Stability-Indicating HPLC Method for the Determination of Cefcapene Pivoxil

The stability-indicating LC assay method was developed and validated for quantitative determination of cefcapene pivoxil in the presence of degradation products formed during forced degradation studies. An isocratic RP-HPLC method was developed with a Lichrospher RP-18 (250 mm × 4.6 mm, 5 μm) column and the mobile phase composed of 45 volumes of acetonitrile and 55 volumes of mixture composed of citric acid 10 mmol L^?1 and potassium chloride 18 mmol L^?1. The flow rate of the mobile phase was 1 mL min^?1. Detection wavelength was 270 nm and temperature was 30 °C. Cefcapene pivoxil, similar to other cephalosporins, was subjected to stress conditions of degradation in aqueous solutions including hydrolysis, oxidation, and thermal degradation. The method was validated with regard to linearity, accuracy, precision, selectivity, and robustness. The method was applied successfully for the determination of cefcapene pivoxil during kinetic studies in aqueous solutions (pH and thermal degradation) and in solid state (oxidative, thermal, and radiolytic degradation).     

LC Method for Studies on the Stability of Sibutramine in Soft Gelatin Capsules

A sensitive and rapid liquid chromatographic method was successfully developed and validated for the determination of sibutramine hydrochloride in bulk and capsules. Sibutramine in the presence of its degradation products was analyzed using UV detection at 225 nm. Chromatography was performed on a reversed-phase C₈ (150 × 4.0 mm I.D., 5 μm) analytical column under isocratic conditions. The mobile phase was composed of acetonitrile:water (aqueous phase containing 0.3% triethylamine and pH adjusted to 7.0) (75:25, v/v) at a flow-rate of 1.1 mL min⁻¹. No chromatographic interference was found during the analysis. Light was the stress condition which most contributed to sibutramine degradation. The method showed a linear response (r > 0.999) from 30 to 90 μg mL⁻¹. The mean recovery for capsules was 101.2%. Inter-day assays showed relative standard deviations of 0.42 and 1.62% for bulk and capsules, respectively. The developed method is able to separate sibutramine from its major degradation products and it may be used in the quality control of this active pharmaceutical ingredient in both bulk and capsules.

     

Stability-Indicating LC Method for the Determination of Olmesartan in Bulk Drug and in Pharmaceutical Dosage Form

A novel stability-indicating LC assay method was developed and validated for quantitative determination of olmesartan in bulk drugs and in pharmaceutical dosage form in the presence of degradation products generated from forced degradation studies. An isocratic, reversed phase LC method was developed to separate the drug from the degradation products, using an Ace5-C18 (250 mm × 4.6 mm, 5 μm) column, and 50 mM ammonium acetate (pH-5.5 by acetic acid) and acetonitrile (70:30 v/v) as a mobile phase. The detection was carried out at the wavelength of 235 nm. The olmesartan was subjected to stress conditions of hydrolysis (acid, base), oxidation, photolysis and thermal degradation. Degradation was observed for olmesartan in acid, base and in 30% H₂O₂ conditions. The drug was found to be stable in the other stress conditions attempted. The degradation products were well resolved from the main peak. The percentage recovery of olmesartan ranged from (99.89 to 100.95%) in pharmaceutical dosage form. The developed method was validated with respect to linearity, accuracy (recovery), precision, specificity and robustness. The forced degradation studies prove the stability-indicating power of the method.

     

An Approach to Transfer Methods from HPLC to UHPLC Techniques in Some Carbapenems

Stability-indicating LC methods were developed and validated for the quantitative determination of doripenem, meropenem and tebipenem in the presence of their degradation products formed during forced degradation studies. Isocratic HPLC and UHPLC separations were performed with a core–shell Kinetex 1.7, 2.6 and 5 µm, all C18, 100A, 100 × 2.1 mm columns and the mobile phase composed of acetonitrile and 12 mmol L⁻¹ ammonium acetate in different ratios. The flow rates of the mobile phase were: 0.5 mL min⁻¹ for 1.7 µm column, and 1.0 mL min⁻¹ for 2.6 and 5 µm ones. Detection wavelength was 298 nm and temperature was set at 30 °C. All analysed drugs were exposed to stress conditions which caused their hydrolysis and thermal degradation. The methods were validated by evaluation of linearity, accuracy, precision, selectivity and robustness. Proposed methods were successfully applied for the determination of investigated antibiotics during kinetic studies in aqueous solutions and in the solid state. The advantages of chromatographic procedures which are based on the use of C18 stationary phases with different particle sizes in the analysis of selected carbapenems were discussed.

