Study of the degradation behavior of dapagliflozin propanediol monohydrate and metformin hydrochloride by a stability-indicating high-performance thin-layer chromatographic method

A simple, selective, precise, rapid and accurate stability-indicating high-performance thin-layer chromatography (HPTLC) method was developed and validated for the estimation of dapagliflozin and metformin in tablet dosage form. In this work, methanol–ethyl acetate–ammonium acetate (6:4:0.1, V/V) as the mobile phase and aluminum-backed TLC plates pre-coated with 250 µm layer of silica gel 60F₂₅₄ as the stationary phase were used for the estimation of dapagliflozin and metformin. The wavelength selected for detection was 220 nm. The linearity range was found to be 20–100 ng/spot (r² = 0.9985) for dapagliflozin and 500–2500 ng/spot (r² = 0.9984) for metformin. Validation of the developed method was performed as per the International Council for Harmonisation (ICH) guidelines. Stress testing of dapagliflozin and metformin was performed under acidic, alkaline, oxidative, photolytic and dry-heat degradation conditions. The chromatographic conditions successfully resolved dapagliflozin and metformin from their degradation products, formed under various stress conditions. From stress testing, dapagliflozin was found to be significantly degrading under acidic, alkaline, oxidative, photolytic and dry-heat degradation conditions, while metformin was found to be significantly degrading in acidic and alkaline degradation conditions and stable under oxidative, photolytic and dry-heat degradation conditions. Tablet dosage form of dapagliflozin and metformin was analyzed by the developed method.

     

Stress Degradation Studies on Tadalafil and Development of a Validated Stability-Indicating LC Assay for Bulk Drug and Pharmaceutical Dosage Form

A stability-indicating HPLC assay method was developed for the quantitative determination of tadalafil in bulk samples and in pharmaceutical dosage forms in the presence of the degradation products. It involved a 250 mm × 4.6 mm, 5 μm C-18 column. The gradient LC method employs solution A and B as mobile phase. Solution A contains a mixture of buffer (phosphate buffer and tetra-n-butyl ammonium hydrogen sulfate) pH 2.5: acetonitrile (80:20, v/v) and solution B contains a mixture of water: acetonitrile (20:80, v/v). The flow rate was 1.0 mL min⁻¹ and the detection wavelength was 220 nm. The retention time of tadalafil is about 17 min. Tadalafil was subjected to different ICH prescribed stress conditions. Degradation was found to occur in hydrolytic and to some extent in oxidative stress conditions, while the drug was stable to photolytic and thermal stress. The drug was particularly labile under alkaline hydrolytic conditions. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The assay of stress samples was calculated against a qualified reference standard and the mass balance was close to 99.5%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and ruggedness.     

Development of stability-indicating method for separation and characterization of benidipine forced degradation products using LC–MS/MS

The present study describes forced degradation of benidipine (BEN) as per  Q1A (R2) and Q1B guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. BEN degraded under hydrolysis (neutral, acidic, and alkaline), hydrogen peroxide induced oxidation, and UV light mediated photolytic degradation. A total of 14 degradation products (DPs) were found in all degradation studies, comprising 4 hydrolytic DPs, 8 oxidative DPs, and 4 photolytic DPs. A selective stability-indicating method was developed using an XBridge BEH C18 column with gradient elution program consisting of ammonium acetate (10 mM, 4.8 pH, acetic acid) and acetonitrile. The flow rate was maintained at 1 ml min⁻¹. All DPs were separated well using the developed HPLC method and were characterized using LC–MS/MS data. As this method is effective in identifying and separating BEN and its DPs with sufficient resolution, it can be used in laboratories for quality control of drugs in daily routine analysis and stability studies.     

