Application of a validated stability-indicating densitometric thin-layer chromatographic method to stress degradation studies on moxifloxacin

A simple, sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for densitometric determination of moxifloxacin both as a bulk drug and from pharmaceutical formulation was developed and validated as per the International Conference on Harmonization (ICH) guidelines. The method employed TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase and the mobile phase consisted of n-propanol-ethanol-6 M ammonia solution (4:1:2, v/v/v). Densitometric analysis of moxifloxacin was carried out in the absorbance mode at 298 nm. Compact spots for moxifloxacin were found at R_f value of 0.58 ± 0.02. The linear regression analysis data for the calibration plots showed good linear relationship with r = 0.9925 in the working concentration range of 100-800 ng spot⁻¹. The method was validated for precision, accuracy, ruggedness, robustness, specificity, recovery, limit of detection (LOD) and limit of quantitation (LOQ). The LOD and LOQ were 3.90 and 11.83 ng spot⁻¹, respectively. Drug was subjected to acid and alkali hydrolysis, oxidation, dry heat, wet heat treatment and photodegradation. All the peaks of degradation products were well resolved from the standard drug with significantly different R_f values. Statistical analysis proves that the developed HPTLC method is reproducible and selective. As the method could effectively separate the drug from its degradation products, it can be employed as stability-indicating one. Moreover, the proposed HPTLC method was utilized to investigate the kinetics of the acidic and alkaline degradation processes at different temperatures. Arrhenius plot was constructed and apparent pseudo-first-order rate constant, half-life and activation energy were calculated. In addition the pH-rate profile for degradation of moxifloxacin in constant ionic strength buffer solutions within the pH range 1.2-10.8 was studied.     

Validated stability-indicating densitometric thin-layer chromatography: application to stress degradation studies of minocycline

A simple, stability-indicating high-performance thin-layer liquid chromatographic (HPTLC) method for analysis of minocycline was developed and validated. The densitometric analysis was carried out at 345 nm using methanol-acetonitrile-isopropyl alcohol-water (5:4:0.5:0.5, v/v/v/v) as mobile phase.The method employed TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase. To achieve good result, plates were sprayed with a 10% (w/v) solution of disodium ethylene diaminetetraacetic acid (EDTA), the pH of which was adjusted to 9.0. Compact spots of minocycline were found at R_f = 0.30 ± 0.02. For proposed procedure, linearity (r = 0.9997), limit of detection (3.7 ng spot⁻¹), recovery (99.23-100.16%), and precision (% R.S.D. ≤ 0.364) was found to be satisfactory. The drug undergoes acidic and basic degradation, oxidation and photodegradation. All the peaks of degradation products were well resolved from the pure drug with significantly different R_f values. The acidic and alkaline degradation kinetics of minocycline, evaluated using this method, is found to be of first order.     

Stability‐indicating HPTLC method for quantitative estimation of silybin in bulk drug and pharmaceutical dosage form

In the present study a novel stability‐indicating high‐performance thin‐layer chromatography (HPTLC) method for quantitative determination of silybin in bulk drug and nanoemulsion formulation has been developed and validated on silica using solvent chloroform–acetone–formic acid (9 : 2 : 1 v/v/v) (R_f of silybin 0.46 ± 0.05) in the absorbance mode at 296 nm. The method showed a good linear relationship (r² ± 0.999) in the concentration range 25–1500 ng per spot. It was found to be linear, accurate, precise, specific, robust and stability‐indicating and can be applied for quality control and standardization of several multi‐component hepatoprotective formulations as well as for stability testing of different dosage forms. The method proposed was also used to investigate the kinetics of acidic and alkaline degradation processes by quantification of drug at different temperature to calculate the activation energy and half‐life for silymarin degradation. Copyright © 2009 John Wiley & Sons, Ltd     

Stability indicating HPTLC determination of clopidogrel bisulphate as bulk drug and in pharmaceutical dosage form

