Degradation Impurities Research Articles

Characterization of a new component and impurities in josamycin by trap-free two-dimensional liquid chromatography coupled to ion trap time-of-flight mass spectrometry.

The toxicities of the impurities of a drug will affect the clinical effects and cause potential health risk; therefore, it is essential to study profiles of the impurities. In this study, a new structural type of component and two acid degradation impurities in josamycin were discovered and characterized for the further improvement of official monographs in pharmacopoeias. The component and acid degradation impurities in josamycin were separated and preliminary characterized by trap-free two-dimensional liquid chromatography coupled to high-resolution ion trap time-of-flight mass spectrometry (2D LC/IT-TOF MS) in both positive and negative electrospray ionization mode. The eluent of each peak from the first dimensional chromatographic system was trapped by a switching valve and subsequently transferred to the second dimensional chromatographic system, which was connected to the mass spectrometer. Full scan MS was firstly conducted to obtain the exact m/z values of the molecules. Then LC/MS/MS and LC/MS/MS/MS experiments were performed on the compounds of interest. A new structural type of component, which was named as josamycin A, and two acid degradation impuritiess, which were identified as impurity I and impurity II, were discovered in josamycin. Their structures and fragmentation pattern were deduced according to MSn data. Furthermore, josamycin A was synthesized and impurity I was separated by preparative HPLC. The structures of josamycin A and the impurities were confirmed by 1 H NMR and 13 C NMR data. Josamycin A was produced when the hydroxyl group on the macrolide of josamycin was oxidized into a carbonyl group. Impurity I and impurity II were produced by the loss of one molecule of acetyl mycaminose from josamycin and josamycin A, respectively. Compared with josamycin, the experimental results showed that josamycin A had a higher antibacterial activity with similar cytotoxicity, while impurity I had no antibacterial activity but a higher cytotoxicity. As a result, the control of impurity I is significant.

The Degradation Chemistry of GSK2879552: Salt Selection and Microenvironmental pH Modulation to Stabilize a Cyclopropyl Amine

The cyclopropyl amine moiety in GSK2879552 (1) degrades hydrolytically in high pH conditions. This degradation pathway was observed during long-term stability studies and impacted the shelf life of the drug product. This article describes the work to identify the degradation impurities, elucidate the degradation mechanism, and design a stable drug product. It was found that salt selection and control of the microenvironmental pH of the drug product formulation blend significantly improved the chemical stability of the molecule in the solid state.

Simultaneous Method for Quantification of Genotoxic Impurity in the Gemcitabine Hydrochloride by RP-HPLC

: Objective: In the pharmaceutical analytical field, effort to analyzing the degradation or related and impurity substances from the complex matrix in the ultra-trace levels is a really a challengeable task. Impurity is any component of the new drug substance that is not the chemical entity defined as the new drug substance. Gemcitabine hydrochloride is a new anticancer bio molecule. According to Indian Pharmacopoeia there is two impurities present in this drug. The literature review exploded that, there is no specific impurity profiling based analytical method developed to estimate the Cytosine. Methods: RP HPLC is developed to quantify and separate Gemcitabine hydrochloride and Cytosine. The Hibar® C18 (250 x 4.6 mm i.d., 5μ), Column choices as stationary phase, tri ethyl ether and Acetonitrile in ratio of 95:5 with 4.35 (with Orthophosphoric acid) as mobile phase, the flow rate fixed as 1ml/ min. at 279 nm. Results: A retention time found to be 3.44min and 8.28min for gemcitabine and cytosine respectively. The developed method was validated as per ICH guidelines and the method was proved as selective, specific and linear. The assay and recovery studies were carried out the assay limit was 99.19 and 99.32 for bulk drug and formulations respectively. The Cytosine were present 0.7821 μg/ml and 0.6531 μg/ml in the bulk drug and formulation respectively. Conclusion: It can be concluded that the developed RP-HPLC method was found to be suitable for the appraisal of cytosine impurities in Gemcitabine HCl. The developed RP-HPLC is economic and suitable for the use.Key words: Gemcitabine HCl (GCH), Cytosine (CYS), RP-HPLC, Genotoxin, Impurity, Anticancer.

