A Stability-Indicating LC Method for Assay of Topotecan Hydrochloride

This paper describes the development of a stability-indicating high-performance liquid chromatographic (HPLC) method for quantitative determination of topotecan hydrochloride, a semi-synthetic derivative of camptothecin and anti-tumor drug with topoisomerase I-inhibitory activity. Chromatographic separation was achieved on a C₁₈ column with a mixture of phosphate buffer and acetonitrile as mobile phase. The method was validated for linearity, accuracy, precision, and robustness. Forced degradation studies were performed by treating bulk samples of topotecan hydrochloride with acid (0.5 M hydrochloric acid), base (0.5 M sodium hydroxide), oxidizing agent (10% v/v hydrogen peroxide), heat (60 °C), and UV light (254 nm).     

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.     

A Stability-Indicating LC Assay Method for Bicalutamide

A stability-indicating HPLC method for the quantitative determination of Bicalutamide is described. Bicalutamide is a nonsteroidal antiandrogen and is an oral medication that is used for treating prostate cancer. Separation was achieved on a Waters Symmetry shield RP18 HPLC column using a mobile phase consists of a mixture of phosphate buffer (Solvent A) and organic modifier acetonitrile (Solvent B). Degradation studies were performed on bulk samples of bicalutamide using acid (0.5 N methanolic hydrochloric acid), base (0.5 N methanolic sodium hydroxide), oxidation (10% v/v methanolic hydrogen peroxide), heat (60 °C) and UV light (254 nm). Degradation was observed under base hydrolysis to give the starting material used during the synthesis of bicalutamide. The degraded samples were assayed and gave a mass balance greater than 99.5%, thus proving the stability-indicating power of the developed method. The method was validated with respect to linearity, accuracy, precision and robustness.     

A Stress Stability Behavior and Development of an LC Assay Method for Anastrozole

This present work narrates the stress stability behavior and development of a liquid chromatographic method for the quantitative determination of anastrozole. Anastrozole is appropriately used when using substantial amounts of aromatizing steroids, or when one is prone to gynecomastia and using moderate amounts of such steroids. A chromatographic separation was achieved on a Hichrom RPB18 (250 × 4.6 mm, 5 μ) column using water and mixture of acetonitrile and methanol (1:1 ratio) as mobile phase. Forced degradation studies were performed on bulk samples of anastrozole using acid, base, hydrogen peroxide, heat and UV light. Degradation of the drug substance was observed in base hydrolysis. Degradation product formed under base hydrolysis was found to be Imp-C. The sample solution and mobile phase were found to be stable up to 48 h. The developed method was validated with respect to linearity, accuracy, precision, robustness and forced degradation studies prove the stability indicating power of the method.     

A Validated Chiral LC Method for the Determination of Enantiomeric Purity of Pemetrexed Disodium on an Amylose-Based Chiral Stationary Phase

A simple and new isocratic normal phase chiral HPLC method has been developed for the determination of enantiomeric purity of pemetrexed disodium (l-enantiomer) in bulk drugs with a short run time of about 20 min. Chromatographic separation of l and d-enantiomers of pemetrexed disodium was achieved on an amylose based chiral stationary phase using a mobile phase consists of hexane, ethanol and trifluoro acetic acid. The resolution between the enantiomers was found to be more than 2.0. The system precision and method precision were found to be within 5% RSD for the distomer (d-enantiomer) at its specification level (i.e. not more than 1.0% w/w). The limit of detection and limit of quantification of distomer were 1.6 and 5 μg mL⁻¹, respectively for 10 μL injection volume. The percentage recovery of distomer was ranged from 90.6 to 105.7 in bulk drug samples. The test solution was found to be stable in the diluent for 48 h. The method was found to be specific for the enantiomers of pemetrexed disodium and can be conveniently used for the quantification of undesired d-enantiomer present in the bulk drug samples of pemetrexed disodium.     

