Unknown Impurities Research Articles

P‐133: Enhancement of Light‐Emitting Efficiency Through Impurity Controls by Using Prep‐NMR Technique

An electroluminescence (EL) material plays a key role in electrical and optical efficiency of an organic light emitting diode (OLED) display. High purity, over 99.9%, EL materials have been required to be deposited onto OLED panel, producing a long‐life time device. Even less than 0.1% of impurities are deposited on the panel, which can drop their efficiency and lifetimes very seriously. In this work, we have developed faster and more accurate evaluation methods to identify unknown impurities in emitting materials. We can separate each other by using semi‐prep liquid chromatography and define their chemical structures by combining mass spectrometry and nuclear magnetic resonance results. The impurities in the in the manufacturing process were identified, which causes the efficiency drop, down to 8%. We find out these were coming from other EL materials in the process by accident. Finally, we can guarantee highquality assurance of our panels and eliminate potential risk factors in the manufacturing process.

The New RP-HPLC Method for Simultaneous Quantification of Cinnarizine, its Five Specified Impurities, Two Degradation Products with Two Antioxidants and Confirmation of all by HPLC-ESI-MS in Different Pharmaceutical Drug Formulations

To determine the safety and efficacy, we have developed and validated the RP-HPLC method for simultaneous quantification of cinnarizine, its five specified impurities and two degradation products in cinnarizine API, tablets and capsules and including two antioxidants in an oral suspension formulation. The chromatographic separation was achieved in gradient elution mode with 1.00mL/min flow on an Ascentis Express C18 (150mm, 4.6mm and 2.7µm particle size) column at 40.0°C column temperature using 0.05% acetic acid in mixtures of 10mM ammonium acetate and acetonitrile. All peaks are eluted within 30 minutes with a 10µL injection volume and detected at a 230nm wavelength. The results of the validation of the proposed RP-HPLC method as per ICH guidelines revealed that the method is specific, accurate, precise, linear and robust for quantification purposes. The recoveries for all specified impurities, degradation products and cinnarizine at a lower concentration were found in the range of 100.0±10.0%. While the assay values were within 100.0±2.0% for cinnarizine and antioxidants (methylparaben and propylparaben). The LOD and LOQ were 0.1125µg/mL and 0.1875µg/mL, respectively. The linearity curves for all the ten analytes mentioned above showed good linearity (r≥0.999). This research work presents the first RP-HPLC method for simultaneous quantification of all ten analytes with unknown impurities, as well as an HPLC-ESI-MS method for correct identification and confirmation of results.

Analysis of the polymerized impurities in cefmetazole sodium based on novel separation principle by liquid chromatography tandem ion trap/time-of-flight mass spectrometry

To effectively control the polymerized impurities in cefmetazole sodium, novel high performance gel filtration chromatography (HPSEC) with TSK-gel G2000SWxl column and RP-HPLC method with C18 column were used in replace of classical gel filtration chromatography with Sephadex G-10 gel. By studying the chromatographic behavior of polymerized impurities in both chromatographic systems with different chromatographic separation principles, the polymerized impurities in cefmetazole sodium were separated and detected effectively. The two-dimensional liquid chromatography tandem ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) was applied to characterize the structures of polymerized impurities eluted from HPSEC method, and liquid chromatography tandem ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities and other unknown impurities eluted from RP-HPLC method. The structures of fourteen unknown impurities in cefmetazole sodium were deduced based on the MS n data, nine of which were polymerized impurities. The corresponding relationship between impurities in the HPSEC method and RP-HPLC method was established, and the specificity of the two methods was evaluated. The RP-HPLC method for analysis of the polymerized impurities has higher column efficiency and specificity than the HPSEC method. The RP-HPLC method is suitable for quality control of the polymerized impurities in cefmetazole sodium. The forming mechanisms of degradation impurities in cefmetazole sodium were also studied.

