Impurity assessment, development and validation of an RP-HPLC method for the determination of eleven potential impurities of eltrombopag precursor.

Eltrombopag is an oral non-peptide thrombopoietin receptor (TPO-R) agonist indicated for the treatment of thrombocytopenia in patients with persistent or chronic immune thrombocytopenia (idiopathic thrombocytopenic purpura, ITP) or chronic hepatitis C infection and the treatment of severe aplastic anemia. The purpose of this research was to assess the possible impurities that may carry over to eltrombopag from its precursor Eltro-1 (3'-amino-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid) and to develop a specific analytical method for the determination of these impurities. Eltro-1 samples synthesized by two different synthesis routes were investigated during the evaluation and method development studies. Besides the expected process-related impurities (Eltro-1A - Eltro-1J), e.g., starting materials, intermediates, and/or compounds formed from their further reactions, an unknown impurity detected above 0.10% was identified by LC-MS, synthesized and fully characterized by NMR, MS and FTIR (Eltro-1K). Accordingly, an HPLC-RP method for the determination of eleven impurities (Eltro-1A - Eltro-1K) in Eltro-1 was developed and validated according to ICH Q2. The control limits for impurities in Eltro-1 were set at ≤ 0.15% for Eltro-1A - Eltro-1J and ≤ 1.0% for Eltro-1K based on fate, spike-purge and carryover studies and in accordance with the ICH M7 classification for impurities in drug substance. Eltro-1 and eleven impurities at the specification limit were separated from each other and the diluent peaks with sufficient resolution without interference. Separation was performed on a Waters XBridge C18 column (150 × 4.6 mm, 3.5 µm) at 40 °C with a 10 µL injection volume at a detection wavelength of 220 nm and 15 °C sample temperature. The gradient elution is performed at a flow rate of 1.0 mL/min for 40 min with mobile phase A (0.1% orthophosphoric acid in water) and B (acetonitrile) according to the following program: Time (min) / Acetonitrile (%): 0/0, 35/70, 36/0, 40/0. Test and standard solutions were prepared at a concentration of 1.0 mg/mL and 1.0 µg/mL, respectively, using a mixture of mobile phase A and acetonitrile (75/25) as diluent. This is the first specific, selective, sensitive, linear, precise, accurate, and robust HPLC method for the determination of Eltro-1A - Eltro-1K in Eltro-1, which showed no significant degradation under thermal stress, photostability (UV and VIS), and standard accelerated and long-term stability conditions.

A specific chiral HPLC method for lifitegrast and determination of enantiomeric impurity in drug substance, ophthalmic product and stressed samples.

Lifitegrast is a lymphocyte function-associated antigen-1 (LFA-1) antagonist used to treat the indications and symptoms associated with dry eye disease (DED), one of the most common ocular surface diseases. Lifitegrast has a chiral center, and the S-enantiomer (S-Lif) is responsible for the therapeutic effects, while the R-enantiomer (R-Lif) lacks efficacy in the treatment of DED. Lifitegrast ophthalmic solution containing 5% lifitegrast was approved by the United States Food and Drug Administration (FDA) in July 2016 for the treatment of DED in patients 17 years of age and older. The objective of this study was to develop a chiral HPLC method for the determination of the enantiomeric impurity of lifitegrast in the drug substance and in the ophthalmic product. In addition, we aimed to investigate the effect of stress and stability conditions on the enantiomeric purity of lifitegrast in both drug substance and ophthalmic solution. During the method development studies, four known lifitegrast impurities (Lif. Imp. A-D) and stressed lifitegrast samples were injected to ensure the specificity of the developed method. The enantiomers of lifitegrast are well separated with a resolution of higher than 4.0. They are also well separated from the peaks of the diluent, impurities, and the placebo used to prepare the ophthalmic solution without interference in 20 min. Chiral separation was achieved using a Chiralpak AD-H column (250 × 4.6 mm, 5.0 µm) at 40 °C with a mobile phase consisting of a mixture of n-hexane, 2-propanol, and formic acid (500:500:2, v/v/v) at a flow rate of 1.0 mL/min and a detection wavelength of 260 nm. Methanol was used as the diluent, and the drug substance solution was found to be stable for 48 h at 15 °C. The optimized chiral HPLC method for lifitegrast was validated according to ICH Q2, and the calibration curves showed excellent linearity for R-Lif (0.0369 - 1.816 µg/mL). This is the first stability-indicating, specific / selective, sensitive, linear, precise, accurate, and robust chiral HPLC method for the determination of R-Lif in S-Lif. The amount of enantiomeric impurity R-Lif in S-Lif increased under all stress and photostability test conditions without exceeding the acceptable impurity limit, with the most significant increase observed at elevated temperatures (105 °C) for both the drug substance in powder form and the ophthalmic drug solution.

