Development and validation of a stability-indicating column high-performance liquid chromatographic assay method for determination of nebivolol in tablet formulation

A simple, precise, and accurate isocratic reversed-phase (RP) stability-indicating column high-performance liquid chromatographic (HPLC) assay method was developed and validated for determination of nebivolol in solid pharmaceutical dosage forms. Isocratic RP-HPLC separation was achieved on a Phenomenex Luna C8 (2) column (250 mm x 4.6 mm id, 5 microm particle size) using mobile phase composed of acetonitrile-pH 3.5 phosphate buffer (35 + 65, v/v) at a flow rate of 1.0 mL/min, and detection was performed at 280 nm using a photodiode array detector. The drug was subjected to oxidation, hydrolysis, photolysis, and heat to apply stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness, and solution stability. The method was linear in the drug concentration range of 40-160 microg/mL with a correlation coefficient of 0.9999. The repeatability relative standard deviation (RSD) for 6 samples was 0.69%, and the intermediate precision (RSD) for 6 samples was 1.39%. The accuracy (recovery) was between 98.57 and 99.55%. Degradation products produced as a result of stress studies did not interfere with detection of nebivolol, and the assay can thus be considered stability-indicating.     

Development and validation of a high-performance liquid chromatographic method for determination of eprosartan in bulk drug and tablets

A simple, precise, and accurate isocratic RP-HPLC method was developed and validated for determination of eprosartan in bulk drug and tablets. Isocratic RP-HPLC separation was achieved on a Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) using the mobile phase 0.5% formic acid-methanol-acetonitrile (80 + 25 + 20, v/v/v, pH 2.80) at a flow rate of 1.0 mL/min. The retention time of eprosartan was 7.64 +/- 0.05 min. The detection was performed at 232 nm. The method was validated for linearity, precision, accuracy, robustness, solution stability, and specificity. The method was linear in the concentration range of 10-400 microg/mL with a correlation coefficient of 0.9999. The repeatability for six samples was 0.253% RSD; the intraday and interday precision were 0.21-0.57 and 0.33-0.71% RSD, respectively. The accuracy (recovery) was found to be in the range of 99.86-100.92%. The drug was subjected to the stress conditions hydrolysis, oxidation, photolysis, and heat. Degradation products produced as a result of the stress conditions did not interfere with detection of eprosartan; therefore, the proposed method can be considered stability-indicating.     

Stability Indicating RP-LC Method for the Determination of Gemifloxacin Mesylate

The present study describes the degradation of gemifloxacin mesylate under different International Conference on Harmonization prescribed stress conditions (hydrolysis, oxidation, dry and wet heat and photolysis) and application of a specific and selective stability-indicating reversed-phase liquid chromatography assay. Separation of drug and degradation products was successfully achieved on a HiQ-SiL C₈ column using 10 mM potassium dihydrogen orthophosphate (pH adjusted to 3.0 with o-phosphoric acid)–acetonitrile (65:35, v/v) at a flow rate of 1 mL min⁻¹ and detection at 273 nm.

     

Development and validation of a stability-indicating HPLC assay method for simultaneous determination of spironolactone and furosemide in tablet formulation

The objective of the current study was to develop and validate a simple, precise and accurate isocratic stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) assay method for the determination of spironolactone and furosemide in solid pharmaceutical dosage forms. Isocratic RP-HPLC separation was achieved on an SGE 150 × 4.6 mm SS Wakosil II 5C8RS 5-μm column using a mobile phase of acetonitrile-ammonium acetate buffer (50:50, v/v) at a flow rate of 1.0 mL/min. The detection was carried out at 254 nm using a photodiode array detector. The drug was subject to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness and solution stability. The method was found to be linear in the drug concentration range of 40-160 μg/mL with correlation coefficients of 0.9977 and 0.9953 for spironolactone and furosemide, respectively. The precision (relative standard deviation; RSD) among a six-sample preparation was 0.87% and 1.1% for spironolactone and furosemide, respectively. Repeatability and intermediate precision (RSD) among a six-sample preparation were 0.46% and 0.20% for spironolactone and furosemide, respectively. The accuracy (recovery) was between 98.05 and 100.17% and 99.07 and 100.58% for spironolactone and furosemide, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of spironolactone and furosemide; therefore, the assay can be considered to be stability-indicating.     