Identification of unknown impurities in triamcinolone acetonide palmitate

Identification of unknown impurities in triamcinolone acetonide palmitate

An inductively coupled plasma mass spectrometry method for the determination of elemental impurities in calcium carbonate mineral medicine

Calcium carbonate mineral medicine contains many unknown elemental impurities; however, the detection strategies of the elemental impurities in calcium carbonate differ in various national pharmacopoeias, hindering accurate quantification and risk assessment. In this study, a simple and rapid inductively coupled plasma mass spectrometry method was developed for the detection of 31 elemental impurities in the calcium carbonate mineral medicine by using the collision-reaction cell technique, which could eliminate the mass spectral interference by the complex polyatomic ion. In addition, the isotope ratios were used as a control parameter parameter. The detection result of the proposed method could meet the United States Pharmacopoeia 223, while the risk assessment was performed following the International Conference on Harmonization (ICH) Q3D guidelines. Our results provided a promising strategy for screening and quantitative detection of 31 elemental impurities in calcium carbonate formulations.

Identification of Process-Related Impurities and Corresponding Control Strategy in Biocatalytic Production of 2-O-α-d-Glucopyranosyl-l-ascorbic Acid Using Sucrose Phosphorylase.

2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) is an ideal substitute for l-ascorbic acid because of its remarkable stability and improved biological activity, which can be easily applied in cosmetic, food, and medicine fields. However, impurity identification and control are significant procedures during the manufacturing of AA-2G. This study assessed a manufacturing routine of AA-2G synthesized by sucrose phosphorylase (SPase). First, three unknown process-related impurities were observed, which were further identified as 3-O-α-d-glucopyranosyl- l-ascorbic acid (impurity I), 2-O-α-d-glucopyranosyl-l-dehydroascorbic acid (impurity II), and 13-O-α-d-glucopyranosyl-2-O-α-d-glucopyranosyl-l-ascorbic acid (impurity III), respectively. Second, a comprehensive formation pathway of impurities was elucidated, and specific strategies corresponding to controlling each impurity were also proposed. Specifically, the content of impurity I can be reduced by 50% by fine tuning reaction conditions. The impurity II-free purification process was also achieved by applying a low concentration of alkali. Finally, a semi-rational design was introduced, and a single mutant L343F was obtained by site-directed mutagenesis, which reduced impurities I and III by 63.9 and 100%, respectively, without affecting the transglycosylation activity. It is expected that the reported impurity identification and control strategies during the AA-2G production will facilitate its industrial production.

Identification of degradation products in flumazenil using LC-Q-TOF/MS and NMR: Degradation pathway elucidation

Flumazenil is an imidazobenzodiazepine derivative that antagonizes the actions of benzodiazepines. The degradation pathway elucidation plays an important role in the drug quality control. In this work, a reliable LC-Q-TOF/MS method was developed to separate and identify the degradation products of flumazenil generated in the stress testing conducted according to the ICH Q1A(R2) guideline. Fifteen degradation products were detected in total including three reported impurities and twelve unknown impurities. Based on the chromatographic and mass spectrometric data acquired, the structures of all the degradation products were identified. Besides, two major degradants were synthetized and further confirmed by NMR. The degradation pathways of flumazenil were elucidated, and the degradation characteristics of benzodiazepines were also discussed. The obtained results are of both great importance for the quality control of flumazenil and good reference for the degradation study of other benzodiazepines.

Favipiravir (SARS-CoV-2) degradation impurities: Identification and route of degradation mechanism in the finished solid dosage form using LC/LC-MS method.

