Impurities In Drug Substances Research Articles

Separation and identification of moxifloxacin impurities in drug substance by high-performance liquid chromatography coupled with ultraviolet detection and Fourier transform ion cyclotron resonance mass spectrometry

Abstract In this paper, a high-performance liquid chromatography coupled with ultraviolet detection and Fourier transform-ion cyclotron resonance mass spectrometry (HPLC–UV/FTICRMS) method was described for the investigation of impurity profile in moxifloxacin (MOX) drug substance and chemical reference substance. Ten impurities were detected by HPLC–UV, while eight impurities were identified by using the high accurate molecular mass combined with multiple-stage mass spectrometric data and fragmentation rules. In addition, to our knowledge, five impurities were founded for the first time in MOX drug substance.

Development of a Stability-Indicating RP-LC Method for the Separation of a Critical Pair of Impurities and their Degradants in Zafirlukast

A high-throughput reverse-phase liquid chromatographic (RP-LC) method is developed for the quantification of zafirlukast and its related impurities in drug substance. The separation of known impurities is accomplished using a short (50 mm) LC column with sub-2-µm particle size in a relatively short run-time. A linear gradient elution involves ammonium formate and acetonitrile as mobile phase. The critical impurity pair is the meta and para isomers of zafirlukast, which are known to be potential impurities of zafirlukast, whose resolution is sensitive to pH. The stability-indicating capability of the developed method is demonstrated using forced degradation samples from stress conditions such as hydrolysis, oxidation, thermal and photolytic degradation. The developed RP-LC method is validated in accordance with International Conference on Harmonization requirements. The results from the validation study indicate that this RP-LC method can be used for the determination of synthetic and degradation impurities in regular quality control analysis for the drug substance.

Development and Validation of Stability-Indicating GC-FID Method for the Quantitation of Memantine Hydrochloride and Its Nonchromophoric Impurities in Bulk and Pharmaceutical Dosages

A stability-indicating method has been developed and validated for the quantitative determination of memantine hydrochloride and its nonchromophoric impurities in drug substance and drug product using gas chromatography coupled with flame ionization detector (GC-FID). The stability-indicating nature of the method has been proved by establishing peak purity and confirming the mass balance of all samples by subjecting them to stress conditions like hydrolysis, oxidation, photolysis, and thermal degradation studies. The chromatographic separation was performed on a fused silica capillary (HP-5, 30 meter, 0.32 mm and 0.25 μm film thickness) column. The method validation results indicate that the method has acceptable specificity, accuracy, linearity, precision, robustness, and high sensitivity with detection limits and quantitation limits ranging from 0.001% to 0.01% and 0.004% to 0.03%, respectively. The effectiveness of the technique was demonstrated by analysis of different bulk sample of Memantine hydrochloride. The proposed GC-FID method was also found to be specific and selective for the analysis of commercial formulation samples.

DEVELOPMENT AND VALIDATION OF AN UPLC METHOD FOR THE RAPID SEPARATION OF POSITIONAL ISOMERS AND POTENTIAL IMPURITIES OF RIVASTIGMINE HYDROGEN TARTRATE IN DRUG SUBSTANCE AND DRUG PRODUCT

Although a number of methods are available for evaluating Rivastigmine hydrogen tartrate (RIV) and its possible impurities, a common method for separation if its potential impurities and positional isomers with good efficiency remains unavailable. With the objective of developing an advanced method for rapid separations with shorter runtimes, a simple, precise, accurate stability-indicating gradient reverse phase ultra-performance liquid chromatographic (RP-UPLC) coupled with a photodiode array detector method was developed for the quantitative determination of RIV and its impurities in drug substance and drug product. The determination was done for an active pharmaceutical ingredient, its pharmaceutical dosage form in the presence of degradation products, and its process-related impurities. Chromatographic separation was achieved on Acquity UPLC BEH Phenyl (100 mm × 2.1 mm, 1.7 µm) column with a mobile phase containing a gradient mixture of solvents A and B. The compounds eluted within a short runtime of 10 min and were monitored at 210 nm with the flow rate and the column oven temperature of 0.4 mL/min and 40°C, respectively. The resolution of RIV and its eleven impurities (positional and potential) was greater than 2.0 for all pairs of components. The newly developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision, and robustness. Forced degradation studies were also performed to demonstrate the stability indicating power of the developed UPLC method.