     

A Stability-Indicating LC Assay and Degradation Behavior of Temozolomide Drug Substances

This study deals with a stability indicating HPLC reverse phase method for quantitative determination of temozolomide. A chromatographic separation was achieved on an Inertsil ODS 3V, 250 × 4.6 mm ID, 5 μm column using mobile phase A (buffer 5 mL glacial acetic acid in 1,000 mL of Milli Q water ) and mobile phase B (methanol). Forced degradation studies were performed on bulk sample of temozolomide using acid (0.5 N hydrochloric acid), base (0.5 N sodium hydroxide), oxidation (10% v/v hydrogen peroxide), heat (60 °C) and UV light (254 nm). Degradation of the drug substance was observed in base hydrolysis and oxidation. Degradation product formed under these conditions was found to be Imp-A. When the stress samples were assayed, the mass balance was close to 99.5%. The sample solution was stable up to 48 h at 5 °C and mobile phase was found to be stable up to 48 h at 25 °C. The developed method was validated with respect to linearity, accuracy, precision, robustness and forced degradation studies prove the stability indicating power of the method.     

LC Method for Studies on the Stability of Sibutramine in Soft Gelatin Capsules

A sensitive and rapid liquid chromatographic method was successfully developed and validated for the determination of sibutramine hydrochloride in bulk and capsules. Sibutramine in the presence of its degradation products was analyzed using UV detection at 225 nm. Chromatography was performed on a reversed-phase C₈ (150 × 4.0 mm I.D., 5 μm) analytical column under isocratic conditions. The mobile phase was composed of acetonitrile:water (aqueous phase containing 0.3% triethylamine and pH adjusted to 7.0) (75:25, v/v) at a flow-rate of 1.1 mL min⁻¹. No chromatographic interference was found during the analysis. Light was the stress condition which most contributed to sibutramine degradation. The method showed a linear response (r > 0.999) from 30 to 90 μg mL⁻¹. The mean recovery for capsules was 101.2%. Inter-day assays showed relative standard deviations of 0.42 and 1.62% for bulk and capsules, respectively. The developed method is able to separate sibutramine from its major degradation products and it may be used in the quality control of this active pharmaceutical ingredient in both bulk and capsules.     

Stability-Indicating LC Determination of Nitazoxanide in Bulk Drug and in Pharmaceutical Dosage Form

A novel stability-indicating high-performance liquid chromatographic assay method was developed and validated for quantitative determination of nitazoxanide in bulk drugs and in pharmaceutical dosage form in the presence of degradation products generated from forced decomposition studies. An isocratic, reversed phase LC method was developed to separate the drug from the degradation products, using an Ace5- C18 (250 mm × 4.6 mm, 5 μm) column, and 50 mM ammonium acetate (pH 5.5 by acetic acid) and acetonitrile (55:45 v/v) as a mobile phase. The detection was carried out at a wavelength of 240 nm. The nitazoxanide was subjected to stress conditions of hydrolysis (acid, base), oxidation, photolysis and thermal degradation. Degradation was observed for nitazoxanide in base, acid and in 30% H₂O₂ conditions. The drug was found to be stable in the other stress conditions attempted. The degradation products were well resolved from the main peak. The percentage recovery of nitazoxanide was from (100.55 to 101.25%) in the pharmaceutical dosage form. The developed method was validated with respect to linearity, accuracy (recovery), precision, system suitability, specificity and robustness. The forced degradation studies prove the stability indicating power of the method.     

Simultaneous LC–MS–MS Determination of Zolmitriptan and Its Active Metabolite N-Desmethylzolmitriptan in Human Plasma

A specific and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for the determination of zolmitriptan and N-desmethylzolmitriptan in human plasma. The analytes and the internal standard (IS) paroxetine were extracted by liquid–liquid extraction with a mixture of saturated ethyl acetate:dichloromethane (4:1) and were separated using an isocratic mobile phase on a XTerra RP18 column. The mobile phase used was acetonitrile: 5 mM ammonium acetate: formic acid (50:50:0.053, v/v/v). Zolmitriptan and N-desmethylzolmitriptan in a range of 0.25–20 ng mL⁻¹ were easily quantified. The validated method can be applied to pharmacokinetic and bioequivalence studies.

