Characterization Of Forced Degradation Products Research Articles

Isolation, identification and structural characterization of forced degradation products of mobocertinib using LC-MS/MS and NMR

The main objective of this study is to develop and validate an LC-MS/MS stability-indicating method for the isolation, identification, and characterization of forced degradation products of mobocertinib under various stress conditions, utilizing LC-MS/MS and NMR techniques. Four degradation products (DP1, DP2, DP3 and DP4) were isolated. Degradation under acidic and oxidative conditions provides DP1 and DP4, respectively. Both DP2 and DP3 were obtained under basic conditions. Chromatographic development and separation were achieved using an ODS C18 column (Chemsil) with a constant flow rate of 1.0 ml/min. The mobile phase consisted of 40 % water containing 0.1 % formic acid in methanol, and detection was performed at a wavelength of 225 nm. The application of LC-MS/MS technology was utilized to separate the above degradation products. The LC-MS/MS method has validated which includes specificity, linearity, LOD and LOQ, precision, accuracy and robustness. The chemical structure of the degradation products were characterized by 1H NMR and mass spectroscopy. The above listed degradation impurities are novel and not listed in the literature. In conclusion, this study provides vital information about mobocertinib’s chemical stability and helpful in further drug development process.

Characterization of forced degradation products of Netarsudil: Optimization and validation of stability-indicating RP-HPLC method for simultaneous quantification of process related impurities of Netarsudil

Characterization of forced degradation products of Netarsudil: Optimization and validation of stability-indicating RP-HPLC method for simultaneous quantification of process related impurities of Netarsudil

Identification and characterization of forced degradation products of 5-hydroxymethyl-2-furaldehyde (5-HMF) by HPLC, LC-LTQ/Orbitrap and NMR studies

5-Hydroxymethyl-2-furaldehyde (5-HMF) is a kind of aldehyde compound with highly active furan ring, which is generated by dehydration of glucose, fructose, and other monosaccharides. It widely exists in drugs, foods, health products, cosmetics, and traditional Chinese medicine preparations with high sugar content. Due to the toxicity, the concentration of 5-HMF was always monitored to identify non-conformities and adulteration, as well as ensure the process efficiency, traceability and safety in foods or drugs in the pharmacopoeias of various countries. Herein, a comprehensive forced degradation study was performed to characterize the degradation products (DPs) of 5-HMF under hydrolytic (neutral, acidic, and alkaline) degradation, oxidative, thermal, humidity, and photolytic degradation conditions. A total of five degradants were identified, and two of them (DP-3 and DP-5) were novel DPs first reported in our study. Major DPs (i.e., DP-1 and DP-2) with relatively high peak areas were isolated using semi-preparative HPLC and characterized by LC-LTQ/Orbitrap and NMR. 5-HMF was only stable in alkaline hydrolysis condition. In addition, the degradation pathways and mechanism of these DPs were also explained using LC-LTQ/Orbitrap. In silico toxicity and metabolism behavior of the DPs were evaluated using Derek Nexus and Meteor Nexus software, respectively. The predicted toxicity data indicated that both the drug 5-HMF and its DPs bear the potential of hepatotoxicity, mutagenicity, chromosome damage, and skin sensitisation. Our research may be beneficial for the quality control and suitable storage conditions of 5-HMF.

Sensitive and validated UPLC-MS/MS method for the identification and characterization of forced degradation products for felodipine

AbstractA rapid, stability indicating reverse phase liquid chromatographic method was developed for the determination of purity of Felodipine in active pharmaceutical substance form in the presence of its impurity and its degradation products. To develop the method which is also compatible to liquid chromatographic mass spectroscopic technique. The developed method is also used to determine the assay of Felodipine in bulk drug form. The drug is subjected to various stress conditions like acidic, basic, oxidation, UV light and thermal conditions. Considerable degradation was observed during base hydrolysis. Two degradation products were identified. The Waters Acquity UPLC BEH C18, 2.1 × 100 mm, 1.7 µm Column was used to achieve chromatographic separation. The gradient conditions, diluent and injection volume were optimized to achieve the acceptable resolution between impurities and its degradation products from Felodipine and to get good peak shapes. The masses were determined for main compound and its identified degradation products. Further, the characterization studies for main compound and its degradation products were performed using LCMSMS Q-TOF.

Identification and characterization of forced degradation products of sertraline hydrochloride using UPLC, ultra-high-performance liquid Chromatography-Quadrupole-Time of flight mass spectrometry (UHPLC-Q-TOF/MS/MS) and NMR

The present study focused on the forced degradation behavior of sertraline hydrochloride (SRT), selective serotonin reuptake inhibitor (SSRI). The drug was exposed to different stressed conditions (hydrolytic, oxidative, thermal and photolytic) according to ICH Q1A (R2) guidelines. The study revealed that SRT was stable in hydrolytic (acidic, basic and neutral) and thermal degradation conditions. In contrast, five degradation products (DPs) were formed under oxidative and photolytic degradation conditions. The chromatographic separation of drug substance and its DPs was achieved on an Acquity HSS T3 column (100 × 2.1 mm, 1.7 μ) using 0.1% formic acid and acetonitrile as the mobile phase in gradient mode using a UHPLC-DAD system. The DPs were identified and characterized by high-resolution LC/MS and LC/MS/MS in ESI positive mode. Two DPs (DP-I and DP-II) were formed when SRT was exposed to oxidative degradation conditions. Three DPs formed (DP III-V) when exposed to photolytic degradation conditions. All the five major DPs were isolated using Preparative HPLC. The structures of major DPs formed were further confirmed using NMR technique (1D and 2D). The proposed mechanism for the formation of the SRT DPs via the photolytic/oxidative stress degradation pathway are discussed and outlined.

ASSAY METHOD DEVELOPMENT AND VALIDATION OF CILNIDIPINE AND RAMIPRIL, CHARACTERIZATION OF ITS DEGRADANTS BY USING LC-MS/MS

Objective: The current study focused on the development, validation of Ramipril and Cilnidipine using HPLC and characterization of forced degradation products using LC-MS/MS. Methods: Simple, accurate, and precise method has been developed for the simultaneous estimation of Cilnidipine and Ramipril in tablet type of dosage form using X-bridge phenyl column (150x4.6 mm, 3.5m) with mobile phase of buffer and Acetonitrile in the 30:70 v/v ratio was pumped through a column with a 1 ml/min flow rate. The buffer used in this process was 0.1 percent tri ethyl amine in 1 lt of water and adjust its pH-2.5 with 0.1 percent ortho phosphoric acid. At ambient temperature, chromatography was isocratically performed and run time was 8 min. 240 nm was the optimized wave length. Results: Cilnidipine and Ramipril retention times were 2.79 min, 5.11 min. respectively. By injecting the norm six times, device parameters of suitability have been studied and the results were found to be well under the acceptance criteria. This approach offers strong linearity over a range of 2-30 µg/ml Cilnidipine 1-15 µg/ml Ramipril concentrations. The regression coefficient R2>0.999 from the calibrated curve implies that the linearity of the system was within the range. Conclusion: The system was validated by using HPLC in terms of accuracy, linearity, method precision, accuracy, limit of detection, limit of quantification, robustness, degradation and the degradation products were characterized by using LC-MS/MS.

LC-MS/MS CHARACTERIZATION OF FORCED DEGRADATION PRODUCTS OF TUCATINIB, A NOVEL TYROSINE KINASE INHIBITOR: DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD

Objective: The current study focused on the development, validation, and characterization of forced degradation products using LC-MS/MS. Methods: A simple, selective, validated and well-defined isocratic HPLC methodology for the quantitative determination of Tucatinib at a wavelength of 239 nm. An isocratic elution of samples was performed on an Inertsil ODS (250x4.6 mm, 5m) column with a mobile phase of 70:30v/v Acetonitrile and formic acid (0.1%) delivered at a flow rate of 1.0 ml/min. MS/MS was used to characterize degradation products formed in the forced degradation study. The validation and characterization of forced degradation products were performed in accordance with ICH guidelines. Results: Over the concentration range of 5-100μg/ml, a good linear response was obtained. Tucatinib's LOD and LOQ were determined to be 0.05 and 0.5, respectively. According to standard guidelines, the method was quantitatively evaluated in terms of system suitability, linearity, precision, accuracy, and robustness, and the results were found to be within acceptable limits. The drug was degraded under acidic, alkaline, and reduction conditions in forced degradation studies. Conclusion: The method was found to be applicable for routine tucatinib analysis. Because no LC-MS/MS method for estimating tucatinib and its degradation products has been reported in the literature. There is a need to develop a method for studying the entire tucatinib degradation pathway.