Validated Densitometric TLC-Method for the Simultaneous Analysis of (R)- and (S)-Citalopram and its Related Substances using Macrocyclic Antibiotic as a Chiral Selector: Application to the Determination of Enantiomeric Purity of Escitalopram

A novel economic procedure for the simultaneous stereospecific separation and analysis of (R)- and (S)-citalopram and its related substances or impurities has been developed and validated. Chromatography was performed on silica gel 60 F254 plates with acetonitrile: methanol: water (15:2.5:2.5: v/v/v) as a mobile phase containing 1.5 mM norvancomycin or 2.5 mM vancomycin as a selector at ambient temperature. (R)- and (S)-citalopram enantiomers in presence of its related substances; citalopram citadiol and citalopram N-oxide were well separated with significant Rf values of 0.33 ± 0.02, 0.85 ± 0.02, 0.45 ± 0.02 and 0.22 ± 0.02, respectively. The spots were detected with either iodine vapor, or by use of a UV lamp followed by densitometric measurement at 239 nm. All variables affecting the resolution, such as concentration of chiral selectors, mobile phase system at different temperatures and pH-values were investigated and the conditions were optimized. Calibration plots for analysis of (R)- and (S)-enantiomers were linear in the range of 0.2-16.8 µg/10 µl (R≥0.9994, n-6) with acceptable precision (%RSD<2.0) and accuracy (99.70 ± 0.85% and 99.51 ± 0.61% for (S)-citalopram and escitalopram, respectively). The limit of detection and quantification were 0.08 µg/10 µl and 0.25 µg/10 µl, respectively, for (R)- and (S)-citalopram. The proposed method is simple, selective, and robust and can be applied for quantitative determination of enantiomeric purity of (R)- and (S)-citalopram (escitalopram) as well as the related impurities in drug substances and pharmaceutical preparations. The method can be useful to investigate adulteration of pure isomer with the cheep racemic form.

Characterization and validation of an in silico toxicology model to predict the mutagenic potential of drug impurities*

Control and minimization of human exposure to potential genotoxic impurities found in drug substances and products is an important part of preclinical safety assessments of new drug products. The FDA's 2008 draft guidance on genotoxic and carcinogenic impurities in drug substances and products allows use of computational quantitative structure–activity relationships (QSAR) to identify structural alerts for known and expected impurities present at levels below qualified thresholds. This study provides the information necessary to establish the practical use of a new in silico toxicology model for predicting Salmonella t. mutagenicity (Ames assay outcome) of drug impurities and other chemicals. We describe the model's chemical content and toxicity fingerprint in terms of compound space, molecular and structural toxicophores, and have rigorously tested its predictive power using both cross-validation and external validation experiments, as well as case studies. Consistent with desired regulatory use, the model performs with high sensitivity (81%) and high negative predictivity (81%) based on external validation with 2368 compounds foreign to the model and having known mutagenicity. A database of drug impurities was created from proprietary FDA submissions and the public literature which found significant overlap between the structural features of drug impurities and training set chemicals in the QSAR model. Overall, the model's predictive performance was found to be acceptable for screening drug impurities for Salmonella mutagenicity.

Development and validation of a stability-indicating LC method for simultaneous determination of related compounds of guaifenesin, terbutaline sulfate and ambroxol HCl in cough syrup formulation