A sensitive, selective, precise and stability indicating high-performance thin layer chromatographic method of analysis of clopidogrel bisulphate both as a bulk drug and in formulations was developed and validated in pharmaceutical dosage form. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of carbon tetrachloride-chloroform-acetone (6:4:0.15, v/v/v). This system was found to give compact spots for clopidogrel bisulphate (R_f value of 0.30±0.01). Clopidogrel bisulphate was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. Also the degraded products were well separated from the pure drug. Densitometric analysis of clopidogrel bisulphate was carried out in the absorbance mode at 230 nm. The linear regression data for the calibration plots showed good linear relationship with r²=0.999±0.001 in the concentration range of 200-1000 ng. The mean value of correlation coefficient, slope and intercept were 0.999±0.001, 0.093±0.011 and 8.83±0.99, respectively. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 40 and 120 ng per spot, respectively. The drug undergoes degradation under acidic and basic conditions, oxidation and dry heat treatment. All the peaks of degraded product were resolved from the standard drug with significantly different R_f values. This indicates that the drug is susceptible to acid-base hydrolysis, oxidation and dry heat degradation. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

Stability-Indicating TLC Method for the Determination of Dutasteride in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability-indicating thin-layer chromatographic method for determination of dutasteride both as a bulk drug and as pharmaceutical tablets was developed and validated as per the International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates precoated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile:methanol:dichloromethane in the ratio of 2.0:1.0:2.0, v/v/v. This solvent system was found to give compact spots for dutasteride (R _f value of 0.64 ± 0.02). Densitometric analysis of dutasteride was carried out in the absorbance mode at 244 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r = 0.9943 with respect to peak area in the concentration range of 100–600 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 7.54 and 22.85 ng per band, respectively. Dutasteride was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug undergoes degradation under acidic, basic conditions, photolytic, oxidative and upon wet and dry heat treatment. The degraded products were well separated from the pure drug. The statistical analysis proves that the developed method for quantification of dutasteride as bulk drug and from pharmaceutical tablets is reproducible and selective. As the method could effectively separate the drug from its degradation products, it can be employed as stability-indicating.     

Determination of Oxazepam in Pharmaceutical Formulation by HPTLC UV-Densitometric and UV-Derivative Spectrophotometry Methods

Abstract

Methods for determination of oxazepam in pharmaceutical formulation by derivative ultraviolet (UV) spectrophotometry as well as high-performance thin-layer chromatography (HPTLC) UV densitometry were described. For UV-derivative spectrophotometry, some derivatives and wavelengths may be recommended for routine quality control of the drug of interest. On the other hand, HPTLC provided good results, but only when the calibration curve was estimated using nonlinear regression analysis. The HPTLC method was developed with silica F₂₅₄ plates, a mobile phase of benzene/ethanol (5:1, v/v), and densitometric detection at 204 nm receiving R _f  = 0.47. Developed methods were validated and found to be sufficiently precise and reproducible for established conditions.     

Study of gliquidone degradation behavior by high‐performance thin‐layer chromatography and ultra‐performance liquid chromatography methods

Gliquidone (GQ) is an oral hypoglycemic agent, belonging to second‐generation sulfonylurea derivatives. New high‐performance thin‐layer chromatography (HPTLC) and ultra‐performance liquid chromatography (UPLC) methods have been developed and validated and used for complete stability study of GQ following International Conference on Harmonization guidelines. GQ was subjected to stress and forced degradation under hydrolytic, oxidative and photolytic conditions. The drug was found to be unstable under acidic, alkaline and oxidative conditions with the formation of gliquidone sulfonamide (GQS), while a marked stability was confirmed under thermal and photolytic stress conditions. GQS is the British pharmacopeial impurity A of GQ and also considered as its synthesis intermediate. The developed chromatographic methods have been utilized for anticipating the degradation behavior of GQ under the studied conditions and then used for quantitation of GQ and GQS either in their pure forms or in laboratory prepared mixtures. The methods were successfully applied to GQ in pharmaceutical formulation. The methods have the advantages of being sensitive and less time consuming compared with the reported methods. The obtained results were statistically compared with a reported HPLC method showing no significant difference regarding both accuracy and precision.     

Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product

Accurate, selective, sensitive and precise HPTLC‐densitometric and RP‐HPLC methods were developed and validated for determination of bumadizone calcium semi‐hydrate in the presence of its alkaline‐induced degradation product and in pharmaceutical formulation. Method A uses HPTLC‐densitometry, depending on separation and quantitation of bumadizone and its alkaline‐induced degradation product on TLC silica gel 60 F₂₅₄ plates, using hexane–ethyl acetate–glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP‐HPLC separation of bumadizone and its alkaline‐induced degradation product using a mobile phase consisting of methanol–water–acetonitrile (20:30:50, v/v/v) on a Phenomenex C₁₈ column at a flow‐rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method. Copyright © 2011 John Wiley & Sons, Ltd.     

Stability-indicating high-performance thin-layer chromatographic determination of levonorgestrel and ethinyloestradiol in bulk drug and in low-dosage oral contraceptives

A stability-indicating high-performance thin-layer chromatography (HPTLC) method was developed and validated for simultaneous determination of steroidal hormones levonorgestrel and ethinyloestradiol both in bulk drug and in low-dosage oral contraceptives. Optimization of conditions for the spectrodensitometric procedure was reached by eluting HPTLC silica gel plates in a 10 cm × 10 cm horizontal chamber. The solvent system consisted of hexane-chloroform-methanol (1.0:3.0:0.25, v/v/v). This system was found to give compact, dense and typical peaks for both levonorgestrel (R_f = 0.65 ± 0.03) and ethinyloestradiol (R_f = 0.43 ± 0.02). Densitometric analysis of the drugs was carried out in the reflectance mode at 225 nm by using a computer controlled densitometric scanner. The calibration curves of levonorgestrel and ethinyloestradiol were linear in the range of 200-800 and 40-160 ng per spot, respectively. The method was validated for precision, robustness and recovery. As the proposed method can effectively separate the drugs from their degradation products, it can be employed as a stability-indicating method.     

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.     

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.

     

Chromatographic methods development,validation and degradation characterization of the antithyroid drug Carbimazole

The current paper reports the development and validation of stability‐indicating HPLC and HPTLC methods for the separation and quantification of main impurity and degradation product of Carbimazole. The structures of the degradation products formed under stress degradation conditions, including hydrolytic and oxidative, photolytic and thermal conditions, were characterized and confirmed by MS and IR analyses. Based on the characterization data, the obtained degradation product from hydrolytic conditions was found to be methimazole—impurity A of Carbimazole as reported by the British Pharmacopeia and the European Pharmacopeia. A stability‐indicating HPLC method was carried out using a Zorbax Eclipse Plus CN column (150 × 4.6 mm i.d, 5 μm particle size) and a mobile phase composed of acetonitrile–0.05 m KH₂PO₄ (20: 80, v/v) in isocratic elution, at a flow rate of 1 mL/min. The method was proved to be sensitive for the determination down to 0.5% of Carbimazole impurity A. Additionally, a stability‐indicating chromatographic HPTLC method was achieved using cyclohexane–ethanol (9:1, v/v) as a developing system on HPTLC plates F₂₅₄ with UV detection at 225 nm. The proposed HPLC and HPTLC methods were successfully applied to Carbimazole^® tablets with mean percentage recoveries of 100.12 and 99.73%, respectively.     

Stability-Indicating HPTLC Method for the Determination of Tamsulosin in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method was developed for the determination of tamsulosin (TAM) in bulk and tablet formulation. Validation was carried out in compliance with International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates pre-coated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile/methanol/dichloromethane (2.0: 1.0: 2.0, v/v/v). This solvent system was found to give compact spots for tamsulosin (R _f = 0.27 ± 0.02). Densitometric analysis of TAM was carried out in the absorbance mode at 286 nm. Linear regression analysis showed good linearity (r ² = 0.9993) with respect to peak area in the concentration range of 300–800 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. Limits of detection and quantitation were 8.49 and 25.72 ng per band, respectively. TAM was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug underwent degradation under acidic, basic and photolytic conditions. The degraded products were well separated from the pure drug. Statistical analysis proved that the developed method, used for quantification of TAM as a bulk drug and present in pharmaceutical tablets, was reproducible and selective.     

Stability-Indicating HPTLC Method for the Determination of Tamsulosin in Pharmaceutical Dosage Forms

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method was developed for the determination of tamsulosin (TAM) in bulk and tablet formulation. Validation was carried out in compliance with International Conference on Harmonization guidelines. The method employed thin-layer chromatography aluminium plates pre-coated with silica gel 60F₂₅₄ as the stationary phase and the mobile phase consisted of acetonitrile/methanol/dichloromethane (2.0: 1.0: 2.0, v/v/v). This solvent system was found to give compact spots for tamsulosin (R _f = 0.27 ± 0.02). Densitometric analysis of TAM was carried out in the absorbance mode at 286 nm. Linear regression analysis showed good linearity (r ² = 0.9993) with respect to peak area in the concentration range of 300–800 ng per band. The method was validated for precision, accuracy, ruggedness and recovery. Limits of detection and quantitation were 8.49 and 25.72 ng per band, respectively. TAM was subjected to acid and alkali hydrolysis, oxidation, photo degradation, dry heat and wet heat treatment. The drug underwent degradation under acidic, basic and photolytic conditions. The degraded products were well separated from the pure drug. Statistical analysis proved that the developed method, used for quantification of TAM as a bulk drug and present in pharmaceutical tablets, was reproducible and selective.

     

Development and validation of a TLC-densitometry method for quantitative analysis of nefopam hydrochloride beside its degradation products

A quantitative densitometric thin-layer chromatographic method for determination of nefopam hydrochloride in pharmaceutical preparations has been established and validated. Nefopam from the formulations was separated and identified on silica gel 60 F₂₅₄ TLC plates with chloroform-methanol-glacial acetic acid (9: 2: 0.1, v/v/v) as mobile phase. Densitometric quantification was performed at absorbance maximum 266 nm. The method was validated for linearity, sensitivity, precision and recovery in accordance with ICH guidelines. The presented method is selective and specific with potential application in pharmaceutical analysis. Nefopam hydrochloride was subjected to acidic and alkaline hydrolysis at different temperatures. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

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.     

Study of forced degradation behavior of idrocilamide and development of stability indicating LC method

The objective of this study was to report the stability profile of novel muscle relaxant drug idrocilamide (Idr) based on information obtained from forced degradation studies. The drug was subjected to acidic (1 M HCl) and alkaline (1 M NaOH) hydrolysis and oxidative decomposition (50% H₂O₂). The products formed under different stress conditions were investigated by LC. The LC method was fine tuned using the samples generated from forced degradation studies. Satisfactory resolution between peaks with the shortest possible analysis time was achieved on C₁₈ 5 μm column (Luna, Phenomenex, USA), with mobile phase methanol-acetonitrile-water-glacial acetic acid (25: 30: 44: 1, v: v: v: v), pumped at 1 mL/min flow rate. Quantification was achieved at 280 nm based on peak area, using DAD detector. The proposed LC method was utilized to investigate the accelerated oxidative degradation of Idr. Besides, Idr’s degradants were identified using IR and MS, and the possible degradation pathway was outlined. The proposed method was validated, and the forced degradation studies proved the stability indicating power of the method. The method was also applied to analyze commercial samples.     

Stability-Indicating HPTLC Method for Analysis of Moclobemide, and Use of the Method to Study Degradation Kinetics

A sensitive, selective, precise, and stability-indicating HPTLC method for analysis of moclobemide in the bulk drug and in formulations has been established and validated. Aluminium TLC plates precoated with silica gel 60 F₂₅₄ were used with benzene–methanol–40% ammonia 7:3:0.1 (v/v) as mobile phase. Densitometric analysis was performed in absorbance mode at 238 nm. Compact bands were obtained for moclobemide (R _F 0.67 ± 0.02; n = 6). The drug was subjected to acidic and alkaline hydrolysis, oxidation, dry heat treatment, and photodegradation. The drug undergoes degradation under acidic, basic, and oxidising conditions. The degradation products were well resolved from the pure drug with significantly different R _F values, so the method can be regarded as stability-indicating. Response to moclobemide was a linear function of amount in the range 50–600 ng per band, with a correlation coefficient, r ², of 0.9967 ± 0.51. LOD and LOQ, determined experimentally, were 10 and 30 ng per band, respectively. Statistical analysis proves the method is repeatable and specific for analysis of moclobemide. The method was used to investigate the kinetics of alkaline degradation. The Arrhenius plot was constructed and the activation energy calculated.     

Diode array detection for stability assessment and evaluation of degradation kinetics of newly introduced sacubitril in its supramolecular complex (LCZ696) with valsartan

A newly FDA-approved heart failure therapy (LCZ696, supramolecular complex of sacubitril (SAC) and valsartan (VAL), Entresto?) is analyzed with a stability indicating HPLC–DAD method. For the newly introduced drug, SAC, there is no sufficient information about its stability under various stress conditions. The proposed chromatographic method was applied to the kinetic investigation of the acidic, alkaline, and oxidative degradation of SAC with the estimation of its activation energy and half-life at room temperature by the aid of Arrhenius plots. Kinetic investigation was conducted using either different strengths of HCl, NaOH, and H₂O₂ at one selected temperature or different temperature degrees at one selected reagent strength, for the acidic, alkaline, and oxidative stress conditions. It was found that the pseudo-first-order kinetics was followed at each case. The half-lives at room temperature using 0.1?M HCl, 0.01?M NaOH, and 15% H₂O₂ were found to be 20.50, 2.76, and 51.58?hr. The chromatographic method was achieved using Zorbax Eclipse plus-C18 (4.6?×?250?mm, 5?µm) with isocratic elution of mobile phase composed of acetonitrile and 0.025?M phosphate buffer (pH3) in a ratio of 60:40 (v/v).     

Optimization and validation of a stability-indicating RP-HPLC method for determination of azithromycin and its related compounds

A validated stability-indicating HPLC method was developed for the analysis of azithromycin (AZ) and its related compounds in raw materials, capsule, and suspension using an Xterra RP C18 column at 50 degrees C with UV detection at 215 nm. Isocratic elution was employed using the mobile phase 14 mM disodium hydrogen phosphate (pH 10.5, adjusted by 1 M NaOH)-methanol-acetonitrile-tetrahydrofuran (40.0 + 30.0 + 30.0 + 0.1, v/v/v/v). AZ and 14 of its related compounds were separated and quantified. The described method was linear over the range of 2-1800 microg/mL AZ with (r = 0.9999). The stability of AZ was studied under accelerated acidic, alkaline, and oxidative conditions. The proposed method was used to investigate the kinetics of acidic and alkaline hydrolysis process of AZ at different temperatures, and the apparent pseudo first-order rate constant, half-life, and activation energy were calculated. The major peak detected from the degradation of AZ in alkaline and acidic conditions was decladinosylazithromycine, while azithromycin N-oxide was detected from the oxidative degradation. Long-term stability studies for capsule and oral suspension were carried out. The proposed stability-indicating method was completely validated according to the U.S. Food and Drug Administration requirements.