Two validated stability-indicating chromatographic methods for the separation of two anti-hypertensive combinations in the presence of their degradation products or impurities

Two RP-HPLC methods were developed, optimized, and validated for the determination of two different anti-hypertensive combinations in the presence of their degradation products or impurities and in their pharmaceutical formulations. The first mixture is Ramipril (RAM) in combination with Amlodipine besylate (AML) [mixture I], while the second one is a combination of Ramipril (RAM), Atorvastatin (ATV), and Aspirin (ASP) [mixture II].The proposed combinations were successfully separated on X-bridge C18column (250 × 4.6 mm i.d, 5 µm p.s.), using a mobile phase of 0.05 M phosphate buffer-acetonitrile-THF (60:40:0.1% by volume) pH 2.5 and an isocratic mobile phase formed of acetonitrile-0.05 M phosphate buffer-THF (60:40:0.1% by volume) pH 2.5 for mixture (I) and (II) at a flow rate of 1 mL/min and 1.2 mL/min, respectively. The compromising components of the mixtures were detected at 218 nm. For the best separation of the mentioned components different parameters were examined and optimized. The two suggested methods were validated in compliance with the ICH guidelines and were successfully applied for the quantification of the cited components in presence of their obtained degradation products as well as in their commercial pharmaceutical formulations. For both methods the obtained results were statistically analyzed and compared to those of the official and reported methods; using Student’s t test and F test showing no significant difference with high accuracy and good precision.

Assay of tipranavir capsules by a simple stability-indicating LC–UV method and cytotoxicity study of degraded samples

Tipranavir (TPV) is a protease inhibitor (PI) specially recommended for treatment-experienced patients who are resistant to other PI drugs. It was approved by FDA in 2005, but, until now, it has not been included in any official compendia. The objective of this study is to develop and validate a simple LC–UV method to assay TPV capsules, and to study the cytotoxicity of TPV and degraded samples over the cell viability. The optimized conditions were C8 Phenomenex® column (Luna®, 150 mm × 4.6 mm, 5 µm); mobile phase composed by methanol, acetonitrile, and acidified water pH 3.5 (40:31:29); flow rate 1.0 mL min−1 and detection at 254 nm. The cytotoxic effects of non-degraded and degraded TPV samples were evaluated in 3T3 cells by means of MTT viability assay. The method was linear in the range of 10–100 µg mL−1 (r = 0.9999) and specific, even in the presence of degradation products and impurities. The method showed suitable accuracy (mean recovery 100.02%), precision (RSD < 1.10%), and a two-level full factorial design indicated the robustness of the method. The degraded samples exhibited cytotoxicity patterns similar to TPV under controlled conditions. The method developed is appropriate for quality control of TPV capsules and stability studies.

The role of mass spectrometry and related techniques in the analysis of extractable and leachable chemicals

In addition to degradation products, impurities, and exogenous contaminants, industries such as pharmaceutical, food, and others must concern themselves with leachables. These chemicals can derive from containers and closures or migrate from labels or secondary containers and packaging to make their way into products. Identification and quantification of extractables (potential leachables) and leachables, typically trace level analytes, is a regulatory expectation intended to ensure consumer safety and product fidelity. Mass spectrometry and related techniques have played a significant role in the analysis of extractables and leachables (E&L). This review provides an overview of how mass spectrometry is used for E&L studies, primarily in the context of the pharmaceutical industry. This review includes work flows, examples of how identification and quantification is done, and the importance of orthogonal data from several different detectors. E&L analyses are driven by the need for consumer safety. These studies are expected to expand in existing areas (e.g., food, textiles, toys, etc.) and into new, currently unregulated product areas. Thus, this topic is of interest to audiences beyond just the pharmaceutical and health care industries. Finally, the potential of universal detector approaches used in other areas is suggested as an opportunity to drive E&L research progress in this arguably understudied, under-published realm.