LC Method for the Determination of the Stability of Levetiracetam Drug Substance under Stressing Conditions

Levetiracetam is used in combination with other medications to treat certain types of seizures in people with epilepsy. Levetiracetam is in a class of medications called anticonvulsants and it works by decreasing abnormal excitement in the brain. A chromatographic separation was achieved on a YMC pack ODS AQ, 250 mm × 4.6 mm, 5 μm column using diluted phosphoric acid and acetonitrile in the ratio 85:15 v/v. Forced degradation studies were performed on the levetiracetam drug substance. The drug substance was degraded to Imp-B during acid and base hydrolysis. When the stress samples were assayed, the mass balance was matching. The sample solution 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 and robustness.     

A Stability-Indicating LC Method for Candesartan Cilexetil

An isocratic reversed-phase liquid chromatographic method has been developed for quantitative determination of candesartan cilexetil, used to treat hypertension, in the bulk drug and in pharmaceutical dosage forms. The method is also applicable to analysis of related substances. Chromatographic separation was achieved on a 250 mm × 4.6 mm, 5 μm particle, CN column with a 50:50 (v/v) mixture of phosphate buffer, pH 3.0, and acetonitrile as mobile phase. The flow rate was 1.0 mL min⁻¹ and the detection wavelength was 210 nm. Resolution of candesartan cilexetil and six potential impurities was greater than 2.0 for all pairs of compounds. The drug was subjected to hydrolytic, oxidative, photolytic, and thermal stress and substantial degradation occurred in alkaline and acidic media and under oxidative and hydrolytic stress conditions. The major product obtained as a result of basic hydrolysis was different from that produced by acid hydrolysis and aqueous hydrolysis. The stress samples were assayed against a reference standard and the mass balance was found to be close to 99.6%. The method was validated for linearity, accuracy, precision, and robustness.     

A Rapid Stability-Indicating LC Method for Ziprasidone Hydrochloride

A simple and rapid reversed-phase liquid chromatographic method was developed for the related substances determination and quantitative evaluation of ziprasidone hydrochloride, which is used as an antipsychotic agent. Forced degradation studies were performed on bulk sample of ziprasidone hydrochloride using acid, base, oxidative hydrolysis, thermal stress and photolytic degradation. Mild degradation of the drug substance was observed during thermal stress and considerable degradation observed during base hydrolysis. The chromatographic method was fine tuned using the samples generated from forced degradation studies. Good resolution between the peaks corresponds to synthetic impurities and degradation products from the analyte were achieved on YMC Pack Pro C18 column using the mobile phase consists of a mixture of 0.05% v/v of phosphoric acid in water and acetonitrile. The stressed test solutions were assayed against the qualified working standard of ziprasidone hydrochloride and the mass balance in each case was close to 99.7% indicating that the developed method was stability-indicating. Validation of the developed method was carried out as per ICH requirements.     

A Validated Stability Indicating RPLC Method for Tazarotene

A simple, isocratic, rapid and accurate reversed phase high performance liquid chromatography method was developed for the quantitative determination of tazarotene. The developed method is also applicable for the related substance determination in bulk drugs. The chromatographic separation was achieved on a Hypersil C18 (250 mm × 4.6 mm 5 μm) column using water pH 2.5 with orthophosphoric acid:acetonitrile (15:85, v/v) as a mobile phase. The chromatographic resolutions between tazarotene and its potential impurity A and B were found greater than three. The limit of detection and limit of quantification of impurities were found to be 25 and 75 ng mL⁻¹. The percentage recovery of impurities in bulk drug sample was ranged from 96.8 to 103.5.The percentage recovery of tazarotene in bulk drug sample was ranged from 98.4 to 100.9. The developed RPLC method was validated with respect to linearity, accuracy, precision and robustness.     

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.     

Development of an HPLC Assay Method for Lenalidomide

Chromatographic separation of lenalidomide and its impurities was achieved on an Inertsil ODS-3 V column using a mobile phase consisting of a mixture of buffer, acetonitrile and methanol in the ratio 80:8:12 v/v. Degradation studies were performed on bulk samples of lenalidomide subjected to 0.5 N hydrochloric acid, 0.5 N sodium hydroxide, 10% v/v hydrogen peroxide, heating to 60 °C and UV light at 254 nm. Degradation was observed only under base hydrolysis conditions. The developed LC method gave a mass balance close to 99.5%, proving it to be suitable for stability studies and was validated with respect to linearity, accuracy, precision and robustness.     