Structural identification of degradants of moexipril by LC–MS/MS

A gradient LC–MS method was developed for the identification and characterization of degradants of moexipril using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS). Moexipril was subjected to hydrolysis (acid, base and neutral), oxidation, photolytic and thermal degradation conditions as mentioned in ICH guidelines Q1A (R2). The drug degraded under hydrolysis, oxidation and photolytic conditions, but it was stable under thermal conditions. In total, five degradants were formed and separated on an Agilent XDB C‐18 column (4.6 × 150 mm, 5 μm) in a gradient elution method. Four degradants ( D1 , D2 , D4 and D5 ) under acidic conditions, three degradants ( D2 , D3 and D4 ) under basic conditions and three degradants ( D1 , D4 and D5 ) under neutral and oxidative stress conditions were formed. In addition, two degradants ( D4 and D5 ) were formed under photolytic stress conditions. To elucidate the structures of degradants, fragmentation of moexipril and its degradants was studied using LC–MS/MS experiments and accurate mass measurements (HRMS) data. The fragment ions in the product ion tandem mass spectra of all the degradants were compared with those of moexipril and assigned the probable structures for the degradants.     

Determination of Atazanavir in the Presence of its Degradation Products by a Stability-Indicating LC Method

A forced degradation study was successfully applied for the development of a stability-indicating assay method for the determination of atazanavir in presence of its degradation products. The method was developed and optimized by analyzing the forcefully degraded samples. Degradation of the drug was done under acidic, alkaline, oxidative, photolytic and thermal stress conditions. The proposed method was able to resolve all of the possible degradation products formed during the stress studies. The major impurities are generated in acidic and alkaline conditions. The product was found stable under thermal, photolytic and oxidative conditions. The developed method was validated for determination of atazanavir, and the method was found to be equality applicable to study the impurities formed during routine and forced degradation of atazanavir.     

LC–MS/MS method for the characterization of the forced degradation products of Entecavir

A rapid, specific, and reliable isocratic LC–MS/MS method has been developed and validated for the identification and characterization of the stressed degradation products of Entecavir (ETV). ETV, an antiviral drug, was subjected to hydrolysis (acidic, alkaline, and neutral), oxidation, photolysis and thermal stress, as per the international conference on harmonization specified conditions. The drug showed extensive degradation under oxidative and acid hydrolysis stress conditions. However, it was stable to thermal, acidic, neutral, and photolysis stress conditions. A total of five degradation products were observed and the chromatographic separation of the drug and its degradation products were achieved on a Waters Symmetry C₁₈ (250 mm × 4.6 mm, id, 5 μm) column using 20 mM ammonium acetate (pH 3)/acetonitrile (50:50, v/v) as a mobile phase. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. The LC–MS method was validated with respect to specificity, linearity, accuracy, and precision. No previous reports were found in the literature regarding the degradation behavior of ETV.     

Development and validation of a stability-indicating liquid chromatographic method for determination of emtricitabine and related impurities in drug substance

A novel stability-indicating high-performance liquid chromatographic (HPLC) method was developed and validated for assay and determination of impurities of emtricitabine in drug substance. Emtricitabine was found to be degraded under acidic, alkaline, and oxidative stress conditions and to be more labile under oxidative conditions. The drug proved to be stable to dry heat and photolytic degradation. Resolution of major and minor degradation impurities was achieved on an Intersil ODS-3V column utilizing 10 mM sodium phosphate buffer and methanol (85:15) as mobile phase. Detection was at 280 nm. Validation studies were performed as per ICH recommended conditions. The developed method was found to be linear, accurate, specific, selective, precise, and robust.     

Characterization of forced degradation products of ketorolac tromethamine using LC/ESI/Q/TOF/MS/MS and in silico toxicity prediction

Ketorolac, a nonsteroidal anti‐inflammatory drug, was subjected to forced degradation studies as per International Conference on Harmonization guidelines. A simple, rapid, precise, and accurate high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (LC/ESI/Q/TOF/MS/MS) method has been developed for the identification and structural characterization of stressed degradation products of ketorolac. The drug was found to degrade in hydrolytic (acidic, basic, and neutral), photolytic (acidic, basic, and neutral solution), and thermal conditions, whereas the solid form of the drug was found to be stable under photolytic conditions. The method has shown adequate separation of ketorolac tromethamine and its degradation products on a Grace Smart C‐18 (250 mm × 4.6 mm i.d., 5 µm) column using 20 mM ammonium formate (pH = 3.2): acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0 ml/min. A total of nine degradation products were identified and characterized by LC/ESI/MS/MS. The most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M + H]⁺ ions of ketorolac and its degradation products. In silico toxicity of the drug and degradation products was investigated by using topkat and derek softwares. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonization guidelines. Copyright © 2014 John Wiley & Sons, Ltd.     

