Stress Degradation Studies Research Articles

Identification and characterization of alkaline hydrolytic degradation products of ivacaftor by UPLC-PDA and UPLC-ESI-QTOF-MS techniques.

The study of inherent stability characteristics of drugs has been advocated to be important by various regulatory agencies like the ICH, USFDA, and others. The current work was envisaged to investigate the forced degradation profile of the drug ivacaftor under ICH-prescribed stress conditions, identification of its potential degradants, and postulation of the degradation routes for their generation. The stress degradation studies were performed on the drug as per the ICH guideline Q1A(R2). A UPLC-photodiode array-based chromatographic method was developed to satisfactorily resolve the drug from its degradation products, validated in accordance with various ICH prescribed parameters, and assessed for its BAGI practicality index. The degradation products were identified and characterized by UPLC-ESI-QTOF-MS studies. The drug was found to significantly degrade under conditions of alkaline hydrolytic stress and it was found to be stable under all other stressor environments including acid/neutral hydrolytic, photolytic, thermal, and oxidative stress. Four alkaline hydrolytic degradation products (I-IV) were revealed by UPLC-QTOF-MS studies which were well-resolved from the drug over a C18 UPLC column by the developed UPLC-PDA method. The detection wavelength was selected as 310 nm. Characterization of the four degradation products (I-IV) was carried out by their mass spectral data and their respective degradation routes were elucidated. A UPLC-PDA method was developed and validated for ivacaftor and its practicality BAGI index was computed. Four alkaline hydrolytic degradation products of ivacaftor were revealed through UPLC-ESI-QTOF-MS studies and corresponding degradation routes were elucidated.

UPLC Estimation of an Antidiabetic Drug by Method Development and Validation Followed by Stability Indicating Studies.

The analytical method development and validation of a common antidiabetic drug, Canaglifozin, by taking the aid of ‘Ultra High-Performance Liquid Chromatography’, along with stability indicating method. The canaglifozin drug was eluted from the optimized mobile phase in 70:30 ratio of water and methanol, at 1 ml/ min flow rate, under separation with C18 column of 100 x 2mm size with 1.8 diameters at isocratic mode. The detection was carried at 286 nm wavelength, resulting in 3.80 retention time, as run time being 10 min. The detection limit and quantification limit, was noticed at ‘0.0039 and 0.0119 µg/ ml’ correspondingly. The percentage RSD and recovery percentage was under ICH guidelines only. The stress degradation studies under acid, basic, neutral, and thermal conditions were carried on.

STABILITY INDICATING HIGH-PERFORMANCE THIN LAYER LIQUID CHROMATOGRAPHIC METHOD FOR EVEROLIMUS

Objective: Developing and validating a stability-indicating method for everolimus by HPTLC and depiction of degradation product of in alkaline conditions by LC-MS. Methods: The chromatographic separation was performed on aluminium plates pre-coated with silica gel 60 F254 as the stationary phase using Toluene: Methanol: Ethyl Acetate (6:2:2v/v/v) as the mobile phase. The evaluation was carried out at 277 nm. For the developed stability indicating method, the ICH Q2 (R1) guidelines were used for validation. Stress degradation studies like hydrolysis under different pH conditions, photolytic degradation, thermal degradation and oxidative degradation as per ICH Q1A (R2) and Q1B guidelines were performed. LC-MS analysis was carried out for the standard everolimus and its alkaline degradation sample using TOF analyser and the degradation pathway was proposed for each degradation product. Results: The Rf value of everolimus was found to be 0.63±0.03. The response was quite linear over the concentration range of 100-500 ng/band, with the regression coefficient value of 0.9921. Under alkaline hydrolytic conditions, everolimus was analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS). The Retention Time (RT) and prominent mass fragmentation (m/z) for the everolimus standard were observed at 9.46 min with m/z values of 980.56 and 908.54. For the degradation products, DP-1 showed an RT of 8.88 min with m/z values of 349.23, 403.24, 574.33, and 646.35, while DP-2 exhibited an RT of 9.10 min with m/z values of 926.55, 614.32, and 542.30. These data were used to propose the structures of the degradation products. Conclusion: The proposed method can conveniently be applied for quantitative analysis of everolimus on routine basis and for stability testing under different stress environments.

