LC-PDA-QTof-MS characterization of the stress degradation products of Zanubrutinib: A novel BTK inhibitor.

Zanubrutinib (ZAN) is an orally administered anti-cancer medication used for the treatment of Mantle cell lymphoma. Recently, it has also been approved by FDA for the treatment of chronic lymphocytic leukemia. Determination of impurities formed in drug substances/products as a result of manufacturing or storage forms an important aspect of drug life cycle management. The current study concentrated on understanding the stability of ZAN under various stress conditions as per the ICH Q1 (R2) guidelines. In total, ZAN produced thirteen degradation products under various hydrolytic (acid, base and neutral) and thermal stress conditions. The stress degradation products were separated by ultra-performance liquid chromatography, chemical structures of these products were characterized by MS/MS experiments combined with accurate mass measurements conducted on a LC-QTof-MS. The mechanism for the formation of these degradation products was also proposed. This study provides comprehensive information on the inherent stability of ZAN which will be useful in the drug development and manufacturing processes.

Application of online liquid chromatography/quadrupole time-of-flight electrospray ionization tandem mass spectrometry for structural characterization of linagliptin degradation products and related impurities.

RATIONALE: Linagliptin is a drug used for the management of type 2 diabetes, which is a leading cause of global ill health and mortality. Impurities can affect the quality and safety of drug products and eventually may affect human health. A robust, sensitive and reliable analytical method is required to detect, characterize, quantify and control the presence of impurities in finished pharmaceutical products such as linagliptin. METHODS: Linagliptin was stressed under harsh conditions as in the ICH Q1A (R2) guidelines to generate degradation products. The degradation products and process-related impurities were separated using an InertSustain C8 column (4.6 mm × 150 mm, 5 µm) and characterized by tandem quadrupole time-of-flight mass spectrometry in positive mode electrospray ionization. The developed method was validated according to the ICH Q2 (R1) guidelines. RESULTS: Upon forced degradation, 12 degradation products were obtained (6 in oxidative stress and 3 in each of acid and alkaline hydrolysis). The special finding here was the presence of a pair of isomeric degradation products in acid hydrolysis and the formation of degradation products in base hydrolysis and oxidative degradation caused by the use of acetonitrile as a diluent. The 12 degradation products and 6 process-related substances were successfully identified using liquid chromatography/tandem mass spectrometry. CONCLUSIONS: A reversed-phase high-performance liquid chromatography method was developed and validated for the separation of the 12 degradation products and 6 process-related impurities. Structural characterization of all impurities was carried out using fragmentation pathways obtained from tandem mass spectrometry. The method was sufficiently sensitive and reproducible for quality control of linagliptin and for further research studies.

Dramatic improvement in pharmacokinetic and pharmacodynamic effects of sustain release curcumin microparticles demonstrated in experimental type 1 diabetes model.

Curcumin (cur) is a well known plant flavonoid with pleiotropic pharmacological activities. However, due to its poor bioavailability those therapeutic benefits are still out of reach for patient community. The main aim of our study was to prepare sustained release cur microparticles (CuMPs) with Poly (lactic-co-glycolic acid) (PLGA), an FDA approved biodegradable polymer and to assess their pharmacological potential in multiple low doses streptozotocin (MLD-STZ) induced type 1 diabetes mellitus (T1DM). CuMPs were formulated and characterized for size (12.71 ±â€¯4.20 µm) and encapsulation efficiency (85.10 ±â€¯2.33%) with 28% drug loading. In vitro release and in vivo pharmacokinetics studies showed promising results of sustained release of cur from CuMPs. With this here we report a strategy that single administration of CuMPs may fill the therapeutic window that is missing from free drug repeated administration and low bioavailability of cur. Moving forward with this concept, we compared the therapeutic effects of CuMPs (equivalent to 7.5 mg/kg cur with free cur orally (100 mg/kg) and intraperitoneally (7.5 mg/kg) administered daily in MLD-STZ challenged animals). CuMPs exhibited superior effects compared to daily administration free drug given either orally or i.p. in terms of lowering the blood glucose levels, improved glucose clearance as evident from results of i.p. glucose tolerance test (IPGTT). Interestingly, we observed a remarkable reduction in diabetes incidence in CuMPs groups (only one out of six animals i.e. 16.6%). Moreover, plasma and tissue levels of insulin indicated superior effect of CuMPs. In addition, immunohistochemical analysis of insulin in pancreatic ß-cells further confirmed the improved therapeutic benefit with significant increase in insulin signal with CuMPs. Amelioration of oxidative stress and inflammation of CuMPs was observed as the molecular mechanism behind the observed superior pharmacological effects with CuMPs. Cumulatively, our sustained release CuMPs formulation may serve as a bridge in overcoming the poor pharmacokinetics issues associated with cur and may hasten the clinical translation of cur.