Designing an electrochemical sensor based on ZnO nanoparticle-supported molecularly imprinted polymer for ultra-sensitive and selective detection of sorafenib.

BACKGROUND: Sorafenib (SOR) is a multikinase inhibitor anticancer drug that is used in treating non-small cell lung cancer. In this work, we focused on developing nanomaterial-supported smart porous interfaces by following the molecular imprinting approach for the selective determination of SOR. Determination-based studies in the literature for SOR are limited, and they are chromatographic techniques-based; hence, there is a need in the literature to elaborate the selective and sensitive analysis/monitoring of SOR in both biological and pharmaceutical samples with more studies. RESULTS: The results showed that adding ZnO NPs enhanced the signal five times compared to the solo molecularly imprinted polymer (MIP). Under the optimized conditions, ZnO/AMPS@MIP-GCE showed a linear response in the concentration range between 1.0 × 10-12 and 1.0 × 10-11 M with LOD and LOQ values of 2.25 × 10-13 M and 7.51 × 10-13 M, respectively, in the serum sample. The selectivity study was conducted against common cations, anions, and compounds such as dopamine, paracetamol, ascorbic acid, and uric acid. Also, the imprinting factor (IF) analysis was performed on selected drug substances having structural similarities to SOR and the relative IF values of regorafenib, leflunomide, teriflunomide, nilotinib, axitinib, and dasatinib indicated the selectivity of the developed sensor for SOR. Finally, ZnO/AMPS@MIP-GCE was implemented to determine SOR in the spiked commercial human serum samples and tablet dosage form with bias% between -0.43 and + 0.66. SIGNIFICANCE AND NOVELTY: This study is the first electrochemical study for the determination of SOR, and thanks to the ZnO NPs supported MIP sensor, it stands out in terms of both high sensitivity and superior selectivity. Also, this designed sensor provides controlled orientation of the template and complete removal of templates in a one-step process, allowing extremely low detection and quantification limits.

Elucidation of DNA-Eltrombopag Binding: Electrochemical, Spectroscopic and Molecular Docking Techniques.

Eltrombopag is a powerful adjuvant anticancer drug used in treating MS (myelodysplastic syndrome) and AML (acute myeloid leukemia) diseases. In this study, the interaction mechanism between eltrombopag and DNA was studied by voltammetry, spectroscopic techniques, and viscosity measurements. We developed a DNA-based biosensor and nano-biosensor using reduced graphene oxide-modified glassy carbon electrode to detect DNA-eltrombopag binding. The reduction of desoxyguanosine (dGuo) and desoxyadenosine (dAdo) oxidation signals in the presence of the drug demonstrated that a strong interaction could be established between the eltrombopag and dsDNA. The eltrombopag-DNA interaction was further investigated by UV absorption and fluorescence emission spectroscopy to gain more quantitative insight on binding. Viscosity measurements were utilized to characterize the binding mode of the drug. To shed light on the noncovalent interactions and binding mechanism of eltrombopag molecular docking and molecular dynamics (MD), simulations were performed. Through simultaneously carried out experimental and in silico studies, it was established that the eltrombopag binds onto the DNA via intercalation.

SnO2 nanoparticles/waste masks carbon hybrid materials for DNA biosensor application on voltammetric detection of anti-cancer drug pazopanib.