Development and Validation of Stability Indicating Liquid Chromatographic Method for Vandetanib: Application to Degradation Studies by LC-MS

Objective: The central goal of this research was to formulate and validate a stability-demonstrating assay approach using reverse-phase high-performance liquid chromatography (RP-HPLC) for the assessment of vandetanib, its primary degradation products, and their respective degradation pathways, which were identified and characterized using liquid chromatography-electrophore spray ionization-mass spectrometry (LC-ESI-MS).Methods: The method was designed and developed by employing a Grace C18 column (250 x 4.6 mm x4.0 μm) and deploying a mixture of methanol and 0.1% formic acid in H2O (in a proportion of 90:10, v/v) as the mobile phase, with a flow rate maintained at 1-mL/min. The analytes were detected at a wavelength of 249 nm. Additionally, vandetanib was subjected to various stress conditions, including acidic, alkaline, oxidative, thermal, and photolytic degradation, to unearth the degradation paths for the principal degradation products.Results: The approach was meticulously formulated and validated to ensure robustness, linearity, precision, accuracy, and linear regression analysis data. A high correlation coefficient of 0.9985 was obtained in the 2 to 12 μg/mL concentration range, demonstrating a robust linear relationship. Moreover, the limit of detection (LoD) and the limit of quantification (LoQ) were found to be 0.166 and 0.503 μg/mL, respectively. In the stress degradation studies, we noticed that vandetanib degraded in alkaline and acidic mediums. The liquid chromatography-mass spectrometry (LC-MS) technique was used to characterize the degradation products.Conclusion: The devised method demonstrated itself to be quick, sensitive, precise, accurate, and robust for the vandetanib analysis, thereby facilitating its routine testing.

Method development and validation of a new stability indicating HPLC and LC-ESI-MS/MS methods for the determination of Tavaborole

Tavaborole, a topical antifungal agent containing Boron is used for the treatment of onychomycosis, an infection of the nail and nail bed caused by Trichophyton rubrum or Trichophyton mentagrophytes infection. Tavaborole is chemically known as 5 - Fluoro -1,3 - dihydro -2,1-benzoxaborol -1-ol. It acts by inhibiting Leucyl-tRNA synthetase an essential fungal enzyme required for protein synthesis. AB SCIEX Instruments LC-ESI-MS/MS (Model no. 5068379-Y) QTRAP Enabled Triple Quad 5500+ with Agilent Zorbox C18 (150 mm x 4.6 mm x 3 µm) column and PDA detector was employed for the present study. The total run time was 10 mins and the detection wavelength was 254 nm. A mixture of 5 mM Ammonium formate: Methanol (30: 70) was used as mobile phase on isocratic mode with 1 ml/min as flow rate. Tavaborole has shown linearity over the concentration range 0.5-100 μg/ml and the proposed method was validated as per ICH guidelines. The proposed method is found to be simple, precise, accurate and suitable for the quantification of the marketed formulations of Tavaborole. Stress degradation studies were performed and the method is found to be selective and specific. Keywords: Tavaborole, Stability indicating, LC-ESI-MS/MS, Validation.

Analytical method development and validation for the evaluation of related substances in Apalutamide by RP-HPLC

Apalutamide is an anti-cancer agent used for the management of prostate cancer. A new stability indicating RP-HPLC method (Gradient mode) has been developed for the estimation of Apalutamide and its related substances using Waters Alliance system (Model no. 2996 and 2695) with Inertsil ODS-3 (250 × 4.6 mm, 5μm) column (PDA detector) was used for the present study. A mixture of Ammonium phosphate buffer solution and Acetonitrile (30: 70, v/v) was used as the mobile phase for the chromatographic study (Flow rate: 1.0 mL/min; Detection wavelength: 243 nm). Stress degradation studies were performed and the method was validated as per ICH guidelines. Keywords: Apalutamide, RP-HPLC, Related substances, Impurities, Stability indicating, Validation, ICH guidelines.

Bioanalytical Method Development and Validation and forced degradation of Sitagliptin and determination of Pharmacokinetic application study in Human Plasma by RP-HPLC method

In the present investigation, an attempt was made for the development, validation, forced degradation and pharmacokinetic application of sitagliptin in the human plasma spiking studies by UV - HPLC method. The experimentation was developed based on the extensive literature survey and ascertained by the statistical parameters of the sampling. A simplified, accurate method was created by the liquid chromatographic system from Shimadazu LC 20AD consisting of manual injection. The optimized chromatogram was obtained with acetonitrile in the isocratic mobile phase method at a 1.0 mL/min flow rate. A thermo C-8 column (4.6X250mm,5μm) was used as a stationary phase, and 265.0nm was selected as the detection wavelength with the aid of a UV-Vis detector. The proposed method was validated as per ICH guidelines. The technique was linear in the range of 10-50μg/mL with correlation coefficient R2 = 0.9746, respectively. Recovery studies postulated that % RSD 19.14, 3 & 9.95 respectively. Injection repeatability values were found to be % RSD 17 & 10.63 for intraday and interday, respectively. Stress degradation studies revealed that sitagliptin degrades more rapidly when subjected to 0.1 NaoH. Human plasma spiking studies reported 3.02 ng/mL at 3.02+/-60 min of C and T max, respectively. Keywords: Sitagliptin, Method development, HPLC, Validation, Stress degradation studies, Human plasma spiking studies