Sustained-Release Curcumin Microparticles for Effective Prophylactic Treatment of Exocrine Dysfunction of Pancreas: A Preclinical Study on Cerulein-Induced Acute Pancreatitis.

Acute pancreatitis (AP) is a serious inflammatory disorder of the pancreas with considerable mortality. The clinical therapy is hampered due to lack of any approved drug for AP. In this study, we developed curcumin (cur)-loaded poly (lactic-co-glycolic acid) cur microparticles (CuMPs) for sustained release. CuMPs were prepared by emulsion solvent evaporation method and characterized for shape, size, compatibility, and entrapment efficiency. The in vitro drug release and in vivo pharmacokinetic studies confirmed sustained release pattern of cur from CuMPs. The pharmacodynamic study was conducted in cerulein induced AP model. Prophylactic treatment was planned with single dose of CuMPs (equivalent to 7.5 mg/kg of cur) and compared with free cur given orally (100 mg/kg) and intraperitoneally (7.5 mg/kg) daily for 7 days. Interestingly, the effects of CuMPs were superior compared to the free drug administered either orally or intraperitoneally through repeated administrations. CuMPs showed significant decrease of serum amylase and lipase levels, oxidative and nitrosative stress was also significantly decreased. Moreover, CuMPs impressively decreased inflammatory cytokines. Our results may pave a way to propose similar strategy for many of promising natural products to combat several oxidative stress-mediated disorders via sustained release microparticle approaches.

Identification of degradation products of saquinavir mesylate by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry and its application to quality control.

RATIONALE: Saquinavir mesylate (SQM) is an antiviral drug used for the treatment of HIV infections. The identification and characterization of all degradation products are essential for achieving the quality in pharmaceutical product development and also for patient safety. METHODS: The drug was subjected to hydrolytic (HCl, NaOH and water), oxidative (H2 O2 ), photolytic (UV and fluorescence light) and thermal (dry heat) forced degradation conditions as per ICH guidelines. The best chromatographic separation of the drug and all degradation products (DPs) was achieved on a CSH-Phenyl Hexyl column (100 × 2.1 mm, 1.7 µm) with ammonium acetate (10 mM, pH 5.0) and methanol as mobile phase in gradient mode at a flow rate of 0.28 mL/min. RESULTS: Nine DPs were obtained under various forced degradation conditions. All the DPs were characterized by using ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/ESI-QTOF MS/MS) and the degradation pathway of the drug was justified by mechanistic explanations. The main DPs were formed by amide hydrolysis, conversion into diastereomers, an N-oxide and dehydration as well as oxidation of the alcohol from the drug. The method was validated and can be used in a quality control (QC) laboratory to assure the quality of SQM in bulk and finished formulations. CONCLUSIONS: A simple UHPLC/photodiode array (PDA) method was developed and successfully transferred to UHPLC/ESI-Q-TOF MS/MS for the identification and characterization of DPs. Very interestingly, diastereomeric DPs were obtained and successfully resolved by the chromatographic method. Copyright © 2017 John Wiley & Sons, Ltd.

Study on the forced degradation behaviour of ledipasvir: Identification of major degradation products using LC-QTOF-MS/MS and NMR.

Ledipasvir, a novel NS5A inhibitor is used in the management of hepatitis C virus infections. The drug was subjected to forced degradation studies as per the conditions prescribed in ICH Q1 (R2) guideline. Ledipasvir degraded in hydrolytic (acid, alkaline and neutral) and oxidative stress conditions. The drug was found to be stable in thermal and photolytic conditions. Eight novel degradation products were obtained and were well separated using an HPLC C18 stationary phase (150×4.6mm, 5µm) and mobile phase composed of formic acid/acetonitrile in gradient elution mode. All the degradation products were characterised using tandem mass spectrometry with a time-of-flight analyser and the major degradation products of hydrolytic and oxidative stress were isolated and their structural confirmation was studied using 1H and 13C NMR. A well resolved chromatographic method proposed in this study suggests that the proposed analytical method finds its application as a stability indicating assay method for the drug and can be used in routine analysis.