LC–MS/MS and NMR characterization of forced degradation products of mirabegron

A rapid, precise, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for the characterization of stressed degradation products of mirabegron. It is used in the treatment of overactive bladder and administered to treat urinary symptoms such as urgency or frequency and incontinence. It also works by relaxing the muscles around bladder.

Mirabegron was subjected to hydrolysis (acidic, alkaline, and neutral) and peroxidation, as per ICH-specified conditions. The drug showed degradation under stress conditions. However, it was stable to neutral conditions. A total of seven degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on X-TerraRP-8 (250 mm × 4.6 mm, i.d., 5 µm) column using 0.01 M ammonium acetate as mobile phase-A and 60:40 ratio of acetonitrile (ACN):water as mobile phase-B. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. Probable possible structures were drawn based on parent and daughter molecular ions. One peroxide degradant impurity was isolated using preparative LC and characterized using liquid chromatography–mass spectrometry and NMR data.     

An Application of Quality by Design and Analytical Greenness Assessment Approach for the Development of Erlotinib Stability Indicating Method

Pharmaceutical regulators are worried about medication quality and stability since drug degradation may result in harmful chemicals. Erlotinib (ERL) is a tyrosine kinase inhibitor associated with the epidermal growth factor receptor (EGFR) containing susceptible functional groups such as quinazoline and amine ketone, methoxy, and ethoxy leads to a reduction in pharmaceutical quality. According to the ICH-Q1A (R2) guideline, the goal of ERL stability studies is to establish its susceptibility to degradation under various environmental conditions. A novel isocratic stability–indicating liquid chromatography method has been developed using systemic quality by design (QbD) approach. The QbD strategy includes screening and optimization as phases. Placket Burman was used for primary parameters screening, and critical factors were optimized with response surface design. The prepared degradation samples (acid, base, neutral hydrolysis, oxidative, photolytic, and thermal) were separated using a Shimadzu GIST C18 column (250 mm?×?4.6 mm, 5 µm) with 15 mM ammonium formate: ACN (58:42% v/v) as mobile phase, 0.9 mL/min flow rate, and 246 nm wavelength, which was found to be LC–MS compatible. A total of six degradation products (DPs) were identified with the optimized chromatography method. The drug was sensitive toward acidic and basic hydrolysis, but it remained stable under neutral, oxidative, thermal, and photolytic stress conditions. The optimized method was sensitive, specific, and robust, with linearity ranging from 10 to 35 µg/mL, with a correlation coefficient (R²?=?0.9997). The analytical method greenness score was calculated and observed that the developed method is green.

     

Stress Studies and Identification of Degradation Products of Cephalexin Using LC–PDA and LC–MS/MS

A stability indicating RP-HPLC method for cephalexin has been developed and validated to identify and characterize potential degradation products. Drug was subjected to hydrolytic (acidic, basic, and neutral), oxidative, thermal, and photolytic stresses as per ICH guidelines Q1A (R2) and Q1B. Chromatographic separation was achieved on C8 column with mixture of ammonium acetate buffer pH 4.5 and acetonitrile in gradient mode as a mobile phase with PDA detection. Specificity of the method was established by peak purity studies. Method was validated as per ICH guideline Q2 (R1) for accuracy, precision, linearity, sensitivity, and robustness. Kinetics for each degradation condition was studied with respect to order of reaction and rate constant. Method was found to comply with acceptance criteria of validation parameters with respect to specificity (peak purity greater than 0.999) linearity (r ² greater than 0.99), accuracy (% recovery in the range of 98–102%), and precision (% RSD not more than 2). A total of six degradation products were generated in different stress conditions; these were identified and structures were proposed using LC–MS/MS. Cephalexin undergoes degradation in almost all the conditions. The developed stability indicating method is suitable for analysis of stability samples as it adequately separates all degradation products. Degradation products generated in photolytic and oxidative conditions are reported for the first time.     

Selective separation and characterization of the stress degradation products of ondansetron hydrochloride by liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Ondansetron hydrochloride was subjected to forced degradation studies under various conditions of hydrolysis (acidic, basic, and neutral), oxidation, photolysis, and thermal as prescribed by International Conference on Harmonisation guideline Q1A (R2). A simple, selective, precise, and accurate high‐performance liquid chromatography method was developed on a Waters Xterra C₁₈ (150 × 4.6 mm id, 3.5 μm) column using 10 mM ammonium formate (pH 3.0)/methanol as a mobile phase in gradient elution mode at a flow rate of 0.6 mL/min. The method was extended to liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry for identification and structural characterization of stress degradation products of ondansetron. The drug showed significant degradation in base hydrolytic and photolytic stress conditions in the liquid state, while it was found to be stable in neutral, acidic, thermal, and oxidative stress conditions. A total of five degradation products were characterized and most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M + H]⁺ ions of the drug and its degradation products. Finally, the developed method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonisation guideline Q2 (R1).     

Study of Forced Degradation Behaviour of Brinzolamide Using LC–ESI–Q-TOF and In Silico Toxicity Prediction

Brinzolamide (BZA) is a topical ophthalmic drug which is generally used to lower the intraocular pressure during glaucoma. It was subjected to forced degradation studies under hydrolytic (acidic, basic), oxidative, photolytic and thermal stress conditions; the drug degraded significantly in hydrolytic and oxidative conditions, leading to the formation of seven degradation products in total. It was stable on exposure to light and dry heat in the solid state. An ultra-performance liquid chromatography (UPLC) method was developed on a Waters CSH phenyl hexyl column (100 × 2.1 mm, 1.7 µm), using gradient elution of 0.1 % formic acid and methanol as mobile phase. The method was extended to quadrupole time-of-flight mass spectrometry (Q-TOF–MS) for the structural characterisation. All degradation products were comprehensively characterised by UHPLC–ESI/MS/MS experiments. The most probable mechanisms for the formation of degradation products were also proposed. In silico toxicity of the drug and its degradation products was determined using TOPKAT toxicity prediction software.

     

Characterization of forced degradation products of pazopanib hydrochloride by UHPLC‐Q‐TOF/MS and in silico toxicity prediction

Pazopanib (PZ), an anti‐cancer drug, was subjected to forced degradation under hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress conditions as per International Conference on Harmonization guidelines. A selective stability indicating validated method was developed using a Waters Acquity UPLC HSS T3 (100 × 2.1 mm, 1.7 µm) column in gradient mode with ammonium acetate buffer (10 m m , pH 5.0) and acetonitrile. PZ was found to degrade only in photolytic conditions to produce six transformation products (TPs). All the TPs were identified and characterized by liquid chromatography/atmospheric pressure chemical ionization–quadrupole‐time of flight mass spectrometry experiments in combination with accurate mass measurements. Plausible mechanisms have been proposed for the formation of TPs. In silico toxicity was predicted using TOPKAT and DEREK softwares for all the TPs. The TP, N4‐(2,3‐dimethyl‐2H‐indazol‐6‐yl)‐N4‐methylpyrimidine‐2,4‐diamine, was found to be genotoxic, whereas all other TPs with sulfonamide moiety were hepatotoxic. The data reported here are expected to be of significance as this study foresees the formation of one potential genotoxic and five hepatotoxic degradation/transformation products. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of stress degradation products of amodiaquine dihydrochloride by liquid chromatography with high‐resolution mass spectrometry and prediction of their properties by using ADMET Predictor™

The degradation behavior of amodiaquine dihydrochloride, an antimalarial drug, was investigated in solution as well as solid states. The drug was subjected to hydrolytic, photolytic, oxidative, and thermal stress conditions, according to International Conference on Harmonization guideline Q1A(R2). It showed extensive hydrolysis in acidic, alkaline, and neutral solutions both with and without light, while it proved to be stable to thermal and oxidative conditions. In total, six degradation products were formed, which were separated on a C8 column, employing a gradient reversed‐phase high‐performance liquid chromatography method in which acetonitrile and 10 mM ammonium formate (pH 3.0) were used in the mobile phase. To characterize the degradation products, mass fragmentation behavior of the drug was established by direct infusion of solution to quadrupole time‐of‐flight and multiple‐stage mass spectrometry systems. Liquid chromatography with high‐resolution mass spectrometry studies were subsequently carried out on the stressed samples using the same gradient high‐performance liquid chromatography method employed for the separation of the degradation products. Hydrogen/deuterium exchange studies were additionally conducted to determine the number of labile hydrogen atoms. The degradation pathway of the drug was delineated, justified by mechanistic explanation. Lastly, ADMET Predictor™ software was employed to predict relevant physicochemical and toxicity data for the degradation products.     

Study of degradation behavior of besifloxacin,characterization of its degradation products by LC–ESI–QTOF–MS and their in silico toxicity prediction

Knowledge and understanding of the stability profile of a drug is important as it affects its safety and efficacy. In the present work, besifloxacin, a new, fourth‐generation fluoroquinolone antibiotic, was subjected to different forced‐degradation conditions as per International Conference on Harmonization (ICH) guidelines such as hydrolysis (acid, base and neutral), oxidation, thermal and photolysis. The drug degraded under acidic, basic, oxidative and photolytic conditions while it was found to be stable under dry heat and neutral hydrolytic conditions. In total, five degradation products (DPs) were formed under different conditions—DP1 and DP2 (photolysis), DP3 (oxidation), DP4 (acidic), DP3 and DP5 (basic). The chromatographic separation of besifloxacin and its degradation products was achieved on a Sunfire C₁₈ (250 mm × 4.6 mm, 5 μm) column with 0.1% aqueous formic acid–acetonitrile as a mobile phase. The gradient RP‐HPLC method was developed and validated as per ICH guidelines. The degradation products were characterized with the help of LC–ESI–QTOF mass spectrometric studies and the most likely degradation pathway of the drug was proposed. In silico toxicity assessment of the drug and its degradation products was carried out, which indicated that DP3 and DP4 carry a mutagenicity alert.     

Development and Validation of a Stability Indicating HPLC Method for Determination of Granisetron

The aim of this study was to study the stress degradation of granisetron and analysis of the drug in the presence of its degradation products. Forced degradation studies were conducted on bulk sample using acidic, alkaline, oxidative, heat and photolytic conditions. Granisetron was relatively unstable under acidic, alkaline and oxidative conditions. Separation of granisetron and degradation products was achieved using a Nova‐Pak C₈ column and acetonitrile‐KH₂PO₄ 25 mM (75:25, v/v) as mobile phase with UV detection at 305 nm. The method was linear over the range of 0.2‐15 μg/mL granisetron (r² > 0.999). The within‐day and between‐day precision values were also in the range of 0.5‐4%. The proposed method was successfully applied for quantitative determination of granisetron in tablets and in vitro dissolution studies.     

Liquid chromatography/mass spectrometric studies on atorvastatin and its stress degradation products

A comprehensive mass fragmentation pathway of atorvastatin, which has not been reported so far, was established by subjecting the drug to multi-stage mass spectrometric (MSn) studies. It was used along with liquid chromatography/mass spectrometric (LC/MS) and liquid chromatography/time-of-flight mass spectrometric (LC/TOFMS) analyses to identify the drug degradation products formed under stress conditions of hydrolysis, oxidation and photolysis. Other than lactone, which is a reported hydrolysis product, six unknown hydrolytic products could be identified, viz., dehydrated drug, dehydrated drug lactone, and diastereomers of the drug, drug lactone, dehydrated drug, and dehydrated drug lactone. Among the two products separated under oxidative conditions, one was lactone, again formed as a result of drug hydrolysis in an acidic environment of peroxide solution. The other was similar to a reported oxidative product. Under photolytic conditions in solution, one new product could be identified, while most of the others matched with those known from the literature. Hence overall a more complete degradation pathway of the drug was established than known at present, by using a stress testing approach and employing LC/MS techniques.     

Optimization of hydrophilic interaction LC by univariate and multivariate methods and its combination with salting‐out liquid–liquid extraction for the determination of antihypertensive drugs in the environmental waters

Hydrophilic interaction LC for the separation of four antihypertensive drugs was optimized by both univariate and multivariate methods. The column efficiency, resolution, and separation time were used as the three assessment parameters. The best separation condition of 97% ACN with 3% aqueous buffer containing 50 mM ammonium acetate at a pH of 3.0 was obtained by the two optimization methods. The multivariate optimization, orthogonal array design herein, was demonstrated to be a little tedious, but afforded a much better understanding of underlying separation factors. The content of ACN in the mobile phase contributed most significantly to separation. Furthermore, sample diluent and injection volume were found to influence the chromatographic performance. To match the hydrophilic interaction LC mobile phase, a proper sample pretreatment method, salting‐out liquid–liquid extraction, in which ACN was the extractant, was chosen. Since reserpine was unstable under both acidic and alkaline conditions, it was not studied in this part. The optimal salting‐out liquid–liquid extraction parameters were as follows: 400 μL ACN was added to 1 mL sample solution containing 500 mg NH₄Cl at a pH of 14.0. The linearity ranged from 0.01 to 1.00 μg/mL with r² > 0.9937. The LODs were between 1.9 and 2.5 ng/mL. The developed method was applied to the environmental water sample with good performance.     

Forced degradation studies of lansoprazole using LC‐ESI HRMS and 1H‐NMR experiments: in vitro toxicity evaluation of major degradation products

Regulatory agencies from all over the world have set up stringent guidelines with regard to drug degradation products due to their toxic effects or carcinogenicity. Lansoprazole, a proton‐pump inhibitor, was subjected to forced degradation studies as per ICH guidelines Q1A (R2). The drug was found to degrade under acidic, basic, neutral hydrolysis and oxidative stress conditions, whereas it was found to be stable under thermal and photolytic conditions. The chromatographic separation of the drug and its degradation products were achieved on a Hiber Purospher, C18 (250 × 4.6 mm, 5 μ) column using 10 mM ammonium acetate and acetonitrile as a mobile phase in a gradient elution mode at a flow rate of 1.0 ml/min. The eight degradation products (DP1–8) were identified and characterized by UPLC/ESI/HRMS with in‐source CID experiments combined with accurate mass measurements. DP‐1, DP‐2 and DP‐3 were formed in acidic, DP‐4 in basic, DP‐5 in neutral and DP‐1, DP‐6, DP‐7 and DP‐8 were in oxidation stress condition Among eight degradation products, five were hitherto unknown degradation products. In addition, one of the major degradation products, DP‐2, was isolated by using semi preparative HPLC and other two, DP‐6 and DP‐7 were synthesized. The cytotoxic effect of these degradation products (DP‐2, DP‐6 and DP‐7) were tested on normal human cells such as HEK 293 (embryonic kidney cells) and RWPE‐1(normal prostate epithelial cells) by MTT assay. From the results of cytotoxicity, it was found that lansoprazole as well as its degradation products (DP‐2, DP‐6 and DP‐7) were nontoxic up to 50‐μM concentrations, and the latter showed slightly higher cytotoxicity when compared with that of lansoprazole. DNA binding studies using spectroscopic techniques indicate that DP‐2, DP‐6 and DP‐7 molecules interact with ctDNA and may bind to its surface. Copyright © 2017 John Wiley & Sons, Ltd.