Stability Indicating Method Development and Validation of Glycyrrhizin Using RP-HPLC-DAD: Application to Glycyrrhiza glabra Extract.

Glycyrrhiza glabra is commonly known as licorice. Licorice is the major source of glycyrrhizin. There is no reported stability indicating method for glycyrrhizin in the literature so far. Therefore, it was proposed to develop a stability indicating method and validate the method for glycyrrhizin and its application in G. glabra root extract. Method validation parameters were performed as per the International Council for Harmonization guidelines. The chromatographic separation was achieved on a Zorbax Extended C-18 (250 × 4.6mm, 5μm) column. The separation achieved using the mobile phase consisted of 0.1% formic acid in water and acetonitrile in gradient elution. The flow rate was kept at 1mL/min, and ultraviolet-visible spectroscopy detection was at 250nm. The average retention time of glycyrrhizin was found to be 7.30min. Stress degradation studies were performed and confirmed that only acidic degradation has shown a degradation profile of glycyrrhizin up to 40%. The percentage of glycyrrhizin was found to be 0.40% in the G. glabra extract. This may be further explored for commercial applications.

Forced Degradation Products of Olmesartan Medoxomil and their In Vitro Genotoxicity Assessment by Comet Assay using HEK Cells

AbstractThis study designed to characterize major forced degradation products of Olmesartan Medoxomil (OM) and to perform genotoxicity profiling using acid, base, and hydrogen peroxide‐induced stress degradation studies. The isolation of degradation products was conducted on flash chromatography using FlashPure ECOFLEX C18 4 g column with gradient elution and diode array detection‐based fraction collection, and characterized by mass spectrometry. Three impurities (impurity‐1, 2, and 3) were identified with respective m/z values of 447.213, 461.229, and 463.208. The genotoxic potential of impurities was assessed by in silico prediction, and in vitro comet assay using HEK cells. The extent of DNA damage was analyzed using Comet Assay IV imaging software. For impurity‐1, head and tail lengths and their respective total intensities were significantly (p<0.001) larger than those of dimethyl sulfoxide (solvent) control and comparable to positive control. Other parameters such as mean grey level and total area of comet were statistically significant as compared with the solvent and positive controls. Overall, impurity‐1 was a possible genotoxic impurity and a known major degradation product in OM tablet dosage form. In silico prediction also revealed impurity‐1 as toxicity class 3, whilst impurities 2 and 3 were of toxicity class 4.

Comprehensive Stress Degradation Studies of Fipronil: Identification and Characterization of Major Degradation Products of Fipronil Including Their Degradation Pathways Using High Resolution-LCMS

Comprehensive Stress Degradation Studies of Fipronil: Identification and Characterization of Major Degradation Products of Fipronil Including Their Degradation Pathways Using High Resolution-LCMS

Rapid RP-HPLC Quantification and Stress Degradation Studies on Alogliptin, Saxagliptin, Metformin in Pharmaceutical Dosage Formulation

Rapid RP-HPLC Quantification and Stress Degradation Studies on Alogliptin, Saxagliptin, Metformin in Pharmaceutical Dosage Formulation

Analysis of Tolfenamic Acid using a Simple, Rapid, and Stability-indicating Validated HPLC Method.

Tolfenamic acid (TA) belongs to the fenamates class of nonsteroidal anti-inflammatory drugs. Insufficient information is available regarding the availability of a reliable and validated stability-indicating method for the assay of TA. A relatively simple, rapid, accurate, precise, economical, robust, and stabilityindicating RP-HPLC method has been developed to determine TA in pure and tablet dosage forms. The method was validated according to the ICH guideline, and parameters like linearity, range, selectivity, accuracy, precision, robustness, specificity, and solution stability were determined. TLC and FTIR spectrometry were used to ascertain the purity of TA. The specificity was determined with known impurities and after performing forced degradation, while the robustness was established by Plackett-Burman's experimental design. The mobile phase used for the analysis was acetonitrile and water (90:10, v/v) at pH 2.5. The detection of the active drug was made at 280 nm using a C18 column (tR = 4.3 min.). The method's applicability was also checked for the yellow polymorphic form of TA. The results indicated that the method is highly accurate (99.39-100.80%), precise (<1.5% RSD), robust (<2% RSD), and statistically comparable to the British Pharmacopoeia method with better sensitivity and specificity. It was observed that the stress degradation studies do not affect the method's accuracy and specificity. Hence the proposed method can be used to assay TA and its tablet dosage form.