A new liquid chromatographic method has been developed and validated for the determination of terbutaline sulfate (TLS), guaifenesin (GFN) and ambroxol HCl (AML), for its potential impurities in drug substances and drug products. Efficient chromatographic separation was achieved on X-Terra RP-18 column with a simple mobile phase combination containing a gradient mixture of solvents A and B at a flow rate of 1.0mLmin−1 and quantitation was carried out using ultraviolet detection at 222nm with column temperature of 35°C. The resolution between TLS, GFN and AML, its associated impurities was found to be greater than 1.5. Regression analysis shows an r value (correlation coefficient) greater than 0.998. This method was capable to detect all the process impurities of TLS, GFN and AML, at a level below 0.015% with respect to a test concentration of 0.125, 5.0 and 1.5mgmL−1, respectively. The % RSD for the inter-day and intra-day precisions for all the impurities of TLS, GFN and AML were found to be less than 3.0. The method has shown good, consistent recoveries. The drugs were subjected to stress conditions of acid, base, water hydrolysis, oxidation, photolysis and thermal degradation, as prescribed by international conference on harmonization (ICH).

Trace-level analysis of mesityl oxide in enalapril maleate by gas chromatography with electron ionization mass spectrometry

The determination of genotoxic impurities in drug substances requires analytical methods with high specificity and sensitivity. Mesityl oxide is a potent genotoxic impurity in drugs because its alpha-unsaturated ketone structure is considered to be a structural alert. A gas chromatograph electron ionization mass spectrometric (GC-EI-MS) method has been established for the determination of mesityl oxide in enalapril maleate in this article. Methyl isobutyl ketone was used as an internal standard (IS). The method was operated in electron ionization/selective ion monitoring (EI/SIM) mode. The mass fragments (m/z) selected for the qualification and quantification were 55, 83 and 98 for mesityl oxide, and 58, 85 and 100 for IS. Quantification was performed by internal standard curve method and the method has been fully validated. This GC-EI-MS method is simple (one-step sample preparation), rapid (chromatographic cycle within 10 min, plus a 10 min postrun), specific (no interference peak shown in blank solution), accurate (mean recoveries within 88.0–97.3%), precise (intra- and inter-day precision ≤2.9%), sensitive (LOD ≤ 0.5 μg g−1 and LOQ ≤ 1.8 μg g−1), and stable (reference standard solution stable over 24 h). This method was successfully applied to the screening of mesityl oxide in three batches of enalapril maleate bulk drug.

In silico and in vitro genotoxicity evaluation of levofloxacin n-oxide, an impurity in levofloxacin

Impurities in drug substances and drug products generally do not have beneficial effects and may impose a risk without associated benefit. Levofloxacin n-oxide is an impurity isolated from levofloxacin. However there is insufficient toxic information about levofloxacin n-oxide. This study investigates the genotoxicity of this impurity by in silico and in vitro methods. We used Derek, a commercial structure-activity relationship software package as an in silico tool. The results showed that there was a structural alert (quinolone-3-carboxylic acid or naphthyridine analogue) in this impurity. Then the mouse lymphoma assay (MLA) and chromosome aberration assay in Chinese hamster lung (CHL) cells were conducted in vitro. Both assays were conducted in the presence or absence of S-9 mix. The test impurity was not mutagenic in the test of MLA. While there was a statistically significant increase in the number of metaphase CHL cells with structural aberrations at the concentration of 1 mg/mL with S-9 mix, and the aberrations rate is 6.5%. It did not significantly increase the number of structural aberration in CHL cells in the presence (at other two doses) or absence of S-9 mix. Based on these assays, levofloxacin n-oxide could be controlled as a non-genotoxic impurity despite the DEREK alert for quinolone-3-carboxylic acid or naphthyridine analogue.

Process Research and Development of a MTP Inhibitor: Another Case of Disappearing Polymorphs upon Scale-up

LAB687 is an inhibitor of microsomal triglyceride transfer protein (MTP) designed to lower triglycerides and LDL cholesterol. The discovery of its polymorphic forms closely intertwines with the synthesis development of the molecule. At the early development stage, LAB687 was known to crystallize in two modifications, Forms A and B. Knowledge of the molecule’s polymorphic nature prompted extensive polymorphic screening using drug substance produced by the earlier synthesis routes. These studies revealed the existence of a third polymorph, Form C. Subsequently, Form C was selected for further development based on data from the additional formulation and polymorphic studies. Surprisingly, a new modification, Form D, appeared when the crystallization process known to routinely produce Form C was scaled up in the pilot plant. Once Form D was introduced to the laboratory, Forms A and C could no longer be made. We hypothesize that a change in drug substance impurity profile due to the changes in synthesis, led to the emergence of the most stable Form D.

