Stability-indicating Method Research Articles

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHIC METHOD FOR BUDESONIDE

Objective: A stability-indicating high-performance thin-layer chromatographic method has been developed for budesonide. Methods: Chromatographic separation was achieved on aluminum plates pre-coated with silica gel 60 F254 as the stationary phase using ethyl acetate: toluene (7:3) v/v as the mobile phase. The densitometric evaluation was carried out at 246 nm. The developed method of stability-indicating was validated as per the ICH Q2 (R1) guidelines. Stress degradation studies such as hydrolysis under different pH conditions, photolytic degradation, thermal degradation, and oxidative degradation were performed as per ICH Q1A (R2) and Q1B guidelines. Results: The Rf value of budesonide was found to be 0.48±0.03. The response in terms of peak area was linear over the concentration range of 500–2500 ng/band, with the regression coefficient value greater than 0.99. The LOD and LOQ were 28.04 ng/band and 84.96 ng/band, respectively. Conclusion: This method can conveniently be used for quantitative analysis of budesonide on a routine basis.

Utility and greenness appraisal of nuclear magnetic resonance for sustainable simultaneous determination of three 1,4-benzodiazepines and their main impurity 2-amino-5-chlorobenzophenone

A robust, stability-indicating, and eco-friendly proton nuclear magnetic resonance (1H-qNMR) method was developed for the concurrent determination of three 1,4-benzodiazepines (BDZs), namely diazepam (DZP), alprazolam (ALP), and chlordiazepoxide (CDP) and their common impurity, synthesis precursor, and degradation product; 2-amino-5-chlorobenzophenone (ACB). In the present method, a novel approach was developed for composing a green and cost-efficient solvent system as an alternative to the common NMR organic solvents utilizing 0.3 M sodium dodecyl sulfate prepared in deuterated water. The conducted method is characterized by simplicity with no need for sample pretreatment or labeling. Phloroglucinol was used as an internal standard. The chosen signals for the determinations of ALP, CDP, DZP and ACB were at 2.35 ppm (singlet), 2.84 ppm (singlet), 3.11 ppm (singlet), and 6.90 ppm (doublet of doublet), respectively. The proposed method possessed linearity over the concentration range of 0.25–15.0 mg ml−1 for DZP, ALP, CDP and of 0.5–25.0 mg ml−1 for ACB with LOD values of 0.06, 0.03, 0.07 and 0.16 mg ml−1 respectively, and LOQ values of 0.18, 0.09, 0.21 and 0.49 mg ml−1, respectively. Accuracy of the method was evidenced by excellent recovery% (99.57–99.90%) and small standard deviation (≥ 1.10) for the three analyzed drugs. Intra- and inter-day precision were determined with coefficient of variation ranging from 0.12 to 1.14 and from 0.72 to 1.67, respectively. For the studied compounds, appraisal of the method greenness was achieved via four approaches: Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), Analytical greenness metric (AGREE), and RGB Additive Color Model. The results proved that the proposed method has the privilege of being a green analytical method.

STABILITY INDICATING HPTLC METHOD DEVELOPMENT AND VALIDATION FOR CERITINIB IN BULK AND FORMULATION

Ceritinib is an anti-cancer of drug used in treatment of non-small cell lung cancer. A high-performance thin layer chromatography (HPTLC) method has been developed for ceritinib, which is stability indicating and simple, precise, and discriminating. The stationary phase used was aluminum-backed silica gel60 F254 plates with chloroform: methanol: glacial acetic acid as the mobile phase in the ratio of 8.5:1.5:0.5 (V/V/V). The retention factor was found to be 0.34 ± 0.02. The densitometric scanning was performed at 277 nm. The linear range for analysis was 100–600 ng band-1, which gave good linear relationship with regression coefficient of 0.998. Method accuracy was proved by the recovery studies. The detection limit and quantification limit were 7.38 and 10.03 ng band-1, respectively. Stress degradation studies like hydrolysis under different pH conditions, photolytic, thermal, and oxidative degradation were carried out as per ICH Q1A (R2) and Q1B guidelines. The method established was found to be robust, and thus it can be used as a stability-indicating method.