Method development and validation for the simultaneous estimation of Doultegravir and Rilpivirine related impurities (Rilpivirine Z Isomer and Dolutegravir hydroxy impurity) using RP HPLC

The combination of Doultegravir and Rilpivirine is used to treat human immunodeficiency (HIV) virus. A new stability indicating RP-HPLC method has been proposed for the quantification of Doultegravir and Rilpivirine along with its impurities Rilpivirine Z Isomer and Doultegravir hydroxy impurity using Water HPLC System (PDA detector) and auto sampler integrated with Empower 2 Software with Inertsil (250 × 4.6 mm, 5 μ) C18 column (PDA detector) was used for the present study. A mixture of 0.01N phosphate buffer and acetonitrile (50: 50, v/v) (pH adjusted to 4.8 with TEA and ortho phosphoric acid) was used as mobile phase for the chromatographic study (Flow rate: 1.0 ml/min; Injection volume: 10 μl; Detection wavelength: 257 nm) with run time 12 mins. Stress degradation studies were performed and the method was validated as per ICH guidelines. The developed method was found to be precise, specific, accurate, linear, stable and robust for the quantification of Dolutegravir and Rilpivirine along with its impurities Rilpivirine Z Isomer and Dolutegravir hydroxy impurity and its bulk drug formulation. The developed method can be applied successfully to quality control and for other analytical purposes.

Method validation and characterization of stress degradation products of azelastine hydrochloride using LC-UV/PDA and LC-Q/TOF-MS studies.

Azelastine HCl is a second-generation H1 -receptor antagonist approved by the US Food and Drug Administration (US FDA) for treating seasonal allergic rhinitis and non-allergic vasomotor rhinitis. This study encompasses the validation of a liquid chromatography-ultra violet photo diode array (LC-UV/PDA) method for the drug and its extension to liquid chromatography/quadrupole time-of-flightmassspectrometry (LC-Q/TOF-MS) studies for identification and characterization of various stress degradation products of the drug. Stress degradation of azelastine HCl was undertaken under the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) prescribed conditions of hydrolytic, photolytic, oxidative, and thermal stress. The degraded drug solutions were analyzed using Ultra Performance Liquid Chromatography (UPLC) employing a C18 (100 × 4.6mm; 2.6μ, Kinetex) column by isocratic elution. Detection wavelength was 241 nm. The degradation products were identified and characterized using UPLC-MS/TOF studies, and an attempt was made to isolate one of the degradation products by solvent extraction. The drug was found to significantly degrade under acidic/alkaline/neutral photolytic, oxidative, and alkaline hydrolytic conditions. Six degradation products (I-VI) were identified through LC-Q/TOF-MS studies that were adequately resolved from the drug with the developed UPLC method. All degradation products (I-VI) were ionized in the total ion chromatogram (TIC) in the LC-MS studies, and these were identified and characterized, and the degradation pathway of the drug was postulated. One of the oxidation products isolated from the degraded drug solution was characterized through differential scanning calorimetry, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance spectral data. Six degradation products generated from stress degradation studies on azelastine HCl were adequately resolved through LC-UV/PDA studies followed by method validation. These were successfully identified and characterized through LC-Q/TOF-MS studies, and the degradation pathways for the generation of these products from the drug have been postulated.

DEGRADATION DETERMINATION OF TINOSORB-S IN SUNSCREEN PREPARATIONS

Objectives: The main objective was to determine the Tinosorb-S degradation in the sunscreen preparations with a simple economic linear and specific analytical method. Methods: The RP-HPLC was achieved with 100% methyl alcohol as the mobile phase, a flow rate of 2.5 mL/min., and an octadecylsilane column (300 × 3.9 cm, 10 µ) at 254 nm. Results: The developed analytical method of Tinosorb-S was statistically validated for accuracy and linearity of 70–130 µg/mL. The correlation coefficient was 0.996, and the % RSD of precision was 0.957. The recovery percentage was 100.01–105.04, and the excellent thermal and photolytic stability of Tinosorb-S was confirmed by the thermal, dry, and wet chromatograms compared with the standard; no such degradation was developed, and the recovery was 99.52%, 99.44%, and 99.45%, respectively. Acid, oxidative, and alkaline decomposition of Tinosorb-S were established by screening the decomposition peaks, and the percentage recovery was 41.31%, 40.38%, and 40.44%, respectively. Conclusions: The stress degradation study of Tinosorb-S is important in stability to evaluate the maximum protecting power of skin from harmful UV rays. Conditions like photolytic degradation, exposure to thermal conditions, acid hydrolysis, oxidation, and alkali hydrolysis were studied with a cost-effective, specific, linear, high-performance liquid chromatographic method with a photodiode array detector.

Stress degradation study of Etoricoxib, isolation, and characterization of major degradation impurity by preparative high‐performance liquid chromatography, liquid chromatography‐mass spectrometry, and nuclear magnetic resonance: Validation of ultra‐performance liquid chromatography method

AbstractEtoricoxib (ETO) is a selective cyclooxygenase‐2 inhibitor, used as a nonsteroidal anti‐inflammatory drug. In this study, a detailed investigation was conducted on the degradation behavior of ETO under acidic, basic, neutral, photolytic, oxidative, and thermal degradation conditions as per the International Conference on Harmonization (ICH) guideline Q1A (R2). A gradient “ultra‐performance liquid chromatography (UPLC)” method was developed for separating the degradation products formed under various degradation conditions along with process‐related impurities. Acquity BEH phenyl column was used for the separation of analytes and 0.1% formic acid in water and 0.05% formic acid in acetonitrile:methanol (8:2 %v/v) were used as mobile phase‐A and mobile phase‐B. A major degradant namely, N‐oxide impurity was observed in oxidative degradation along with two minor degradants where N‐oxide was confirmed based on LC‐mass spectrometry (LC‐MS) studies. Further, the major degradant was enriched and isolated using preparative HPLC and the conformation of the N‐oxide impurity was established using nuclear magnetic resonance techniques including two‐dimensional studies and high‐resolution MS. In addition, the method for related substances by UPLC has been validated by considering a major degradant, namely N‐oxide along with process‐related impurities as per the ICH guidelines validation parameters.

Stability Indicating UV Spectrophotometric Method for Loxoprofen Sodium: Method Development and Validation

Loxoprofen sodium was analyzed using UV spectrophotometry, and the method was designed and validated in the present study in accordance with ICH Q2 recommendations. Loxoprofen sodium’s stress degradation behavior was investigated using a newly discovered technique. Analysis was carried out at 223 nm by using distilled water as solvent. Stress degradation of drug was studied at acidic, basic and oxidative conditions. With a correlation coefficient of 0.999, linearity was found between 5 and 25 μg/mL. Intraday and interday precision studies showed RSD values of &lt;0%, demonstrating the accuracy of the presented approach. Results obtained for LoD and LoQ are 0.012 and 0.037 μg/mL, respectively. For the ruggedness study, the absorbance value of six replicates was found to be 0.477 ± 0.004 with %RSD &gt; 2.0% states that the developed method was found to be rugged. During the stress degradation study, it was observed that the drug is susceptible to basic and oxidative conditions with degradation of more than 10%.

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.

A new stability indicating HPLC and LC-APCI-MS methods for the estimation of Flucytosine in pharmaceutical dosage forms

Flucytosine is an anti-fungal agent which acts against mycotic infections. The anti-mycotic activity of Flucytosine is due to its conversion in to 5-fluoro uracil. In the present study the authors have proposed a new stability indicating isocratic RP-HPLC as well as LC-APCI-MS methods for the estimation of Flucytosine and the methods are validated as per ICH guidelines. Thermo scientific - TSQ Quantis with Vanquish HPLC coupled with MS was employed for the study with Simpack C18 (250 mm x 4.6 mm x 5µm) column. A triple quadrupole mass spectrometer with atmospheric pressure chemical ionization (APCI) source, running in the positive mode (as well as negative mode) was used for the detection. A mixture of 0.1% Acetic acid: Methanol was used as mobile phase (50:50, v/v) (UV detection at 235 nm). A wide linearity concentration range 1.0-120 μg/ml was observed and the method is found to be precise and accurate and can be used for the estimation of marketed formulations. Stress degradation studies were performed and the method is found to be selective and specific.

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.