This present study is the first investigation of pazopanib-dsDNA binding using bare and modified GCE. The interaction was mainly evaluated based on the decrease of voltammetric signal of deoxyadenosine by differential pulse voltammetry using three different ways, including the incubated solutions, dsDNA biosensor, and nanobiosensor. The nanobiosensor was fabricated with the help of SnO2 nanoparticles and carbon hybrid material. The carbon material is derived from the waste mask, the most used personal protective equipment for the ongoing COVID-19 pandemic. Both materials were synthesized via the green synthesis technique and characterized by various techniques, including BET, TEM, SEM-EDX, AFM, XPS, and XRD. Spectrophotometric and molecular docking studies also evaluated the pazopanib-dsDNA binding. All calculations showed that pazopanib (PZB) was active in the minor grove region of DNA.

A sensitive and selective electrochemical sensor based on molecularly imprinted polymer for the assay of teriflunomide.

In this work, pyrrole-histidine has been designed, synthesized and, used as a novel functional monomer to fabricate a molecularly imprinted electrochemical sensor for the selective and sensitive detection of teriflunomide (TER). The molecularly imprinted thin film of electrochemical sensor was constructed by directly electropolymerization of co-polymer of pyrrole-histidine (PyHis) with pyrrole in the presence of a template, TER, on a glassy carbon electrode (GCE). After electropolymerization, the structure and morphology of the fabricated MIP sensor were characterized by Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) and its electrochemical parameters such as cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS). The poly (pyrrole-co-pyrrole-histidine) [Poly (Py-co-PyHis)]@MIP/GCE sensor have a linear TER concentration in the of 0.1-1.0 pM with a low detection limit of 11.38 fM. The present strategy for electrochemical sensor have been also showed excellent recovery in synthetic serum samples and tablet dosage form with the recoveries 97.56% and 100.35%, respectively. The developed [Poly (Py-co-PyHis)]@MIP/GCE sensor exhibited an excellent electrochemical response for TER due to the synergistic effect of conducting polymer and molecularly imprinting techniques.

Synthesis, characterization and control of eight process-related and two genotoxic fingolimod impurities by a validated RP-UPLC method.

An HPLC method has been described in the European Pharmacopoeia and United States Pharmacopeia for the determination of nine organic impurities (imp A-I) in fingolimod hydrochloride, a synthetic sphingosine-1-phosphate receptor modulator. The manufacturing process of fingolimod hydrochloride consists of multistep chemical synthesis wherein controls of precursors, intermediates and process steps should be performed to assure the final quality of the drug substance. We synthesized and isolated eight process-related impurities (FINI imp A-H) of fingolimod, which were different from the pharmacopoeial impurities. One unknown process-related impurity was found as a key intermediate (FINI) and was identified by LC-MS. Characterization of all of the impurities were done using spectroscopic techniques (1 H and 13 C NMR, FTIR, MS), and the mechanistic pathways to the formation of these impurities were also discussed. Two of these impurities were evaluated as potential genotoxic impurities owing to their alerting structures and alkylating properties (alkyl sulfonates and alkyl halides, class 3, ICH M7). We also developed and validated an RP-UPLC method in line with ICH Q2 guidelines for control these impurities (FINI imp A-H) and to assure the pharmacopoeial quality drug substance.

Correction to "Target-Driven Design of a Coumarinyl Chalcone Scaffold Based Novel EF2 Kinase Inhibitor Suppresses Breast Cancer Growth In Vivo".

[This corrects the article DOI: 10.1021/acsptsci.1c00030.].

Target-Driven Design of a Coumarinyl Chalcone Scaffold Based Novel EF2 Kinase Inhibitor Suppresses Breast Cancer Growth In Vivo.

