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.     

LC Method for Studies on the Stability of Lopinavir and Ritonavir in Soft Gelatin Capsules

This study describes the development and full validation of a stability-indicating HPLC method to quantify ritonavir (RTV) and lopinavir (LPV) in soft gelatin capsules. The method uses a LiChrospher^® 100 RP-18 (250 mm × 4.6 mm, 5 µm, Merck) column and isocratic elution. The mobile phase consisted of a mixture of acetonitrile-water-methanol (53:37:10, v/v/v), pumped at a flow-rate of 1.0 mL min^?1 and UV detection at 210 nm using a photodiode array detector. LPV and RTV were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. The response was linear over a range of 40-360 µg mL^?1 for LPV and 10–90 µg mL^?1 for RTV (r > 0,999 for both drugs). The mean recoveries were 99.46 and 100.81% for LPV and RTV, respectively. The RSD values for intra- and inter-day precision studies were < 0.70% for both drugs. Degradation studies showed that lopinavir is stable in thermal, alkaline and oxidative conditions, while ritonavir degraded under these conditions. The method was found to be stability-indicating and can be used for the routine analysis of the association LPV/RTV in soft gelatin capsules.     

LC Determination of Ritonavir, a HIV Protease Inhibitor, in Soft Gelatin Capsules

A rapid, simple and sensitive high-performance liquid chromatographic method (HPLC) has been developed to assay ritonavir in semisolid capsules. The HPLC analysis used a reversed-phase C₈ (125 × 4.0 mm i.d., 5 μm particle size) analytical column and a mobile phase consisting of methanol and water (67:33, v/v), with UV detection at 210 nm. Specificity was evaluated using a photodiode array detector (PDA). The validation data showed that the assay is sensitive, specific and reproducible for determination of ritonavir in this dosage form. Calibration curves were linear from 100–300 μm L^?1 (R² ≥ 0.999). The accuracy of the method ranged from 98.8 to 102.0%. Mean inter- and intra-assay relative standard deviations (RSD) were less than 1.0%. The proposed method provided an accurate and precise analysis of ritonavir in soft capsules, requiring neither the use of a buffered mobile phase, nor the addition of amine modifiers.     

Stress Degradation Studies on Aripiprazole and Development of a Validated Stability Indicating LC Method

The present paper describes the development of a stability indicating reversed phase column liquid chromatographic method for aripiprazole in the presence of its impurities and degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of aqueous hydrolysis, oxidative, photolytic and thermal stress degradation. The degradation of aripiprazole was observed under acid hydrolysis and peroxide. The drug was found to be stable to other stress conditions attempted. Successful separation of the drug from the synthetic impurities and degradation products formed under stress conditions was achieved on an Inertsil phenyl column using a mixture of 0.2% trifluoroacetic acid and acetonitrile (55:45, v/v). The developed LC method was validated with respect to linearity, accuracy, precision, specificity and robustness. The assay method was found linear in the range of 25–200 μg mL^?1 with a correlation coefficient of 0.9999 and the linearity of the impurities were established from LOQ to 0.3%. Recoveries of the assay and impurities were found between 97.2 and 104.6%. The developed LC method for the related substances and assay determination of aripiprazole can be used to evaluate the quality of regular production samples. It can also be used to test the stability samples of aripiprazole. To the best of our knowledge, the validated stability indicating LC method which separates all the impurities disclosed in this investigation was not published elsewhere.     

A Stability Indicating LC Method for Vardenafil HCl

A simple, sensitive gradient RP-LC assay method has been developed for the quantitative determination of vardenafil HCl in bulk drug and in pharmaceutical dosage forms, used to treat erectile dysfunction. The developed method is also applicable for the related substances determination. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in a gradient mode and quantification was carried out using ultraviolet detection at a flow rate of 1.0 mL min^?1. In the developed LC method the resolution between vardenafil and its four potential impurities was found to be greater than 3.0. Regression analysis shows an r ² value (correlation coefficient) greater than 0.99 for vardenafil and its four impurities. This method was capable of detecting all four impurities of vardenafil at a level of 0.009% with respect to test concentration of 1.0 mg mL^?1 for a 10 μL injection volume. The method has shown good and consistent recoveries for vardenafil (98.4–100.6%) and its four impurities (93.5–106.2%). The test solution was found to be stable in the diluent for 48 h. Mass balance was found close to 99.4%.     

