Stress Degradation Studies Research Articles

Isolation and structural characterization of degradation products of afatinib dimaleate by LC-Q-TOF/MS/MS and NMR: cytotoxicity evaluation of afatinib and isolated degradation products

Afatinib is an irreversible tyrosine kinase receptor inhibitor which was approved lately by USFDA for the treatment of metastatic non-small cell lung cancer (NSCLC). AFT was subjected to stress degradation studies under hydrolytic (acid, base and neutral), oxidative, thermal and photolytic conditions to investigate the inherent stability of the drug. The present study describes the simple and rapid HPLC method development for the selective separation of the AFT and its degradation products. The drug and degradation products were separated on Agilent Eclipse plus C18 (150 × 4.6 mm, 5 μ) column with ammonium acetate buffer (10 mM, pH 6.7) in gradient elution mode. The drug was found to be unstable in all the conditions studied. The developed chromatographic method was extended to tandem mass spectrometry (QTOF-MS) for the characterization of the degradation products. A total of 11 unknown degradation products were characterized using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS/MS). Two major degradation products (DP2 and DP3) were isolated using preparative HPLC and their structures were confirmed by conducting 1H and 13C NMR experiments. The isolated DPs were evaluated for their anticancer potential using non small cell lung cancer cell line A549. The IC50 values for AFT, DP2 and DP3 were found to be 15.02 ± 1.49, 25.00 ± 1.26 and 32.56 ± 0.11 respectively. The in silico toxicity studies were performed employing ProTox-II software for the assessment of toxicity potential of drug and its degradation products. Finally, the developed chromatographic method was validated as per the International Conference on Harmonization guideline Q2 (R1).

UPLC Method for Simultaneous Estimation of Ledipasvir and Sofosbuvir in Bulk and Dosage Forms and Their Stress Degradation Studies

UPLC Method for Simultaneous Estimation of Ledipasvir and Sofosbuvir in Bulk and Dosage Forms and Their Stress Degradation Studies

Stability indicating RP-UPLC method for the simultaneous determination of Atorvastatin and Amlodipine

Amlodipine is a calcium channel blocker. Atorvastatin is used for lowering the cholesterol. A new stability indicating liquid chromatographic method (RP-UPLC) has been proposed for the simultaneous estimation of Atorvastatin and Atorvastatin. Acquity UPLC HSS C18 column was used for the chromatographic study and a mobile phase mixture consisting of 0.1% Formic acid: Acetonitrile: Methanol (0.1:45: 55, v/v)/ 0.3/220 (Flow rate: 0.3 mL/min) (PDA detector 220 nm) was used for the elution of these two drugs. Stress degradation studies were performed and the method was validated as per ICH guidelines.

Stress degradation studies of hydrochlorothiazide and development of validated method by UV spectroscopy

To develop a simple, precise, accurate, and stability indicating a UV-method for estimation of Hydrochlorothiazide(HCT)in bulk and formulated dosage form.The drug was alsosubjected to stress degradation at different conditions recommended by the International Conference on Harmonization (ICH). The samples are generated and used for the degradation studies.The λmax of the HCTwas found to be 273nm.The linearity of calibration curve (Absorbance Vs Concentration) in pure solution was checked over the concentration ranges of about 5-30μg/mLwith the correlation coefficient higher than 0.99. The regression equation of the curve was Y = 0.598x + 0.0042.% RSD was found to be within the limit as per ICH guidelines. The obtained percentage recovery of HCTwas found to be within the limit100% ± SD.  Stress degradation studies revealed thatitwas within the limit(5-20%).

STABILITY-INDICATING REVERSED-PHASE HPLC METHOD FOR THE SEPARATION AND ESTIMATION OF RELATED IMPURITIES OF CILNIDIPINE IN PHARMACEUTICAL FORMULATIONS