A novel UPLC-PDA isocratic method for the quantification fulvestrant in oil-based pre-filled syringe injection matrix formulations

BackgroundFulvestrant is a new estrogen receptor antagonist available in the market globally for the treatment of hormone receptor-positive metastatic breast cancer in postmenopausal women.MethodsThe chromatographic separation of fulvestrant was carried out by using ACQUITY UPLC and a BEH Shield RP18, 50 mm × 2.1 mm, i.d 1.7-μm column with a prepared mobile phase consisting of water, acetonitrile, and methanol in the ratio of 300:400:300 (v/v/v), respectively. 1.0 mL orthophosphoric acid was added to the prepared mobile phase. The wavelength for detection was made at 220.0 nm using a PDA-UV detector with a flow rate of 0.3 mL min− 1.ResultsThe system suitability parameters were found within the limits. The coefficient of correlation was found not less than 0.999. The percent recoveries of fulvestrant from 80, 100, to 120% levels are 100.1, 100.4, and 99.7 respectively. The LOD (0.51 μg mL− 1) and LOQ (1.54 μg mL− 1) values from the study demonstrate that the method is sensitive. The samples were subjected to forced degradation conditions of acidic and alkaline hydrolysis, oxidation, photolysis, metallic and thermal degradation in all conditions; peak was found pure (purity angle less than that of threshold).ConclusionA rapid, simple, stability-indicating, and validated RP-UPLC method was developed with 6 min of run time for the quantification of fulvestrant in oil-based injection formulations. This is the first stability-indicating method with the capability of resolving all the fulvestrant degradation impurities in the drug products. The method was validated for system suitability, linearity, precision, accuracy, specificity, intermediate precision, ruggedness, robustness, and solution stability.

Identification and characterization of unknown degradation impurities in beclomethasone dipropionate cream formulation using HPLC, ESI-MS and NMR

The present study focuses on identifying the degradation profile and pathways of unknown impurities from beclomethasone dipropionate (BDP) topical cream formulation reported under accelerated stability conditions. Six degradation impurities were observed during the accelerated stability testing of BDP topical cream formulation, and these thermally labile degradation impurities were primarily identified using a simple, effective and mass compatible isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection method. The degradation impurities found in this sample were of very low concentration levels, thus the concentration of these impurities in the sample was enriched by mimicking the thermal degradation conditions to structurally elucidate the unknown impurities. These BDP thermal degradation impurities were isolated using preparative liquid chromatography and followed by pre-concentration using rota-vapour. Further, the collected thermal degradation impurities were characterized using ESI-MS, and the major impurity was identified using 1H and C13 NMR spectroscopy, and DEPT technique. Plausible degradation pathway and mechanism of each impurity from BDP has been proposed based on the obtained mass and NMR spectral data. Thus, the present method is simple and suitable to be applied towards BDP assay in various formulations, and also to investigate the thermal stability and degradation kinetics of the final drug product.

Synthesis of High Purified Tulobuterol and Its Study of the Organic Impurities.

The synthetic condition of tulobuterol was optimized to gain lower impurity content. Two intermediates were analyzed, and three degradation impurities were isolated using preparative liquid chromatography for the first time and subsequently characterized by various techniques. Possible degradation impurities were deduced by an oxidative mechanism. Two intermediate impurities were detected: α-bromo-2-chloroacetophenone and 1-(2-chlorophenyl)-2-bromoethanol. Three unreported degradation impurities were found and characterized as N-tert-butyl glycine, o-chloro-benzoic acid and chlorobenzene. The single crystal structure of tulobuterol was firstly reported.

A controlled, efficient and robust process for the synthesis of an epidermal growth factor receptor inhibitor: Afatinib Dimaleate

A simple, controlled, robust and scalable three-stage manufacturing process of Afatinib Dimaleate was assessed and optimized leading to improved yield and quality. The synthetic process involves sequence of reactions as nitro-reduction, amidation and salification. The developed and optimized route was demonstrated on 300g scale with over all isolated yield of 84% for Afatinib free base. The developed process has the capability to control not only the process related impurities but also the degradation impurities. One new impurity was identified during the process development studies and characterized as acetamide impunity, chemically known as (S)-N-(4-((3-chloro-4-fluorophenyl) amino)-7-((tetrahydrofuran-3-yl) oxy) quinazolin-6-yl) acetamide. Other impurities were identified as degradation impurities, Process impurity impurities and were labeled as 1-(4-((3-chloro-4-fluorophenyl) amino)-7-(((S)-tetrahydrofuran-3-yl) oxy) quinazoline-6-yl)-5-Hydroxypyrrolidin-2-one (hydroxy impurity), Afatinib N-Oxide impurity and N4-(3-chloro-4-fluorophenyl)-7-[[(3S)-tetrahydro-3-furanyl] oxy]-4,6-quinazolinediamine (Intermediate-1).

