP’+2HONO and 2HONO→NO+NO2+H2O). It is shown from this reaction series that water slows HONO decomposition and therefore in small quantities, 100 s of ppm, water actually stabilizes NP against hydrolysis by equilibrium and reducing acidity.

Eco-friendly stability-indicating HPTLC micro-determination of the first FDA approved SARS-CoV-2 antiviral prodrug Remdesivir: Study of degradation kinetics and structural elucidation of the degradants using HPTLC-MS

The worldwide spread coronavirus (covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a global health crisis. The world was forced to face a great challenge to control and overcome this health disaster through various containment measures including efficient vaccination side by side with effective medication. Remdesivir (RMD) is the first FDA approved antiviral agent for treatment of covid-19 pandemic and hence regarded as the first-in-class medication of this highly contagious respiratory disease. The current study represents the first stability indicating HPTLC method for the estimation of RMD in bulk form and pharmaceutical formulation. The method employed TLC silica gel aluminum plates 60 F254 as stationary phase and green mobile phase composed of ethyl acetate and ethanol (96: 4, v/v) with densitometric detection at 245 nm. Comprehensive validation of the adopted method was accomplished according to the ICH guidelines regarding linearity, ranges, detection and quantification limits, precision, accuracy and robustness. The developed method offered a neat separation of the drug in presence of pharmaceutical excipients as well as in presence of acidic, alkaline, neutral hydrolytic, oxidative and photolytic degradants. Additionally, structural elucidation of alkaline and hydrolytic oxidation degradation products was carried out using HPTLC-MS. Furthermore, for the first time the acidic and alkaline degradation kinetics of RMD were studied and its degradation rate constants and half-lives were calculated. Moreover, greenness appraisal of the developed method as well as comparison with previously published stability indicating HPLC methods were performed using analytical Eco-scale, GAPI and AGREE metrics.

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.

Univariate versus multivariate spectrophotometric methods for the simultaneous determination of omarigliptin and two of its degradation products

Six selective spectrophotometric techniques, four univariate and two multivariate ones were developed for the determination of the antidiabetic drug omarigliptin (OMR) along with its hydrolytic and oxidative degradation products. The proposed univariate spectrophotometric methods were ratio subtraction, first derivative, derivative ratio and ratio difference. Linearities were constructed in the range of 10.0–180.0 μg mL−1 for both OMR & its hydrolytic degradation product and 10.0–110.0 μg mL−1 for the oxidative degradation one. On the other hand, partial least squares and artificial neural networks were the chosen multivariate approaches. Their linearity ranges were 20.0–60.0 μg mL−1 for OMR and 10.0–30.0 μg mL−1 for the two degradation products. All the methods were validated, effectively applied for quantification of the intact drug in its tablet formulation and favorably compared to the reported one.

Fast determination of paracetamol and its hydrolytic degradation product p-aminophenol by capillary and microchip electrophoresis with contactless conductivity detection.

Paracetamol (PAC) is one of the most extensively used analgesics and antipyretic drugs to treat mild and moderate pain. P-aminophenol (PAP), the main hydrolytic degradation product of PAC, can be found in environmental water. Recently, CE has been developed for the detection of a wide variety of chemical substances. The purpose of this study is to develop a simple and fast method for the detection and separation of PAC and its main hydrolysis product PAP using CE and microchip electrophoresis with capacitively coupled contactless conductivity detection. The determination of these compounds using microchip electrophoresis with capacitively coupled contactless conductivity detection is being reported for the first time. The separation was run for all analytes using a BGE (20mM β-alanine, pH 11) containing 14% (v/v) methanol. The RSDs obtained for migration time were less than 4%, and RSDs obtained for peak area were less than 7%. The detection limits (S/N = 3) that were achieved ranged from 0.3 to 0.6mg/L without sample preconcentration. The presented method showed rapid analysis time (less than 1min), high efficiency and precision, low cost, and a significant decrease in the consumption of reagents. The microchip system has proved to be an excellent analytical technique for fast and reliable environmental applications.

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.

LC-HRMS studies on ruxolitinib degradation: a comprehensive approach during drug development.

Ruxolitinib, a kinase inhibitor, was subjected to stress studies as described in the ICH Q1A(R2) guidelines. Solution state hydrolytic and solid state oxidative and thermal stress studies were carried out to understand its degradation behaviour. The drug showed significant instability in the hydrolytic condition in comparison with other conditions. HPLC and UHPLC methods were developed for the separation of the drug and its hydrolytic degradation products. Mass fragmentation pathway of the drug was established as the first step of the LC-MS characterization of the degradation products. MS/MS analysis of the drug and MS3 of selected fragments were achieved through QTOF and QTRAP by varying the collision energy and performing an H/D exchange. LC-MS/MS QTOF studies were subsequently carried out on stress samples and the structures of the degradation products were identified through comparison of the drug fragmentation pathways. The four hydrolytic products viz. 4-(1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine, 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanoic acid, 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanamide, and 3-(4-(6-amino-5-formylpyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile were formed under acidic and basic conditions. The degradation pathway was delineated through a mechanistic explanation. The in silico tools preADMET and Protox-II predictor were used to compare the toxicity of the impurities with respect to the drug.