Development and validation of a method for studying Relugolix and its impurities by UPLC-MS

Understanding of cabotegravir degradation through isolation and characterization of key degradation products and kinetic studies

The present study investigated the degradation behaviour of tivozanib (TVZ), a potent tyrosine kinase inhibitor, under various stress conditions, including hydrolytic (acid, base, and neutral), oxidative, thermal, and photolytic (UV and visible light) conditions. The resulting degradation products (DPs) were characterized by LC-Q-TOF-MS/MS and NMR. Additionally, in silico toxicity assessment and mutagenicity studies were accomplished by using DEREK Nexus and SARAH Nexus for TVZ and its DPs. The in silico degradation profile of TVZ was predicted using Zeneth software. Separation of the DPs was executed by RP-HPLC with gradient elution mode using Shim-pack Solar C18 column (250 × 4.6 mm, 5 μm). The mobile phase consists of solvent A as 10 mM ammonium acetate buffer (pH 3.8) and solvent B as a mixture of acetonitrile:methanol (70%:30% v/v). Structural characterization was performed for TVZ and its DPs using LC-Q-TOF-MS/MS and NMR spectroscopy. TVZ was sensitive to hydrolytic (acid, base, and neutral), oxidative, and photolytic (UV light) degradation conditions, wherein seven DPs were observed and separated by RP-HPLC. A plausible degradation pathway of TVZ was proposed based on mass spectrometry data. The preparative HPLC was used to isolate DP-III, DP-IV, and DP-VI, and the structures were confirmed by NMR spectroscopy. Furthermore, the 2D NMR data helped to differentiate the chemical structure of DP-IV, which had the same molecular mass asTVZ. Forced degradation studies were conducted in accordance with ICH Q1A (R2) and Q1B guidelines. Totally, seven DPs were observed in various stress conditions. DP-IV, a structural isomer of TVZ, was detected in basic hydrolytic conditions. The developed RP-HPLC method was validated following the ICH Q2(R1). This study helps in the stability testing and routine quality control analysis during the formulation development of TVZ.

A Validated, Specific Stability-Indicating RP-LC Method for Moxifloxacin and Its Related Substances

To assess the stability of Relugolix under stress conditions, identify and characterize the degradation products, it was subjected to hydrolytic and oxidative stress conditions, according to the International Council for Harmonization guideline Q1A (R2). The drug showed degradation in basic and oxidative conditions, while it was stable in acid conditions. Four degradation products were obtained, two from each basic and oxidative degradation, which was separated on a C18 column by using Auto Purification Mass Spectrometer. The four degradant products have not been reported earlier in any of the literature and are being reported here for the first time. HIGHLIGHTS Present work captures force degradation of Relugolix under acidic, basic, oxidative, photolytic, and thermal conditions as per International Council for Harmonization guidelines. Two degradation products were observed in basic condition marked as Degradation Product-1, Degradation Product-2 and two degradation products were formed in oxidative condition marked as Degradation Product-3, Degradation Product-4. All the four degradants are novel and are not published in any of the literature. Four degradation products were identified and characterized using various high-end analytical techniques such as Liquid Chromatography and Mass Spectrometer, Reversed Phase–High Performance Liquid Chromatography, High Resolution Mass Spectrometer and Nuclear Magnetic Resonance spectroscopy.

Forced degradation studies are useful for better understanding of the stability of active pharmaceutical ingredients and drugs and to generate information about drug degradation pathways and formation of degradation products (DPs). Identification of DPs plays a vital role in establishing the safety and therapeutic benefit of a drug. Canagliflozin (CAN) was subjected to different stress conditions as per International Conference on Harmonization guidelines (Q1A R2). All the DPs and the drug were well separated on an Aquity CSH C18 (100 × 2.1mm, 1.7 μm) column using acetonitrile-methanol (70:30, v/v) and formic acid in gradient mode. The same UPLC method was employed for LC/HRMS for the characterization of DPs. In addition, in silico toxicity was predicted for all the DPs by using TOPKAT and DEREK software tools. CAN was found to degrade under oxidative stress condition and formed DP1 and DP2. This is a typical case of degradation where co-solvents acetonitrile-water (50:50, v/v) and methanol-water (50:50, v/v) react with CAN under acid hydrolytic conditions leading to the formation of pseudo-DPs, DP3 and DP4, respectively. Among these, DP2 and DP3 showed ocular irritancy whereas DP1 showed skin sensitization. The drug was labile under oxidative stress condition. CAN reacted with co-solvent under acid hydrolytic conditions and gave pseudo-DPs. All the DPs were separated using UPLC and characterized by LC/QTOF/MS/MS. Toxicity of DPs was evaluated using TOPKAT and DEREK software tools.