A Stability Indicating LC Method for Amtolmetin Guacyl

A simple, sensitive gradient RP-LC assay method has been developed for the quantitative determination of amtolmetin guacyl in bulk drug, used as anti-inflammatory drug. The developed method is also applicable for related substances determination. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in a gradient mode and quantification was carried out using ultraviolet detection at 313 nm at a flow rate of 1.0 mL min^?1. In the developed LC method the resolution between Amtolmetin Guacyl and its three potential impurities was found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) greater than 0.99 for amtolmetin guacyl and its three impurities. This method was capable to detect all three impurities of amtolmetin guacyl at a level of 0.002% with respect to test concentration of 0.5 mg mL^?1 for a 10 μL injection volume. The inter- and intra-day precision values for all three impurities and for amtolmetin guacyl was found to be within 2.0% RSD at its specification level. The method has shown good and consistent recoveries for amtolmetin guacyl (99.2–101.5%) and its three impurities (94.5–104.8%). The test solution was found to be stable in diluent for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.6%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

UV-Derivative Spectrophotometric Determination of Ritonavir Capsules and Comparison with LC Method

Abstract

An ultraviolet-derivative spectrophotometric method (UV-D) has been proposed as an alternative to a previously described liquid chromatographic (LC) method for the quantitative determination of ritonavir in soft gelatin capsules. The spectrophotometric method is based on recording the second-derivative spectra for ritonavir at 222.3 nm of its solutions in methanol. The linear dynamic range was 10.0–30.0 µg · mL^?1 with a correlation coefficient of 0.9995. Mean recoveries were between 99.2% and 100.2% for the tested capsules samples. Mean intra- and interassay relative standard deviations (RSDs) were less than 2.0%. The statistic analysis showed that LC and UV-D methods were equivalent to assay ritonavir capsules.     

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.     

Validation of a Stability Indicating LC Method for Amiodarone HCL and Related Substances

A simple, rapid and sensitive isocratic high performance liquid chromatographic (HPLC) method has been developed for the estimation of purity and quantitative determination of Amiodarone HCl active pharmaceutical ingredient (API).The method describes a quantitative estimation of five process related impurities of Amiodarone HCl with a resolution of more than or near to 3.0 between each impurity. These five known related substances are estimated by a simple, rapid and accurate reverse phase isocratic HPLC method. The method has been validated for the determination of assay and related substances in Amiodarone HCl API, using a C₈ column. The elution is carried out using a mobile phase consisting of water-methanol-acetic acid with a pH 5.8. For the quantitative determination of these relative substances, a relative response factors have been determined for all five related substances with respect to Amiodarone HCl. The precision (system precision, method precision and intermediated precision) is demonstrated for both the assay as well as related substances on six independent sample preparations. Accuracy of the method (recovery) is demonstrated for both Amiodarone and each of the five related substances. Specificity of the method is demonstrated by forced degradation study of Amiodarone HCl API under various stress conditions. The method is found to be stability indicating and useful for the analysis of assay and related substances of Amiodarone HCl API in a routine quality control laboratory and for the stability studies of drug substance.     

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 Stability Indicating LC Method for Deferasirox in Bulk Drugs and Pharmaceutical Dosage Forms

A novel, sensitive, stability indicating RP-LC method has been developed for the quantitative determination of deferasirox, its related impurities in both bulk drugs and pharmaceutical dosage forms. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in an isocratic mode and quantitation was by ultraviolet detection at 245 nm. The mobile phase consisted of buffer, acetonitrile and methanol (50:45:5, v/v) delivered at a flow rate of 1.0 mL min^?1. Buffer consisted of 10 mM potassium dihydrogen orthophosphate monohydrate, pH adjusted to 3.0 by using orthophosphoric acid. In the developed LC method the resolution (R _s ) between deferasirox and its four potential impurities was found to be greater than 2.0. Regression analysis showed an r value (correlation coefficient) greater than 0.999 for deferasirox and its four impurities. This method was capable to detect all four impurities of deferasirox at a level of 0.002% with respect to test concentration of 0.5 mg mL^?1 for a 10 μL injection volume. The inter- and intra-day precision values for all four impurities and for deferasirox was found to be within 2.0% RSD. The method showed good and consistent recoveries for deferasirox in bulk drugs (98.3–101.1%), pharmaceutical dosage forms (100.2–103.1%) and for its all the four impurities (99.7–102.1%). The test solution was found to be stable in methanol for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid stress hydrolysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.95%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

Development and Validation of a Stability Indicating LC Method for the Determination of Faropenem in Pharmaceutical Formulations

A simple, selective and sensitive stability indicating LC method has been developed and validated for the determination of faropenem in bulk drug and pharmaceutical formulations in the presence of degradation products. The separation was achieved by using an isocratic mobile phase mixture of acetate buffer of pH 3.5 and methanol (65:35, v/v) and 250 mm × 4.6 mm I.D., 5 μm particle size SGE make Wakosil C-18 AR column at flow rate of 1.0 mL min^?1 with detection at 305 nm. The retention time of faropenem is 6.63 min and was linear in the range of 5–75 μg mL^?1 (r = 0.9999). The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation and was found to be unstable in all the stress conditions. The proposed method was successfully employed for quantification of faropenem in bulk drug and its pharmaceutical formulations.     

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 LC Method for Mitotane in Bulk Drugs and Pharmaceutical Dosage Forms

A novel, sensitive, stability indicating RP-LC method has been developed for the quantitative determination of mitotane, its impurity in both bulk drugs and pharmaceutical dosage forms. Efficient chromatographic separation was achieved using a C18 stationary phase with simple mobile phase combination delivered in an isocratic mode and quantitation was by ultraviolet detection at a wavelength of 230 nm. The mobile phase consisted of buffer and acetonitrile (25:75, v/v) delivered at a flow rate of 1.0 mL min^?1. Buffer consisted of 10 mM potassium dihydrogen orthophosphate monohydrate, pH adjusted to 2.5 by orthophosphoric acid. In the developed LC method the resolution (R _s ) between mitotane and its impurity namely Imp-1 was found to be greater than 2.5. Regression analysis shows an r value (correlation coefficient) greater than 0.999 for mitotane and its impurity. This method was capable to detect the impurity of mitotane at a level of 0.003% with respect to test a concentration of 0.2 mg mL^?1 for a 10 μL injection volume. The inter- and intra-day precision values for mitotane and its impurity was found to be within 2.0% RSD. The method has shown good and consistent recoveries for mitotane in bulk drugs (99.2–101.5%), pharmaceutical dosage forms (98.2–103.1%) and for its impurity (99.7–102.1%). The test solution was found to be stable in diluent for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in basic stress hydrolysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.97%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

A Validated Stability Indicating LC Method for Mitotane in Bulk Drugs and Pharmaceutical Dosage Forms

A novel, sensitive, stability indicating RP-LC method has been developed for the quantitative determination of mitotane, its impurity in both bulk drugs and pharmaceutical dosage forms. Efficient chromatographic separation was achieved using a C18 stationary phase with simple mobile phase combination delivered in an isocratic mode and quantitation was by ultraviolet detection at a wavelength of 230 nm. The mobile phase consisted of buffer and acetonitrile (25:75, v/v) delivered at a flow rate of 1.0 mL min⁻¹. Buffer consisted of 10 mM potassium dihydrogen orthophosphate monohydrate, pH adjusted to 2.5 by orthophosphoric acid. In the developed LC method the resolution (R _s ) between mitotane and its impurity namely Imp-1 was found to be greater than 2.5. Regression analysis shows an r value (correlation coefficient) greater than 0.999 for mitotane and its impurity. This method was capable to detect the impurity of mitotane at a level of 0.003% with respect to test a concentration of 0.2 mg mL⁻¹ for a 10 μL injection volume. The inter- and intra-day precision values for mitotane and its impurity was found to be within 2.0% RSD. The method has shown good and consistent recoveries for mitotane in bulk drugs (99.2–101.5%), pharmaceutical dosage forms (98.2–103.1%) and for its impurity (99.7–102.1%). The test solution was found to be stable in diluent for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in basic stress hydrolysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.97%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.