Stability Indicating HPTLC Method Development and Validation for Acebrophylline in Bulk and its Tablet Dosage Form

A simple, precise, accurate stability-indicating method was developed and validated for Acebrophylline in bulk and tablet dosage form. The chromatographic separation was achieved by using Methanol: Acetonitrile: Acetone: Formic acid (4:4:2:0.1 v/v/v/v) as mobile phase and UV detection at 272nm. The retention factor for Acebrophylline was found to be 0.74±0.10. The developed method was validated with respect to linearity, accuracy, precision, limit of detection, limit of quantitation, and robustness as per ICH guidelines. Results were found to be linear in the concentration range of 400-2400ng band -1 with a correlation coefficient of 0.9921. The % assay was found to be 102.4±5. The drug was subjected to stress conditions of hydrolysis (acid, base), oxidation, photolysis, and thermal degradation. The developed method can be used for the quantification of drug in the dosage form, bulk drug as well as for routine analysis in quality control laboratories.

Development of electrochemical and stability-indicating chromatographic methods for the determination of cefprozil

Development of electrochemical and stability-indicating chromatographic methods for the determination of cefprozil

Development of stability-indicating HPLC method for quantification of pharmacopeia impurities of Zuclopenthixol and characterization of its stress degradation products by LCMS/MS

BackgroundThe present study focused to develop a simple and sensitive HPLC method for resolution and estimation process-related impurities of zuclopenthixol and further assessment of forced degradation behavior of zuclopenthixol.ResultsThe chromatographic separation of drug substance, process-related impurities and its degradation products (DPs) was achieved on KNAUER C18 (250 mm × 4.6 mm, 5µ id) column at that was maintained at 35 °C temperature using 0.1 M sodium acetate buffer at pH 4.3 and methanol in 20:80 (v/v) as mobile phase A, 0.1% formic acid and acetonitrile in 75:25 (v/v) as mobile phase B. Equal volume of mobile phase A and B was pumped in isocratic elution at 0.8 mL/min. Detection wavelength was selected as 257 nm. In the proposed conditions, zuclopenthixol is identified at 6.91 and 1.91 min and 2.89 min, respectively, for impurity B and A min with acceptable system suitability and specificity. The method produces LOD at 0.009 for impurities with calibration range of 30–180 µg/mL for zuclopenthixol and 0.03–0.18 µg/mL for impurities. The other validation parameters were notices to be with in the acceptable levels for zuclopenthixol and its impurities. The drug was exposed to different stressed conditions (acid, base, peroxide, thermal and UV light) according to ICH Q1A (R2) guidelines. The DPs formed during the stress study were identified and characterized by LCMS/MS in ESI positive mode.ConclusionThe analysis involved a comparison of collision-induced dissociation mass spectrometry data between the degradation products and zuclopenthixol. As a result, potential structures for six degradation compounds were suggested. The results from additional validation studies were similarly pleasing and demonstrated their suitability for the routine analysis of zuclopenthixol and its associated impurities in both bulk drug and pharmaceutical dosage forms. Additionally, these findings can be extended to assess the mechanism of stress degradation in zuclopenthixol.

Optimization of Stability-Indicating HPLC Method for Analyzing Process Related Impurities of Penfluridol and Structural Elucidation of Stress Degradation Products by LCMS/MSucidation of Stress Degradation Products by LCMS/MS

This study focused on the development of a simple and sensitive HPLC method for resolution and quantification of process-related impurities of penfluridol and further assessment of forced degradation behavior of penfluridol. The chromatographic separation was achieved on XTerra™ C18 (250×4.6 mm, 5.0μm) column and UV detection at 245nm. The mobile phase comprises of methanol and tetrahydrofuran in 55:45 (v/v) as solvent A and acetonitrile and tetrahydrofuran in 80:20 (v/v) as solvent B. The 60:40 (v/v) composition of solvent A and B were pumped isocratically at 1.0mL/min. In the proposed conditions, the retention time identified as 5.29 min for penfluridol, 4.51 min, 9.95 min and 7.64 min respectively for impurity 1, 2 and 3 with acceptable system suitability. The method produces sensitive detection limit of 0.008μg/mL for impurity 1, 2 and 0.004 μg/mL for impurity 3 with calibration range of 25-150 μg/mL for penfluridol and 0.025-0.150 μg/mL for impurities. The drug was exposed to different stressed conditions (acid, base, peroxide, thermal and UV light) according to ICH Q1A (R2) guidelines. The Degradation Products (DPs) formed during the stress study was characterized by LCMS/MS in ESI positive mode and the possible structures of five DPs with possible degradation pathways were proposed. The outcomes of other validation studies were likewise satisfactory and proven adequate for regular analysis of penfluridol and its process-related impurities in bulk drug and pharmaceutical dosage forms and can also applicable for evaluation of stress degradation mechanism of penfluridol.

Delineation of prototypical degradation mechanism, characterization of unknown degradation impurities by liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry and stability-indicating analytical method of selumetinib.

Selumetinib (SELU) was recently approved by the US Food and Drug Administration (US FDA) in 2020. However, the degradation impurities of SELU have not been characterized or identified to date. The mechanism for impurity formation and the degradation behavior have not been previously studied. This study aims to elucidate the prototypical degradation mechanism of SELU. Furthermore, the degradation impurities have been identified using LC-quadrupole-time-of-flight tandem mass spectrometry and are reported in this article for the first time. In addition, a stability-indicating analytical method (SIAM) has been developed for this drug. Forced degradation studies revealed the degradation of SELU under various stress conditions, including hydrolytic stress (acid and base), oxidative stress, and photolytic stress (ultraviolet and visible). Three degradation impurities were identified. This article presents the first validated SIAM, capable of accurately quantifying SELU in the presence of its degradation impurities. Furthermore, we have proposed the degradation pathway for SELU and its degradation impurities, a first in the field. The developed SIAM can find applications in process development and quality assurance of SELU in both research laboratories and pharmaceutical industries. Moreover, the identified degradation impurities may serve as impurity standards for quality control testing in pharmaceutical industries.

Analysing the In-Use Stability of mRNA-LNP COVID-19 Vaccines Comirnaty™ (Pfizer) and Spikevax™ (Moderna): A Comparative Study of the Particulate.

Comirnaty™ and Spikevax™ were the first vaccines approved for human use based on modified non-replicating mRNA lipophilic nanoparticle (mRNA-LNP) technology, with great success in the treatment of COVID-19. They have been used massively worldwide. One of the major inconveniences of these vaccines is related to pharmaceutical stability issues. Proper transportation, storage, and in-use handling before administration to patients are critical steps since failures can potentially reduce potency. In this research, the in-use stability of Comirnaty™ and Spikevax™ clinical samples was analysed and the results were compared. As changes in the size of the mRNA-LNPs are related to potency, these modifications were analysed by qualitative Dynamic Light Scattering (DLS) as a stability-indicating method for control and stressed vaccine samples. Strong stress factors (accelerated light irradiation, manual shaking, and vortex vibration) and conditions that mimic in-use handling (exposure to natural light and room temperature, repeated cycles of injections, and 24 h storage in syringes) were checked. The morphology of the mRNA-LNPs was analysed by Transmission Electron Microscopy (TEM) to better interpret and support the DLS results. Although the two vaccines are based on the same mRNA-LNP technology, the results demonstrate that they are characterised by very different particle size profiles and behaviours against different handling/stress conditions.