The current research work envisages an analytical quality by design-enabled development of a simple, rapid, sensitive, specific, robust and cost-effective stability-indicating reversed-phase high-performance liquid chromatographic method for determining stress-induced forced-degradation products of sorafenib tosylate (SFN). An Ishikawa fishbone diagram was constructed to embark upon analytical target profile and critical analytical attributes, i.e. peak area, theoretical plates, retention time and peak tailing. Factor screening using Taguchi orthogonal arrays and quality risk assessment studies carried out using failure mode effect analysis aided the selection of critical method parameters, i.e. mobile phase ratio and flow rate potentially affecting the chosen critical analytical attributes. Systematic optimization using response surface methodology of the chosen critical method parameters was carried out employing a two-factor-three-level-13-run, face-centered cubic design. A method operable design region was earmarked providing optimum method performance using numerical and graphical optimization. The optimum method employed a mobile phase composition consisting of acetonitrile and water (containing orthophosphoric acid, pH 4.1) at 65:35 v/v at a flow rate of 0.8 mL/min with UV detection at 265 nm using a C18 column. Response surface methodology validation studies confirmed good efficiency and sensitivity of the developed method for analysis of SFN in mobile phase as well as in human plasma matrix. The forced degradation studies were conducted under different recommended stress conditions as per ICH Q1A (R2). Mass spectroscopy studies showed that SFN degrades in strongly acidic, alkaline and oxidative hydrolytic conditions at elevated temperature, while the drug was per se found to be photostable. Oxidative hydrolysis using 30% H2 O2 showed maximum degradation with products at retention times of 3.35, 3.65, 4.20 and 5.67 min. The absence of any significant change in the retention time of SFN and degradation products, formed under different stress conditions, ratified selectivity and specificity of the systematically developed method.

Sunitinib belongs to the tyrosine kinase inhibitors class of medical drugs prescribed to treat various types of cancers, including renal cell carcinoma, gastrointestinal stromal tumors, and pancreatic neuroendocrine tumors by blocking the action of enzymes that promote cancer cell growth. Forced degradation studies are crucial for assessing drug stability and will be helpful in drug discovery and development. In this study, sunitinib underwent different stress testing according to ICH guidelines to identify and analyze its degradation products (DPs). The stress study findings proved that sunitinib is highly sensitive to acid (2DPs), base (1 DP), and oxidation (1 DP) degradation but remains stable in other stress conditions with significantly less degradation. The separation of sunitinib and its impurities and four DPs was achieved using a Waters XBridge C18 column (250 mm × 4.6 mm, 5 µm) with 0.7 mL/min gradient flow of ammonium formate buffer (pH 4.8) and acetonitrile with a detection wavelength of 258 nm. These conditions produce well‐retained and resolved peaks corresponding to sunitinib at 8.4 min, whereas 4.8, 3.5, and 1.9 min are noticed for impurities 1, 2, and 3, respectively. These study findings provide a comprehensive analysis of sunitinib, its impurities, and DPs, offering crucial insights into its stability and toxicity that can enhance drug development and safety profiling. The study also detailed the degradation pathway of sunitinib, its impurities, and the fragmentation patterns of DPs, which had not been previously reported in any other studies. All these four DPs belong to Class 4 (moderate) toxicity with distinct pharmacokinetic profiles. Hence, it can be concluded that these study findings provide a comprehensive analysis of sunitinib, its impurities, and DPs, offering crucial insights into its stability and toxicity that can enhance drug development and safety profiling.

LC and LC–MS/MS studies for the identification and characterization of degradation products of acebutolol