A novel HPLC method was developed and validated for the determination of cilnidipine (CLDP) along with its related impurities A and B in pharmaceutical formulations. The chromatographic separation was carried out by isocratic elution using an X Terra C18 column (250×4.6mm; 5μ). The mobile phase was composed of methanol and phosphate buffer at a pH of 5.8 in the ratio of 10:90 (V/V) at a flow rate of 1.0 mL/min. The eluents were detected and quantified at a UV detection wavelength of 229 nm. Calibration curves of all analytes in the range of 2-12μg/mL showed a good correlation linearly (r ≥ 0.999) with recovery rate of more than 98% for each analyte. The percentage RSD in intraday, interday precision and ruggedness were found to be less than 2. Small variations in the developed conditions like mobile phase ratio, flow rate, pH and UV wavelength do not influence the results. This proves the precise and robust nature of the developed method. Stress degradation studies were conducted for standard drug and high amount of degradation was observed under UV light exposure. After 24hours, the molecules degraded up to 9.967%. In base hydrolysis, the CLDP molecule degrades up to 6.223%. In other stress conditions like acidic (5.347%), oxidative (4.916%) and thermal (4.319%) conditions, CLDP was found to be highly labile. In stress degradation study, there is no interference of both known impurities and other degradation products formed and were separated from CLDP. Therefore, this method was found to be selective and specific. The method development and validation result confirms that this method is suitable for determination and quantification of process impurities (A and B) of CLDP in pharmaceutical formulations. Key

Stability-indicating high-performance thin-layer chromatographic method for the estimation of antipsychotic drug combination clozapine and aripiprazole

A sensitive and precise high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous estimation of clozapine and aripiprazole in combination. The method employed HPTLC aluminum plates pre-coated with silica gel 60 F254 as the stationary phase, while the solvent system was toluene‒methanol‒ethyl acetate‒ammonia (6.5:2.5:1:0.1, v/v). The RF values were observed to be 0.43 and 0.60 for clozapine and aripiprazole, respectively. Densitometric analysis was carried out in absorbance mode at 218 nm. The method was linear in the range of 200–1600 ng per band for clozapine and 100-800 ng per band for aripiprazole. The stress degradation study was performed, and it was found that clozapine was susceptible to acid hydrolysis, base hydrolysis, and photolytic degradation study. Aripiprazole was susceptible to oxidative stress degradation study. The method was validated and applied successfully for the estimation of both drugs in the synthetic mixture.

Stress Degradation Studies of Anagliptin, Development of Validated Stability Indicating Method, Degradation Kinetics Study, Identification and Isolation of Degradation Products

A gradient-specific stability indicating HPLC method was developed and validated for the determination of the antidiabetic agent anagliptin in laboratory mixtures. Reversed-phase chromatography was performed using a Shimadzu LC-20 AD pump (binary), Shimadzu PDA M-20A diode array detector, and Waters Symmetry C-18 column (150 × 4.6 mm, 3.5 µm) maintained at a column oven temperature of 40 °C with UV detection at 247 nm. A gradient program was run at flow rate of 1 mL min−1. Mobile phase A consisted of a mixture of acetate buffer(10 mm) pH 5/methanol/acetonitrile in the ratio of 90:5:5. Mobile phase B consisted of a mixture of acetate buffer (10 mm) pH 5/methanol/acetonitrile in the ratio of 50:25:25. The method was validated according International Conference of Harmonization (ICH) guidelines. Linearity was observed in the concentration range of 10–120 µg/mL with regression coefficient r2(0.999). The LOD was found to be 7.8 µg/mL and LOQ was found to be 22.68 µg/mL. Anagliptin was subjected to stresses such as acidic, alkali, oxidation, photolysis, and thermal conditions. The proposed method was validated as per ICH guidelines and was found to be accurate, precise, and specific. The drug showed significant degradation in alkaline and oxidative conditions. Alkaline and oxidative degradation followed first-order kinetics. Degradation rate constant and half-lives were determined. Degradation products in alkaline and oxidative conditions were identified by LC–MS. One major degradation product was isolated from each condition by preparative HPLC. These degradation products were characterized by 1H NMR, 13C NMR, DEPT, D2O exchange, MS/MS, HRMS, and IR techniques. From the spectral data the alkaline degradation product was characterized as 1-{2-[1-(2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamido)-methyl-propan-2-yl-amino]acetyl}pyrrolidine-2-carboxamide. The oxidative degradation product was characterized as N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo-[1,5-a]pyrimidine-N-oxido-6-carboxamide.