Eukaryotic elongation factor 2 kinase (eEF-2K) is an unusual alpha kinase involved in protein synthesis through phosphorylation of elongation factor 2 (EF2). eEF-2K is highly overexpressed in breast cancer, and its activity is associated with significantly shortened patient survival and proven to be a potential molecular target in breast cancer. The crystal structure of eEF-2K remains unknown, and there is no potent, safe, and effective inhibitor available for clinical applications. We designed and synthesized several generations of potential inhibitors. The effect of the inhibitors at the binding pocket of eEF-2K was analyzed after developing a 3D target model by using a domain of another α-kinase called myosin heavy-chain kinase A (MHCKA) that closely resembles eEF-2K. In silico studies showed that compounds with a coumarin-chalcone core have high predicted binding affinities for eEF-2K. Using in vitro studies in highly aggressive and invasive (MDA-MB-436, MDA-MB-231, and BT20) and noninvazive (MCF-7) breast cancer cells, we identified a lead compound that was highly effective in inhibiting eEF-2K activity at submicromolar concentrations and at inhibiting cell proliferation by induction of apoptosis with no toxicity in normal breast epithelial cells. In vivo systemic administration of the lead compound encapsulated in single lipid-based liposomal nanoparticles twice a week significantly suppressed growth of MDA-MB-231 tumors in orthotopic breast cancer models in nude mice with no observed toxicity. In conclusion, our study provides a highly potent and in vivo effective novel small-molecule eEF-2K inhibitor that may be used as a molecularly targeted therapy breast cancer or other eEF-2K-dependent tumors.

Synthesis and characterization of oxitropium bromide related substances and novel stability indicating HPLC methods.

The European Pharmacopoeia (Ph. Eur.) described two separate HPLC methods for determination of organic impurities in oxitropium bromide, a synthetic anticholinergic agent used by inhalation in the treatment of asthma and other bronchial disorders, and a potentiometric titration assay method which is not a stability indicating method. During synthetic process development and analytical studies of oxitropium; besides known Ph. Eur.-impurities new process related and degradation impurities were determined, identified by LC-MS, synthesized, characterized, and then used in development and validation studies of oxitropium analytical methods. As a result of these studies, a single HPLC related substances method was developed and validated according to international conference on harmonisation (ICH) guidelines for determination of all oxitropium related substances by using an inertsil ODS-4 (250 mm × 4.6 mm, 5 µm) column at 15 °C with 50 µL injection volume at a wavelength of 210 nm with gradient elution of phosphate-buffer/acetonitrile mixture flowing at a rate of 1.2 mL/min during 60 min. Also, a stability indicating HPLC assay method was developed and validated by using an XBridge C18 (150 mm × 4.6 mm, 3.5 µm) column at 25 °C with 10 µL injection volume at a wavelength of 210 nm and with phosphate-buffer/acetonitrile (85/15) mixture flowing at a rate of 1.0 mL/min during 10 min. Stress-testing and stability studies of oxitropium bromide was carried out and samples were analyzed by using newly developed stability-indicating HPLC assay and related substances methods.

Development and validation of stability indicating HPLC methods for related substances and assay analyses of amoxicillin and potassium clavulanate mixtures.

Antibacterial combinations consisting of the semisynthetic antibiotic amoxicillin (amox) and the ß-lactamase inhibitor potassium clavulanate (clav) are commonly used and several chromatographic methods were reported for their quantification in mixtures. In the present work, single HPLC method for related substances analyses of amoxicillin and potassium clavulanate mixtures was developed and validated according to international conference on harmonization (ICH) guidelines. Eighteen amoxicillin and six potassium clavulanate impurities were successfully separated from each other by using triple gradient elution using a Thermo Hypersil Zorbax BDS C18 (250 mm×4.6mm, 3µm) column with 50µL injection volumes at a wavelength of 215nm. Commercially unavailable impurities were formed by degradation of amoxicillin and potassium clavulanate, identified by LC-MS studies and used during analytical method development and validation studies. Also, process related amoxicillin impurity-P was synthesized and characterized by using nuclear magnetic resonance (NMR) and mass spectroscopy (MS) for the first time. As complementary of this work, an assay method for amoxicillin and potassium clavulanate mixtures was developed and validated; stress-testing and stability studies of amox/clav mixtures was carried out under specified conditions according to ICH and analyzed by using validated stability-indicating assay and related substances methods.

Quantitative determination of two polymorphic forms of imatinib mesylate in a drug substance and tablet formulation by X-ray powder diffraction, differential scanning calorimetry and attenuated total reflectance Fourier transform infrared spectroscopy.