     

Stress Degradation Behavior of Desipramine Hydrochloride and Development of Suitable Stability-Indicating LC Method

A simple reverse phase liquid chromatographic method was developed for the quantitative determination of desipramine hydrochloride and its related impurities in bulk drugs which is also stability-indicating. During the forced degradation at hydrolysis, oxidative, photolytic and thermal stressed conditions, the degradation results were only observed in the oxidative stress condition. The blend of the degradation product and potential impurities were used to optimize the method by an YMC Pack Pro C₁₈ stationary phase. The LC method employs a linear gradient elution with the water–acetonitrile–trifluoroacetic acid as mobile phase. The flow rate was 1.0 mL min⁻¹ and the detection wavelength 215 nm. The stressed samples were quantified against a qualified reference standard and the mass balance was found close to 99.0% (w/w) when the response of the degradant was considered to be equal to the analyte (i.e. desipramine). The developed RP-LC method was validated in agreement with ICH requirements.

     

Development and validation of a novel RP-HPLC method for stability-indicating assay of Abiraterone acetate

Abiraterone acetate is a prodrug of Abiraterone widely used for the treatment of metastatic castration resistant prostate cancer. In this study, a simple, sensitive, and rapid stability-indicating reverse phase HPLC method was developed and validated for the determination of Abiraterone acetate in bulk and its pharmaceutical formulation. The method was developed by HPLC using a Hypersil ODS C-18 (150 mm × 4.6 mm, 5 µm) column in a isocratic mode with mobile phase constituted by potassium phosphate buffer and acetonitrile (40:60, v/v%) flow rate was 1.0 mL min^?1, column temperature of 30°C, UV detection wavelength 235 nm, and injection volume of 20 µL. The validated parameters were in accordance with FDA and ICH specifications, assay exhibited a linear range of 25–250 µg mL^?1 with regression (r²) coefficient 0.9998. The limits of detection and quantification were 0.23 and 0.70 µg mL. Accuracy was between 99.34 and 100.07%. The drug was subjected to various stress conditions like acidic, base hydrolysis, oxidation, thermal, and photolytic degradation. Stress study Abiraterone acetate was found susceptible to degrade under hydrolytic (acid and base) conditions. The proposed method has stability indicating the resolution of the main peak from their degradation peaks. The validated method is suitable for quality control application and reduced analysis time.     

Favipiravir (SARS-CoV-2) degradation impurities: Identification and route of degradation mechanism in the finished solid dosage form using LC/LC–MS method

Favipiravir finished dosage was approved for emergency use in many countries to treat SARS-CoV-2 patients. A specific, accurate, linear, robust, simple, and stability-indicating HPLC method was developed and validated for the determination of degradation impurities present in favipiravir film-coated tablets. The separation of all impurities was achieved from the stationary phase (Inert sustain AQ-C18, 250 × 4.6 mm, 5-μm particle) and mobile phase. Mobile phase A contained KH₂PO₄ buffer (pH 2.5 ± 0.05) and acetonitrile in the ratio of 98:2 (v/v), and mobile phase B contained water and acetonitrile in the ratio of 50:50 (v/v). The chromatographic conditions were optimized as follows: flow rate, 0.7 mL/min; UV detection, 210 nm; injection volume, 20 μL; and column temperature, 33°C. The proposed method was validated per the current International Conference on Harmonization Q2 (R1) guidelines. The recovery study and linearity ranges were established from the limit of quantification to 150% optimal concentrations. The method validation results were found to be between 98.6 and 106.2% for recovery and r² = 0.9995–0.9999 for linearity of all identified impurities. The method precision results were achieved below 5% of relative standard deviation. Forced degradation studies were performed in chemical and physical stress conditions. The compound was sensitive to chemical stress conditions. During the study, the analyte degraded and converted to unknown degradation impurities, and its molecular mass was found using the LC–MS technique and established degradation pathways supported by reaction of mechanism. The developed method was found to be suitable for routine analysis of research and development and quality control.     