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.     

Development and validation of stability indicating method for the determination of exemestane by reverse phase high performance liquid chromatography

Exemestane is an aromatase inhibitor used in the treatment of breast cancer. A selective stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed which can separate and accurately quantitate low levels of exemestane. The stability-indicating capability of the method was demonstrated by adequate separation of exemestane and all the degradation product peaks from exemestane peak and also from each other in stability samples of exemestane. Chromatographic separation of exemestane and its degraded products were achieved by using isocratic elution at a flow rate of 1.0 mL/min on a C18 reverse phase column (Phenomenex, size: 250 × 4.60 mm, particle size 5 μm) at ambient temperature. The mobile phase used for the analysis was acetonitrile-water (60:40, %v/v) with UV visible detection at 242 nm. The proposed method was used to study the degradation behavior of drug under various stress conditions as per ICH recommended guidelines.     

HPTLC method for determination of nevirapine in pharmaceutical dosage form

A sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic method of analysis of nevirapine both as a bulk drug and in formulations was developed and validated. The solvent system consisted of toluene-carbon tetrachloride-methanol-acetone-ammonia (3.5:3.5:2.0:1.0:0.05, v/v/v/v/v). Densitometric analysis of nevirapine was carried out in the absorbance mode at 289nm. This system was found to give compact spots for nevirapine (R(f) value of 0.44+/-0.02). Nevirapine was subjected to acid and alkali hydrolysis, oxidation, dry heat and wet heat treatment and photodegradation. The drug undergoes degradation under acidic, basic conditions and oxidation. Also the degraded products were well resolved from the pure drug with significantly different R(f) values. Linearity was found to be in the range of 30-1000ng/spot with significantly high value of correlation coefficient. The linear regression analysis data for the calibration plots showed good linear relationship with r(2)=0.998+/-0.002 in the working concentration range of 300ng/spot to 1000ng/spot. The mean value of slope and intercept were 0.073+/-0.005 and 36.78+/-1.50, respectively. The method was validated for precision, robustness and recovery. The limit of detection and quantitation were 5 and 10ng/spot, respectively. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one. Moreover, the proposed HPTLC method was utilized to investigate the kinetics of acid degradation process. Arrhenius plot was constructed and activation energy was calculated.     

Stability-indicating reversed-phase liquid chromatographic method for simultaneous determination of atorvastatin and ezetimibe from their combination drug products

A simple, precise, and rapid stability-indicating reversed-phase column liquid chromatographic (RP-LC) method has been developed and subsequently validated for simultaneous estimation of atorvastatin (ATV) and ezetimibe (EZE) from their combination drug product. The proposed RP-LC method utilizes a LiChrospher 100 C18, 5 microm, 250 x 4.0 mm id column at ambient temperature; the optimum mobile phase consists of acetonitrile-water-methanol (45 + 40 + 15, v/v/v) with apparent pH adjusted to 4.0 +/- 0.1; mobile phase flow rate of 1.0 mL/min; and UV detection at 250 nm. ATV, EZE, and their combination drug product were exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. There were no other coeluting, interfering peaks from excipients, impurities, or degradation products due to variable stress conditions, and the method is specific for the estimation of ATV and EZE in the presence of degradation products. The response was linear over the concentration range of 1-80 microg/mL for ATV and EZE. The mean recoveries were 99.27 and 98.5% for ATV and EZE, respectively. The intermediate precision data were obtained under different experimental conditions, and the calculated value of the coefficient of variation was found to be less than the critical value. The proposed method can be useful in the quality control of bulk manufacturing and pharmaceutical dosage forms.     

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.

