Developed LC Method Research Articles

Development and validation of a LC method for the separation and determination of the anticancer-active FeIII(4-methoxy-salophene) using the new second-generation monolith

LC method with the newly introduced second-generation monolithic silica RP-18e column has been developed for the separation of Fe(III) (salophene) and four methoxy-substituted Fe(III) (salophene) complexes. The method has been validated for the quantitation of Fe(III) (4-OMe-salophene), a highly active anticancer substance in vitro, bound to serum albumin. Our routinely used high-resolution continuum-source atomic absorption spectroscopy method based on the determination of the central iron atom was unsuitable in this case because serum originally contains significant amounts of iron as revealed by a blank sample of serum albumin. The developed LC method depends on detecting the whole complex rather than the bound iron. Two morphologically different first- and second-generation HPLC monolithic columns have been compared for this purpose. The newly introduced second-generation monolithic silica column Chromolith(®) HighResolution RP-18e column (100 × 4.6 mm, Merck) separated the mixture successful within 13 min. A mobile phase consisting of 25 mM phosphate buffer pH 3/methanol (60:40, v/v) was used at a flow rate of 1 mL/min. The dynamic linear working range of the calibration curve for Fe(III) (4-OMe-salophene) was found to be between 1 and 200 μg/mL. Detection and quantitation limits were 0.3 and 1 μg/mL, respectively.

Degradation Pathway for Eplerenone by Validated Stability Indicating UP-LC Method.

Degradation pathway for eplerenone is established as per ICH recommendations by validated and stability-indicating reverse phase liquid chromatographic method. Eplerenone is subjected to stress conditions of acid, base, oxidation, and thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Four impurities are studied and the major degradant (RRT about 0.31) was identified by LC-MS and spectral analysis. The stress samples are assayed against a qualified reference standard and the mass balance is found close to 99.5%. Efficient chromatographic separation is achieved on a Waters symmetry C18 stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 240 nm at a flow rate of 1.0 mL min−1. In the developed LC method the resolution between eplerenone and four potential impurities (imp-1, imp-2, imp-3, and imp-4) is found to be greater than 4.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.999 for eplerenone and four potential impurities. This method is capable to detect the impurities of eplerenone at a level of 0.020% with respect to test concentration of 1.0 mg mL−1 for a 20 μL injection volume. The developed UPLC method is validated with respect to specificity, linearity and range, accuracy, precision, and robustness for impurities and assay determination.

Degradation Pathway for Rilpinavir Hydrochloride by Validated Stability Indicating UP-LC Method

Degradation pathway for Rilpivirine Hydrochoride is established as per ICH recommendations by validated and stability indicating reverse phase liquid chromatographic method. Rilpivirine Hydrochoride is subjected to stress conditions of acid, base, oxidation, thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Six impurities are studied and the major degradant (RRT about 0.52) is identified by LC-MS and spectral analysis. The stress samples are assayed against a qualified reference standard and the mass balance is found close to 99.5 %. Efficient chromatographic separation is achieved on a Shimpack XR –ODS-II stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 295 nm at a flow rate of 1.0 mL min-1.In the developed LC method the resolution between Rilpivirine Hydrochoride and six potential impurities (imp-1, imp-2, imp-3, imp-4, imp-5 and imp-6) is found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.995 for Rilpivirine Hydrochoride and six potential impurities. This method is capable to detect the impurities of Rilpivirine Hydrochoride at a level of 0.006 % with respect to test concentration of 1.0 mg mL-1 for a 4-µL injection volume. The developed LC method is validated with respect to specificity, linearity & range, accuracy, precision and robustness for impurities and degradant determination.

Development of Stability Indicating RP-HPLC Method for Determination of Levosulpiride Hydrochloride In Bulk And Pharmaceutical Dosage Form

A rapid, specific and sensitive stability indicating reverse phase high performance liquid chromatographic method has been developed and validated for analysis of levosulpiride hydrochloride in both bulk and pharmaceutical dosage form.An isocratic stability indicating reversed-phase liquid chromatographic determination was developed for the quantitative determination of levosulpiride in the pharmaceutical dosage form. A sunfire C-18, 4.5mm column with mobile phase containing methanol-water (10:90, v/v) was used. The flow rate was 1.0 mL min-1and effluents were monitored at 232 nm. The retention time of Levosulpiride was 5.5 min. Levosulpiride stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation, wet hydrolysis, dry heat degradation and sun light degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time values. Stressed samples were assayed using developed LC method. The proposed method was validated with respect to linearity, accuracy, precision and robustness. The method was successfully applied to the estimation of Levosulpiride in tablet dosage forms.The proposed study describes stability indicating LC method for the estimation of Levosulpiride in bulk and their pharmaceutical dosage form. The method is suitable for the routine analysis of Levosulpiride in tablets.