Differential role of potential stressors, underlying degradation mechanism, characterization of degradants using LC-MS/MS, and establishment of a stability-indicating analytical method for duvelisib.

Duvelisib (DUV) was first approved globally in 2018. An extensive literature search revealed that the differential role of a potential degradation medium in altering the shelf-life of DUV due to its exposure during storage has not been identified till date. Moreover, its degradation impurities and degradation mechanism are not known. In addition, no analytical method has been reported for the quantification of DUV in the presence of its degradation impurities. Therefore, the aim of this study was to identify the impact of different potential degradation media on the stability of DUV, establish the degradation mechanism, and identify its major degradation impurities. The aim was also to establish a stability-indicating analytical method for the quantification of DUV in the presence of its degradation impurities. This study is the first to report the structure of degradation impurities and the step-by-step degradation mechanism of DUV. This information will be useful for the scientific community and manufacturers in optimizing the formulation parameters and/or storage conditions. The validated method can be employed for analysis of stability study and routine quality control samples of newer DUV formulations in pharmaceutical industries. The identified impurities may serve as impurity standards for specifying their limits in the drug after required qualification studies.

Development and Validation of a Stability-indicating UPLC-DAD Method for the Simultaneous Determination of Ivermectin and Praziquantel in Pharmaceutical Tablets and Dissolution Media.

Currently, there is no single rapid and accurate stability-indicating quantitative method that can simultaneously determine both ivermectin and praziquantel and their related compounds. Thus, the goal of this research is to develop and validate a new rapid, accurate, and stability-indicating ultra-performance liquid chromatography (UPLC) method. The method uses a water, acetonitrile, and methanol gradient. The chromatographic separation was achieved on a C18 (1.7 μm, 2.1 × 50 mm) column with a flow rate of 0.7 mL/min, and the column temperature was maintained at 40°C. Analytes are detected at 245 nm. The method was validated in accordance with ICH Q2R1 guidelines. The linearity (R2) was >0.9987 and 0.9997 for praziquantel and ivermectin, respectively. The corresponding accuracy ranged between 98.0 and 102.0%. Intermediate precision (assessed as inter-day precision) was determined by calculating the cumulative %CV of eighteen assay preparations and was less than 2.0% for both praziquantel and ivermectin. The specificity of the method was shown by the resolution of the two active pharmaceutical ingredients (APIs) from any interfering excipients, impurities, or degradation products. The limit of detection and quantitation for ivermectin was 26.80 ng/mL and 81.22 ng/mL, respectively. The limit of detection and quantitation for praziquantel was 1.39 μg/mL and 4.22 μg/mL, respectively. The robustness study proved that method performance is stable against small variations in sample processing parameters (shaking, sonication time, and acetonitrile % in solvent solution) and also against small variations in the initial % of mobile phase components and gradient slope. Using ICH Q2R2 criteria, the method was demonstrated to be specific, accurate, stability indicating, and robust to small variations of chromatographic variables.

Potential degradation products of abemaciclib: Identification and structural characterization employing LC-Q/TOF-MS and NMR including mechanistic explanation

Degradation products are the potential drug impurities that can be generated during transport and storage of pharmaceuticals. Before this study, degradation chemistry and potential degradation products of abemaciclib (ABM) were unknown. Moreover, no stability-indicating analytical method was available that can be used to analyse ABM in presence of its degradation products. In this study, stress testing on ABM was carried out under oxidative, thermal, photolytic (UV & visible), and hydrolytic (acid, alkaline, and neutral) degradation conditions. The study revealed that ABM is susceptible to photolytic, oxidative, and thermal stress leading to the formation of five degradation products (DPs). ABM and its degradation products were chromatographically separated employing a developed RP-HPLC-based stability-indicating analytical method. The method was transferred to an LC-Q-TOF system for further analysis. To elucidate the structure of degradation products, fragmentation pathway of ABM was initially established through high-resolution mass spectrometry (HRMS). Subsequently, mass fragmentation pathways of all the DPs have been established through HRMS and MSn based analysis. The major degradation product was isolated and fully characterized using atmospheric chemical ionization-mass spectrometry and nuclear magnetic resonance techniques. ABM showed extensive degradation under oxidative and photolytic systems. Therefore, special care may be sought during storage and transport of ABM or its formulations to avoid photolytic and oxidative stress exposure to the drug. Lastly, in silico toxicity of the characterized degradation products was assessed employing ProTox ІІ online web predictor freeware in which some of them were found to have the potential of hepatotoxicity, immunogenicity and mutagenicity.