In silico approaches to predicting cancer potency for risk assessment of genotoxic impurities in drug substances

The current risk assessment approach for addressing the safety of very small concentrations of genotoxic impurities (GTIs) in drug substances is the threshold of toxicological concern (TTC). The TTC is based on several conservative assumptions because of the uncertainty associated with deriving an excess cancer risk when no carcinogenicity data are available for the impurity. It is a default approach derived from a distribution of carcinogens and does not take into account the properties of a specific chemical. The purpose of the study was to use in silico tools to predict the cancer potency (TD50) of a compound based on its structure. Structure activity relationship (SAR) models (classification/regression) were developed from the carcinogenicity potency database using MultiCASE and VISDOM. The MultiCASE classification models allowed the prediction of carcinogenic potency class, while the VISDOM regression models predicted a numerical TD50. A step-wise approach is proposed to calculate predicted numerical TD50 values for compounds categorized as not potent. This approach for non-potent compounds can be used to establish safe levels greater than the TTC for GTIs in a drug substance.

Stability Studies of Microparticulate System with Piroxicam as Model Drug

Non-steroidal anti-inflammatory agent, piroxicam (PIR), is particularly employed in treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, pain in musculoskeletal disorders, and acute gout (1). This drug has also been in clinical practice as an analgesic in dental, postoperative and postpartum pain (2). According to the biopharmaceutical classification system, PIR is included in class 2, having characteristics of low solubility and high permeability characteristics. The pharmacokinetic data reveal that this molecule undergoes slow and gradual absorption pattern when given orally. This leads to a delayed onset of anti-inflammatory and analgesic effect. Poly(lactide-co-glycolide) (PLGA) has been the polymer of choice for injectable microparticulate systems due to its biocompatible nature (3). Release behavior from PLGA polymers are either by diffusion, erosion, or a combination of both (4). The dissolution kinetics can be affected by a number of significant factors like polymer molecular weight, copolymer ratio and crystallinity (5–8), properties related to the molecule (9–11), dissolution conditions employed in the study (12,13), and formulation factors like preparative conditions, particle size, and encapsulation efficiency (14–18). Depending upon the type of PLGA, the drug release profiles from PLGA microspheres can range from days to months. Hence, accelerated release testing of such systems especially for in vitro drug release kinetics becomes imperative. However, it should be noted that in ideal situation, the drug release from accelerated test and “real-time” study should follow the same release mechanism and a 1:1 correlation is expected. Exaggerated conditions like temperature, pH of media, etc. are generally employed in the test to achieve rapid release. It is possible that the release mechanism may alter due to these exaggerated conditions. Literature study shows that an increase in drug release from PLGA has been achieved either by increase in temperature (due to increased mobility of polymer and hence drug diffusion), by acid or alkali catalyzed hydrolysis, by addition of a surfactant that would enhance the dissolution, by addition of a cosolvent, or by radiation (19–24). Degradation of polylactic acid and PLGA polymers of different copolymer ratios and molecular weights at 37°C and 60°C with change in mass loss, molecular weight, and formation of respective acid components has been reported (25). Stability of a pharmaceutical dosage form is very significant as it reflects the quality of the dosage form during its shelf life. The stability studies give proof on how the quality of a drug substance or product varies with time under the influence of environmental factors like temperature, humidity, and light. Regulatory bodies lay emphasis on performing stability studies and is a part of the product submission requirements. International conference on harmonization (ICH) has laid guidelines for stability studies. The ICH Q1A(R2) is for stability testing of new drug substances and products. ICH Q3A, Q3B, and Q3C are based on the impurities in drug substance, drug product, and residual solvents, respectively. The specifications, test procedures, and acceptance criteria for new drug substances and products are dealt in ICH Q6A. The stability studies should be an integral part of the development cycle for new dosage forms. Often it is observed that the stability aspect is often neglected during the development of a new pharmaceutical product. To get more insight on how the product fairs during shelf life, we performed stability studies on the final system. Extensive literature search indicates that not much attention has been given to evaluate the quality of microsphere based products on storage for prolonged time period along with real-time release profiling for long-term release products. In the above context, a strong need was felt to conduct real-time stability studies for the developed PIR–PLGA microparticulate formulation. A sensitive stability indicating high-performance thin-layer chromatographic (HPTLC) method developed and validated in our lab was employed for estimation of drug. An attempt was made in the present investigation to focus on the stability aspects of PIR–PLGA microsphere system.

