Development of forced degradation and stability indicating studies of drugs——A review

Forced degradation is a degradation of new drug substance and drug product at conditions more severe than accelerated conditions. It is required to demonstrate specificity of stability indicating methods and also provides an insight into degradation pathways and degradation products of the drug substance and helps in elucidation of the structure of the degradation products. Forced degradation studies show the chemical behavior of the molecule which in turn helps in the development of formulation and package. In addition, the regulatory guidance is very general and does not explain about the performance of forced degradation studies. Thus, this review discusses the current trends in performance of forced degradation studies by providing a strategy for conducting studies on degradation mechanisms and also describes the analytical methods helpful for development of stability indicating method.

Forced Degradation Studies on Sodium Picosulfate and Separation of 15 Process Related Impurities/Degradants by HPLC

A selective, precise and stability-indicating, high performance liquid chromatographic method was developed for the analysis of active ingredient sodium Picosulfate and forced degradation behavior was studied. The current article describes forced degradation behavior of the Sodium Picosulfate drug substance in detail by analyzing 15 process related/degradants in a single HPLC method under ICH recommended stressed conditions. Mobile phase comprised of 0.01 M of Disodium hydrogen phosphate and 0.01 M of potassium phosphate monobasic buffer and 1 mL of triethyl amine in 1000 mL water adjusted to pH 7.5 with 10% phosphoric acid. Acetonitrile was used as Mobile Phase B. The separation was achieved on a gradient method. The reversed phase chromatography was performed in Hypersil BDS C18 5.0 μm, 4.6 × 250 mm column maintained at temperature 35°C. Injection volume was 60 μL. Milli-Q water used as diluent. The mobile phase was pumped at 0.9 mL/min-1. The eluted compounds were monitored at 220 nm. Secondary wavelength of the 263 nm was studied to check any further degradants during the forced degradation studies. New additional degradants Sodium Picosulfate Benzyl alcohol Impurity and N oxide degradations were discussed and studied during the forced degradation to understand the chemical stability of the drug substance.

Identification, Isolation and Structure Confirmation of Forced Degradation Products of Sofosbuvir

A new stability indicating reverse phase chromatographic method was developed for the analysis of Heptisis C Drug Sofosbuvir. The developed UPLC method was superior in technology to conventional RP-HPLC with respect to resolution, speed, solvent consumption and analysis cost. Sofosbuvir was subjected to the thermal, hydrolytic, oxidative, and photolytic degradation, according to ICH guidelines. The drug depicted degradation in acidic, basic and oxidative conditions and it was stable to other stress conditions (thermal and photolytic). Identified degradation products were isolated using mass supported auto purification system and characterized by NMR techniques (1H NMR, D2O Exchange, 13C NMR, 31P NMR, 19F NMR, HSQC and HMBC) and HRMS experiments. Isolated acid degradation impurity was showing molecular weight of 416.08, molecular formula C16H18FN2O8P and its name as (R)-((2R, 3R, 4R, 5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methyl phenyl hydrogen phosphate. Isolated base degradation impurity-A was showing molecular weight of 453.13, molecular formula C16H25FN3O9P and its name as (S)-isopropyl 2-((R)-(((2R, 3R, 4R, 5R)-5-(2, 4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(hydroxy)phosphorylamino)propanoate. Isolated base degradation impurity-B was showing molecular weight of 411.08, molecular formula C13H19FN3O9 P and its name as (S)-2-((R)-(((2R, 3R, 4R, 5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methox-y)(hydroxy)phos-phorylamino)propanoic acid. In oxidative degradation study, degradation was observed very less and with obtained quantity (less than 2 mg) recorded 1H NMR and HRMS analysis. From the spectral data degradation product was showing molecular weight of 527.15, molecular formula C22H27FN3O9P and its name as (S)-isopropyl 2-((S)-(((2R, 4S, 5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-4-methyl-3-oxotetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate. The developed chromatographic method was validated as per ICH guidelines and proved suitable for the stability testing and quality control of the drug Sofosbuvir

Investigation of the long-term stability and forced degradation of valtrate by high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry

Valtrate is the main drug quality control for the qualitative and quantitative analysis of Valerian medicines in the Chinese Pharmacopoeia 2010. However, valtrate is unstable under some conditions. We, for the first time, systemically evaluated the stability of two bath reference standards （RS） by high performance liquid chromatography coupled with a triple quadrupole mass spectrometer （HPLC-MS/MS）. The forced degradations of valtrate were performed to evaluate its optimal storage, transportation and experiment conditions according to ICH guideline. The developed HPLC method was validated to determine the degradation products. Valtrate RS was sensitive to alkaline and thermal conditions, but it was relatively stable under acidic, oxidation and photolysis conditions. A total of nine degradation components were identified under alkaline hydrolysis （N1-N4） and thermal degradation （B1-B5）. The information obtained in this work would be valuable to minimize the decomposition of valtrate during the processes of preparation, storage, distribution and utilization. It was highly suggested to store valtrate with a single dose packing in brown closed ampoule at -20℃. Under the above-mentioned storage condition, valtrate could be stable for up to 3 years.

Isolation and characterization of a degradation product in leflunomide and a validated selective stability-indicating HPLC-UV method for their quantification

Leflunomide （LLM） is subjected to forced degradation under conditions of hydrolysis, oxidation, dry heat, and photolysis as recommended by International Conference on Harmonization guideline Q1A（R2）. In total, four degradation products （I-IV） were formed under different conditions. Products I, II and IV were formed in alkaline hydrolytic, acidic hydrolytic and alkaline photolytic conditions. LLM and all degradation products were optimally resolved by gradient elution over a C18 column. The major degradation product （IV） formed in hydrolytic alkaline conditions was isolated through column chromatography. Based on its IH NMR, IR and mass spectral data, it was characterized as a British Pharmacopoeial impurity B. The HPLC method was found to be linear, accurate, precise, sensitive, specific, rugged and robust for quantification of LLM as well as product IV. Finally, the method was applied to stability testing of the commercially available LLM tablets.

Stability-indicating assay method for determination of actarit,its process related impurities and degradation products:Insight into stability profile and degradation pathways

The stability of the drug actarit was studied under different stress conditions like hydrolysis（acid,alkaline and neutral）,oxidation,photolysis and thermal degradation as recommended hy International Conference on Harmonization（ICH） guidelines.Drug was found to be unstable in acidic,basic and photolytic conditions and produced a common degradation product while oxidative stress condition produced three additional degradation products.Drug was impassive to neutral hydrolysis,dry thermal and accelerated stability conditions.Degradation products were identified,isolated and characterized by different spectroscopic analyses.Drug and the degradation products were synthesized by a new route using green chemistry.The chromatographic separation of the drug and its impurities was achieved in a phenomenex luna C18 column employing a step gradient elution by high performance liquid chromatography coupled to photodiode array and mass spectrometry detectors（HPLC-PDAMS）.A specilic and sensitive stability-indicating assay method for the simultaneous determination of the drug actarit.its process related impurities and degradation products was developed and validated.

Drug-Excipient Interactions: Case Studies and Overview of Drug Degradation Pathways

The objective of the current research article is to provide a comprehensive review of excipients impact on the stability of the drug product and their implications during the product development. Recent developments in the understanding of the degradation pathways further impact methodologies used in the pharmaceutical industry for potential stability assessment. The formation of drug excipient adducts was very common based on the sensitive chemical moieties in the drugs and the excipients. The formation of the impurities was not limited to drug related impurities but there were several possibilities of the drug-excipient adduct formations as well as excipient impurities reaction with Active Pharmaceutical Ingredients. Identification of drug degradation in presence of excipients/excipient impurities requires extensive knowledge and adequate analytical characterization data. Systematic literature review and understanding about the drug formulation process, give you a smooth platform in establishing the finished product in the drug market. This paper discusses mechanistic basis of known drug-excipient interactions with case studies and provides an overview of common underlying themes in solid, semisolid and parenteral dosage forms.