     

Stress studies on acyclovir

Acyclovir is an antiviral drug of choice in the treatment of many types of herpes virus infections, including genital herpes simplex infections, herpetic conjunctivitis, herpes simplex encephalitis, etc. The present study describes the degradation behavior of acyclovir under different International Conference on Harmonization recommended stress conditions (hydrolysis, oxidation, photolysis, and thermal decomposition) in order to establish a validated stability-indicating high-performance liquid chromatography method. Acyclovir is found to degrade extensively in acidic conditions and oxidative stress. Mild degradation of the drug occurs in alkaline and neutral conditions. The drug is stable to dry heat. The drug is found to be sufficiently stable after light exposure in a solid state; however, photolytic degradation is observed when the drug is exposed as a solution in water. The major degradation product in acidic hydrolysis and photolysis is identified as guanine through comparison with the standard. Separation of drug and the degradation products under various conditions is successfully achieved on a C-18 column utilizing water-methanol in the ratio of 90:10. The flow rate is 1 mL/min, and the detection wavelength is 252 nm. The method is validated with respect to linearity, precision, accuracy, selectivity, specificity, and robustness. The mean values of slope and correlation coefficient are 39.307 and 0.9998 with relative standard deviation values less than 2%. The recovery of the drug is found to be in the range of 97.34% to 102.35%. From the previous study it is concluded that the stability-indicating method developed for acyclovir can be used for analysis of the drug in various stability samples.     

Structural Elucidation of Alkali Degradation Impurities of Favipiravir from the Oral Suspension: UPLC-TQ-ESI-MS/MS and NMR

A novel stability-indicating, reversed-phase, high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination of favipiravir in an oral suspension. The effective separation of favipiravir and its degradation products was achieved on a Zorbax Eclipse Plus C18 column (5 μm particle size, 150 mm length × 4.6 mm diameter). The mobile phase was prepared by mixing 5 mM of phosphate buffer (pH 3.5) and methanol in a 75:25 v/v ratio delivered at a 1.0 mL/min flow rate. The eluents were monitored using a photodiode array detector at a wavelength of 322 nm. The stability-indicating nature of this method was evaluated by performing force degradation studies under various stress conditions, such as acidic, alkali, oxidative, thermal, and photolytic degradation. Significant degradation was observed during the alkali stress degradation condition. The degradation products generated during various stress conditions were well separated from the favipiravir peak. In addition, the major degradation product formed under alkali stress conditions was identified using UPLC-ESI-TQ-MS/MS and NMR. Method validation was performed according to the ICH Q2 (R1) guideline requirements. The developed method is simple, accurate, robust, and reliable for routine quality control analysis of favipiravir oral suspensions.     

Stability-indicating high-performance liquid chromatographic assay of atorvastatin with fluorescence detection

The purpose of this work was to develop a sensitive, selective, and validated stability-indicating high-performance liquid chromatographic (LC) assay of atorvastatin (ATV) in bulk drug and tablet form. ATV was subjected to different stress conditions, including UV light, oxidation, acid-base hydrolysis, and temperature. ATV and its degradation products were analyzed on an Agilent Zorbax XDB C18 column using isocratic elution with acetonitrile-0.02 M sodium acetate, pH 4.2 (45 + 55, v/v) for 25 min. The samples were monitored with fluorescence (FL) detection at 282 nm (excitation)/400 nm (emission). The response ratio of FL to UV detection (at 247 nm) for ATV was 1.66. The method showed good resolution of ATV from its decomposition products. The photodegradation products were separated by silica gel thin-layer chromatography using double development with ethyl acetate-n-hexane-glacial acetic acid-methanol (40 + 55 + 0.5 + 4.5, v/v/v/v) followed by (39 + 55 + 0.5 + 5.5, v/v/v/v), and confirmed by LC-FL analysis. The FL response was linear over the investigated range for ATV. The linear range was 10-1200 ng/injection, and the limit of quantitation was 2.0 ng/injection.     