Liquid Chromatographic Method for the Quantification of Antipsychotic Agent Iloperidone in Pharmaceutical Formulation

Liquid chromatographic method for the quantitative determination of iloperidone in the pharmaceutical dosage form was developed. A reversed phase column with mobile phase containing 0.02 M potassium dihydrogen phosphate: methanol: acetonitrile (55 : 25 : 20, v/v/v, pH 4 adjusted with phosphoric acid) was used. The flow rate was 1.0 mL min−1, and effluents were monitored at 230 nm. The retention time of iloperidone was 11.6 min. Iloperidone stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation, and dry heat degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time values. Stressed samples were assayed using developed LC method. The proposed method was validated with respect to linearity, accuracy, precision, and robustness. The method was successfully applied to the estimation of iloperidone in tablet dosage forms.

Simultaneous estimation of atorvastatin calcium and aspirin in combined dosage form by liquid chromatographic method

Summary An isocratic reversed-phase liquid chromatograpic assay method was developed for the quantitative determination of atorvastatin and aspirin (ASP) in combined dosage form. A Phenomenex Gemini C-18, 5-μm column with mobile phase containing 0.02 M potassium dihydrogen phosphate-acetonitrile-methanol (30:30:40, v/v/v) adjusted to pH 3 using o-phosphoric acid was used. The flow rate was 1.0 mL min−1 and effluents were monitored at 240 nm. The retention times (RTs) of atorvastatin calcium (ATV) and ASP were 10.5 and 3.8 min, respectively. ATV and ASP stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation, and dry heat degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their RT values. Stressed samples were assayed using developed LC method. The proposed method was validated with respect to linearity, accuracy, precision, and robustness. The method was successfully applied to the estimation of ATV and ASP in combined...

A VALIDATED STABILITY INDICATING ION EXCHANGE CHROMATOGRAPHIC METHOD FOR THE QUANTIFICATION OF ALENDRONATE, PHOSPHITE AND PHOSPHATE IN ALENDRONATE SODIUM TABLETS AND ANALYSIS OF IN-VITRO DISSOLUTION SAMPLES

A stability-indicating LC method was developed on ion exchange chromatography equipped with refractive index detector for the quantification of phosphite, phosphate content and assay of alendronic acid in pharmaceutical dosage forms. The isocratic separation was achieved by using Allsep anion (150mm x 4.6mm) 7 µm column with mobile phase containing a 5.3 mM formic acid, pH adjusted to 3.5 with sodium hydroxide solution at a flow rate of 1.2 mL/min. Alendronate sodium was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Alendronate sodium was found to degrade significantly in oxidative stress conditions only. The developed method was extensively validated as per ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision and robustness. The developed LC method was successfully applied for analysis of in-vitro dissolution samples of marketed alendronate sodium tablets.

Development and Validation of Stability Indicating LC Method for Olmesartan Medoxomil

The present method provides the detailed description of development and validation of a simple stability indicating re- verse phase column liquid chromatographic method for Olmesartan in the presence of its impurities namely Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G and degradation products generated from forced degradation studies. The drug substance was subjected to stress conditions of aqueous hydrolysis, Oxidative, photolytic and thermal stress degradation. The degradation of Olmesartan was observed under acid hydrolysis, base hydrolysis and peroxide. The drug was found to be stable to other stress conditions attempted. Successful separation of the drug from synthetic impu- rities and degradation products formed under stress conditions was achieved on symmetry C18, 150 mm × 4.6 mm, 5μ column using a phosphate buffer, Acetonitrile and Milli Q water. The developed LC method was validated with respect to specificity, linearity, accuracy, precision, raggedness and robustness. The assay method was found to be linear in the range of 250 μg?mL–1 to with 1000 μg?mL–1 correlation coefficient of 0.9999 and the linearity of the impurities was es- tablished from LOQ to 0.4%. Recoveries of assay and impurities were found between 98.5% and 101.2%. The devel- oped LC method to determine the related substances and assay determinations of Olmesartan can be used to evaluate the quality of regular production samples and stability samples. To best of our knowledge, the validated stability indi- cating LC method which separates all the impurities disclosed in this investigation was not published elsewhere.