Taxifolin stability: In silico prediction and in vitro degradation with HPLC-UV/UPLCe ESI-MS monitoring

Taxifolin has a plethora of therapeutic activities and is currently isolated from the stem bark of the tree Larix gmelinni(Dahurian larch). It is a flavonoid of high commercial interest for its use in supplements or in antioxidant-rich functional foods. However, its poor stability and low bioavailability hinder the use of flavonoid in nutritional or pharmaceutical formulations. In this work, taxifolin isolated from the seeds of Mimusops balata, was evaluated by in silico stability prediction studies and in vitro forced degradation studies(acid and alkaline hydrolysis, oxidation, visible/UV radiation, dry/humid heating) monitored by high performance liquid chromatography with ultraviolet detection(HPLC-UV) and ultrahigh performance liquid chromatography-electrospray ionization-mass spectrometry(UPLC-ESI-MS). The in silico stability prediction studies indicated the most susceptible regions in the molecule to nucleophilic and electrophilic attacks, as well as the sites susceptible to oxidation. The in vitro forced degradation tests were in agreement with the in silico stability prediction, indicating that taxifolin is extremely unstable(class 1) under alkaline hydrolysis. In addition, taxifolin thermal degradation was increased by humidity.On the other hand, with respect to photosensitivity, taxifolin can be classified as class 4(stable).Moreover, the alkaline degradation products were characterized by UPLC-ESI-MS/MS as dimers of taxifolin. These results enabled an understanding of the intrinsic lability of taxifolin, contributing to the development of stability-indicating methods, and of appropriate drug release systems, with the aims of preserving its stability and improving its bioavailability.

Force degradation behavior of glucocorticoid deflazacort by UPLC：isolation,identification and characterization of degradant by FTIR,NMR and mass analysis

In this investigation,sensitive and reproducible methods are described for quantitative determination of deflazacort in the presence of its degradation product.The method was based on high performance liquid chromatography of the drug from its degradation product on reverse phase using Acquity UPLC BEH C18columns（1.7 urn,2.1 mm ×150 mm） using acetonitrile and water（40：60 V/V） at a flow rate of 0.2 mL/minute in UPLC.UV detection was performed at 240.1 nm.Deflazacort was subjected to oxidative,acid,base,hydrolytic,thermal and photolytic degradation.The drug was found to be stable in water and thermal stress,as well as under neutral stress conditions.However,forced-degradation study performed on deflazacort showed that the drug degraded under alkaline,acid and photolytic stress.The degradation products were well resolved from the main peak,which proved the stability-indicating power of the method.The developed method was validated as per ICH guidelines with respect to accuracy,linearity,limit of detection,limit of quantification,accuracy,precision and robustness,selectivity and specificity.Apart from the aforementioned,the results of the present study also emphasize the importance of isolation characterization and identification of degradant.Hence,an attempt was made to identify the degradants in deflazacort.One of the degradation products of deflazacort was isolated and identified by the FTIR,NMR and LC-MS study.

Identification, Isolation and Characterization of Unknown Acid Degradation Product of Nevirapine

A Novel stability indicating RP-UPLC chromatographic method was developed for analysis of Nevirapine in pharmaceutical formulations. The developed RP-UPLC method is superior in technology to conventional RP-HPLC with respect to speed, resolution, solvent consumption and cost of analysis. Nevirapine was subjected to the stress conditions like acid, base, thermal, oxidative and photolytic degradation. Nevirapine was found to degrade significantly in acid and thermal degradation. In acid degradation relative retention time with 0.42 is found as unknown impurity. New impurity was identified, isolated using mass based auto purification system and characterized by 1H NMR (1D and 2D) and HRMS experiments. Isolated impurity was showing molecular weight of 244.10, molecular formula C12H12N4O2 and its name as 2-(3-Amino-4-methylpyridin-2-ylamino)nicotinic acid. The calibration graph was linear and the method showed less deviation in accuracy results. The test solution was found to be stable for 20 days when stored in the refrigerator between 2°C to 8°C. The developed RP-UPLC method was validated and meets the requirements delineated by the International Conference on Harmonization (ICH) guidelines. The intra-day and inter-day variation was less than 1%. The method was reproducible and selective for the estimation of Nevirapine. Because the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating method.