Analysis of unstable degradation impurities of a benzodiazepine and their quantification without isolation using multiple linear regression

This manuscript presents a novel methodology for calculating the relative response factors (RRFs) of unstable degradation impurities of molibresib (1). The degradation impurities were observed by HPLC during stress testing and were accompanied by large mass balance deficits. However, the impurities could not be isolated for traditional RRF determination due to their instability. The RRFs of two degradation impurities were determined without isolation by multiple linear regression analysis of HPLC-UV data. The results permitted accurate quantification of the degradants. The benefits and drawbacks of the approach are discussed, including suggested validation acceptance criteria.

Enantiospecific UPC2 SFC-MS method for separation and quantification of R &amp; S-Eliglustat tartrate in presence of its stereo isomers and degradation impurities

Abstract Eliglustat tartrate is an oral inhibitor of glucosylceramide synthase used in the treatment of Gaucher’s disease. This drug is administered as enantio-specific trans-(R,R) isomer and there is a possibility to have three more potential stereo isomers due to two chiral centers, owing to the toxicological difference between stereoisomers chiral method with high sensitivity is required to detect low level contaminations. A method for enantiomeric separation and quantification of trans-(R,R) Eliglustat tartrate in presence of other isomers trans-(S,S) Eliglustat, cis-(R,S) Eliglustat, cis-(S,R) Eliglustat was developed and validated using ultra-high performance supercritical fluid chromatography with tandem mass spectrometry (UPC2 SFC-MS) which is termed as a green chromatographic technique. Chiral separation was developed using Chiralcel OX-3 (4.6 * 150 mm) 3 µm column which has 4 chloro, 3 methyl phenyl carbamate as chiral selector, with a flow rate of 3 g/min using CO2 and 0.5% diethyl amine in ethanol: methanol (1:1) as a co-solvent. The effect on the enantioseparation by varying chiral additives, gradient conditions and content of organic modifier were investigated. The method has good linearity with concentration ranges from 0.25 to 7.5 µg mL−1. The detection limit was 0.08 µg mL−1 and quantification limit was 0.25 µg mL−1. Forced degradation studies were performed and all the degradation impurities were separated from product peak, the molecular weight of impurities were identified by using UPC2-Mass spectrometer. The method was validated in line to ICH guidelines in terms of linearity, accuracy, precision, LOQ, LOD and robustness.

Characterization and in silico Mutagenic Assessment of a New Betahistine Degradation Impurity

Currently, the pharmaceutical industry devotes great attention to drug degradation products because these compounds can offer risks to patients. A previous degradation study of betahistine (N-α-methyl-2-pyridylethylamine) conducted under different stress conditions detected three main impurities named A, B and C. Degradation products were analyzed by high-resolution mass spectrometry in electrospray source and time of flight analyzer (ESI-TOF) and nuclear magnetic resonance (NMR). Impurity mutagenicity was evaluated by Derek Nexus and Sarah Nexus softwares. Liquid chromatography hyphenate with tandem mass spectrometry (LC-MS/MS) analysis of the betahistine forced degradation sample indicated the presence of a new impurity, which was named impurity C1. 2D NMR experiments allowed the complete structural characterization of the new entity. The active pharmaceutical ingredient and degradation impurities were classified as inactive in the in silico mutagenic studies. Systematic investigation of a forced degradation sample led to the characterization of a new betahistine impurity. The in silico mutagenicity study of the betahistine degradation impurities may be useful in the risk assessment of the drug products.