     

LC Stability Studies of Voriconazole and Structural Elucidation of Its Major Degradation Product

Voriconazole is a broad spectrum agent used to treat serious fungal infections. Stability studies conducted so far refer to the stability of the injectable formulation in different solvents, packaging materials and on storage but studies on the inherent chemical stability of the drug are not available. The purpose of this study was to evaluate the stability of the drug under stress conditions, in solution and in the solid state; isolate and elucidate the structure of the major degradation product and evaluate the antifungal activity of the degradation products. The quantification of the drug after exposure to degradation conditions was studied by a validated LC method. Among the conditions tested, it was found that the drug is more rapidly degraded in an alkaline medium, exposure to UVC radiation (254 nm) and elevated temperatures (60 °C). Degradation was greater under the first two conditions and in solution. Tablets exposed to UVC radiation for 14 days remained chemically and physically stable. For the isolation of the major degradation product, semi-preparative LC was employed and for the structural elucidation, spectroscopic techniques (¹H and ¹³C NMR spectroscopy, IR spectroscopy and mass spectrometry) were used, and the major degradation product identified as 1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)-1-ethanone. The degraded samples were evaluated through microbiological assay and found to have no antifungal activity.     

LC Stability Studies of Voriconazole and Structural Elucidation of Its Major Degradation Product

Voriconazole is a broad spectrum agent used to treat serious fungal infections. Stability studies conducted so far refer to the stability of the injectable formulation in different solvents, packaging materials and on storage but studies on the inherent chemical stability of the drug are not available. The purpose of this study was to evaluate the stability of the drug under stress conditions, in solution and in the solid state; isolate and elucidate the structure of the major degradation product and evaluate the antifungal activity of the degradation products. The quantification of the drug after exposure to degradation conditions was studied by a validated LC method. Among the conditions tested, it was found that the drug is more rapidly degraded in an alkaline medium, exposure to UVC radiation (254 nm) and elevated temperatures (60 °C). Degradation was greater under the first two conditions and in solution. Tablets exposed to UVC radiation for 14 days remained chemically and physically stable. For the isolation of the major degradation product, semi-preparative LC was employed and for the structural elucidation, spectroscopic techniques (¹H and ¹³C NMR spectroscopy, IR spectroscopy and mass spectrometry) were used, and the major degradation product identified as 1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)-1-ethanone. The degraded samples were evaluated through microbiological assay and found to have no antifungal activity.     

LC Method for the Determination of Rhaponticin in Rat Plasma, Faeces and Urine for Application to Pharmacokinetic Studies

A simple liquid chromatography (LC) method has been developed and validated to determine rhaponticin in rat plasma, faeces and urine. Chromatographic separation was achieved through mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL min^?1. Rhaponticin was quantified using UV detection at 324 nm. The assay was linear over the concentration range of 50–4,000 ng mL^?1 for plasma, faeces and urine. The intra- and inter-day RSD were less than 10%. The plasma, faeces and urine rhaponticin levels were monitored in rats after oral administration. This simple LC method appears to be useful in the pharmacokinetic investigation of rhaponticin.     

Stress Degradation Studies of Tebipenem and a Validated Stability-Indicating LC Method

A inexpensive and rapid isocratic LC method has been developed for the quantitative determination of tebipenem—a new β-lactam antibiotic. Stress degradation studies were performed on tebipenem in acidic (0.2 N hydrochloric acid) and basic (0.02 N sodium hydroxide) solutions, in a solution with oxidizing agent (3 % hydrogen peroxide), and in the solid state, during thermolysis and photolysis. For a chromatographic separation of tebipenem and its degradation products, a C-18 stationary phase and 12 mM ammonium acetate-acetonitrile (96:4 v/v) were used. A quantitative determination of tebipenem was carried out by using a PDA detector at 298 nm, with a flow rate of 1.2 mL min^?1. The linear regression analysis for the calibration plots showed a good linear relationship (r = 0.999) in the concentration range 0.041–0.240 mg mL^?1. The method demonstrated good precision (1.14–1.96 % RSD) and recovery (99.60–101.90 %). The limits of detection and quantitation were 9.69 and 29.36 μg mL^?1, respectively. The analysis of tebipenem reactivity was supported by quantum chemical calculations based on the density functional theory (DFT). The analysis of the electron density of the HOMO and LUMO of tebipenem suggested the possibility of electron transport in the molecule during the degradation of bi-cyclic 4:5 fused penem rings.