Development of a Validated Stability-Indicating LC Method for Nitazoxanide in Pharmaceutical Formulations

A reversed-phase liquid chromatographic (LC) method was developed for the assay of nitazoxanide (NTZ) in solid dosage formulations. An isocratic LC separation was performed on a Phenomenex Synergi Fusion C₁₈ column (250 mm × 4.6 mm, i.d., 4 μm particle size) using a mobile phase of 0.1% o-phosphoric acid solution, pH 6.0: acetonitrile (45:55, v/v) at a flow rate of 1.0 mL min⁻¹. Detection was achieved with a photodiode array detector at 240 nm. The detector response for NTZ was linear over the concentration range from 2 to 100 μg mL⁻¹ (r = 0.9999). The specificity and stability-indicating capability of the method were proved using stress conditions. The RSD values for intra-day precision were less than 1.0% for tablets and powder for oral suspension. The RSD values for inter-day precision were 0.6 and 0.7% for tablets and powder for oral suspension. The accuracy was 100.4% (RSD = 1.8%) for tablets and 100.9% (RSD = 0.3%) for powder for oral suspension. The limits of quantitation and detection were 0.4 and 0.1 μg mL⁻¹. There was no interference of the excipients on the determination of the active pharmaceutical ingredient. The proposed method was precise, accurate, specific, and sensitive. It can be applied to the quantitative determination of drug in tablets and powder for oral suspension.     

Development of a New Analytical Method for Determination of Related Components in Nateglinide

An isocratic reverse phase liquid chromatographic (RP-LC) assay method has been developed for the quantitative determination of nateglinide and its related components namely imp-1 and imp-2 in bulk drug and in pharmaceutical dosage form, used for the treatment of type II diabetes mellitus. The developed method is stability indicating and also can be used for stability testing. The chromatographic separation was achieved on C-8, 150 × 4.6 mm, 3.5 μm stationary phase. The LC method employs solution A as mobile phase. Solution A contains a mixture of phosphate buffer pH 3.0: acetonitrile (50:50 v/v). The flow rate was 1.0 mL min⁻¹ and the detection wavelength was 210 nm. In the developed LC method the resolution between nateglinide and its potential impurities namely imp-1 and imp-2 was found to be greater than 5.0. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid medium, alkaline medium and oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.2%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

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.     

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.     

A Validated, Stability-Indicating, LC Method for Rabeprazole Sodium

We present a simple and reliable method for simultaneous determination of voriconazole and its main metabolite resulting from N-oxidation (UK-121,265), in human plasma. The work-up procedure used acetonitrile and potassium salts to precipitate plasma proteins. No internal standard was used. The chromatographic system used a LiChroCART^® 250-4 cartridge packed with LiChrospher^® 100 RP-8 (diameter particules, 5 μm). The UV monochromatic detector was set on 260 nm. The mobile phase consisted of a 60/40 (v/v) mixture of acetonitrile and water. The flow rate was 1 mL min^?1. The retention times for voriconazole and its metabolite were 8.98 and 4.02 min respectively, and total run time was 12 min. The linearity of the method was investigated from 0.31 to 10.0 mg L^?1; the lowest limit of quantification was 0.30 mg L^?1. Precision ranged from 2.41% to 6.32% for voriconazole and 0.80% to 11.6% for the N-oxide voriconazole metabolite. Accuracy was between 93.0% and 101% for voriconazole and 90.0% and 101% for the N-oxide voriconazole metabolite. This rapid and accurate method could be interesting to investigate metabolite/voriconazole ratio with respect to CYP2C19 genetic status and CYP3A4 activity changes.     