Imatinib has been identified as a tyrosine kinase inhibitor that selectively inhibits the Abl tyrosine kinases, including Bcr-Abl. The active substance used in drug product is the mesylate salt form of imatinib, a phenylaminopyrimidine derivative and chemically named as N-(3-(4-(pyridin-3-yl) pyrimidin-2-ylamino)-4-methylphenyl)-4-((4-methylpiperazin-1-yl) methyl)-benzamide methanesulfonic acid salt. It exhibits many polymorphic forms and most stable and commercialized polymorphs are known as α and ß forms. Molecules in α and ß polymorphic forms exhibit significant conformational differences due to their different intra- and intermolecular interactions, which stabilize their molecular conformations and affect their physicochemical properties such as bulk density, melting point, solubility, stability, and processability. The manufacturing process of a drug tablet included granulation, compression, coating, and drying may cause polymorphic conversions. Therefore, polymorphic content of the drug substance should be controlled during quality control and stability testing. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD) methods were evaluated for determination of the polymorphic content of the drug substance and drug product; and PXRD was the most accurate technique and selected as preferred method and validated. Prior to development of a quantification method, pure α and ß polymorphs were characterized and used throughout the method development and validation studies. Mixtures with different ratios of α and ß forms were scanned using X-ray diffractometer with a scan rate of 0.250°/min over an angular range of 19.5-21.0° 2θ and the peak heights for characteristic peak of ß form at 20.5 ± 0.2° 2θ diffraction angle were used to generate a calibration curve. The detection limit of ß polymorph in α form imatinib mesylate tablets was found as 4% and the linear regression analysis data for the calibration plots showed good linear relationship with correlation coefficient of 0.992 with respect to relative peak height in the concentration range of 12-75 wt% ß form containing tablet mixtures. The obtained results at each stage of the validation study proved that the method is specific, repeatable, precise and accurate, and could be used for determination of ß polymorph content in tablets produced by using α polymorph of imatinib mesylate. The developed PXRD quantification method was used to monitor the polymorphic purity of α form drug substance and corresponding drug products during the quality control analyses and stability studies, and the results indicated that α form was stable and not converted to ß form during the manufacturing process and stability period.

Identification, synthesis and characterization of process related impurities of benidipine hydrochloride, stress-testing/stability studies and HPLC/UPLC method validations.

Benidipine hydrochloride, used as an antihypertensive agent and long-acting calcium antagonist, is synthesized for commercial use as a drug substance in highly pure form. During the synthetic process development studies of benidipine, process related impurities were detected. These impurities were identified, synthesized and characterized and mechanisms of their formation were discussed in detail. After all standardization procedures, they were used as reference standards for analytical studies. In addition, a separate HPLC method was developed and validated for detection of residual 1-benzylpiperidin-3-ol (Ben-2), which is used during benidipine synthesis and controlled as a potential process related impurity. As complementary of this work, stress-testing studies of benidipine were carried out under specified conditions and a stability-indicating UPLC assay method was developed, validated and used during stability studies of benidipine.

Identification, synthesis and characterization of process related desfluoro impurity of ezetimibe and HPLC method validations.

Ezetimibe, which selectively inhibits cholesterol absorption across the intestinal wall and is used as an antihyperlipidemic agent, is synthesized for commercial use as a drug substance in highly pure form. During the synthetic process development studies of ezetimibe, an impurity was detected in the final product at levels ranging from 0.05% to 0.15% in reverse phase gradient high performance liquid chromatography (HPLC) method and its molecular weight was determined by LC-MS analysis. The impurity was identified as (3R,4S)-3-((S)-3-(4-fluorophenyl)-3-hydroxypropyl)-4-(4-hydroxyphenyl)-1-phenylazetidin-2-one which is called desfluoro ezetimibe (lactam-related) impurity, synthesized and characterized, the mechanism of its formation was discussed in detail. After all standardization procedures, it was used as a reference standard during validation of HPLC method and routine analyses. In addition, content of Eze-1 desfluoro impurity in Eze-1 intermediates was specified as 0.10% to keep the formation of desfluoro ezetimibe impurity under control and the related substances HPLC method was validated accordingly.