An eco-friendly and alternative method of forced degradation of fluoroquinolone drugs by microwave irradiation: a new application for analytical eco-scale

Forced degradation studies are essential to determine the drug stability, elucidate the main degradation routes, and monitor the degradation products in qualitative/quantitative terms. Over the years, the pharmaceutical industry has been using a traditional and conventional method that employs a stove as the heating source; this requires a lengthy process and high energy. Recently, a new forced degradation method using modern microwave reactors has been reported as a greener, more economical and efficient alternative. The present work reports for the first time degradation of fluoroquinolone drugs (levofloxacin and norfloxacin) under microwave irradiation. Also for the first time, it presents the utilization of analytical eco-scale as a novel comprehensive approach to evaluating the greenness of analytical methodology for studies on the forced degradation. The objective of this study was to design and validate a forced degradation method assisted by microwave irradiation, alongside a conventional stress degradation study, and to compare the two methods. Microwave irradiation showed excellent performance as it yielded similar amounts of specific degradation products for both drugs, equivalent to what is produced in the conventional procedure. Therefore, there were some advantages to the new eco-friendly degradation method with respect to the significantly reduced time (72 times), energy (360 times), reagents and waste.

Abstract CT063: BAT1706 demonstrates high similarity to Bevacizumab in comparative analysis with originator and phase I clinical trials in New Zealand and China

Abstract Bevacizumab, a humanized monoclonal antibody against VEGF, has been approved by FDA to treat colon, lung, renal cancers and glioblastoma. We have developed BAT1706 as a biosimilar to Bevacizumab. Their evaluation was performed using several analytical assays and stress degradation studies. Both products BAT1706 shows similar binding affinity and biological function as Bevacizumab. In additionAdditionally, , several analytical assays and stress degradation studies have been conducted to compare multiple lots of BAT1706 and Bevacizumab. Tthe data indicate that BAT1706 is at least as pure as Bevacizumab and that the following attributes are consistent highly similar between both products: isoelectric point, peptide mapping profile, amino acid sequence, disulfide bond numbers and location, molecular weight and size, binding affinity towards the antigen and relevant immunologic receptors, isoform ratios, levels of impurities, glycosylation parameters, and secondary and tertiary structures. The degradation pathways and rates were evaluated using two different conditions (high temperature and strong light exposure) by means of several stability indicating methods. All Both products showed similar reactions during the incubating conditions and similar degradation rates. These data demonstrated that BAT1706 is highly similar to Bevacizumab, in structure, function, process impurities, and stability attributes. We have also completed two phase I studies with BAT1706 in New Zealand and China. Data revealed that a single IV infusion of BAT1706 (1 mg/kg) is safe and well tolerated in healthy subjects. No death was reported during either study and there was only 1 SAE of fibula fracture, which was not considered as related to BAT1706 by the investigator. The key PK parameters of both Phase I studies also revealed that BAT1706 is a bioequivalent to Bevacizumab. No positive results of anti-drug antibodiesy werewas reported for any subject in either study. Currently, a global randomized, double blind phase III study is on-going, to compare the efficacy, and to evaluate the safety and immunogenicity of BAT1706 to EU Bevacizumab in patients with previously untreated advanced non-squamous NSCLC. Citation Format: Jin-Chen Yu. BAT1706 demonstrates high similarity to Bevacizumab in comparative analysis with originator and phase I clinical trials in New Zealand and China [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT063.

Studies of Stress Degradation of Ramosetron Hydrochloride, a 5-HT3 Antagonist

Three issues of fundamental importance in drug therapy are safety, efficacy and stability. Extensive information derived from stress degradation studies of Ramosetron HCl will expand the scientific thought further to ensure to achieve the intended quality of Ramosetron HCl as drug substance and drug products available in the market. Stress degradation screening of Ramosetron HCl was conducted in aqueous, 0.1N, 0.5N, 1.0N and 2.0N acid-base and oxidative (3, 5 and 10 % H2O2) conditions and photo degradation. No degradation was found in aqueous condition at 60°C for 7 days. Acceptable degradations were found in 0.5 N, 1N and 2N HCl at 70ºC for 7 days, 0.1N NaOH at 60°C for 2 days, 3% hydrogen peroxide at room temperature for 1, 2 and 3 hours in dark and 3.6 million lux fluorescence light or 600 watts hour/m2 UV light. Among other applied stress conditions, the base hydrolysis and oxidative methods degraded Ramosetron hydrochloride more drastically than the other stressed conditions.Dhaka Univ. J. Pharm. Sci. 17(1): 9-17, 2018 (June)

Liquid chromatography/electrospray ionization tandem mass spectrometry study of repaglinide and its forced degradation products.