Development of a stability-indicating assay method by HPLC-DAD and MS characterization of forced degradation products of ravuconazole.

Ravuconazole (RAV) is a triazole antifungal with broad spectrum and a novel alternative in the treatment of systemic fungal infections. A stability-indicating method by high-performance liquid chromatography-diode array detection was developed and fully validated to assay ravuconazole in the presence of its degradation products. Separation was achieved with a Sunfire C18 column (250 mm × 4.6 mm id, 5 μm), mobile phase composed of acetonitrile and water (80:20), at 1 mL/min. The volume of injection was 5 μL and DAD detection was performed at 287 nm. RAV was well resolved from its degradation products and the method proved to be linear, selective, accurate, precise and robust. A forced degradation study was conducted on the pure drug under oxidative conditions in presence of H2O2 and metallic ions and under acid, alkaline and neutral hydrolysis. RAV was degraded mainly under alkaline hydrolysis, forming two main degradation products. The chemical structures were proposed according to the data obtained by liquid chromatography coupled to mass spectrometry (LC-MS) analysis. This study provided a new and selective stability-indicating method to evaluate the intrinsic stability of ravuconazole in active pharmaceutical ingredients. The developed method was found to be suitable for quality control routine analysis and to stability studies of ravuconazole.

Identification and characterization of forced degradation products of vortioxetine by LC/MS/MS and NMR

Vortioxetine (VTX) is a novel multimodal antidepressant drug that affects the serotoninergic and noradrenergic systems. In this work, the forced degradation of VTX was studied according to (ICH) Q1A (R2) guidelines. The study revealed that VTX was stable under thermal stress conditions and hydrolytic stress conditions i.e., acidic, basic and neutral conditions. In contrast, six degradation products (DPs) were formed under photolytic and oxidative stress conditions. The DPs were identified and characterized by high-resolution LC/MS and LC/MS/MS. The structures of major DPs were further confirmed by the synthesis and characterization by 1H and 13C NMR data. A possible mechanism for the formation of the VTX DPs via photolytic/oxidative stress degradation pathway was proposed.

Identification and Characterization of Forced Degradation Products for Dofetilide using Rapid and Sensitive UPLC-MS/MS Method and HRMS Studies

A simple, rapid and efficient method has been developed and validated using ultra UPLC combined with Q-ToF MS system for recognition and characterization of forced degradation products obtained from dofetilide degradation studies. The dofetilide drug is an antiarrhythmic and belongs to Class III and it was treated with various stress conditions like acidic, basic, oxidative, photolytic and thermal conditions as per ICH guidelines. The main drug shows extensive degradation towards oxidative degradation conditions and single degradation product was identified through chromatogram. The chromatographic separation among main and its impurities were attained through 2.1 × 150, 1.8 μm column from gradient elution using UPLC and its detection at wavelength 230 nm. The validation was performed for the developed method using various parameters like specificity, linearity and robustness studies. Waters Synapt G2 Q TOF system was used and performed MSn studies to establish mass spectral fragmentation pathway for drug and its degradation products and determined accurate masses study. The efficiency of this method was helpful to identify and characterize the drug and degradation products using LC/MSn techniques.