Favipiravir finished dosage was approved for emergency use in many countries to treat SARS‐CoV‐2 patients. A specific, accurate, linear, robust, simple, and stability‐indicating HPLC method was developed and validated for the determination of degradation impurities present in favipiravir film‐coated tablets. The separation of all impurities was achieved from the stationary phase (Inert sustain AQ‐C18, 250 × 4.6 mm, 5‐μm particle) and mobile phase. Mobile phase A contained KH2PO4 buffer (pH 2.5 ± 0.05) and acetonitrile in the ratio of 98:2 (v/v), and mobile phase B contained water and acetonitrile in the ratio of 50:50 (v/v). The chromatographic conditions were optimized as follows: flow rate, 0.7 mL/min; UV detection, 210 nm; injection volume, 20 μL; and column temperature, 33°C. The proposed method was validated per the current International Conference on Harmonization Q2 (R1) guidelines. The recovery study and linearity ranges were established from the limit of quantification to 150% optimal concentrations. The method validation results were found to be between 98.6 and 106.2% for recovery and r 2 = 0.9995–0.9999 for linearity of all identified impurities. The method precision results were achieved below 5% of relative standard deviation. Forced degradation studies were performed in chemical and physical stress conditions. The compound was sensitive to chemical stress conditions. During the study, the analyte degraded and converted to unknown degradation impurities, and its molecular mass was found using the LC–MS technique and established degradation pathways supported by reaction of mechanism. The developed method was found to be suitable for routine analysis of research and development and quality control.

Stability of a calibrant as certified reference material for determination of trans-zearalenone by high performance liquid chromatography-diode array detection-triple quadrupole tandem mass spectrometry.

In this study, a trans-zearalenone (trans-ZEN) calibrant in acetonitrile as certified reference material (CRM) was prepared and intensively investigated the stability by high performance liquid chromatography coupled diode array detection and triple quadrupole tandem mass spectrometry (HPLC-DAD-MS/MS). The photoisomerization and degradation of main component and related impurities in trans-ZEN calibrant CRM was studied in detail under different light conditions such as UV light (254nm), sunlight, and visible light. Trans-ZEN in acetonitrile was confirmed a significant shift toward cis-ZEN up to a 52% cis-isomerization rate after exposing to UV light (254nm) in transparent ampule for 1day. The unsaturated double bond photosensitive groups of trans-ZEN and cis-ZEN will further undergo photoreaction to generate more isomers and related products with the increase of UV irradiation time. The calibrant in amber ampules was relatively stable after exposing to sunlight for 28days, with only 0.35% cis-isomer observed. The results indicated that trans-ZEN solution calibrant should be packed in amber ampules to avoid UV rays. Thermal stability test exhibited this calibrant was stable over 6weeks even at 60°C. Trans-ZEN was found to be more stable in acetonitrile than in methanol since an unknown impurity was observed in methanol after 6weeks placed at 25°C. The stability study of trans-ZEN calibrant provided a basis for the usage, storage, and transportation of the CRM. A concentration and expanded uncertainty of the trans-ZEN calibrant CRM of 11.01 ± 0.18µg/mL was developed.

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.

Identification, isolation, and characterization of two novel impurities from phenothiazine heterocycle derivative: Perphenazine and their control in the process

AbstractDuring the process development of perphenazine, two unknown impurities were found by high‐performance liquid chromatography (HPLC) analysis. These two impurities were further identified, isolated, and characterized form the mother liquor. These two impurities were found novel in the process of perphenazine. Also the reaction conditions were controlled for commercial manufacturing of perphenazine with overall good yield and minimal side reactions with control of these two novel impurities.