Stress stability studies of drugs have been recognized as an essential part of the drug development process. These studies are used to investigate the intrinsic stability of the drugs and for the development of a selective stability indicating assay method (SIAM). Stress testing is also useful for the formulation and packaging development, shelf-life determination and designing of manufacturing processes. As per regulatory guidelines, stress degradation studies and structural characterization should be carried out to establish degradation pathways of the drug, which is essential from both the efficacy and safety point of view. As the stress stability studies of repaglinide have not been reported in the literature, the present study has been undertaken. Repaglinide (RP), an oral anti-diabetic drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress conditions as per International Conference on Harmonization (ICH) guidelines Q1A (R2). The chromatographic separation of the drug and its degradation products (DPs) was achieved on an Agilent XDB C-18 column using the gradient elution method with a mobile phase consisting of 20mM ammonium acetate and acetonitrile at flow rate of 1.0mLmin-1 . The DPs were characterized using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with accurate mass measurements. The drug degraded under hydrolytic and oxidative stress, while it was stable under thermal and photolytic stress conditions. In total, six DPs were formed and the LC/MS method described here can resolve all DPs from the parent as well as from each other under various stress conditions. To elucidate the structures of DPs, fragmentation of the [M+H]+ ions of RP and its DPs was studied by using LC/ESI-MS/MS combined with accurate mass measurements. The forced degradation of RP carried out as per ICH guidelines results in the formation of six degradation products which have been characterized using LC/MS/MS in combination with accurate mass measurements.

Quantitative Determination of thymoquinone in Nigella Sativa and its nano formulation using validated stability indicating HPTLC densiometric method

An accurate and stability indicating high-performance thin layer chromatographic method (HPTLC) was developed and validated for the quantification of thymoquinone (TQ) as per the ICH guidelines. The analysis was carried out on the aluminum plate using n-hexane: ethyl acetate: methanol (7:2:1 v/v/v) as the mobile phase and the densitometric determination was carried out by TLC scanner (CAMAG) at 254 nm. The developed method was validated for different parameters like linearity, precision, recovery, robustness, and stressed stability study. The developed analytical method was found to be linear in the concentration range of 75-500 ng band-1 with regression value closer to unity (r2 = 0.997). The developed system was found to give compact spots for thymoquinone (Rf 0.77) with the limit of detection and limit of quantification (18 and 54 ng band-1) respectively. Further, the study showed accuracy, precision and repeatability were all within the required limits. The stress degradation study of TQ showed well separated degraded peak from the pure thymoquinone. The mean recoveries measured at three concentrations were more than 95% with RSD ≤ 3%. The method has been successfully applied in the analysis and routine quality control of herbal material and formulations containing TQ.Taleuzzaman et al., International Current Pharmaceutical Journal, September 2017, 6(10): 53-60http://www.icpjonline.com/documents/Vol6Issue10/01.pdf

Separation, Characterization and quantification of Impurities and Identification of stress degradants of Cabazitaxel by RP-HPLC and LC-ESI-MS techniques

Cabazitaxel is a natural taxoid used to treat prostate cancer in advanced stage. The authors have separated and identified seven impurities of Cabazitaxel using RP-HPLC and LC-MS techniques. Gradient mode was chosen for the separation of impurities in presence of Cabazitaxel and the method was validated as per the International Conference on Harmonization guidelines. The impurities were quantified in the present study and the plausible degradation path was also proposed. Stress degradation studies were performed and the degradants obtained were characterized with the help of mass spectra. Shimadzu HPLC prominence coupled with MS Spectrometry (THERMO) with Sunfire C18 column (100x 4.6 mm, 3.5 μm particle size) was used for performing the chromatographic separation with 0.05% formic acid and acetonitrile mixture as mobile phase with flow rate 1.0 ml/min. The injection volume 10μL and wavelength was monitored at 220 nm). The method was applied to the pharmaceutical formulation to study the suitability of the method. The method is very much useful for the pharmacokinetic studies as well as the metabolites study in vitro and in vivo of cancer patients.