The present research work was carried out to determine the stability of erlotinib hydrochloride (ERLO) drug under different stress conditions recommended by International Conference on Harmonization (ICH) guideline Q1A (R2). The stability of the drug was studied under hydrolytic, oxidative, photolytic, and thermal stress conditions. The drug was found susceptible to degradation under acidic, basic, and photolytic stress conditions, while it was stable under neutral, oxidative, and thermal stress conditions. A total of three degradation products (DPs) were formed. Separation was carried out by using high-performance liquid chromatography system (HPLC). Better separation was achieved on Kromasil®(150 mm × 4.6 mm, 5 µm) C18 column using gradient elution program. A mixture of stressed samples was subjected to LC-MS/TOF studies to obtain mass spectral data. The information obtained from accurate mass study was first utilized to build a complete mass fragmentation pathway of the drug, and later it was used for its degradants. Structures were proposed for each fragment based on the best possible molecular formula. Three novel DPs 6-(2-hydroxyethoxy)-7-(2-methoxyethoxy)quinazolin-4-amine, 6,7-bis(2-methoxyethoxy)quinazolin-4-amine, and N-(3-ethynylphenyl)-6, 7-bis (2-methoxyethoxy)-2-oxy-quinazolin-4-amine were identified.

The investigation's objective was to access the stability of the drug substance Pimozide under the stress conditions specified in the International Conference on Harmonization Q1A(R2) guideline and to separate, identify, and characterize the degradation products by using liquid chromatography‐tandem mass spectrometry studies without isolating them from the reaction mixture. It was observed that the drug was susceptible to oxidative degradation (15% hydrogen peroxide, 48 h, room temperature) forming four novel degradation products while being stable under hydrolytic, Photolytic, and thermal conditions. The separation of the drug and the degradation products was achieved with good resolution on a Phenomenex C18 column (150 mm × 4.6 mm, 5 µm) using gradient elution. The fragmentation pattern constructed from the liquid chromatography‐quadrupole‐time of flight mass spectrometry data was used for structural characterization of the degradation products. Lastly, the in‐silico absorption, distribution, metabolism, excretion, and toxicity properties prediction was performed by using in‐silico tools like the pKCSM webserver, ToxTree, and OSIRIS property explorer. The primary screening through the pKCSM webserver exposed the hepatoxic and the mutagenic potential of Pimozide, 1‐(1‐(4‐(4‐fluorophenyl)‐4‐phenylbutyl) piperidin‐4‐yl)‐1,3‐dihydro‐2H‐benzo[d]imidazol‐2‐one (degradation product 3) and 1‐(4,4‐bis(4‐fluorophenyl)butyl)‐4‐(2‐oxo‐2,3‐dihydro‐1H‐benzo[d]imidazol‐1‐yl)piperidine 1‐oxide (degradation product 5) whereas, 1‐(1‐(4,4‐diphenylbutyl) piperidin‐4‐yl)‐2,3‐dihydro‐1H‐benzo[d]imidazol‐2‐ol (degradation product 1) was predicted to only possess mutagenic potential. Secondary screening for mutagenicity was done by using ToxTree and OSIRIS property explorer.

A simple, robust, and reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantitative determination and stability assessment of Remdesivir. Forced degradation studies were conducted under acidic, alkaline, oxidative, thermal, and photolytic conditions to evaluate the intrinsic stability of the drug. Chromatographic separation was achieved on an Eclipse Plus C18 column (150 × 4.6 mm, 5 μm) using a gradient elution of 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 1.0 mL/min. Detection was performed at 245 nm using a diode array detector. The method demonstrated excellent linearity over the concentration range of 2–40 µg/mL, with recovery between 98.78% and 102.05%. Validation parameters complied with ICH Q2(R1) guidelines. Degradation products generated under acidic, alkaline, and oxidative conditions were further Identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Based on the m/z values of the degradants, probable degradation pathways were proposed. The method was successfully applied for the quantitative estimation of Remdesivir in bulk drug and injectable formulations, confirming its utility as a stability-indicating analytical tool.

The stability of a new drug substances and new drug products is a vital parameter which may affect purity, safety & potency. Changes in drug stability can threat patient safety by formation of toxic degradation products or deliver to lower dose than expected. Therefore it is to know the purity profile & behaviour of a drug substances under the various environmental condition. Forced Degradation studies show the chemical behavior of the molecule which in turn helps in the development of new formulation & package . Degradation study is required to the design of a regulatory compliant stability program for the both drug substances & products, and formalized as a regulatory requirement in ICH Guideline Q1A in 1993. Forced degradation studies (chemical and physical stress testing) of new chemical entities and drug product which is required to develop and demonstrate the specificity i.e stability indicating method. Forced degradation studies used to determination of the degradation pathways and degradation product of drug substances i.e during storage, development, manufacturing and packaging Thus , this review discusses the current trends in performance of forced degradation studies by provide the information about strategy for conducting the studies of forced degradation
 Keywords: - Regulatory Guidelines (ICH, FDA, EMA), Degradation condition, Forced degradation, Degradation product.