Drug-Excipient Interaction of Methylphenidate with Glycerin in Methylphenidate Oral Solution and Identification of its Transesterification Products by UPLC-MS/MS

Reactions between active drug substances and excipients are of interest in the drug formulation process should be checked for the interactions during the storage conditions. Some excipients react with certain chemical groups in drug substances which will form new impurities in the finished product formulations. In the present paper transesterification reaction of methylphenidate with glycerin to form different structural isomeric products was described. These impurities identified in forced degradation studies, excipient compatibility studies and stability analysis of the finished product. Stability samples were analyzed and observed that about ~0.6% of the Methylphenidate content was transformed into methylphenidate-glycerin isomers within 3 Months at 40°C/75% RH and 18 Months at 25°C/60% RH conditions. Analysis of two lots of marketed preparations having expiry dates in 2012 and 2013 showed content of the Methylphenidate esters corresponding to ~0.6% of the declared Methylphenidate content. The samples of this impurity were investigated by HPLC, UPLC-MS/MS to generate the mechanism of the impurity formation.

Establishment of inherent stability of pramipexole and development of validated stability indicating LC-UV and LC-MS method

Pramipexole belongs to a class of nonergot dopamine agonist recently approved for the treatment of early and advanced Parkinson＇s disease.A validated specific stability indicating reversed-phase liquid chromatographic method has been developed for the quantitative determination of pramipexole in bulk as well as in pharmaceutical dosage forms in the presence of degradation products.Forced degradation studies were performed by exposition of drug to hydrolytic（acidic and basic）,oxidative and photolytic stress conditions,as defined under ICH guideline Q1A（R2）.Significant degradation was observed under hydrolytic,oxidative and photolytic conditions and the degradation products formed were identified by LC-MS.

Stability indicating high performance thin-layer chromatographic method for simultaneous estimation of pantoprazole sodium and itopride hydrochloride in combined dosage form

A specific, precise and stability indicating high-performance thin-layer chromatographic method for simultaneous estimation of pantoprazole sodium and itopride hydrochloride in pharmaceutical formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F254 as the stationary phase. The solvent system consisted of methanol：water：ammonium acetate; 4.0：1.0：0.5 （v/v/v）. This system was found to give compact and dense spots for both itopride hydrochloride （Rf value of 0.55__＋0.02） and pantoprazole sodium （Rf value of 0.85＋0.04）. Densitometric analysis of both drugs was carried out in the reflectance- absorbance mode at 289 nm. The linear regression analysis data for the calibration plots showed a good linear relationship with R2=0.9988___0.0012 in the concentration range of 100--400 ng for pantoprazole sodium. Also, the linear regression analysis data for the calibration plots showed a good linear relationship with R2=0.9990_＋0.0008 in the concentration range of 200-1200 ng for itopride hydrochloride. The method was validated for specificity, precision, robustness and recovery. Statistical analysis proves that the method is repeatable and selective for the estimation of both the said drugs. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating method.

Development and validation of a stability-indicating HPLC method for the determination of retigabine and its related substances in drug substances

A stability-indicating high-performance liquid chromatography（HPLC） method has been developed and validated for the separation and determination of Retigabine and its related substances. The chromatographic separation was achieved on Agilent Eclipse Plus C18 column（4.6 mm×150 mm, 5 μm）. The mobile phase was constituted of triethylamine-phosphate buffer as A and acetonitrile as B. The analysates were then eluted under the gradient conditions as description in this paper. The forced degradation study validated that the newly developed method was specific and selective to the degraded products. The performance of the method was verified according to the present International Conference on Harmonisation（ICH） guidelines for specificity, linearity, accuracy, precision and robustness. The correlation coefficients for Retigabine and its six impurities were greater than 0.999, which was shown in the regression analysis. Limits of detection（LOD） of these impurities were in the range of 0.0092%–0.0103%, indicating the high sensitivity of the newly developed method. Accuracy of the method was determined on the basis of recoveries to be between 96.49% and 118.35% for all impurities. Relative standard derivation（RSD） receiving in the repeatability and intermediate precision experiment, was less than 1.0%. The method can be successfully applied to routine quantify and stability testing Retigabine and its related substances in bulk drugs.