Development and Validation of a New Analytical Method for the Determination of Related Components and Assay of Ranolazine in Bulk Drug and Pharmaceutical Dosage Forms by LC

A novel liquid chromatographic method has been developed and validated for the determination of ranolazine, its potential four impurities in drug substance and drug products. Efficient chromatographic separation was achieved on a C18 stationary phase (150 × 4.6 mm, 3.0 microns particles) with simple mobile phase combination delivered in gradient mode at a flow rate of 1.0 mL min−1 at 210 nm. In the developed method, the resolution between ranolazine and its four potential impurities was found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) greater than 0.999 for ranolazine and for its four impurities. This method was capable to detect all four impurities of ranolazine at a level below 0.004% with respect to test concentration of 1.0 mg mL−1 for a 10 μL injection volume. The method has shown good, consistent recoveries for ranolazine (98.8–101.1%) and for its four impurities (97.2–100.3). The test solution was found to be stable in the diluent for 48 h. The drug was subjected to stress conditions. The mass balance was found close to 99.5%.

Response to the Comments by Snodin on Our Article “Approaches to Assessment, Testing Decisions, and Analytical Determination of Genotoxic Impurities in Drug Substances” [Org. Process Res. Dev. Web publication November 13, 2008; DOI: 10.1021/op8002129; 2009, 13, 285−291.

Response to the Comments by Snodin on Our Article “Approaches to Assessment, Testing Decisions, and Analytical Determination of Genotoxic Impurities in Drug Substances” [<i>Org. Process Res. Dev</i>. Web publication November 13, 2008; DOI: 10.1021/op8002129; <b>2009</b>, <i>13</i>, 285−291.

Genotoxic Impurities in Drug Substances

Genotoxic Impurities in Drug Substances

Analytical control of genotoxic impurities in the pazopanib hydrochloride manufacturing process

Pharmaceutical regulatory agencies are increasingly concerned with trace-level genotoxic impurities in drug substances, requiring manufacturers to deliver innovative approaches for their analysis and control. The need to control most genotoxic impurities in the low ppm level relative to the active pharmaceutical ingredient (API), combined with the often reactive and labile nature of genotoxic impurities, poses significant analytical challenges. Therefore, sophisticated analytical methodologies are often developed to test and control genotoxic impurities in drug substances. From a quality-by-design perspective, product quality (genotoxic impurity levels in this case) should be built into the manufacturing process. This necessitates a practical analysis and control strategy derived on the premise of in-depth process understanding. General guidance on how to develop strategies for the analysis and control of genotoxic impurities is currently lacking in the pharmaceutical industry. In this work, we demonstrate practical examples for the analytical control of five genotoxic impurities in the manufacturing process of pazopanib hydrochloride, an anticancer drug currently in Phase III clinical development, which may serve as a model for the other products in development. Through detailed process understanding, we implemented an analysis and control strategy that enables the control of the five genotoxic impurities upstream in the manufacturing process at the starting materials or intermediates rather than at the final API. This allows the control limits to be set at percent levels rather than ppm levels, thereby simplifying the analytical testing and the analytical toolkits to be used in quality control laboratories.