Separation and characterization of forced degradation products in homoharringtonine injection by UHPLC-Q-TOF-MS

A rapid ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method was developed for separation and characterization of the degradation products in HHT injection. Chromatographic separation was achieved on a ZORBAX Eclipse XDB-C18 HD column (2.1 × 100 mm, 1.8μm) using methanol- ammonium formate (pH 3.0; 30 mM) (30:70, v/v) as mobile phase in an isocratic mode of elution. Forced degradation studies were conducted under hydrolytic (acidic and alkaline), oxidative, photolytic and thermal stress conditions as described in ICH. A total of eleven forced degradation products were detected and the drug was found to be susceptible to all the tested stress conditions. The degradation products were characterized through Q-TOF fragmentation studies and their fragmentation pathways were proposed. Seven of them have not been reported or described as degradation product before, and one of them was further confirmed by reference substance. In addition, plausible mechanisms for the formation of the degradation products were proposed.

Structural characterization of forced degradation products of empagliflozin by high resolution mass spectrometry

Eleven unique degradation products (DPs) of empagliflozin (EGF), a sodium glucose cotransporter (SGLT) 2 inhibitor have been reported for the first time. These DPs were generated as per International Conference on Harmonization (ICH) guidelines Q1 (R2) using stress conditions such as acid, base hydrolysis and oxidative environment. The sum of 12 DPs have been successfully resolved by Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) method involving Eclipse plus C18 RRHD (2.1 × 50 mm, 1.8µm) column and mobile phase comprising of water with 0.1% formic acid and methanol in 1:1 quotient. Structure of DPs and the degradation pathways have been proposed on the basis of accurate mass and MS/MS fragmentation pattern acquired through Liquid Chromatography-Electrospray Ionization Quadrupole Time of Flight Mass Spectrometer (LC-ESI-QTOF-MS). It has been observed that maximum DPs were formed during acid hydrolysis and the major ones were (2S,3R,4R,5S,6R)-2-(4-chloro-3-(4-(((S)-1,4-dihydroxybutan-2-yl)oxy)benzyl)phenyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (DP1) and (2S,3R,4R,5S,6R)-2-(4-chloro-3-(4-hydroxybenzyl)phenyl)-6-(hydroxymethyl) tetrahydro-2H-pyran-3,4,5-triol (DP2) generated due to ring opening of tetrahydrofuran moiety and elimination of tetrahydrofuran ring respectively. The proposed structures of DPs and their associated pathways will be essential for optimization of manufacturing and quality control parameters of EGF.

Characterization of forced degradation products of toloxatone by LC-ESI-MS/MS

Forced degradation of toloxatone in solutions under basic, acidic, neutral, photo UV–VIS, photo UVC and oxidative stress conditions was investigated and structural elucidation of its degradation products was performed with the use of UHPLC system coupled ESI-Q-TOF mass spectrometer. Eight degradation products were found and their masses and formulas were obtained with high accuracy (0.09–3.79 ppm). The structure of unknown degradation products were elucidated from MS/MS fragmentation spectra of all analyzed compounds. Additionally, whole signals of decomposed substances were compared chemometrically. It was found that toloxatone is fragile towards basic hydrolysis, oxidative conditions and UVC irradiation. Finally, the toxicity of transformation products was computationally evaluated and compared in multivariate manner.

Characterization of forced degradation products of canagliflozine by liquid chromatography/quadrupole time-of-flight tandem mass spectrometry and in silico toxicity predictions.

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.

A Sensitive, Stability indicating UPLC method for the identification and characterization of forced degradation products for Drometrizole Trisiloxane through MSn studies

A Sensitive, Stability indicating UPLC method for the identification and characterization of forced degradation products for Drometrizole Trisiloxane through MSn studies

Characterization of forced degradation products of torasemide through MS tools and explanation of unusual losses observed during mass fragmentation of drug and degradation products through density functional theory

Mass spectrometry tools (HRMS/LC-HRMS, MSn, and/or on-line H/D exchange) were employed to establish mass fragmentation pattern of torasemide and to characterize its degradation products. During collision-induced dissociation, multiple rearrangement processes and unusual losses of sulfur (S), sulfanyl (HS), sulfur dioxide (SO2), sulphinic acid radical (HSO2), sulfur monoxide (SO), carbon monoxide (CO), formyl radical (CHO) and C5H3NOS were observed. The same were successfully explained by study of energy profiles, established by application of density functional theory (DFT).