LC-ESI-MS/MS evaluation of forced degradation behaviour of silodosin: In vitro anti cancer activity evaluation of silodosin and major degradation products.

Silodosin (SLD) a novel α1-adrenoceptor antagonist was subjected to forced degradation involving hydrolysis (acidic, alkaline and neutral), oxidative, photolysis and thermal stress, as per ICH specified conditions. The drug underwent significant degradation under hydrolytic (acidic, alkaline and neutral) and oxidative stress conditions whereas, it was found to be stable under other stress conditions. A rapid, precise, accurate and robust chromatographic method for the separation of the drug and its degradation products (DPs) was developed on a Fortis C18 analytical column (150×4.6mm, 5µm) using 0.1% formic acid and acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0mL/min. A total of 5 (DP 1 to DP 5) hitherto unknown DPs were identified by LC-ESI-TOF-MS/MS experiments and accurate mass measurements. The most probable mechanisms for the formation of DPs have been proposed based on a comparison of the fragmentation of the [M+H]+ ions of silodosin and its DPs. The major DPs (DP 1 and DP 2) were isolated and evaluated for anticancer activity using PC3 (human prostate cancer) cell lines by MTT assay. The results revealed that silodosin, DP 1 and DP 2 have potential anticancer activity with IC50 values (µM) 72.74 (±4.51), 25.21 (±2.36), and, 114.07 (±11.90) respectively.

Characterization of forced degradation products and in silico toxicity prediction of Sofosbuvir: A novel HCV NS5B polymerase inhibitor.

Sofosbuvir is a direct acting antiviral medication used to treat Hepatitis C viral infection. The present study focuses on the degradation behavior of the drug under various stress conditions (hydrolysis, oxidative, thermal and photolytic) as per International Conference on Harmonization (ICH Q1A (R2)) guidelines. A high performance liquid chromatographic system (HPLC) was used to develop a selective, precise and accurate method for separating all the degradation products. The separation was achieved on a Sunfire™ C18 (150mm×4.6mm×5µm) stationary phase with a mobile phase of 10mM ammonium acetate (pH 5.0) buffer and acetonitrile in gradient elution mode. A quadrupole-time of flight mass analyzer equipped with an electrospray ionization technique was used to propose the structural information based on the MS/MS and accurate mass measurements. Seven degradation products were identified and characterised by LC-ESI-QTOF-MS/MS. In silico toxicity of the drug and its degradation products was determined using TOPKAT and DEREK toxicity prediction softwares. The proposed method was validated as per the ICH Q2 guidelines.

Liquid chromatography/tandem mass spectrometry study of forced degradation of azilsartan medoxomil potassium.

RATIONALE: Azilsartan medoxomil potassium (AZM) is a new antihypertensive drug introduced in the year 2011. The presence of degradation products not only affects the quality, but also the safety aspects of the drug. Thus, it is essential to develop an efficient analytical method which could be useful to selectively separate and identify the degradation products of azilsartan medoxomil potassium. METHODS: AZM was subjected to forced degradation under hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress conditions. Separation of the drug and degradation products was achieved by a liquid chromatography (LC) method using an Acquity UPLC(®) C18 CSH column with mobile phase consisting of 0.02% trifluoroacetic acid and acetonitrile using a gradient method. Identification and characterization of the degradation products was carried out using LC/electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOFMS). RESULTS: A total of five degradation products (DP 1 to DP 5) were formed under various stress conditions and their structures were proposed with the help of tandem mass spectrometry (MS/MS) experiments and accurate mass data. A common degradation product (DP 4) was observed under all the degradation conditions. DP 1, DP 2 and DP 5 were observed under acid hydrolytic conditions whereas DP 3 was observed under alkaline conditions. CONCLUSIONS: AZM was found to degrade under hydrolytic, oxidative and photolytic stress conditions. The structures of all the degradation products were proposed. The degradation pathway for the formation of degradation products was also hypothesized. A selective method was developed to quantify the drug in the presence of degradation products which is useful to monitor the quality of AZM.