Approaches to Assessment, Testing Decisions, and Analytical Determination of Genotoxic Impurities in Drug Substances

The assessment and control of genotoxic impurities (GTI) in pharmaceutical products has received considerable attention in recent years. Molecular functional groups that render starting materials and synthetic intermediates useful as reactive building blocks for small molecules may also be responsible for their genotoxicity. As a potential safety concern, it is important to understand the various issues related to GTIs and how they can be addressed for clinical and commercial phases of development. Justification that these impurities are controlled to safe levels must be obtained during development. This article will briefly discuss the multiple sources of anticipated impurities in a drug substance (also known as active pharmaceutical ingredient or API) synthetic route and how they are identified as GTIs in early chemical process development. A risk-based approach consistent with regulatory expectations is described for establishing control of GTIs. The approach includes process design considerations, imp...

Development and Validation of a New Analytical Method for the Determination of Related Components in Tolterodine Tartarate Using LC

A novel liquid chromatographic method has been developed, and validated for the determination of tolterodine tartarate, for its potential three impurities in drug substances and drug products. Efficient chromatographic separation was achieved on a C8 stationary phase (150 × 4.6 mm, 3.5 μm particles) with a simple mobile phase combination delivered in an isocratic mode at a flow rate of 0.8 mL min−1 and quantitation was carried out using ultraviolet detection. Microwave assisted degradation procedure was employed for stress testing studies in addition to the conventional way of a refluxing method. The results of both studies were compared. In the developed LC method, the resolution between tolterodine and its three potential impurities was found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) greater than 0.999 for tolterodine and for its three impurities. This method was capable to detect all three impurities of tolterodine at a level below 0.0038% with respect to a test concentration of 0.5 mg mL−1 for a 10 μL injection volume. The inter- and intra-day precisions for all three impurities and for tolterodine were found to be within 1.1% RSD at its specification level. The method has shown good, consistent recoveries for tolterodine (98.9–101.6%) and for its three impurities (94.5–103.0%). The test solution was found to be stable in the diluent for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation, as prescribed by ICH. Degradation was found to occur in alkaline stress condition, while the drug was stable to water hydrolysis, acid hydrolysis, oxidative stress, photolytic and thermal stress. The assay of stressed samples was calculated against a qualified reference standard and the mass balance was found close to 99.5%. Microwave degradations were very fast and comparable to the conventional way of the refluxing method. Robustness studies were carried out and suggested that system suitability parameters were unaffected by small changes in critical factors. The validated method was successfully applied for the determination of tolterodine tartarate in drug substances and drug products.

Harmonization of strategies for the validation of quantitative analytical procedures: A SFSTP proposal: Part IV. Examples of application

A harmonized approach for the validation of analytical methods based on accuracy profile was introduced by a SFSTP commission on the validation of analytical procedure. This fourth and last document aims at illustrating this methodology and the statistics used. Therefore the validation of real case methods are proposed such as methods for the quality control of drugs, for the quantitation of impurities in drug substances, for bioanalysis or for the determination of nutriments. Furthermore, different types of analytical methods are used in order to demonstrate the applicability of the proposed approach to a wide range of methods such as liquid chromatography (LC-UV, LC–MS), spectrophotometry or ELISA.

A practical derivatization LC/MS approach for determination of trace level alkyl sulfonates and dialkyl sulfates genotoxic impurities in drug substances

Derivatization LC/MS methodology has been developed for the determination of a group of commonly encountered alkyl esters of sulfonates or sulfates in drug substances at low ppm levels. This general method uses trimethylamine as the derivatizing reagent for ethyl/propyl/isopropyl esters and triethylamine for methyl esters. The resulting quaternary ammonium derivatization products are highly polar (ionic) and can be retained by a hydrophilic interaction liquid chromatography (HILIC) column and readily separated from the main interfering active pharmaceutical ingredient (API) peak that is usually present at very high concentration. The method gives excellent sensitivity for all the alkyl esters at typical target analyte level of 1–2 ppm when the API samples were prepared at 5 mg/mL. The recoveries at 1–2 ppm were generally above 85% for all the alkyl esters in the various APIs tested. The injection precisions of the lowest concentration standards were excellent with R.S.D. = 0.4–4%. A linear range for concentrations from 0.2 to 20 ppm has been established with R 2 ≥ 0.99. This general method has been tested in a number of API matrices and used successfully for determination of alkyl sulfonates or dialkyl sulfates in support of API batch releases at GlaxoSmithKline.