DEGRADATION KINETICS OF CARVEDILOL PHARMACEUTICAL DOSAGE FORMS (TABLETS) THROUGH STRESS DEGRADATION STUDY

Objective: The aim of the research work to monitor impurities profiling and degradation kinetics of Carvedilol Pharmaceutical Dosage Form (Tablets) through stress degradation study.
 Methods: To study impurity profiling and degradation kinetics Chromatographic condition used as, Inertsil ODS 3V column (150 mm x 4.6 mm, 5μm) with mobile phase consisting Mobile phase-A (Water, Acetonitrile and Trifluroacetic acid in the ratio of 80:20:0.1 v/v/v respectively and pH adjusted to 2.0 with dilute potassium hydroxide solution) and Mobile phase-B (Water and acetonitrile in the ratio of 100:900 v/v respectively) delivered at flow rate of 1.0 ml min-1 and the detection wavelength 240 nm. The column compartment temperature maintained at 40 °C.
 Results: Stress degradation study conducted using Acid, Alkali, Oxidation, Humidity, Thermal and Photolytic stress degradation conditions. Known, unknown and degradant impurities nature and degradation kinetics in different stressed degradation conditions were monitored through stability indicated method reverse phase HPLC method. Carvedilol molecule found sensitive to Oxidation and Alkali conditions. Impurity-A significantly increased from its not detected level.
 Carvedilol molecule found stable in Acid, Humidity, Thermal and Photolytic stress degradation condition. In all stressed conditions, mass balance was found between 95% to 105% and peak purity of carvedilol peak was found pure.
 Conclusion: Stress degradation study is required to know the degradation pathway of product and to prove the stability indicating nature of the analytical method. Study provide information pertaining to the intrinsic stability of drug product.

FORMULATION AND DEVELOPMENT OF FIXED-DOSE COMBINATION OF BI-LAYER TABLETS OF EFAVIRENZ, LAMIVUDINE AND TENOFOVIR DISOPROXIL FUMARATE TABLETS 600 MG/300 MG/300 MG

Objective: This study is to formulate bi-layer tablet as a multidrug regimen against each reference listed drugs of Brand SUSTIVA® (efavirenz tablets 600 mg), EPIVER®(lamivudine tablets 300 mg), and VIREAD®(tenofovir disoproxil tablets 300 mg) to treat human immunodeficiency virus (HIV) infections. Which provides highly active antiretroviral therapy to provide effective treatment. Methods: Bilayer formulation was developed with each blend of layer-I (efavirenz) and layer-II (lamivudine and tenofovir disoproxil fumarate) through wet granulation process and roller compaction process, respectively. Further, both layers were compressed by using bi-layer compression followed by film coating. Layer-I and II formulations were developed by using various concentrations of diluents, surfactants, and disintegrants to improve the solubility of efavirenz and improve the flowability and uniformity of layer-II. Finally, the optimum formulation was developed to compare the in vitro dissolution with each branded formulation. Results: Drug-excipients interaction results revealed that the mixtures of three drug substances in 50 °C/75 % relative humidity (RH) resulted in an increase in tenofovir IMP-E and the highest unknown impurity was significantly increased and additionally decreased tenofovir assay in the presence of efavirenz. Sodium lauryl sulfate is very critical and it acts as a wetting agent and increases the solubility of efavirenz, and directly influences the dissolution of a drug product. Microcrystalline and croscarmellose sodium have a chance to affect the dissolution and friability of tenofovir. Powdered cellulose was acting as a diluent and flow property of the lamivudine part and it also affects the uniformity and dissolution. So, these ranges were optimized. X-ray diffraction (XRD) indicates there are no polymorphic changes for the optimized formulation and there is no interaction between the three active substances, and finally, in vitro dissolution results for the optimized formulation against the reference drugs. Conclusion: Optimum formulation yielded consistent drug release against each branded drug to treat human immunodeficiency virus (HIV1) infections. This formulation is robust and easily scale up for the next stage.

Management of validation of HPLC method for determination of acetylsalicylic acid impurities in a new pharmaceutical product

The work mainly focused on a validation of the method for determining the content of salicylic acid and individual unknown impurities in new pharmaceutical product—tablets containing: 75, 100 or 150 mg of acetylsalicylic acid and glycine in the amount of 40 mg for each dosage. The separation of the components was carried out by means of HPLC, using a Waters Symmetry C18 column (4.6 × 250 mm, 5 μm) as the stationary phase. The mobile phase consisted of a mixture of 85% orthophosphoric acid, acetonitrile and purified water (2:400:600 V/V/V). Detection was carried out at a wavelength of 237 nm, with a constant flow rate of 1.0 ml min−1. In order to verify the method, linearity, precision (repeatability and reproducibility), accuracy, specificity, range, robustness, system precision, stability of the test and standard solution, limit of quantification and forced degradation were determined. Validation tests were performed in accordance with ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) guidelines. The method was validated successfully. It was confirmed that the method in a tested range of 0.005–0.40% salicylic acid with respect to acetylsalicylic acid content is linear, precise and accurate.

New Stability Indicating Rp-Hplc Method for the Determination of Potential Process Related Impurites and its Degradation Products of Dasatinib and Identification, Isolation and Chracterization of Unknown Acid Degradation Impurity of Dasatinib

New Stability Indicating Rp-Hplc Method for the Determination of Potential Process Related Impurites and its Degradation Products of Dasatinib and Identification, Isolation and Chracterization of Unknown Acid Degradation Impurity of Dasatinib

USING PREPARATIVE CHROMATOGRAPHY AND NMR/LCMS/FT-IR, ISOLATION, IDENTIFICATION, AND CHARACTERIZATION OF POSACONAZOLE OXIDATIVE DEGRADATION IMPURITIES

Posaconazole is a pharmacological ingredient that is used to treat fungal infections. It comes in the form of delayedrelease tablets. During the forced degradation studies of Tablets, two unknown oxidative degradation impurities were discovered. The impurities were made from Posaconazole using meta-Chloroperoxybenzoic acid as an oxidant in the degradation process. Preparative chromatography was used to separate impurities, and spectroscopic techniques such as IR, NMR, and MS were used to deduce the structures. For the measurement of the total of six associated impurities of Posaconazole in Delayed-Release Tablets, a reverse phase HPLC technique was developed and validated according to ICH Q2 criteria

Glabrol impurity exacerbates glabridin toxicity in zebrafish embryos by increasing myofibril disorganization

Ethnopharmacological relevanceGlabridin, extracted from Glycyrrhiza glabra L., is widely used for the treatment of hyperpigmentation because of its anti-inflammatory and antioxidant activities and its ability to inhibit melanin synthesis. This led to the strict regulation of its quality and safety. However, traditional quality control methods used for plant extracts cannot reflect the product quality owing to multiple unknown impurities, which necessitates the further analysis of impurities. Aim of the studyThe study identified the toxic impurities of glabridin and their toxicological mechanism. Materials and methodsIn total, 10 glabridin samples from different sources were quantified using high-performance liquid chromatography. Sample toxicities were evaluated using zebrafish and cell models. To identify impurities, samples with different toxicity were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry. The toxicity of related impurities was verified in the zebrafish model. Phalloidin stain was used to evaluate subtle changes in myofibril alignment. ResultsAlthough glabridin content in the samples was similar, there were significant differences in toxicity. The results were verified using four different mammalian cell lines. Higher contents of glabrone and glabrol were identified in the sample with the highest toxicity. In the zebrafish model, the addition of glabrol reduced the LC50 of glabridin to 9.224, 6.229, and 5.370 μM at 48, 72, and 96 h post-fertilization, respectively, whereas glabrone did not have any toxic effect. Phalloidin staining indicated that a glabrol impurity exacerbates the myotoxicity of glabridin in zebrafish embryos. ConclusionGlabrol, but not glabrone, was identified as a key impurity that increased glabridin toxicity. This finding indicates that controlling glabrol content is necessary during glabridin product production.

Detection and Characterization of an Unknown Impurity in Levothyroxine Sodium Tablets by UHPLC, LC/MS, MS/MS and MS/MS/MS

Impurities in the active ingredient of a drug are very important for the quality and reliability of the finished product. Therefore, the identification of these impurities constitutes reliability for patients using the drug. Levothyroxine sodium has known but also unknown impurities. Liothyronine sodium is known as the most common impurity of Levothyroxine sodium. Thought to be more active than levothyroxine and with a rapid (few hours) onset and short duration of action, liothyronine sodium is used in the treatment of hypothyroidism, particularly in the cases of hypothyroid coma. In our study, besides known impurities of Levothyroxine sodium, some unknown impurities were observed in samples that were exposed to heat. In these unknown impurities, the most important one that increases the fastest with the temperature that will affect the quality of the product is the impurity at RRT 0.6, according to our impurity method which was developed in-house. The impurity that increases with temperature has been defined by various identification studies, including MS/MS analysis, LC-UV detection etc. The degradation product, which was initially characterized by its retention time as RRT 0.6, was subsequently identified to be a lactose adduct of Levothyroxine API, which is formed by a Maillard reaction between Levothyroxine and lactose.

Stability Indicating Method for Known and Unknown Impurities Profiling for Vildagliptin in Vildagliptin Tablet

Background: Vildagliptin is a drug for the treatment of diabetes. DPP-IV inhibitor represents a new class of oral antihyperglycemic agents to treat patients with type 2 diabetes. Several RP-HPLC methods have been reported to determine Vildagliptin alone. However, it has been noted that there are no available stability-indicating methods in pharmacopeias (USP/BP/EP/JP) nor in the available literature to quantify known and unknown impurity patterns for vildagliptin in vildagliptin tablets. Objective: The aim of this study is to develop a new single, sensitive, robust and specific gradient RP-HPLC method to quantify known and unknown impurities and degradants of Vildagliptin in Vildagliptin tablets. Methods: Chromatographic separation has been accomplished on the Hypersil ODS column (250 x 4.6) mm, 5 μm with a mobile phase consisting of a mixture of Perchloric acid Buffer, methanol, acetonitrile and Triethylamine delivered at a flow rate of 1.0 mL minute-1 and the detection wavelength 210 nm. The developed method was validated as per ICH guidelines. Results: Vildagliptin was found degraded significantly under oxidative and alkaline stress conditions. The degradation products were well resolved from Vildagliptin and its impurities. An analytical method was found linear, accurate and precise from LOQ (Limit of Quantification) level to 150% of impurity specification limit (0.5%). Conclusion: The method found sensitive, rapid and accurate quantification of known, unknown impurities and degradants. The peak purity results confirmed that the Vildagliptin peak was homogeneous and pure in all stress samples, thus proving the stability-indicating nature of the method.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR KNOWN AND UNKNOWN IMPURITIES PROFILING FOR CARVEDILOL PHARMACEUTICAL DOSAGE FORM (TABLETS)

Objective: The aim of the research work to develop a simple, sensitive, rugged, robust and specific novel gradient stability indicating reverse phase HPLC method for quantitative determination of known and unknown impurities profiling of Carvedilol pharmaceutical dosage forms (Tablets).
 Methods: Chromatographic separation has been achieved on an Inertsil ODS 3V column (150 mm x 4.6 mm, 5μm) with mobile phase consisting Mobile phase-A (Water, Acetonitrile and Trifluroacetic acid in the ratio of 80:20:0.1 v/v/v respectively and pH adjusted to 2.0 with dilute potassium hydroxide solution) and Mobile phase-B (Water and acetonitrile in the ratio of 100:900 v/v respectively) delivered at flow rate of 1.0 ml min-1 and the detection wavelength 240 nm. The column compartment temperature maintained at 40 °C.
 Results: Resolution between Carvedilol and its impurities has been achieved greater than 1.5. The developed method was validated as per ICH guidelines. Analytical method found Precise, Linear, accurate, specific, rugged and robust.
 Conclusion: Developed and validated novel analytical method can be used to for impurity profile analysis of Carvedilol Pharmaceutical dosage form (Tablets).