A validated stability-indicating high-performance liquid chromatography-photodiode array method for the evaluation of extemporaneous compounded formulations prepared from isoniazid substances and tablets

A validated stability-indicating high-performance liquid chromatography-photodiode array method for the evaluation of extemporaneous compounded formulations prepared from isoniazid substances and tablets

Quantification of Drospirenone- and Ethinyl Estradiol-Related Impurities in a Combined Pharmaceutical Dosage Form by a Chromatography Method With a QbD Robustness Study.

The estimation of drugs containing drospirenone (DRSP) and ethinyl estradiol (EE), and their related impurities, in low-dose oral contraceptive drug products is an extremely challenging target. The proposed research sought to develop and validate a stability-indicating method for quantifying drug substances and their related impurities in tablet formulation. To develop and validate a simple, specific, accurate, precise, and stability-indicating reverse-phase (RP)-HPLC method for quantification of DRSP, EE, and their impurities in accordance with International Conference on Harmonisation (ICH) guidelines. The separation was achieved using an Agilent Zorbax SB C18 column (4.6 mm × 250 mm, 5 µm) with a detection wavelength of 215 nm and mobile phases A (100% acetonitrile) and B (acetonitrile-water, 1 + 3, v/v) at a flow rate of 1.3 mL/min and a column temperature of 40°C. The recovery study of each impurity was conducted in the range of 24 to 72 µg/mL for DRSP-related impurities and 0.2 to 0.6 µg/mL for EE-related impurities with respect to the specification limit. A linearity study was conducted over a range of 1.5 to 90 µg/mL for DRSP and DRSP-related impurities, and 0.125 to 0.75 µg/mL for EE-related impurities. A Quality by Design (QbD) study demonstrated the method's robustness. As per current guidelines, a stability-indicating method has been developed for the determination of impurities in DRSP/EE film-coated tablets. A QbD-based robustness test was performed and the method was found to be robust. An accurate, precise, stability-indicating, gradient RP-HPLC method has been developed and validated to determine DRSP, EE, and nine related impurities in tablet formulation. A QbD technique was used to establish a robustness study.

Development and Validation of a Stability-Indicating RP-HPLC Method for Estimation of Glibenclamidei Bulk and Pharmaceutical Formulation

Glibenclamide is the choice of medicine for diabetes type II. There are numerous methods for estimation; however the methods are either not suitable or expensive. A simple, cheap, precise, and accurate stability-indicating HPLC method was developed and further validated. The wavelength of 229 nm with a constant flow rate of (Methanol: Mixed Phosphate Buffer, 70:30% v/v (pH 7.3)) 1 mL/min was used during the experimentation. The run time was (run time). The internal standard used was (Internal standard). The sample eluted and showed a sharp peak at 2.1 minutes. It was discovered that calibration curve experiments had a 5–50 ppm linearity. The precision of the developed method was lower than 2% RSD. Recovery studies showed that the method’s accuracy and precision ranged from 99.25 to 101.81%. Stress testing was used to determine specificity. The developed method was capable of separating the drug peak from any probable degradation products. There was no evidence of recipient or impurity interference. The method can be used to anticipate how long glibenclamide will be stable. The method was feasible and appropriate for analyzing the presence of glibenclamide in a drug substance as well as a drug product.