Characterization of forced degradation products of clozapine by LC-DAD/ESI-Q-TOF

Forced degradation of clozapine in solutions under acidic, basic, neutral, photo UV–vis, photo UVC and oxidative stress conditions was investigated and structural elucidation of its degradation products was performed with the use of the UHPLC-DAD system coupled with accurate hybrid ESI-Q-TOF mass spectrometer. The developed method allows to collect all essential data for the determination of degradation kinetics and for the structural elucidation of the formed products. Six degradation products were found and their masses and formulas were obtained with high accuracy (0.61–3.75ppm). For all the analyzed compounds MS/MS fragmentation spectra were also obtained allowing structural elucidation of the unknown degradation products. It was found that the decomposition of clozapine yields the first-order kinetic reaction in all stress conditions and it is fragile towards acidic hydrolysis and oxidative conditions. Additionally, PCA analysis of registered TOF (MS) forced degradation profiles of clozapine shows no differences between the samples obtained from pharmaceutical formulation and bulk substance.

Identification and characterization of forced degradation products of pralatrexate injection by LC-PDA and LC–MS

Pralatrexate (PTXT) is an antineoplastic folate analog and the chemical name is (2S)-2-[[4-[(1RS)-1-[(2,4-diaminopteridin-6-yl)methyl]but-3-ynyl] benzoyl] amino] pentanedioic acid. Degradation products of PTXT drug product (DP) under different forced degradation conditions have been studied using LC-PDA and LC–MS techniques. PTXT DP was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, and heat in accordance with ICH guidelines. The LC–MS compatible HPLC method was developed and stressed solutions were chromatographed on reversed phase HPLC. The degradation products were monitored at a wavelength of 242nm. Stress study revealed that PTXT was sensitive towards acid, alkali, peroxide, light and heat. The degradation impurities (I–IX) were identified and characterized using LC-PDA and mass spectral data.

LC–MS/MS and NMR characterization of forced degradation products of mirabegron

ABSTRACTA rapid, precise, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for the characterization of stressed degradation products of mirabegron. It is used in the treatment of overactive bladder and administered to treat urinary symptoms such as urgency or frequency and incontinence. It also works by relaxing the muscles around bladder.Mirabegron was subjected to hydrolysis (acidic, alkaline, and neutral) and peroxidation, as per ICH-specified conditions. The drug showed degradation under stress conditions. However, it was stable to neutral conditions. A total of seven degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on X-TerraRP-8 (250 mm × 4.6 mm, i.d., 5 µm) column using 0.01 M ammonium acetate as mobile phase-A and 60:40 ratio of acetonitrile (ACN):water as mobile phase-B. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. Probable possible structures were drawn based on parent and daughter molecular ions. One peroxide degradant impurity was isolated using preparative LC and characterized using liquid chromatography–mass spectrometry and NMR data.

Stability-Indicating RP-UPLC Method Development and Validation for the Process Related Impurities of Nebivolol and Structural Characterization of Its Forced Degradation Products by LC-MS/MS

The present study reports the development and validation of a stability-indicating reverse phase ultra performance liquid chromatographic method for the determination of nebivolol (NEB) in the presence of its process related impurities in bulk drugs and also demonstrates the structural characterization of forced degradation products (DP) of nebivolol by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Forced decomposition study of nebivolol was carried out and studied under acidic, basic, aqueous, oxidative, photolytic and thermal conditions according to the ICH guidelines. Nebivolol was found to be stable under photolytic, thermal and aqueous conditions applied in our study. However, nebivolol degradation was observed under basic and acidic conditions. Successful chromatographic separation of the drug and its process related impurities and degradation products were achieved on a waters acquity BEHC18 stationary phase (50 × 2.1 mm, 1.7µm) with a gradient elution of 10 mM ammonium acetate and acetonitrile at a flow rate of 0.3 mL min-1. Detection of analytes was carried out using a PDA detector with its wavelength set at 222 nm. The developed UPLC method was validated for all validation parameters according to the ICH guidelines. The degradation products were characterized by LC-MS/MS study and the most possible degradation and fragmentation pathways were proposed.