New Rapid Stability indicating RP-UPLC Method for the Determination of Olaparib, its Related Substances and Degradation Products in Bulk drug and Dosage Form

Abstract A rapid, specific, sensitive, and precise reverse-phase UPLC method was developed for the quantitative determination of a typical anticancer drug. Olaparib and its eight potential impurities in drug substances is described in this report. Statistical experimental designs (DOE) are used as advancement over conventional experimental approach .It provides more accurate results in fewer runs compared with conventional methods. Chromatographic separation was achieved on a Waters Acquity UPLC® BEH C18 (2.1 mm × 100 mm, 1.7 micron) column, using buffer (1.0ml Orthophosphoric acid in 1000ml water) and acetonitrile in gradient elution mode. The flow rate was kept at 0.4mL/min; thermostated at 35˚C with a short runtime of 15 min. The optimized method was found to produce symmetric and sharp peaks with good separation between processes related impurities and degradation impurities. Quantification is achieved with photodiode array detection at 220 nm over the concentration range of 10- 20 μg/mL. Forced degradation study was carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to demonstrate the stability-indicating capability of the developed UPLC method. Developed method is validated as per ICH guidelines and found to be linear, accurate, specific, selective, precise and robust. The method is useful during process development and quality of bulk manufacturing.

Isolation and Characterization Photo Degradation Impurities of Drug Product Olopatadine Hydrochloride by Spectral Techniques

Unknown impurities were detected during Photo degradation of Olopatadine Hydrochloride ophthalmic solution 0.5% (w/v) when analyzed using the High performance liquid chromatographic technique with Photo Diode Array Detection. For further investigation was carried out by isolating these impurities from impurity rich sample of Olopatadine Hydrochloride ophthalmic solution 0.5% (w/v) using preparative isolation technique. The Olopatadine Hydrochloride ophthalmic solution 0.5% (w/v) was subjected to photolytic forced degradation in the presence of Benzalkonium chloride and other excipients like Hypromellose, Mannitol, Hydroxypropyl, Boric acid, Kollidon 30 LP and mixture of solvents (Acetonitrile: Methanol; 1: 1 (v/v) under Ultra violet visible light. This led to the formation of the said impurities in higher concentration. This sample was then subjected to preparative HPLC for isolation of these unknown impurities. The structure of these unknown impurities was further elucidated using a different technique like Infra Ray Spectroscopy, Direct infusion (DI) Mass Spectroscopy, Ultra violet-Visible Spectroscopy, Proton Nuclear Magnetic Resonance Spectroscopy, carbon Nuclear Magnetic Resonance and Distortionless Enhancement by Polarization Transfer (DEPT) Spectroscopy which helped to confirm the structure of the impurities. Structure elucidation of the two impurities revealed that these are E and Z isomers of the Olopatadine hydrochloride Carbaldehyde. Olopatadine Z- isomer is used in the formulation of the Olopatadine Hydrochloride Ophthalmic Solution 0.5% (w/v). The minor amount of E- isomer also remains present in this solution as a potential impurity. However, the amount of E-isomer may increase in the solution form due to racemization. Hence, the respective Carbaldehyde impurities (both E and Z isomers) are forming during Photolytic degradation. This formation is happening through photolytic Norrish type-1 reaction which is elaborated in the paper.

Synthesis and characterization of potential stereoisomeric and degradation impurities of ulipristal acetate

Potential stereoisomeric and degradation impurities of UPA has been synthesized through practical approaches which would be valuable for the quality control of UPA.

Oligosaccharide Chromatographic Techniques for Quantitation of Structural Process-Related Impurities in Heparin Resulting From 2-O Desulfation.

Heparin is a widely-used intravenous anticoagulant comprising a complex mixture of highly-sulfated linear polysaccharides of repeating sequences of uronic acids (either iduronic or glucuronic) 1->4 linked to D-glucosamine with specific sulfation patterns. Preparation of crude heparin from mammalian mucosa involves protease digestion with alcalase under basic conditions (pH ≥ 9) and high temperature (>50°C) and also oxidation. Under such conditions, side reactions including the ubiquitous 2-O desulfation occur on the heparin backbone yielding non-endogenous disaccharides within polysaccharide chains. Whatever the process used for its manufacture, some level of corresponding degradation impurities is therefore expected to be found in heparin and the derived Low Molecular Weight Heparins. These impurities should be monitored to control the quality of the final therapeutic product. Two anion exchange chromatography techniques were used to analyze heparin samples exhaustively or partially depolymerized with heparinases and determine the proportions of non-endogenous disaccharides generated by side reactions during the manufacturing process (epoxides and galacturonic moieties). We also present data from a case study of marketed heparin. Current heparin sodium monographs do not directly address process impurities related to modification of the structure of heparin. Although desulfation reduces the overall biological potency, we found that heparin with an average of one modified disaccharide per chain can still comply with the USP or Ph. Eur. heparin sodium monographs requirements. We have implemented disaccharide analysis to monitor the quality of this product on a risk basis.