Analytical Method and Stability Study for Oral Suspension of Idebenone in Syrspend

Idebenone (IDB) (2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone) is a compound synthesized in the early 1980s. Initially developed for the treatment of cognitive disturbances and Alzheimer’s disease, IDB is now studied for Friedreich’s ataxia, Leber’s hereditary optic neuropathy (LHON), or Duchenne muscular dystrophy. The greatest disadvantage of IDB is its low solubility in water, resulting in low bioavailability. Galenic preparations of IDB in customized doses are common for pediatric patients, which can often prove to be the only option for access to therapy. As an antioxidant, the chemical stability of IDB is an essential guarantee for exerting the desired antioxidant action. Stability studies are essential to know the effect of storage conditions of a galenic product. For the first time, a stability-indicating LC-MS method has been developed to define the stability of IDB suspensions in SyrSpend® Sugar-Free Unflavored (Fagron), a carrier phase formulated for setting up suspensions of active pharmaceutical ingredients (APIs) insoluble or poorly insoluble in water and compatible with it. The proposed method was validated for linearity, accuracy, specificity, robustness, matrix effect and recovery, limit of detection (LOD), limit of quantification (LOQ), and repeatability.

Greens appraisal of validated stability indicating RP-HPLC method and forced degradation study for quantification of Ebastine in wastewater and dosage form

Allergic rhinitis and chronic idiopathic urticaria are common conditions triggered by environmental irritants, stress, and certain foods. The FDA has recently announced that the efficacy and safety of Ebastine (EBS) have been thoroughly evaluated and confirmed. This study considered using various tools to assess their greenness. We used AGREEprep, analytical eco-scale (ESA), and analytical method volume intensity (AMVI) to evaluate the greenness of the validated stability-indicating method and a forced degradation study. This allowed for easy determination and quantitation of EBS in wastewater and dosage form. The method was established on Symmetry RP-C18 (150mm×4.6mm,5μm) using mobile phase, which can be prepared by mixing buffer solution of pH 3 with acetonitrile in a ratio of (37.5: 62.5, v/v) in addition to dissolving 0.72 gm of sodium lauryl sulfate in the final solution. The separation process was executed at a flow rate of 1.5mL/min and 5μL injection volume with UV detection at 254nm. Linearity was conducted for EBS in the 5-50μg/mL range. Different validation parameters were investigated, including accuracy, precision, robustness, and specificity. The limits of both detection and quantification were 0.84μg/mL and 2.57μg/mL for EBS. The recovery percentages of EBS were found to be 101.01% and 101.02% for wastewater and pharmaceutical formulations, respectively. According to International Council for Harmonisation (ICH) guidelines, a forced degradation study of EBS was evaluated, including acid, base hydrolysis, and oxidative hydrolysis using hydrogen peroxide and photolytic and thermal degradation. The highest degradation was achieved by acid hydrolysis. The safety and efficacy of EBS were evaluated via a safety comparative profile study.

Design, development and in vitro quantification of novel electrosprayed everolimus-loaded Soluplus®/Polyvinyl alcohol nanoparticles via stability-indicating HPLC method in cancer therapy

