Degradation Impurities Research Articles

Stability-Indicating LC-UV Method for the Determination of Eszopiclone and Degradation Impurities in Tablet Dosage Form

A sensitive, stability-indicating reversed-phase high-performance liquid chromatographic method was developed for the determination of eszopiclone and related impurities in tablet dosage form. The chromatographic separation was achieved on an Inertsil C18 column (250 × 4.6 mm, 5 µm), using a mobile phase consisting of 0.05M monobasic sodium phosphate buffer containing 0.8% sodium lauryl sulfate (pH 3.5) and acetonitrile in the ratio of 60:40 (v/v), at a flow rate of 1.5 mL/min and temperature of 40°C. Quantification was achieved with photodiode array detection at 303 nm. The described method showed excellent linearity over a range of limits of quantification to 4.8 µg/mL (150% of specification limit; i.e., 3.2 µg/mL). The drug product was subjected to the stress conditions of oxidative, acid, base, thermal and photolytic degradation. Eszopiclone degradation was observed in acid hydrolysis, base hydrolysis and peroxide stress conditions. Eszopiclone was stable in thermal and photolytic degradation conditions. The developed method is simple, selective and accurate for the quantification of impurities and degradation products of eszopiclone in tablet dosage form.

VALIDATED GRADIENT STABILITY-INDICATING UPLC METHOD FOR THE DETERMINATION OF LIDOCAINE AND ITS DEGRADATION IMPURITIES IN PHARMACEUTICAL DOSAGE FORM

Objective: Aim of the present work was to develop a stability indicating ultra performance liquid chromatography (UPLC) method to determine Lidocaine and its degradation impurities in pharmaceutical dosage forms. Method: Chromatographic separation was achieved by gradient elution on Agilent eclipse plus C18 (100x4.6) mm, and 1.8m column with potassium dihydrogen phosphate buffer (pH 4.50) and acetonitrile within a short runtime of 14.0 min. The eluted compounds were monitored at 230 nm using photodiode array (PDA) detector, the flow rate was 1.0 mL/min, and the column oven temperature was maintained at 40 ?C. Result: The resolution of Lidocaine and six (potential, bi-products and degradation) impurities was greater than 2.0 for all pairs of components. The repeatability and intermediate precision, expressed by the RSD, were less than 1.0%. The accuracy and validity of the method were further ascertained by performing recovery studies. The specificity of the method was investigated under different stress conditions including hydrolytic, oxidative, photolytic and thermal as recommended by ICH guidelines. Relevant degradation was found to take place under oxidative condition. Conclusion: Method was Robustness against small modification in pH, column oven temperature, flow rate and percentage of the mobile phase composition was ascertained. All these results provide that the method has stability indicating properties being fit for its intended purpose; it may find application for the routine analysis of the related substances of Lidocaine formulations.

DEVELOPMENT OF A STABILITY-INDICATING UPLC METHOD FOR DETERMINING ZIPRASIDONE HYDROCHLORIDE AND ITS ASSOCIATED DEGRADATION PRODUCTS PRESENT IN ACTIVE PHARMACEUTICAL INGREDIENTS AND PHARMACEUTICAL DOSAGE FORMS

A simple, sensitive, and reproducible ultra-performance liquid chromatography (UPLC) coupled with a photodiode array detector method was developed for the quantitative determination of Ziprasidone Hydrochloride (ZIP) in API and pharmaceutical dosage forms. Chromatographic separation was achieved on an Acquity UPLC BEH 100-mm, 2.1-mm, and 1.7-µm Shield RP-18 columns, and within a short runtime, that is, within 8.0 min. The eluted compounds were monitored at 230 nm, the flow rate was 0.3 mL/min, and the column oven temperature was maintained at 27°C. The resolution of ZIP and seven (potential, bi-products, and degradation) impurities were greater than 2.0 for all pairs of components. The high correlation coefficient (r2 > 0.9992) values indicated clear correlations between the investigated compound concentrations and their peak areas within the test ranges. The repeatability and intermediate precision, expressed by the RSD, were less than 2.0%. The accuracy and validity of the method were further ascertained by performing recovery studies via a spike method. The accuracy of the method expressed as relative error was satisfactory. The drug was subjected to the International Conference on Harmonization (ICH)-prescribed hydrolytic, oxidative, photolytic, and thermal stress conditions. The performance of the method was validated according to the present ICH guidelines.

Kinetics and mechanism of solid state imidapril hydrochloride degradation and its degradation impurities identification

Abstract A detailed stability testing of solid state imidapril hydrochloride (IMD) was performed and its degradation products were identified. The analysis was conducted according to ICH guidelines Q1A(R2). Pure IMD samples were exposed to stress conditions of elevated temperature and relative humidity (T = 363 K, RH = 76.4%) in order to accelerate degradation. The regular loss of IMD content with time, and the formation of two degradation impurities were observed. The appropriate reaction rate constants k (for IMD degradation and for the formation of product I and II) were calculated using Prout-Tompkins equation. The obtained degradation products were separated and identified by means of LC-MS technique. Based on the obtained m/z values, the masses and the structures of the formed degradation impurities were established. Also IMD degradation scheme was constructed. It was demonstrated that under the applied analytical conditions, IMD degradation follows an autocatalytic reaction model with the rate constant k = (4.764 ± 0.34)×10 −6 s −1 and with the parallel formation of two degradation products: imidaprilat and the diketopiperazine derivative. The obtained experimental results are in agreement with IMD degradation pathways proposed theoretically.

A Validated Stability-Indicating UHPLC Method for Determination of Naproxen and Its Related Compounds in Bulk Drug Samples

A simple, rapid, precise, accurate, rugged and robust stability-indicating ultra-fast high performance liquid chromatographic (UHPLC) method has been developed for the estimation of related compounds (imp-A, imp-B, imp-C, imp-D and imp-E) in Naproxen and also the assay of Naproxen from bulk drug samples. The stability indicating capability of the method was proven by subjecting the samples to stress conditions such as acid, base, oxidation, photolysis and thermal degradation. The efficient chromatographic separation was achieved using mobile phase solution A prepared as buffer solution 10 mM monobasic potassium phosphate pH 4.0 ± 0.05 adjusted with diluted ortho phosphoric acid solution and solution B acetonitrile with linear gradient elution on poroshell 120 EC-C18 shot column (50 mm × 4.6 mm, 2.7 μm) and UV detection at 235 nm at a flow rate 1.0 mL/min, column oven temperature was set to 25?C. The above are all known impurities and degradation impurities are well resolved with Naproxen peak and these are eluted within a 10 min runtime of HPLC. The photo diode array detector was used for peak homogeneity testing during stress study experiments and the overall mass balance was found to be 99.2% to 100.2% in all stress conditions. The linear calibration range was found to be 0.05 μg/mL to 0.75 μg/mL for related compounds and 50 μg/mL to 150 μg/mL for Naproxen and the accuracy of the method was found to be 91.5% to 98.5% recovery for the related substance method and 95.4% to 97.4% recovery for the assay method. The Naproxen and related compounds were found to be stable up to 48 hours and the method validation data show excellent results for precision, linearity, specificity, limit of detection, limit of quantitation and robustness. The present method can be successfully used for routine QC and stability studies and it will help to reduce the analysis cost, time and effluent load compared to conventional HPLC methods.

Determination of Dapiprazole Hydrochloride in the Presence of Process and Degradation Impurities by a Validated Stability Indicating RP-HPLC Method

Determination of Dapiprazole Hydrochloride in the Presence of Process and Degradation Impurities by a Validated Stability Indicating RP-HPLC Method

Isolation and Characterisation of Degradation Impurities in the Cefazolin Sodium Drug Substance

Two unknown impurities were detected in the cefazolin sodium bulk drug substance using gradient reversed-phase high-performance liquid chromategraphy (HPLC). These impurities were isolated by preparative HPLC and characterized by using spectroscopic techniques like LC-MS, LC-MS/MS, 1D, 2D NMR, and FT-IR. Based on the spectral data, the impurities have been characterized as N-(2,2-dihydroxyethyl)-2-(1H-tetrazol-1-yl)acetamide (Impurity-I) and 2-{carboxy[(1H-tetrazol-1-ylacetyl)amino]methyl}-5-methylidene-5,6-dihydro-2H-1,3-thiazine-4-carboxylic acid (Impurity-II). The structures of these impurities were also established unambiguously by co-injection into HPLC to confirm the retention time. To the best of our knowledge, these two impurities were not reported elsewhere.

Rapid Quantitative Determination of Related Substances and Degradants in Milnacipran

A simple, sensitive, precise and accurate stability-indicating ultra-performance liquid chromatography (UPLC) method was developed for the quantitative determination of the purity of the active pharmaceutical ingredient of milnacipran hydrochloride (MCP) and its pharmaceutical dosage form in the presence of its process-related impurities and degradation products. The proposed UPLC method utilizes Acquity UPLC BEH 100 mm, 2.1 mm and 1.7 µm phenyl columns at 27°C with a gradient program of 12.0 min at a flow rate of 0.5 mL/min. The compounds of interest were monitored at 210 nm. The resolution for MCP and 12 (potential byproducts and degradation) impurities was found to be greater than 2.0 for any pair of components. The high correlation coefficients (r(2) >0.9995) indicate clear correlations between the concentrations of investigated compounds and their peak areas. The relative standard deviations obtained for the repeatability and intermediate precision experiments were less than 5.0%. The accuracy of the method was further ascertained by performing recovery studies through spiking experiments. The drug substance was subjected to hydrolytic, oxidative, photolytic and thermal stress conditions, as per the International Conference on Harmonization (ICH). The developed method was validated according to the current ICH guidelines for specificity, limits of detection and quantitation, linearity, accuracy, precision, ruggedness and robustness. The method is also suitable for the assay determination of MCP in pharmaceutical dosage forms.

A Novel, Validated Stability-Indicating UPLC Method for the Estimation of Lansoprazole and its Impurities in Bulk Drug and Pharmaceutical Dosage Forms

A novel, reversed-phase ultra-performance liquid chromatographic method was developed and validated for the determination of the assay and related substances of Lansoprazole (LAN) in bulk drug and capsule dosage forms. The related substances include degradation and process-related impurities. The method was developed using the Waters Acquity BEH C18 column and gradient program with mobile phase A as a pH 7.0 phosphate buffer and methanol in the ratio of 90: 10 (v/v), and mobile phase B as methanol and acetonitrile in the ratio of 50:50 (v/v). Lansoprazole and its impurities were monitored at 285 nm. Lansoprazole was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, humidity, and photolytic degradation and found to degrade significantly under acid and oxidative stress conditions. The degradation products were well-resolved from the main peak and its impurities, proving the stability-indicating power of the method. The performance of the method was validated according to the present ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness, and robustness.

Stress Degradation Behavior of Atorvastatin Calcium and Development of Suitable Stability-Indicating LC Method for the Determination of Atorvastatin, its Related Impurities and its Degradation Products

A rapid, reversed-phase liquid chromatographic method was developed for the quantitative determination of Atorvastatin calcium, its related substances (12 impurities), and degradation impurities in bulk drugs. The chromatographic separation was achieved on a Zorbax Bonus-RP column by employing a gradient elution with water–acetonitrile–trifluoroacetic acid as the mobile phase in a shorter run time of 25 min. The flow rate was 1.0 mL/min and the detection wavelength was 245 nm. The drug substance was subjected to stress studies such as hydrolysis, oxidation, photolysis, and thermal degradation, and considerable degradation was observed in acidic hydrolysis, oxidative, thermal, and photolytic stress conditions. The formed degradation products were reported and were well-resolved from the Atorvastatin and its related substances. The stressed samples were quantified against a qualified reference standard and the mass balance was found to be close to 99.5% (w/w) when the response of the degradant was considered to be equal to the analyte (i.e. Atorvastatin), which demonstrates the stability-indicating capability of the method. The method was validated in agreement with ICH requirements. The method developed here was single and shorter (25 min method for the determination of all 12 related impurities of Atorvastatin and its degradation products), with clearly better resolution and higher sensitivity than the European (85 min method for the determination of six impurities) and United States pharmacopeia (115 min and 55 min, two different methods for the determination of six related substances).

Identification of Degradation Components in Lindane Pharmaceutical Dosage Forms by GC-MS and their Quantification by GC-ECD

Chemical degradation of drugs often results in altered therapeutic efficacy and can lead to toxic side effects. A single, short and sensitive GC-ECD method for the quantification of degradation and process related impurities of lindane has been developed and validated in all of its pharmaceutical topical dosage forms (shampoo, cream and lotion). Three major degradants were observed during forced degradation (stress stability) studies of lindane dosage forms under various conditions recommended by International Conference on Harmonization (ICH). These were identified and confirmed by using both chemical ionization (CI) and electron ionization (EI) techniques of GC-MS analysis. One major degradation component was identified to be the same as the one that was enhanced during the accelerated and long term stability studies of the dosage forms. A dehydrohalogenation mechanism was proposed for this degradation process, which should considerably ease the pharmaceutical development of lindane dosage forms. The chromatographic conditions were optimized using an impurity-spiked solution and the samples that were generated from forced degradation studies. The best chromatographic separation was achieved on a USP-G27 column using electron capture detector (ECD). The newly developed GC-ECD method was validated with respect to linearity, accuracy, precision and robustness. The limit of detection for the lindane and its impurities were found to be 0.004,0.005,0.005 and 0.004 μg/mL respectively. The accuracy (%recovery) was observed to be 100.0±3.0% for lindane and 100.0±5.0% for its impurities respectively in all-three dosage forms. The current method provides a significant improvement in monitoring stability, quality and therapeutic efficacy of lindane pharmaceutical dosage forms. Keywords: Forced degradation, GC-ECD, GC-MS, Lindane Lotion, Lindane Shampoo, Method validation

Stress Degradation Assessment of Lamotrigine Using a Validated Stability‐Indicating HPTLC Method

In this work, a sensitive and stability‐indicating HPTLC method for the determination of lamotrigine is presented. According to the International Conference on Harmonization guidelines Q1A, lamotrigine was exposed to a variety of stress conditions; these include heating in acidic, basic and neutral media. Its stability towards oxidative stress, humidity, high temperature and direct sunlight was also examined. Separation of the drug from its forced degradation impurities was achieved using TLC silica gel plates and a mobile phase composed of ethyl acetate: methanol: ammonia. The linear regression analysis of the data obtained for the correlation plots showed good linearity over the concentration range of 10–300 ng/spot. The forced degradation studies showed that lamotrigine is susceptible to degradation under acidic, basic, neutral and oxidative conditions, among which alkaline‐induced hydrolysis had the highest degradative potential. Alternatively, the drug was stable under heat, humidity, and daylight stress factors. In order to assess the purity and stability of the drug in tablet formulations, the developed method was applied to the analysis of commercial tablets in brand and generic products. The obtained results showed that the degradation of the drug has not occurred in the marketed formulations that were analyzed by the described methodology.

VALIDATED RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ALISKIREN AND VALSARTAN IN TABLET DOSAGE FORM

A simple, precise and accurate stability indicating RP-HPLC method has been developed and Subsequently validated for simultaneous estimation of Aliskiren (ALN) and Valsartan (VAL) from their combination dosage form. Water’s HPLC equipped with UV-Visible and Diode Array detectors, with Empower -2 software was used. Column used was XTerra® RP8, 5 μm, 100 mm × 4.6 mm i.d., at ambient temprature.Mobile phase consisting of 0.05M Ammonium Acetate and 0.5% TEA buffer having pH 5.5and Acetonitrile in the ratio of 68:32 v/v at a flow rate of 1.0 mL/ min and UV detection was carried out at 238 nm and 271 nm for ALN and VAL, respectively. ALN, VAL and their combined dosage form were exposed to thermal, photolytic, oxidative, acid-base hydrolytic stress conditions, the stressed samples were analyzed by proposed method. Peak purity results suggested no other co-eluting, interfering peaks from excipients, impurities, or degradation products due to variable stress condition, and the method is specific for the estimation of ALN and VAL in presence of their degradation products and impurities within 6 minutes. The retention time of Aliskiren and Valsartan were 1.98 and 4.03 minutes respectively. The method was found linear over the range of 1-20 μg per ml for Aliskiren and 1.6-240 μg per ml for Valsartan. The proposed method was validated as per the ICH and USP guidelines.

Identification, Synthesis, and Strategy For Minimization of Potential Impurities Observed In Raltegravir Potassium Drug Substance

Multiple sources of anticipated degradation and process impurities of raltegravir potassium drug substance observed during the laboratory optimization and later during its bulk synthesis are described in this article. The impurities were monitored by UPLC, and their structures are tentatively assigned on the basis of fragmentation patterns in LC–MS and NMR spectroscopy. Most of the impurities are synthesized, and their assigned constitutions were confirmed by co-injection in UPLC. In addition to the formation, synthesis, and characterization, strategy for minimizing these impurities to the level accepted by ICH is also described. We feel that our study will be helpful to the generic industry for obtaining chemically pure raltegravir potassium.

Development of a Stability-Indicating RP-LC Method for the Separation of a Critical Pair of Impurities and their Degradants in Zafirlukast

A high-throughput reverse-phase liquid chromatographic (RP-LC) method is developed for the quantification of zafirlukast and its related impurities in drug substance. The separation of known impurities is accomplished using a short (50 mm) LC column with sub-2-µm particle size in a relatively short run-time. A linear gradient elution involves ammonium formate and acetonitrile as mobile phase. The critical impurity pair is the meta and para isomers of zafirlukast, which are known to be potential impurities of zafirlukast, whose resolution is sensitive to pH. The stability-indicating capability of the developed method is demonstrated using forced degradation samples from stress conditions such as hydrolysis, oxidation, thermal and photolytic degradation. The developed RP-LC method is validated in accordance with International Conference on Harmonization requirements. The results from the validation study indicate that this RP-LC method can be used for the determination of synthetic and degradation impurities in regular quality control analysis for the drug substance.

Analysis of Cyclosporin A and Main Degradation Impurities by Cyclodextrin–Modified Micellar Electrokinetic Chromatography

Separation of the immunosuppressive drug cyclosporin A (CyA) from the closely related degradation impurities cyclosporin H (CyH, a CyA diastereomer) and isocyclosporin A (IsoCyA) was accomplished by means of cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC). Heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin (TM-βCD) showed to be an effective modifiers of the sodium dodecyl sulfate (SDS) micellar system, allowing for the CD-MEKC separation of CyA from CyH and IsoCyA. By means of electric current measurements carried out using the capillary electrophoretic apparatus, the critical micelle concentration of SDS in the presence of different neutral cyclodextrins was estimated. Interestingly, it was found that TM-βCD strongly inhibited the micellization of SDS compared to β-, and hydroxypropyl-β-cyclodextrin. This unusual behavior was considered to be a key factor of the specific ability of the methylated cyclodextrin in providing CD-MEKC separation of hydrophobic cyclosporins. The optimized method consisted of: 50 mM sodium dodecyl sulfate (SDS) in 50 mM tetraborate buffer (pH 9.2) supplemented with 15 mM TM-βCD. The electrophoretic runs were performed at 30°C under the application of 22 kV in a fused-silica capillary (50 µm id, 64.5 cm total length, 56 cm length to the detector). The method was validated for linearity of CyA (within 0.25–5.00 mg/mL) and CyH and IsoCyA (within 0.25–5.0%, w/w with respect to CyA), sensitivity (LOQ 5.0 µg/mL), accuracy, and precision. The applicability of the method was proved by analysis of a commercially available pharmaceutical (gelatin capsules).

Use of Radioactive Compounds and Autoradiography to Determine Drug Tissue Distribution

Radioactivity has been used in drug discovery and development for several decades because it offers researchers a highly sensitive way to quantitatively assess the absorption, distribution, metabolism, and/or excretion (ADME) of chemical entities by incorporating a radioactive isotope into the structure of the drug molecule. Regulatory agencies around the world require drug makers to characterize the ADME properties of prospective new drugs as one way to help ensure that patients are not exposed to dangerous drug and/or drug metabolite levels before they can be approved for human use. Radiolabeled compounds have consistently proved to be the most efficient tool for determining that information, even though attempts have been made to use nonradioactive techniques. The techniques of quantitative whole-body autoradiography (QWBA) and microautoradiography (MARG), which rely on the use of radiolabeled drugs, are two techniques that are routinely used to examine tissue distribution of drugs in discovery and development. These techniques provide drug researchers with quantitative tissue concentration data and a visual location of those concentrations in intact organs, tissues, and cells of laboratory animals. It is important for readers to realize that these techniques visualize total radioactivity, which can include the parent molecule along with its metabolites, and/or degradation products or impurities. This requires investigators to treat the quantitative data with caution unless the identity of the radioactivity is determined using some type of other bioanalytical techniques, such as mass spectroscopy and/or radio-HPLC, which can be easily performed on the tissue obtained from the animals used for QWBA and/or MARG. Nevertheless, these data are used in drug discovery and development to answer questions related to tissue penetration, fetal/placental transfer, tissue retention, routes of elimination, drug-drug interactions, enzyme induction/inhibition, formulation comparisons, in vivo compound solubility, differential metabolite distribution, interspecies comparisons, and to predict human exposure to parent drugs, metabolites, and radiation during clinical studies. This review will consider the strategic use of WBA, QWBA, and MARG in the pharmaceutical industry. Case studies and anecdotal information will also be presented; however, readers should realize that these are general examples and that some details have been omitted for brevity and/or because the data is proprietary and could not be presented at this time. Nevertheless, the images and discussions are provided to demonstrate how the techniques can and have been used to examine in situ tissue distribution of therapeutic compounds.

A VALIDATED STABILITY INDICATING UPLC METHOD FOR SIMULTANEOUS DETERMINATION OF ASSAY, RELATED SUBSTANCES, AND DEGRADATION PRODUCTS OF PALIPERIDONE PALMITATE ACTIVE PHARMACEUTICAL INGREDIENT AND ITS PHARMACEUTICAL INJECTION FORMS

A simple, sensitive, and reproducible ultra-performance liquid chromatography (UPLC) coupled with a photodiode array detector method was developed for the quantitative determination of Paliperidone palmitate in API and pharmaceutical injectable forms. The method is applicable to the quantification of related substances and assay of Paliperidone palmitate. Chromatographic separation was achieved on Acquity UPLC BEH (50 mm, 2.1 mm, and 1.7 µm) C-18 column, and the impurities are eluted with a gradient program of runtime about 10.0 min. The eluted compounds were monitored at 238 nm, the flow rate was 0.5 mL/min, and the column oven temperature was maintained at 35°C. The resolution of Paliperidone palmitate and eight (potential, bi-products and degradation) impurities was greater than 2.0 for all pairs of components. The high correlation coefficient (r2 > 0.999) values indicated clear correlations between the investigated compound concentrations and their peak areas within the test ranges. The repeatability and intermediate precision, expressed by RSD, were less than 10.0%. The accuracy and validity of the method were further ascertained by performing recovery studies via a spike method. The accuracy of the method expressed as relative error was satisfactory. The drug was subjected to the International Conference on Harmonization (ICH) prescribed hydrolytic, oxidative, photolytic, and thermal stress conditions. The performance of the method was validated according to the present ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness, and robustness.

A Validated Stability-Indicating Liquid Chromatographic Method for Determination of Degradation Impurities and Diastereomers in Voriconazole Tablets

A reversed-phase gradient liquid chromatographic method has been developed for the quantitative determination of Voriconazole, along with its degradation and diastereomeric impurities in tablet dosage form. Chromatographic separation has been achieved on an Inertsil ODS 3V, 150 × 4.6 mm, 5 μm column. The mobile phase consisting of solvent A 0.05 molar (M) potassium dihydrogen phosphate (pH 2.5 buffer) and solvent B (mixture of acetonitrile and methanol in the ratio 90:10 (v/v)), was delivered at a flow rate of 1.2 mL min−1 with the detection wavelength at 256 nm. Resolution of Voriconazole and all five potential impurities was achieved at greater than 2.0 for all pairs of compounds. The drug was subjected to stress conditions such as oxidative, acid and base hydrolysis, and thermal and photolytic degradation. Voriconazole was found to degrade significantly under base hydrolysis stress conditions compared to acid hydrolysis stress conditions. The degradation products were well-resolved from the main peak and its impurities, thus proving the stability-indicating power of the method. The stressed samples were assayed against a reference standard and the mass balance was found to be close to 99.0%. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision, and robustness.

A Novel Validated Stability Indicative UP-LC Method for Etravirine for the Determination of Process Related and Degradation Impurities

A novel stability indicating reverse phase ultra performance liquid chromatographic (UP-LC) method has been developed for Etravirine along with eight impurities (imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and imp-8) and validated as per ICH recommendations. Stress degradation conditions were established for Etravirine by subjecting it to stress conditions of acid, base, oxidation, humidity, thermal and photolysis. Significant degradation is observed in base stress condition and the major degradant (RRT at about 0.94) is identified by LC-MS and spectral analysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.0%. Efficient chromatographic separation was achieved on a Shimpack ODS-II stationary phase with a gradient mobile phase combination. Quantification was carried at 303 nm at a flow rate of 0.6 mL?min–1. The resolution between Etravirine and eight potential impurities is found to be greater than 2.0. Regression analysis shows as r value (correlation coefficient) of greater than 0.999 for Etravirine and eight potential impurities. This method is capable to detect the impurities of Etravirine at a level of 0.003% with respect to test concentration of 1.0 mg·mL–1.