Stability-Indicating Liquid Chromatographic Method for the Quantification of the New Antipsychotic Agent Asenapine in Bulk and in Pharmaceutical Formulation

A simple, specific and stability-indicating reversed phase high performance liquid chromatographic method was developed for the quantitative determination of asenapine in tablet dosage form. A SunFire C18, 5 μm column having 250×4.6 mm i.d. in isocratic mode, with mobile phase containing 0.02 M potassium dihydrogen phosphate: acetonitrile (95:05, v/v, pH 3.5 adjusted with 1% o-phosphoric acid) was used. The flow rate was 1.0 mL min−1 and effluents were monitored at 232 nm. The retention time of asenapine was 5.51 min. The linearity for asenapine was in the range of 0.1–20 μg/ml. The recoveries obtained for asenapine were 98.31–101.51%. Asenapine stock solutions were subjected to acid and alkali hydrolysis, chemical oxidation, sunlight and dry heat degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time values. Stressed samples were assayed using developed LC method. The proposed method was validated with respect to linearity, accuracy, precision and robustness. The method was successfully applied to the estimation of asenapine in tablet dosage form.

A Validated Stability-Indicating UF LC Method for Bortezomib in the Presence of Degradation Products and its Process-Related Impurities

Degradation pathway for bortezomib is established as per ICH recommendations by validated and stability indicating reverse phase liquid chromatographic method. Bortezomib is subjected to stress conditions of acid, base, oxidation, thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Five impurities are studied and the major degradant (RRT about 1.19) was identified by LC-MS and spectral analysis. The stress samples are assayed against a qualified reference standard and the mass balance is found close to 99.5%. Efficient chromatographic separation is achieved on a Shim pack XR-ODS-II(100X3 mmx2.2 μm) stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 270 nm at a flow rate of 0.6 mL min. In the developed LC method the resolution between bortezomib and five potential impurities (imp-1, imp-2, imp-3, imp-4 and imp-5) is found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.999 for bortezomib and five potential impurities. This method is capable to detect the impurities of bortezomib at a level of 0.020 % with respect to test concentration of 2.0 mg mL for an 8-μL injection volume. The developed rapid LC method is validated with respect to specificity, linearity & range, accuracy, precision and robustness for impurities determination.

Determination of NiII(3-OMe-salophene) in MCF7 and HT29 cancer cell lines using HR-CS-AAS and in serum albumin using LC with monolithic silica

A high-resolution continuum source atomic absorption spectrometric method was developed and validated for the determination of NiII(3-OMe-salophene) (a complex with anticancer activity in vitro) in MCF7 and HT29 cancer cell lines. The primarily most sensitive line 232.003nm was selected for analysis. Compared to the standard nickel, the absorbance values obtained for NiII(3-OMe-salophene) complex was at least 93% at the upper end of linear range of the calibration curve. The use of common matrix modifiers including magnesium nitrate, palladium nitrate, ammonium hydrogen phosphate, lanthanum chloride and calcium nitrate brought no significant improvement in the GF AAS measurement. The dynamic linear working range of the calibration curve was found to be between 2.16 and 12.0μgL−1 (ppb). This covers a concentration range of the complex from 0.036μM to 0.204μM. Typical coefficients of variation (n=6) ranged from 0.2% to 6.7% for Ni in NiII(3-OMe-salophene). Detection and quantitation limits were 0.65 and 2.16μgL−1 (ppb), respectively. The proposed method has been successfully applied to the analysis of NiII(3-OMe-salophene) complex in cell lines of breast cancer (MCF7) and colon cancer (HT29). However, being based on the determination of nickel in the salophene complex, the method was unsuitable for the quantitation of NiII(3-OMe-salophene) in serum albumin, which originally contains significant amount of nickel. For this purpose, a high performance liquid chromatographic method with a monolithic silica RP-18e column has been developed to quantitate the complex in serum albumin. The developed chromatographic method depends on detecting the whole complex in serum rather than the bounded nickel. A mobile phase consisting of 25mM phosphate buffer pH 3/methanol (30:70, v/v) was pumped at a flow rate of 1mLmin−1. The eluted complex was monitored at a wavelength of 250nm. The dynamic linear working range of the calibration curve for the developed LC method was found to be between 100 and 20,000μgL−1 (0.23–46.18μM). Detection and quantitation limits were 30 and 100μgL−1 (ppb), respectively.

A Validated LC Method for Determination of Trace Impurities in Technical Triadimefon

A simple and sensitive liquid chromatography with ultraviolet detection (LC–UV) method was developed for the determination of three impurities with a content over 0.1% (w/w) in technical triadimefon. A Gemini C18 column (5 μm, 250 mm × 4.6 mm i.d.) was used for the chromatographic separations. The samples were separated by gradient elution with water (solvent A) and methanol (solvent B) using the following conditions: 70% A isocratic for 12 min, linear to 0% A within 8 min, and isocratic for 10 min at 0% A with a flow rate of 1.0 mL min−1. Chromatograms were recorded at an absorption wavelength of 280 nm. The chromatographic resolutions between triadimefon and its potential impurities A, B, and C were greater than 3. The developed LC method was validated with respect to linearity, accuracy, precision, and robustness. This method was successfully applied to analyze the impurities in commercial technical triadimefon. In addition, the structures of the three impurities were identified to be (A) 4-chlorophenol, (B) 1-(2,4-dichlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone, and (C) 1,1-bis(4-chlorophenoxy)-3,3-dimethyl-2-butanone.

DEVELOPMENT OF A VALIDATED STABILITY INDICATING LC METHOD FOR QUANTITATIVE DETERMINATION OF AN ANTISPASMODIC DRUG AND ITS RELATED SUBSTANCES

Present article describes development and validation of stability indicating liquid chromatographic method for quantitative determination of α-cyclohexyl-α-hydroxy-benzeneacetic acid-4-(diethylamino)-2-butynyl ester hydrochloride a potential antispasmodic and anticholinergic drug and its four impurities (related substances) using a Zorbax SB-Cyano column and mobile phase consisting of aqueous buffer (0.20% triethylamine) and acetonitrile in the ratio 49:51 and with a pH 6.3. The developed LC method was validated with respect to linearity, specificity, accuracy, precision, limit of detection, and limit of quantitation. For the quantitative determination of the impurities, a relative response factor approach was applied with respect to the drug substance. The developed LC method for the related substances and assay determination of drug can be used to evaluate quality of regular production samples. It can also be used to test the stability samples of the drug substance.

A Validated Stability-indicating Reverse Phase HPLC Assay Method for the Determination of Memantine Hydrochloride Drug Substance with UV-Detection Using Precolumn Derivatization Technique

This present paper deals with the development and validation of a stability indicating high performance liquid chromatographic method for the quantitative determination of Memantine hydrochloride. Memantine hydrochloride was derivatized with 0.015 M 9-fluorenylmethyl chloroformate (FMOC) and 0.5 M borate buffer solution by keeping it at room temperature for about 20 minutes and the chromatographic separation achieved by injecting 10 muL of the derivatized mixture into a Waters HPLC system with photodiode array detector using a kromasil C18 column (150 x 4.6 mm), 5 mu. The mobile phase consisting of 80% acetonitrile and 20% phosphate buffer solution and a flow rate of 2 milliliter/minute. The Memantine was eluted at approximately 7.5 minutes. The volume of FMOC used in derivatization, concentration of FMOC and derivatization time was optimized and used. Forced degradation studies were performed on bulk sample of Memantine hydrochloride using acid (5.0 Normal (N) hydrochloric acid), base (1.0 N sodium hydroxide), oxidation (30% hydrogen peroxide), thermal (105 degrees C), photolytic and humidity conditions. The developed LC method was validated with respect to specificity, precision (% RSD about 0.70%), linearity (linearity of range about 70-130 mug/mL), ruggedness (Overall % RSD about 0.35%), stability in analytical solution (Cumulative % RSD about 0.11% after 1450 min.) and robustness.

LC Separation of para and meta Isomers of Zafirlukast in Bulk Drug Samples and Pharmaceutical Dosage Forms Using a Chiral Stationary Phase

A simple, rapid and sensitive high performance liquid chromatographic method was developed for the separation and quantification of positional isomers of zafirlukast in bulk drugs and dosage forms using a chiral column. Elution time was 20 min in normal phase mode and ultra violet detection was carried out at 240 nm. Efficient separation was achieved on an immobilized amylose-based Chiralpak-IA column using n-hexane/ethanol/trifluoroacetic acid/diethyl amine (65:35:0.1:0.1, v/v) as the mobile phase. Resolutions between ortho, meta and para isomers of zafirlukast were found to be >3.0. The active pharmaceutical ingredient was extracted from tablets using tetrahydrofuran. The calibration graphs for meta and para isomers of zafirlukast were linear (r 2 > 0.999) when ranging from the limit of quantitation to 0.3%. The method showed excellent recoveries for both zafirlukast isomers identified in bulk and formulated products. The test solution was found to be stable in the mobile phase for 48 h after preparation. The developed LC method was validated with respect to linearity, accuracy, precision and robustness.

Development and Validation of a New Analytical Method for the Determination of Related Components in Quetiapine Hemifumarate

A simple, sensitive isocratic rapid resolution liquid chromatographic assay method has been developed for the quantitative determination of quetiapine hemifumarate in bulk active pharmaceutical ingredient, used for the treatment of schizophrenia. The developed method is also applicable for the process related impurities determination. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in a isocratic mode and quantification was by ultraviolet detection at 225 nm at a flow rate of 1.0 mL min−1. In the developed LC method the resolution between quetiapine hemifumarate and its three potential impurities was found to be greater than 2.0. Regression analysis showed an r value (correlation coefficient) greater than 0.99 for quetiapine hemifumarate and its three impurities. This method was capable to detect all three impurities of quetiapine hemifumarate at a level of 0.003% with respect to test concentration of 1.0 mg mL−1 for a 3 μL injection volume. The bulk active pharmaceutical ingredient 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 99.5%. The developed RR-LC method was validated with respect to linearity, accuracy, precision and robustness.

A validated stability-indicating RP-HPLC method for levofloxacin in the presence of degradation products, its process related impurities and identification of oxidative degradant

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of levofloxacin as well as its related substances determination in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its process related impurities. Forced degradation studies were performed on bulk sample of levofloxacin as per ICH prescribed stress conditions using acid, base, oxidative, water hydrolysis, thermal stress and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during oxidative stress and the degradation product formed was identified by LCMS/MS, slight degradation in acidic stress and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process related impurities and degradation products from the analyte were achieved on ACE C18 column using the mobile phase consists a mixture of 0.5% (v/v) triethyl amine in sodium dihydrogen orthophosphate dihydrate (25 mM; pH 6.0) and methanol using a simple linear gradient. The detection was carried out at 294 nm. The limit of detection and the limit of quantitation for the levofloxacin and its process related impurities were established. The stressed test solutions were assayed against the qualified working standard of levofloxacin and the mass balance in each case was in between 99.4 and 99.8% indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of levofloxacin at the time of batch release and also during its stability studies (long term and accelerated stability).

Development of a Validated Stability Indicating LC Method for Lamotrigine

The present method provides the detailed description of development of a simple, time efficient stability indicating reversed phase column liquid chromatographic method for lamotrigine in the presence of its impurities namely Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F 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 lamotrigine was observed under acid hydrolysis, base 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 product formed under stress conditions was achieved on Symmetry C18, 75 mm × 4.6 mm column using aqueous trifluoroacetic acid and acetonitrile. 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 5–60 μg mL−1 with correlation coefficient of 0.9999 and the linearity of the impurities were established from LOQ to 0.2%. Recoveries of assay and impurities were found between 99.1 and 101.4%. The developed LC method to determine the related substances and assay determination of lamotrigine can be used to evaluate the quality of regular production samples and stability samples. 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.

Chemical synthesis of bile acid acyl-adenylates and formation by a rat liver microsomal fraction

In mammals, unconjugated bile acids formed in the intestine by bacterial deconjugation are reconjugated ( N-acylamidated) with taurine or glycine during hepatocyte transport. Activation of the carboxyl group of bile acids to form acyl-adenylates is a likely key intermediate step in bile acid N-acylamidation. To gain more insight into the process of bile acid adenylate formation, we first synthesized the adenylates of five common, natural bile acids (cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and lithocholic acid), and confirmed their structure by proton NMR. We then investigated adenylate formation by subcellular fractions of rat liver (microsomes, mitochondria, cytosol) using a newly developed LC method for quantifying adenylate formation. The highest activity was observed in the microsomal fraction. The reaction required Mg 2+ and its optimum pH was about pH 7.0. In term of maximum velocity ( V max) and the Michaelis constant ( K m), the catalytic efficiency of the enzyme under the conditions used was highest with cholic acid of the bile acids tested. The formation of cholyl-adenylate was strongly inhibited by lithocholic and deoxycholic acid, as well as by palmitic acid; ibuprofen and valproic acid were weak inhibitors. In cholestatic disease, such adenylate formation might lead to subsequent bile acid conjugation with glutathione or proteins.

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 (Rs) 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.