Everolimus (RAD001) a mammalian target of rapamycin has been hampered by poor solubility, affecting its dissolution rate, a relationship that extends to low bioavailability. Nanoparticles (NP) based on Soluplus (SOL®) and Polyvinyl alcohol (PVA) was fabricated by electrospraying (ES) for the delivery of RAD001 to improve anti-tumour efficacy. Electrospraying with established experimental conditions produced PVA-SOL®-RAD001 NP with 71 nm mean diameter, smaller particle size distribution and >90 % encapsulation efficiency. Various polymer-drug concentrations exposed to various freeze–thaw (F/T) cycles were studied for NP optimisation and to enhance its mechanical properties. The optimised NP formulation demonstrated complete encapsulation as well as a sustained and pH dependent drug release profile for in vitro release test. In addition, to specifically study the degradation profile of RAD001 and to quantify RAD001 in the fabricated NP, a new HPLC method was developed and validated. The purpose and novelty of the HPLC method was also to ensure that RAD001 can be detected at low amounts where other conventional characterisation methods are unable to detect. The developed HPLC method was accurate, precise, robust and sensitive with LOD and LOQ values of 4.149 and 12.575 μg/mL. In conclusion, the novel developed HPLC system can be applied for the quantification of different chemotherapeutic agents and the novel electrosprayed hydrogel NP is a potential drug delivery system to increase solubility and bioavailability of RAD001 in cancer therapy.

Understanding the Stress Testing Characteristics of Apixaban, Structural Elucidation of a Novel Degradation Impurity, and Stability-Indicating Method Development for Quantification of Related Substances.

People who have non-valvular atrial fibrillation may benefit from taking a new oral anticoagulant called apixaban, which has recently been given the green light by the U.S. Food and Drug Administration. During stress testing, apixaban was found to have a high degree of degradability when subjected to both acidic and basic conditions, and one significant unknown impurity was observed in addition to the major known impurities. Our aim is the isolation and characterization of degradation product observed in stress/forced degradation studies, and also the development of a single HPLC method that is both reliable and accurate for quantifying all 10 related impurities of apixaban. Preparative HPLC was used to isolate the degradation product, and 1H NMR, 13C NMR, and MS were used to elucidate the structure of the product. Additionally, a single reverse-phase (RP) HPLC method was developed for quantification of all related impurities of apixaban. Based on the spectral characterization data, the identified unknown degradation impurity was found to be a pH-independent hydrolysis degradation impurity of apixaban. The developed method is specific, linear, accurate, robust, and rugged. The isolated and characterized impurities were the same as those found during stress testing. The developed method has been validated for its intended purpose in accordance with the regulatory requirements that were outlined. The unknown impurity is a new apixaban degradation impurity that helps us understand its toxicity. The scientific community will benefit from the developed analytical method information as it relates to understanding drug product impurity profiling.

Pharmaceutical Forced Degradation (Stress Testing) Endpoints: A Scientific Rationale and Industry Perspective

Forced degradation (i.e., stress testing) of small molecule drug substances and products is a critical part of the drug development process, providing insight into the intrinsic stability of a drug that is foundational to the development and validation of stability-indicating analytical methods. There is a lack of clarity in the scientific literature and regulatory guidance as to what constitutes an “appropriate” endpoint to a set of stress experiments. That is, there is no clear agreement regarding how to determine if a sample has been sufficiently stressed. Notably, it is unclear what represents a suitable justification for declaring a drug substance (DS) or drug product (DP) “stable” to a specific forced degradation condition. To address these concerns and to ensure all pharmaceutically-relevant, potential degradation pathways have been suitably evaluated, we introduce a two-endpoint classification designation supported by experimental data. These two endpoints are 1) a % total degradation target outcome (e.g., for “reactive” drugs) or, 2) a specified amount of stress, even in the absence of any degradation (e.g., for “stable” drugs). These recommended endpoints are based on a review of the scientific literature, regulatory guidance, and a forced degradation data set from ten global pharmaceutical companies. The experimental data set, derived from the Campbell et al. (2022) benchmarking study,1 provides justification for the recommendations. Herein we provide a single source reference for small molecule DS and DP forced degradation stress conditions and endpoint best practices to support regulatory submissions (e.g., marketing applications). Application of these forced degradation conditions and endpoints, as part of a well-designed, comprehensive and a sufficiently rigorous study plan that includes both the DS and DP, provides comprehensive coverage of pharmaceutically-relevant degradation and avoids unreasonably extreme stress conditions and drastic endpoint recommendations sometimes found in the literature.