A Validated and Stability-Indicating LC Assay Method for Valdecoxib

A gradient reversed-phase liquid chromatographic assay was developed for the quantitative determination of the non-steroidal anti-inflammatory drug valdecoxib. The developed method was also applicable to the determination of related substances in the bulk drug. Forced degradation studies were performed on bulk valdecoxib using acid (2.0 N hydrochloric acid), base (2.0 N sodium hydroxide), oxidation (6.0% v/v hydrogen peroxide), water hydrolysis, heat (60 °C) and photolysis. Mild degradation was observed using alkaline conditions and considerable degradation observed during oxidative stress. Chromatographic separation of process-related impurities and degradation products was achieved using a 5 micron Zorbax SB-CN LC column. The mobile phase consisted of aqueous potassium dihydrogen phosphate (pH 3.0) and acetonitrile. Stressed samples were assayed using the developed LC method and determination of the mass balance accounted for 99.5%, thus indicating the suitability of this stability-indicating method. Linearity, accuracy, precision and robustness have also been evaluated.     

Development and validation of stability indicating analytical method for Ilaprazole

The objective of the present study was to develop and validate a rapid and simple stability indicating analytical method for estimating Ilaprazole. Ilaprazole was subjected to different stress conditions prescribed by International Conference on Harmonization (ICH) such as hydrolysis, oxidation, photolytic and dry heat degradation conditions. The drug was very susceptible to degradation under hydrolysis and photolytic conditions, less susceptible to oxidation and stable under dry heat degradation condition. An acceptable separation of drug and its degradants was achieved by using a C-18 column and mobile phase composed of ammonium acetate buffer (pH 3.2)—acetonitrile (55: 45, v/v). Flow rate was 1 mL/min and detection wavelength was set at 303 nm. Retention time of drug was found to be 6.6 min and analysis can be completed within 10 min. The method was validated with respect to linearity, precision, accuracy, robustness, LOD and LOQ as per ICH. The method was linear (R² = 0.996) in the range of 2.5–250 μg/mL. The recovery was in the range from 99.2–100.2%.     

The International Conference on Harmonisation Guidance in Practice: Stress Degradation Studies on Lamivudine and Development of a Validated Specific Stability-Indicating HPTLC Assay Method

A sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic method of analysis of lamivudine both as a bulk drug and in formulations was developed and validated. The solvent system consisted of carbon tetrachloride – methanol – chloroform - acetonitrile (7.0: 3.0: 2.0: 1.5, v/v/v/v). Densitometric analysis of lamivudine was carried out in the absorbance mode at 275 nm. This system was found to give compact spots for lamivudine (R_F value of 0.36 ± 0.02) following double development of chromatoplates with the same mobile phase. Lamivudine was subjected to acid and alkali hydrolysis, oxidation, dry heat and wet heat treatment and photo degradation. The drug undergoes degradation under acidic, basic conditions, oxidation, wet heat and photo degradation. Also the degraded products were well resolved from the pure drug with significantly different R_F values. Linearity was found to be in the range of 50 – 1000 ng spot^–1 with significantly high value of correlation coefficient. The linear regression analysis data for the calibration plots showed good linear relationship with r² = 0.9994 ± 0.05 in the working concentration range of 300 ng spot^–1 to 1000 ng spot^–1. The mean value of slope and intercept were 0.11 ± 0.08 and 10.47 ± 1.21, respectively. The method was validated for precision, robustness and recovery. The limit of detection and quantitation were 15 ng spot^–1 and 40 ng spot^–1 respectively. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one. Moreover, the proposed HPTLC method was utilized to investigate the kinetics of acid degradation process. Arrhenius plot was constructed and activation energy was calculated.     

Stability-indicating reversed-phase liquid chromatographic method for simultaneous determination of simvastatin and ezetimibe from their combination drug products