Identification and Characterisation of Rucaparib Degradation Products and Their Comparison with Known Impurities

A reversed-phase HPLC (high-performance liquid chromatographic) method is employed to identify and quantify the degradation impurities and related substances of Rucaparib (RCB), API bulk drug. There is no literature available that elucidates degradation phenomenon of RCB. Thus, the present study focuses on understanding the degradation phenomenon of RCB. The chromatographic separation is achieved within 25 min run time, where Zorbax Bonus RP column is used with a gradient elution of trifluoroacetic acid–ACN–water as the mobile phase. In this study, various forced degradation conditions such as photolysis, hydrolysis, oxidation, and thermal degradation were employed on RCB. Oxidative, basic, and acidic stress conditions showed a significant impact on degradation compared to thermal and photolysis. LC–MS technique was used for the characterization of degradation products and the plausible pathways of fragmentation were proposed. Qualified reference standards were used to quantify the stressed samples and the mass balance was found close to 99.5% (w/w) considering the response of the degradants to be equivalent to RCB. Results of this investigation confirmed the efficacy of the proposed novel method to determine drug stability of RCB, and ICH guidelines were also considered to authenticate our results.

Identification, Characterization and Quantification of Process-Related and Degradation Impurities in Lisdexamfetamine Dimesylate: Identifiction of Two New Compounds.

Twelve impurities (process-related and degradation) in lisdexamfetamine dimesylate (LDX), a central nervous system (CNS) stimulant drug, were first separated and quantified by high-performance liquid chromatography (HPLC) and then identified by liquid chromatography mass spectrometry (LC-MS). The structures of the twelve impurities were further confirmed and characterized by IR, HRMS and NMR analyses. Based on the characterization data, two previously unknown impurities formed during the process development and forced degradation were proposed to be (2S)-2,6-di-(lysyl)-amino-N-[(1S)-1-methyl-2-phenyl ethyl]hexanamide (Imp-H) and (2S)-2,6-diamino-N-[(1S)-1-methyl-2-(2-hydroxyphenyl)ethyl] hexanamide (Imp-M). Furthermore, these two compounds are new. Probable mechanisms for the formation of the twelve impurities were discussed based on the synthesis route of LDX. Superior separation was achieved on a YMC-Pack ODS-AQ S5 120A silica column (250 × 4.6 mm × 5 μm) using a gradient of a mixture of acetonitrile and 0.1% aqueous methanesulfonic acid solution. The HPLC method was optimized in order to separate, selectively detect, and quantify all the impurities. The full identification and characterization of these impurities should prove useful for quality control in the manufacture of lisdexamfetamine dimesylate.

LIQUID CHROMATOGRAPHY METHOD DEVELOPMENT AND VALIDATION OF RELATED IMPURITIES OF LURASIDONE AND ITS FORMULATION

An RP-HPLC method was developed for the quantification of related impurities of lurasidone and its formulation. The chromatographic separation employs gradient elution using an Inertsil ODS C18 (150x4.6) mm, 5μm columns. Mobile phase consisting of solvent A-buffer (pH 3.0): methanol (90:10 %v/v) and solvent B-acetonitrile: water (80:20 % v/v) delivered at a flow rate of 1.0 mL/min. The analytes were detected and quantified at 210 nm using PDA. The method was validated as per ICH guidelines, demonstrating to be a simple, precise, selective, linear and accurate within the corresponding range of impurities of lurasidone. Linearity was observed in the concentration range of 2-6 µg/mL. The RT for Lurasidone was about 18.5 min and three known impurities at RRT about 0.15, 0.21 and 0.36. The specificity of the method was investigated under different stress conditions including hydrolytic, oxidative, photolytic and thermal as recommended by ICH guidelines. Relevant degradation was found to take place under oxidative conditions. Degradation impurities did not interfere with the RT of drug. The peak purity obtained with the aid of PDA detection and satisfactory resolution between related impurities established the specificity of the determination. All these results provide the stability indicating capability of the method.