Rimegepant is a type of medication classified as a calcitonin gene-related peptide (CGRP) receptor antagonist, used to treat migraines. The present research focused to identify and characterize its degradation products using the liquid chromatography-high-resolution mass spectrometry (Q-TOF-HRMS) and nuclear magnetic resonance spectroscopy (NMR). The degradation profile of rimegepant was evaluated using forced degradation studies, which included exposure to hydrolysis, oxidation, photolysis, and thermal conditions. The drug degraded under hydrolytic and oxidative conditions whereas it was found to be stable under photolytic and thermal conditions. An Acquity CSH C18 column (2.1 × 100mm; 1.7µm) was used to separate the analytes using a mobile phase of 0.1% formic acid in water and 0.1% formic acid in acetonitrile eluting in gradient mode at a flow rate of 0.3mL/min. The degradation products were separated and identified using the mass fragmentation and accurate mass information obtained from LC-MS. A preparative HPLC was used to isolate the major degradation products for structural identification through 1D and 2D NMR studies. According to ICH Q2 (R1) guidelines, the developed UHPLC-PDA method was validated for its accuracy, precision, linearity, and specificity.

Venetoclax is an orally bioavailable, B-cell lymphoma-2 (BCL-2) selective inhibitor, used for the treatment of various types of blood cancers, such as chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). In this study we investigated the degradation of venetoclax under various stress conditions including acidic, basic, oxidative, photolytic and thermolytic conditions. We isolated and identified six of its main degradation products produced in forced degradation studies. The structures of the isolated degradation products were determined by using nuclear magnetic resonance (NMR) spectroscopy, high resolution mass spectrometry (HRMS) and infrared (IR) spectroscopy. Additionally, one oxidation degradation product was identified with comparison to a commercially obtained venetoclax impurity. We proposed the key degradation pathways of venetoclax in solution. To the best of our knowledge, no structures of degradation products of venetoclax have been previously published. The study provides novel and primary knowledge of the stability characteristics of venetoclax under stress conditions. Venetoclax is currently the only BCL-2 protein inhibitor on the market. In addition to single agent treatment, it is effective in combinational therapy, so future drug development involving venetoclax can be expected. A better insight into the stability properties of the therapeutic can facilitate future studies involving venetoclax and aid in the search of new similar therapeutics.

BackgroundNateglinide and metformin HCl are used in combination for the treatment of type 2 diabetes. A simple, sensitive and reliable UPLC method was developed for simultaneous estimation of nateglinide and metformin HCl using Phenomenox C18 (50*2.1 mm, 3.5 µm) column at ambient temperature as stationary phase in addition to mobile phase containing 75 volumes of ammonium formate buffer (pH = 3) along with 25 volumes of acetonitrile with a flow rate of 0.2 mL/min with UV detection at 260 nm and a run time of 3 min. The developed method was validated as per ICH Q2(R1) guidelines.ResultsThe separation of metformin HCl and nateglinide was done at retention times of 1.014 min and 1.435 min, respectively. The mean % recovery for nateglinide and metformin HCl in the accuracy study was observed to be 99.9% and 99.2%, respectively. LOD and LOQ values were determined considering the S/N ratio and were found to be 0.09 µg/mL and 0.3 µg/mL, respectively, for nateglinide and 0.75 µg/mL and 2.5 µg/mL, respectively, for metformin HCl. The method was found to be precise with % RSD values of 0.58 and 0.45, respectively, for repeatability and intermediate precision of nateglinide and 0.43 and 0.43, respectively, for repeatability and intermediate precision of metformin HCl which were within acceptance criteria. The method was found to be linear in the range of 7.5–45 µg/mL and 62.5–375 μg/mL for nateglinide and metformin HCl, respectively. The regression equations for nateglinide and metformin HCl were found to be y = 17377x + 6543.4 and y = 18439x + 43,537, respectively. The method was found to be robust by deliberate changes in the method parameters like flow rate and mobile phase composition. Forced degradation studies were performed as per ICH Q1A(R2) and Q1B guidelines, and peak purity was observed in all types of degradation studies for both the drugs.ConclusionThe developed method was found to be satisfactory as it is simple, sensitive, accurate, precise, robust, rapid, economical and yet stability indicating and can be applied successfully in the routine laboratory analysis for the simultaneous estimation of nateglinide and metformin HCl in the bulk and pharmaceutical dosage forms.