Stability Indicating LC Assay Method for the Determination of Famciclovir in Bulk Drug and Pharmaceutical Dosage Forms

A simple isocratic stability indicating LC method was developed and validated for the determination of famciclovir in bulk drug and pharmaceutical dosage form. A mixture of 0.05 M potassium dihydrogen orthophosphate buffer and acetonitrile (80:20 v/v) was used as mobile phase at a flow rate of 1.0 mL min^?1. Hypersil BDS C18 (250 mm × 4.6 mm × 5 μm) column was used and the eluents were monitored at 220 nm. Forced degradation studies were performed for famciclovir active substance, reconstituted matrix and 500 mg tablets using the parameters like acid, base, peroxide, temperature, light, and relative humidity. Peak purity index was checked using PDA detection to demonstrate the specificity and stability indicating nature of the method. The developed method was validated for precision, ruggedness, linearity, LOD, LOQ, range, robustness and accuracy. The developed method can be used for regular quality control and stability study applications of famciclovir bulk drug and tablet dosage forms.     

A Validated Stability Indicating RP-LC Method for Nitazoxanide, a New Antiparasitic Compound

The present paper describes stability indicating reverse phase high-performance liquid chromatography (RP-HPLC) assay method for nitazoxanide in bulk drugs. The developed method is also applicable for the related substances determination in bulk drug. The drug substance was subjected to stress conditions of hydrolysis, photolysis and thermal degradation. The considerable degradation of nitazoxanide was observed under base and peroxide hydrolysis. The drug was found to be stable in other stress conditions attempted. The chromatographic separation of the drug was achieved on reversed-phase C-18 column. Eluents were monitored on photo-diode array detector at a wavelength of 240 nm. The mobile phase was aqueous 0.005 M tetra butyl ammonium hydrogen sulphate and acetonitrile (45:55, v/v). In the developed HPLC method, resolution between nitazoxanide and its potential impurities, namely Imp-A (5-nitro-1,3-thiazol-2-amine), Imp-B (N-(5-nitro-1,3-thiazol-2-yl) acetamide) and Imp-C (2-{[(5-nitro-1,3-thiazol-2-yl) amino] carbonyl} phenyl 2-(acetyloxy) benzoate) was found greater than three. The developed RP-HPLC method was validated with respect to response function, accuracy, precision, specificity, stability of analytical solutions and robustness. Also to determine related substances and assay determination of nitazoxanide that can be used to evaluate the quality of regular production samples. The developed method can also be conveniently used for the assay determination of nitazoxanide in pharmaceutical formulations.     

An Approach to Transfer Methods from HPLC to UHPLC Techniques in Some Carbapenems

Stability-indicating LC methods were developed and validated for the quantitative determination of doripenem, meropenem and tebipenem in the presence of their degradation products formed during forced degradation studies. Isocratic HPLC and UHPLC separations were performed with a core–shell Kinetex 1.7, 2.6 and 5 µm, all C18, 100A, 100 × 2.1 mm columns and the mobile phase composed of acetonitrile and 12 mmol L^?1 ammonium acetate in different ratios. The flow rates of the mobile phase were: 0.5 mL min^?1 for 1.7 µm column, and 1.0 mL min^?1 for 2.6 and 5 µm ones. Detection wavelength was 298 nm and temperature was set at 30 °C. All analysed drugs were exposed to stress conditions which caused their hydrolysis and thermal degradation. The methods were validated by evaluation of linearity, accuracy, precision, selectivity and robustness. Proposed methods were successfully applied for the determination of investigated antibiotics during kinetic studies in aqueous solutions and in the solid state. The advantages of chromatographic procedures which are based on the use of C18 stationary phases with different particle sizes in the analysis of selected carbapenems were discussed.     