A simple, precise, and rapid stability-indicating reversed-phase column liquid chromatographic (RP-LC) method has been developed and subsequently validated for simultaneous estimation of simvastatin (SIM) and ezetimibe (EZE) from their combination drug product. The proposed RP-LC method utilizes a LiChrospher 100 C18, 5 microm, 250 x 4.0 mm id column at ambient temperature; optimum mobile phase consisting of acetonitrile-water-methanol (60 + 25 + 15, v/v/v) with apparent pH adjusted to 4.0 +/- 0.1; mobile phase flow rate of 1.5 mL/min; and ultraviolet detection at 238 nm. SIM, EZE, and their combination drug product were exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. There were no other coeluting, interfering peaks from excipients, impurities, or degradation products due to variable stress conditions, and the method is specific for the estimation of SIM and EZE in the presence of degradation products. The described method was linear over the range of 1-80 and 3-80 microg/mL for SIM and EZE, respectively. The mean recoveries were 99.17 and 100.43% for SIM and EZE, respectively. The intermediate precision data were obtained under different experimental conditions, and the calculated value of the coefficient of variation was found to be less than the critical value. The proposed method can be useful in the quality control of bulk manufacturing and pharmaceutical dosage forms.     

HPLC, TLC, and first-derivative spectrophotometry stability-indicating methods for the determination of tropisetron in the presence of its acid degradates

Three stability-indicating assay methods were developed for the determination of tropisetron in a pharmaceutical dosage form in the presence of its degradation products. The proposed techniques are HPLC, TLC, and first-derivative spectrophotometry (1D). Acid degradation was carried out, and the degradation products were separated by TLC and identified by IR, NMR, and MS techniques. The HPLC method was based on determination of tropisetron in the presence of its acid-induced degradation product on an RP Nucleosil C18 column using methanol-water-acetonitrile-trimethylamine (65 + 20 + 15 + 0.2, v/v/v/v) mobile phase and UV detection at 285 nm. The TLC method was based on the separation of tropisetron and its acid-induced degradation products, followed by densitometric measurement of the intact spot at 285 nm. The separation was carried out on silica gel 60 F254 aluminum sheets using methanol-glacial acetic acid (22 + 3, v/v) mobile phase. The 1D method was based on the measurement of first-derivative amplitudes of tropisetron in H2O at the zero-crossing point of its acid-induced degradation product at 271.9 nm. Linearity, accuracy, and precision were found to be acceptable over concentration ranges of 40-240 microg/mL, 1-10 microg/spot, and 6-36 micro/mL for the HPLC, TLC, and 1D methods, respectively. The suggested methods were successfully applied for the determination of the drug in bulk powder, laboratory-prepared mixtures, and a commercial sample.     

Development and validation of the stability-indicating LC-UV method for the determination of cefoselis sulphate

The stability-indicating LC assay method was developed and validated for quantitative determination of cefoselis sulphate in the presence of degradation products formed during the forced degradation studies. An isocratic, RP-HPLC method was developed with C-18 (250 × 4.6 mm, 5 μm) column and 12 mM ammonium acetate-acetonitrile (95:5 V/V) as a mobile phase. The flow rate of the mobile phase was 1.0 mL min⁻¹. Detection wavelength was 260 nm and temperature was 30°C. Cefoselis similarly to other cephalosporins was subjected to stress conditions of degradation in aqueous solutions including hydrolysis, oxidation, photolysis and thermal degradation. The developed method was validated with regard to linearity, accuracy, precision, selectivity and robustness. The method was applied successfully for identification and determination of cefoselis sulphate in pharmaceuticals and during kinetic studies.     

Stress degradation study and structure characterization of oxidation degradation product of dexlansoprazole using liquid chromatography-mass spectrometry/time of flight,liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance

The present study deals with the forced degradation behavior of dexlansoprazole under International Conference on Harmonisation (ICH) prescribed stress conditions. The drug was found to be more labile under acid, base, neutral, oxidative hydrolysis and thermal stress, while it was moderately stable under photolytic conditions. The known and unknown degradation products were separated on a C-18 column using a stability-indicating method. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed for all the degradation studies. Isolation and structure characterization of oxidation degradation products were executed using sophisticated tools, viz. preparative high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry/time of flight (LC-MS/TOF), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and nuclear magnetic resonance (NMR). This study demonstrates an ample methodology of degradation studies and structure elucidation of unknown degradation products of dexlansoprazole, which helps in the development and stability study of active pharmaceutical ingredients and formulated products.     

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.