Development and Validation of a Stability-Indicating LC Method to Quantify Hydrochlorothiazide in Oral Suspension for Pediatric Use

A stability-indicating reversed-phase high-performance liquid chromatography (LC) method was developed and validated for the determination of hydrochlorothiazide in an oral suspension. Isocratic chromatography was performed on a C₁₈ column with 0.1 M sodium phosphate buffer pH 3.0/acetonitrile (70:30 v/v) as mobile phase, at a flow rate of 1.3 mL min⁻¹, and UV detection at 254 nm. The method was linear (r ² = 0.9998), accurate (mean recovery = 100.3%), and precise (RSD <2%). It was also validated for specificity and robustness. The method was successfully applied for the quality control analysis of a new pharmaceutical formulation of HCTZ for pediatric use.     

Validation of a Stability-Indicating LC Method for Assay of Ezetimibe in Tablets and for Determination of Content Uniformity

A simple, precise, and accurate HPLC method has been developed and validated for assay of ezetimibe in tablets and for determination of content uniformity. Reversed-phase liquid chromatographic separation was achieved by use of phosphoric acid (0.1%, v/v)–acetonitrile 50:50 (v/v) as mobile phase. The method was validated for specificity, linearity, precision, accuracy, robustness, and solution stability. The specificity of the method was determined by assessing interference from the placebo and by stress testing of the drug (forced degradation). Response was a linear function of drug concentration in the range 20–80 μg mL⁻¹ (r = 0.9999). Intraday and interday system and method precision were determined. Accuracy was between 100.8 and 102.7%. The method was found to be robust, and was suitable for assay of ezetimibe in a tablet formulation and for determination of content uniformity. An erratum to this article can be found at     

A Stability-Indicating LC Method for the Simultaneous Determination of Telmisartan and Ramipril in Dosage Form

A simple, rapid, and precise method is developed for the quantitative simultaneous estimation of telmisartan and ramipril in combined pharmaceutical dosage form. A chromatographic separation of the two drugs was achieved with an ACE 5 C₁₈, 25-cm analytical column using buffer–acetonitrile (55:45 v/v). The buffer used in mobile phase contains 0.1 M sodium perchlorate monohydrate in double distilled water pH adjusted 3.0 with trifluoroacetic acid. The instrumental settings are flow rate of 1.5 mL min⁻¹, column temperature at 30 °C, and detector wavelength of 215 nm using a photodiode array detector. The resolution between ramipril and telmisartan were found to be more than 5. Theoretical plates for ramipril and telmisartan were 13,022 and 6,629. Tailing factor for ramipril and telmisartan was 0.94 and 0.98. Telmisartan, ramipril and their combination drug product were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were analysed by the proposed method. Peak homogeneity data of telmisartan and ramipril is obtained using photodiode array detector, in the stressed sample chromatograms, demonstrated the specificity of the method for their estimation in presence of degradants. The described method shows excellent linearity over a range of 20–400 μg mL⁻¹ for telmisartan and 2.5–50 μg mL⁻¹ for ramipril. The correlation coefficient for telmisartan and ramipril are 1. The relative standard deviation for six measurements in two sets of each drug in tablets was always less than 2%. The proposed method was found to be suitable and accurate for quantitative determination and the stability study of telmisartan and ramipril in pharmaceutical preparations.     

A Validated LC Method for Separation and Quantification of Voriconazole and Its Enantiomer

A simple liquid chromatographic method was developed for the separation and quantification of voriconazole and its enantiomer in drug substance. The separation was achieved on Chiral cel-OD (250 mm × 4.6 mm × 10 μm) using mobile phase consisting of n-hexane and ethanol in the ratio 9:1 (v/v) with a flow rate of 1.0 mL min⁻¹, at 27 °C column temperature and detection at 254 nm with an injection volume of 20 μL. Ethanol was used as diluent. The method is capable of detecting the (2S, 3R) enantiomer down to 0.0075% and can quantify down to 0.021% with respect to sample concentration. The method is rapid and the resolution achieved was about 3.0. This method can be employed for the quantification of (2S, 3R) enantiomer in voriconazole drug substance.