Stability-Indicating LC Determination of a New Antihypertensive,Olmesartan Medoxomil in Tablets

A stability-indicating liquid chromatographic method was developed and validated for quantitative determination of olmesartan medoxomil (OLM) in coated tablets in the presence of degradation products generated under stress conditions. An isocratic LC separation was performed using a Phenomenex RP-18 column using a mobile phase consisting of water:triethylamine:acetonitrile (60:0.3:40 v/v/v, pH adjusted to 6.3 with phosphoric acid). The flow rate was 1.2 mL min⁻¹ and the detection was achieved with a photodiode array detector set at 257 nm. The response was linear over a range of 10.0 to 30.0 μg mL⁻¹ (r = 0.9999). The specificity and stability-indicating capability of the method was verified subjecting the reference substance and drug product to hydrolytic, oxidative, photolytic, and thermal stress conditions. The method showed a good and consistent recovery (100.2%) with low intra- and inter-day relative standard deviation (RSD) (≤1.0%). A considerable degradation occurred in all stress conditions and the degradation product was well resolved from the main peak. There was no interference of the excipients in the determination of the active pharmaceutical ingredient. Thus, the proposed method was found to be stability-indicating and can be used for routine analysis for quantitative determination of OLM in coated tablets without the interference of major degradation products.

     

Simultaneous LC Estimation of Glimepiride and Metformin in Glimepiride Immediate Release and Metformin Sustained Release Tablets

In the present study, a simple, rapid and precise liquid chromatographic method was developed and validated for the simultaneous determination of glimepiride and metformin in sustained release formulation. The separation was achieved on a Nucleosil 100-5SA column, 250 × 4.6 mm i.d., 5 μm using a mobile phase composed of 1.7 % ammonium dihydrogen phosphate buffer pH 3.0: acetonitrile (70:30 v/v).The instrumental settings were a flow rate of 1.0 ml min⁻¹ and a detector wavelength of 230 nm. The retention time of glimepiride and metformin were 5.1 and 11.3, respectively. The developed method was validated in terms of specificity, sensitivity, linearity, range, accuracy, precision and ruggedness. The proposed method was successfully applied to the sustained release pharmaceutical dosage form for the simultaneous determination of glimepiride and metformin without any interference by excipients.

     

LC Separation and Determination of Five Diester-Diterpenoid Alkaloids in the Unprocessed and Processed Aconite Roots

A reliable liquid chromatographic method with photodiode array detector (DAD) was developed and validated for simultaneous separation and determination of five diester-diterpenoid alkaloids in the aconite roots. The separation was successfully performed on a Zorbax Extend-C₁₈ column with a mobile phase gradient prepared from methanol and ammonia solution at a flow rate of 1.0 mL min⁻¹. Good linearity (r > 0.999) was observed over the concentration ranges investigated, and intra-day and inter-day precision were high. The mean recoveries of five components ranged from 90.45 to 102.63% and relative standard deviations were always <5%. The validated method was successfully used for simultaneous determination of the five diester-diterpenoid alkaloids of unprocessed and processed aconite roots. The quantitative method provided a scientific basis for safety assurance and clinical application of aconite roots.

     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min⁻¹ for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.

     

Evaluation of Sempervivum tectorum L. Flavonoids by LC and LC–MS

A reversed-phase high-performance liquid chromatography method with UV detection was developed for determination of [(N-morpholine)methylene]daunorubicin hydrochloride (DD-M) during studies of its stability. In this LC method the following were used: an RP-column, the mobile phase—acetonitrile:methanol:solution A (9:1:10 v/v/v) [solution A contains 2.88 g of sodium laurilsulfate and 1.6 mL of phosphoric acid(V)] with a flow rate of 1.4 mol L⁻¹ and quinine hydrochloride as an internal standard. The detection wavelength was 254 nm. The method was validated with regard to linearity, limit of detection, limit of quantitation, selectivity and precision. Hydrolysis of the DD-M catalyzed by hydrogen ions in hydrochloric acid and a spontaneous reaction of the DD-M degradation under the influence of the water in sodium hydroxide took place. The thermodynamic parameters of these reactions—energy, enthalpy and entropy of activation—were calculated. It was observed that a positive salt effect occurred in hydrochloric acid.

     

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog

Nitrosylcobalamin (NO-Cbl), a novel vitamin B₁₂ analog and anti-tumor agent, functions as a biologic ‘Trojan horse’, utilizing the vitamin B₁₂ transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. A stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an Ascentis^® RP-Amide (150 mm × 4.6 mm, 5 μm) column at 35 °C with a mobile phase (1.0 mL min^?1) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. Additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B₁₂ therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl.