Oxidative Degradation Conditions Research Articles

Development and Validation of Fast and Sensitive RP-HPLC Stability-Indicating Method for Quantification of Piroxicam in Bulk Drug.

A simple, rapid, sensitive, and cost-effective green solvent-assisted reverse-phase high-performance liquid chromatographic technique, coupled with a photodiode array detector, was developed and validated for the estimation of piroxicam (PRXM). The chromatographic separation was achieved by using a C-18 (250 × 4.6) mm, 5-μm stationary phase and a mobile phase consisting of methanol and 0.1% ortho-phosphoric acid in water in a ratio of (80:20) v/v at a flow rate of 1ml/min. The detection was carried out at a wavelength of 254nm with a constant injection volume of 10μL throughout the analysis. The calibration curve was observed to be linear over the optimum concentration range of 50-300μgmL-1, with an R2 value of 0.9995. The developed method was validated as per the International Council for Harmonisation (ICH) Q2 (R1) guideline. Various parameters like selectivity/specificity, accuracy/recovery, linearity, precision, detection limit, quantitation limit, robustness and stability of analyte in solution were performed for the method validation. The PRXM was evaluated under stressed conditions, including acidic, basic, oxidative, thermal and photolytic, as per ICH Q1 (R2) guidelines. Significant degradation was observed in acidic and basic degradation conditions. Conversely, the drug substance showed stability when exposed to oxidative, photolytic and thermal degradation conditions.

Quality by Design-driven Analytical Method: A Quality Risk Management-Based Liquid Chromatography Method for Daclatasvir and Characterization of its Putative Degradants by LC-MS/MS

Background:: Antiviral drugs can cure more than 95 percent of people with hepatitis C, but the inaccessibility of quality affordable medicines and the lack of their uninterrupted supply pose a major challenge. Impurities in drugs have a significant impact on their quality and are one of the substantial causes of drug recalls, ultimately leading to the unavailability of the drug in the market. Hence, there is a need for a robust, quality risk management and quality by design-driven analytical method that can detect the antiviral drug, Daclatasvir dihydrochloride, in the presence of its probable impurities. Objective:: This study aimed to develop a Quality by Design-driven stability- indicating liquid chromatography method for Daclatasvir dihydrochloride and the characterization of its putative degradants by LC-MS. Method:: The fishbone diagram and quality risk assessment investigated twenty-four process parameters and concluded that three risk parameters, i.e., flow rate, buffer pH, and stationary phase type, were the critical process parameters. The critical quality attributes viz. resolution between impurity 6 and DCV and impurity 2 & 3 (Rs˃1.5), the shape of the peak of DCV which is decided by the Number of Theoretical Plates (NTP˃5000), and the retention time of Daclatasvir (tR14-23 mins) were optimized using a two-level three-factor full factorial design with five center points. Results:: The optimized method is stability-indicating in its true sense as it can separate the sample with its degradants generated in basic (three), acidic (two), oxidative (H2O2: three, Azobisisobutyronitrile: one), photo (three), and dry heat (one) conditions. Degradants structures were elucidated, and degradation routes were established, using LC-MS and LC-MS/MS analyses. Conclusion:: The drug is highly susceptible to acid, base hydrolysis, and oxidation degradation conditions and poses a significant risk to the analytical method to fail in system suitability criteria. Hence, a robust and flexible chromatographic method with the capacity for continuous improvement was developed and successfully validated within the criteria of design space.

LC-HRMS and NMR studies for the characterization of degradation impurities of ubrogepant along with the in silico approaches for the prediction of degradation and toxicity

Ubrogepant is the first oral calcitonin gene-related peptide (CGRP) receptor antagonist which is used for the acute treatment of migraine in adults. The present study employs liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR) techniques for the identification and characterization of degradation impurities of ubrogepant. The forced degradation study of ubrogepant was performed as per the International Council for Harmonisation (ICH) Q1A and Q1B guidelines. The in silico degradation profile of ubrogepant was predicted by Zeneth. It was observed that ubrogepant was labile to acidic hydrolysis, basic hydrolysis, and oxidative degradation conditions (H2O2), although it was stable in neutral hydrolysis and photolytic (UV light and visible light) conditions. Eight degradation impurities were formed, which were separated on reversed-phase HPLC with a gradient program on an InertSustain C8 column (4.6 × 250 mm, 5 µm) using 10 mM ammonium formate (pH unadjusted) and acetonitrile as the mobile phase. The structures of all the degradation impurities were characterized using the exact masses obtained from the HRMS/MS. Further, NMR studies were conducted on two major degradation impurities (UB-4 and UB-7). A plausible mechanism was proposed to support the structures of all the degradation impurities of UBR. In silico toxicity and mutagenicity assessment were done by DEREK Nexus, SARAH Nexus, and ProTox-II.

Degradation of diazine dye safranin T using potassium persulfate activated by ultrasonic treatment and MnFe2O4 spinel nanoparticles

An advanced oxidation process of ultrasound/MnFe2O4/K2S2O8 was developed for the degradation of diazine dye safranin T, according to which potassium persulfate was co-activated by ultrasonic (US) cavitation and MnFe2O4 spinel nanoparticles synthesized via co-precipitation in the ultrasonic field. A synthesis product annealed at a temperature of 4000C, with an average size of MnFe2O4 crystallites of about 7 nm, was used as a catalyst for the decomposition of potassium persulfate. Based on the results of experimental studies on the influence of various factors on the degree and rate constant of safranin T oxidative degradation, as well as considering energy and resource-saving principles, the rational conditions of oxidative degradation were determined as follows: the reaction medium temperature of 600C, the molar ratio of safranin T:K2S2O8=1:100, the catalyst loading of 0.1 g/l, and the specific power of the ultrasonic cavitation treatment of 51.0 W/l. It was established that under such conditions the oxidative degradation degree of safranin T was equal to 98.3%, and the rate constant was 1.510–3 s–1. The changes in the UV-Vis spectra of safranin T, namely a decrease in the intensity of absorption bands, both in the visible (at a wavelength of 520 nm) and in the UV (at a wavelength of 275 nm) regions of the spectrum, confirmed the degradation of safranin T. In addition, the absence of the appearance of new peaks in the visible and UV regions of the spectrum indicated mineralization of the dye.

Stress degradation study of Etoricoxib, isolation, and characterization of major degradation impurity by preparative high‐performance liquid chromatography, liquid chromatography‐mass spectrometry, and nuclear magnetic resonance: Validation of ultra‐performance liquid chromatography method

AbstractEtoricoxib (ETO) is a selective cyclooxygenase‐2 inhibitor, used as a nonsteroidal anti‐inflammatory drug. In this study, a detailed investigation was conducted on the degradation behavior of ETO under acidic, basic, neutral, photolytic, oxidative, and thermal degradation conditions as per the International Conference on Harmonization (ICH) guideline Q1A (R2). A gradient “ultra‐performance liquid chromatography (UPLC)” method was developed for separating the degradation products formed under various degradation conditions along with process‐related impurities. Acquity BEH phenyl column was used for the separation of analytes and 0.1% formic acid in water and 0.05% formic acid in acetonitrile:methanol (8:2 %v/v) were used as mobile phase‐A and mobile phase‐B. A major degradant namely, N‐oxide impurity was observed in oxidative degradation along with two minor degradants where N‐oxide was confirmed based on LC‐mass spectrometry (LC‐MS) studies. Further, the major degradant was enriched and isolated using preparative HPLC and the conformation of the N‐oxide impurity was established using nuclear magnetic resonance techniques including two‐dimensional studies and high‐resolution MS. In addition, the method for related substances by UPLC has been validated by considering a major degradant, namely N‐oxide along with process‐related impurities as per the ICH guidelines validation parameters.

Identification, characterisation and in silico ADMET prediction of ozenoxacin and its degradation products using high‐performance liquid chromatography‐photodiode array and liquid chromatography‐quadrupole time‐of‐flight‐tandem mass spectrometry

RationaleOzenoxacin (OXC) is an antibiotic used topically to treat impetigo. This study aimed to evaluate the degradation products (DP) of OXC drug substance under different stress conditions, including hydrolysis, oxidation, thermal and photolysis, in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines Q1A(R2) and Q1B. The analytical technique was validated in compliance with ICH Q2(R1) requirements.MethodsThe drug substance underwent degradation under various forced degradation conditions, including thermal, oxidative, photolytic and hydrolytic (neutral, acidic and basic) degradation. Overall, four DPs were formed under oxidative stress conditions with AIBN. The formed DPs were identified and separated using a Shimadzu LC system with a reversed‐phase Phenomenex Kinetex C18 column (4.6 × 250 mm, 5 μm), using 10 mM NH4CH3COOH buffer (pH −5.0) as mobile phase A and acetonitrile as mobile phase B at a detection wavelength of 254 nm.Results and ConclusionThe drug was found to be stable in neutral, acidic, basic and oxidative degradation conditions with hydrogen peroxide. Liquid chromatography‐electrospray ionisation‐quadrupole time‐of‐flight‐tandem mass spectrometry‐ was employed in positive ionisation mode to analyse both the drug and the mass of the identified DP. The mechanism and the pathway of mass fragmentation have been proposed. The developed method was accurate, repeatable, linear and selective for further research. The ADMET Predictor software was applied to predict the in silico toxicity of the drugs and its DPs as well as their physicochemical characteristics.

Potential degradation products of abemaciclib: Identification and structural characterization employing LC-Q/TOF-MS and NMR including mechanistic explanation

Degradation products are the potential drug impurities that can be generated during transport and storage of pharmaceuticals. Before this study, degradation chemistry and potential degradation products of abemaciclib (ABM) were unknown. Moreover, no stability-indicating analytical method was available that can be used to analyse ABM in presence of its degradation products. In this study, stress testing on ABM was carried out under oxidative, thermal, photolytic (UV & visible), and hydrolytic (acid, alkaline, and neutral) degradation conditions. The study revealed that ABM is susceptible to photolytic, oxidative, and thermal stress leading to the formation of five degradation products (DPs). ABM and its degradation products were chromatographically separated employing a developed RP-HPLC-based stability-indicating analytical method. The method was transferred to an LC-Q-TOF system for further analysis. To elucidate the structure of degradation products, fragmentation pathway of ABM was initially established through high-resolution mass spectrometry (HRMS). Subsequently, mass fragmentation pathways of all the DPs have been established through HRMS and MSn based analysis. The major degradation product was isolated and fully characterized using atmospheric chemical ionization-mass spectrometry and nuclear magnetic resonance techniques. ABM showed extensive degradation under oxidative and photolytic systems. Therefore, special care may be sought during storage and transport of ABM or its formulations to avoid photolytic and oxidative stress exposure to the drug. Lastly, in silico toxicity of the characterized degradation products was assessed employing ProTox ІІ online web predictor freeware in which some of them were found to have the potential of hepatotoxicity, immunogenicity and mutagenicity.

RP-HPLC Method Development, Validation Including Stability Indicating and Forced Degradation Studies for the Estimation of Pemetrexed in API and Pharmaceutical Dosage Form

Stability indicating analytical RP-HPLC (Reverse Phase- High performance Liquid Chromatography) method was developed for the analysis of the drug in the presence of its degradation products and subsequent validation of Pemetrexed that was conducted in API and pharmaceutical dosage form. Pemetrexed was subjected to different stress conditions as per International Conference on Harmonization (ICH) guidelines, stress conditions applied were including the effect of acid, base, oxidative, hydrolysis, thermal and photolytic degradation conditions. Chromatographic separation of API (Active Pharmaceutical ingredient) was achieved on Water’s HPLC instrument and ODS C18, 250x4.6 mm, 5µm particle size column with gradient elution of 0.1% ortho phosphoric acid buffer: Acetonitrile 80:20 ratio was taken as the eluent (mobile phase). The buffer pH was maintained at 3, the flow rate was 1.0 ml/min and wavelength were detected at 225 nm. The Retention time (RT) of Pemetrexed was Found at 2.9 minutes, column temperature was kept ambient and the runtime was 6 min. System suitability parameters like theoretical plates, tailing factor, and Asymmetry values were showed at 3391, 1.41, and 1.101 respectively. The percentage drug purity was found to be 99.99%. Validation of the method was proved that linear relationship over the range of 25-150 µg/mL, with linear regression curve (correlation coefficient) r2 value was noticed at 0.999 and LOD (limit of detection), LOQ (limit of quantification) values was established at 0.33µg/mL, 0.99µg/mL respectively. Recovery studies carried out at the concentrations of 50%, 100% and 150% with percentage recovery values of 100.06, 99.8 and 99.87 respectively. The precision of intraday was 0.64% and inter-day was 0.95%. Robustness of flow rate and mobile phase was changed even though the method was Robust. Forced degradation studies were also conducted to check the stability and suitability of this method to resolve the degradation products. Method validation was proved to be accurate, precise, linear, repeatable and robust. Thus, the accepted method can be proceeded for the estimation of Pemetrexed in pharmaceutical dosage form in a daily basis, due to their simplicity, suitability, accuracy, robustness and reproducibility.

A Different Perspective on the Characterization of a New Degradation Product of Flibanserin with HPLC-DAD-ESI-IT-TOF-MSn and its Pharmaceutical Formulation Analysis with Inter-Laboratory Comparison.

Flibanserin (FLB) was firstly synthesized as an antidepressant drug; however, due to its enhancing effects on sexual activity, it was approved for treatment of hypoactive sexual desire disorder in women in 2015. The aim of this study was to develop a new and fully validated HPLC method for analysis of FLB in pharmaceutical formulations besides its degradation products, and identification of possible formation mechanisms by using HPLC-DAD-ESI-IT-TOF-MSn. The HPLC separation was achieved in an Supelco Ascentis® Express series phenyl hexyl column (100 × 4.6 mm, ID 2.7µm). The mobile phase was acetonitrile-ammonium acetate solution (50:50, v/v, 10 mM, pH 5.4) mixture, which was pumped at the rate of 0.5 mL/min. Chromatography, detection and structural identification was performed by using LCMS-IT-TOF instrument (Shimadzu, Japan). 1-(2-(4-(3-hydroxy-5-(trifluoromethyl)phenyl)piperazine-1-yl)ethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one is proposed as a novel degradation product, with a mass of 407.1695, and a formula of C20H21F3N4O2 with a margin of error about 0.001 ppm. The developed method is applicable with 98% accuracy within 2.5-50.0 µg/mL range. The LOD and LOQ was about 500 ng/mL and 1.50 µg/mL, respectively. The transferability and variation between laboratories were tested by inter-laboratory comparison and evaluated with one-way analysis of variance. A novel FLB degradation product, which was produced under oxidative forced degradation condition was observed and identified for the first time; in addition, the formation kinetics of the degradation product, besides decomposition of FLB was studied. Furthermore, an inter-laboratory comparison was carried out and application of the proposed method on pseudo Addyi® sample was tested using both instrument configurations. A novel stability indicating assay method was developed and fully validated according to ICH (Q2)R1 for the analysis of FLB in the pharmaceutical preparations. A new degradation product was identified in the oxidative forced degradation condition and characterized using HPLC-DAD-ESI-IT-TOF-MS3. Moreover, the possible mechanism and the formation kinetic of the degradation product were revealed. In addition, the developed method was transferred to another LC-PDA instrument for inter-laboratory comparison. Finally, the current method was applied to pseudo formulation of Addy® in both instruments and ANOVA was applied for evaluation.

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.

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.

Separation and Identification of Related Substances in Candesartan Cilexetil Tablets by UHPLC-Q-TOF–MS

Related substances, such as process-related substances and degradation products, may affect the efficacy of drugs and cause adverse reactions. Therefore, identifying and controlling them is of the importance. A rapid ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF–MS) method has been developed for the separation and characterization of related substances in candesartan cilexetil tablets. After optimization, the method validation was followed according to ICH guidelines. The developed UHPLC method showed adequate specificity, sensitivity, accuracy, linearity, precision, stability and robustness for validation of analytical procedures. Commercial candesartan cilexetil tablets was subjected to stress testing (60 ℃, 90% RH and 4500 lx for ten days) and forced degradation studies (acidic, alkaline, oxidative and photolytic degradation conditions). A total of eleven related substances were detected and characterized. Among them, four related substances have not been reported in the literature yet, and one of them (RS7) was confirmed as candesartan cilexetil methoxy analog by reference substance. In addition, plausible mechanisms for the formation of these related substances are discussed. This study provides a useful reference for the quality control of candesartan cilexetil tablets.

ПРОЦЕС “СОНОПЕРОКСАТ” ДЛЯ ОКИСНЮВАЛЬНОЇ ДЕГРАДАЦІЇ МЕТИЛОВОГО ОРАНЖЕВОГО

The efficiency of application of advanced oxidation processes for removal of methyl orange mono azo dye from wastewater is considered. For intensive oxidative degradation of methyl orange, it was proposed to use a combination of ultrasonic cavitation and “Peroxate” process – the “Sonoperoxate” process. Rational conditions for oxidative degradation of methyl orange at its initial concentration in an aqueous solution of 25 mg/dm3 (76.5·10-6 mol/dm3) were established: the molar ratio of methyl orange:hydrogen pero- xide:potassium metaperiodate = 1:50:10; pH of the reaction medium – 3; temperature – 293 K; specific power of cavitation ultrasonic treatment – 68 W/dm3. Under such conditions, the degradation degree of methyl orange of 89.4 % was achieved for 1800 s.

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.

Development and validation of sensitive high‐performance liquid chromatography‐photodiode array method for determination of three sulfonated esters and N‐methyl‐O‐phenyldiamine dihydrochloride as potential genotoxic impurities in Amlodipine and Telmisartan fixed‐dose combination

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

LC and LC-MS/MS studies for identification and characterization of degradation products of d-tubocurarine chloride

Forced degradation studies of d-tubocurarine (DTC) was carried out in hydrolytic (acidic, alkaline and neutral), thermal, photolytic and oxidative degradation conditions as per International Conference on Harmonization (ICH) guideline Q1A (R2). The present study revealed that DTC is highly sensitive to oxidative degradation conditions even at room temperature whereas the drug was found to be stable in hydrolytic, photolytic and thermal stress conditions.Separation of DTC and its four degradation products (DPs) (DP-I to DP-IV) formed during stress degradation conditions were achieved on Waters Acquity CSH C18 (1.7 µm, 2.1 mm × 100 mm) column using gradient elution with a mobile phase consisting of Eluent-A: 0.1 % Formic acid Eluent-B: acetonitrile achieved successfully. The detection was carried out at 210 nm wavelength and the flow rate was kept at 0.3 mL/min with a 5 μL injection volume.Also, a highly sensitive and robust HRMS/MS/TOF method was established for the identification and characterization of four DPs formed during the stress study. ESI +ve mode was used throughout the study for ionization of all the DPs. The degradation pathway was also established in the study that is never reported earlier.

HSC70 mediated autophagic degradation of oxidized PRL2 is responsible for osteoclastogenesis and inflammatory bone destruction.

Inflammation leads to systemic osteoporosis or local bone destruction, however, the underlying molecular mechanisms are still poorly understood. In this study, we report that PRL2 is a negative regulator of osteoclastogenesis and bone absorption. Mice with PRL2 deficiency exhibit a decrease in bone volume and an increase in osteoclast numbers. PRL2 negatively regulates RANKL-induced reactive oxygen species production through the activation of RAC1, thus PRL2 deficient osteoclast precursors have both increased osteoclast differentiation ability and bone resorptive capacity. During inflammation, oxidized PRL2 is a selected substrate of HSC70 and conditions of oxidative stress trigger rapid degradation of PRL2 by HSC70 mediated endosomal microautophagy and chaperone-mediated autophagy. Ablation of PRL2 in mouse models of inflammatory bone disease leads to an increase in the number of osteoclasts and exacerbation of bone damage. Moreover, reduced PRL2 protein levels in peripheral myeloid cells are highly correlated with bone destruction in a mouse arthritis model and in human rheumatoid arthritis, while the autophagy inhibitor hydroxychloroquine blocked inflammation-induced PRL2 degradation and bone destruction in vivo. Therefore, our findings identify PRL2 as a new regulator in osteoimmunity, providing a link between inflammation and osteoporosis. As such, PRL2 is a potential therapeutic target for inflammatory bone disease and inhibition of HSC70 mediated autophagic degradation of PRL2 may offer new therapeutic tools for the treatment of inflammatory bone disease.

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.

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 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.