Analytical Method Development and Validation for Simultaneous Estimation of Candesartan Cilexetil and Hydrochlorothiazide in Tablet Dosage form

A simple, precise and reproducible stability indicating HPLC method has been developed and validated for simultaneous determination of Candesartan and Hydrochlorothiazide on an Hypersil BDS C18 column (150*4.6*5um) using a mobile phase consisting of phosphate buffer pH 5.4 and acetonitrile (65:35) at flow 1.0 ml/min. Detection was carried out at 232nm . The method was validated with respect to specificity, accuracy linearity, precession, ruggedness and robustness parameters as per ICH guidelines. Linearity of the method was found to be 0.999; %RSD of precession was below 2.0%, recovery of added drug within 98-102 %, System suitability parameters were within limit for Candesartan and Hydrochlorothiazide. This method is simple, accurate and precise, sensitive and applicable for analysis of Candesartan and Hydrochlorothiazide in tablet formulation. In Stress degradation studies were also carried out under stress testing conditions of acidic, basic, oxidative, photolytic, thermal and neutral degradation.

Development and validation of a new stability indicating ultrafast liquid chromatographic method for the determination of Exemestane tablets

Exemestane is an irreversible steroidal aromatase inactivator. Exemestane is used for postmenopausal women who are suffering with estrogen receptor positive early breast cancer as an adjuvant treatment. A new stability indicating ultrafast liquid chromatographic method was developed and validated for the determination of Exemestane. Shimadzu Model UFLC system with PDA detector was used for the present study. Mobile phase consisting of tetra butyl ammonium hydrogen sulphate and acetonitrile (30: 70, v/v) was used with a flow rate of 1.2 mL/min (Detection wavelength 250 nm). Exemestane shows linearity 0.5–100 µg/mL with linear regression equation y = 62584 x + 599.1 (R2 = 0.9999). The LOD and LOQ were found to be 0.1401 and 0.4348 µg/mL. Stress degradation studies were performed and method was validated as per ICH guidelines.

Estimation of Anti Diabetic Teneligliptin in Bulk and Formulation by Densitometric and Spectrophotometric Method

A simple, accurate, precise and economical HPTLC and UV method has been developed and validated for the estimation of teneligliptin hydrobromide hydrate (THH) in bulk and tablet dosage form. The chromatographic method employed pre-coated silica gel 60F254 plates using toluene: methanol: triethylamine (8:2:0.2 v/v/v) as mobile phase. The plates were developed to a distance of 8.0 cm at ambient temperature. Experimental conditions such as band size, chamber saturation time, migration of solvent front, slit width, etc were critically studied and the optimum conditions were selected. A TLC scanner set at 254 nm was used for direct evaluation of the chromatograms in reflectance/absorbance mode. The system was found to give good result for Teneligliptin at Rf 0.51. The calibration plot was found linear between concentration range 0.5-3 μg/ band and r2 = 0.9993. Method was validated according to the ICH guidelines. In stability testing, teneligliptin was found susceptible to alkali hydrolysis and oxidatative degradation. Because the method could effectively separate the drug from its degradation products, it can be used as a stability indicating method. A UV spectrophotometric method was also developed using methanol as solvent at λ max 247 nm. Beer's law was obeyed in the concentration range of 5-50 μg/ml and r2 = 0.9997. The proposed method was validated according to the ICH guidelines. Therefore, both the methods and stress degradation study can be used for routine quality control analysis of Teneligliptin in bulk and pharmaceutical formulation.

Stability-Indicating TLC-Densitometric Assay for Methyltestosterone and Quantum Chemical Calculations.

Methyltestosterone is a synthetic testosterone derivative commonly used for the treatment of testosterone deficiency in males and one the anabolic steroids whose use is banned by World Anti-Doping Agency (WADA). This study presents a simple, cost-effective and rapid stability-indicating assay for densitometric quantification of methyltestosterone in pharmaceutical formulation. The developed method employed pre-coated TLC plates with mobile phase hexane:acetone (6.5:3.5 v/v). Limit of detection and limit of quantitation were found to be 2.06 and 6.24 ng/spot, respectively. Stress degradation study of methyltestosterone was conducted by applying various stress conditions such as hydrolysis under acidic, basic and neutral conditions, heating in anhydrous conditions and exposure to light. Methyltestosterone was found to be susceptible to photodegradation, acidic and basic hydrolysis. Degraded products were well resolved with significantly different Rf values. Acid degraded product was identified as 17,17-dimethyl-18-norandrosta-4,13(14)-dien-3-one through spectroscopic methods. The reactivity of methyltestosterone under applied stress conditions was also explained by quantum chemical calculations. The developed method is found to be repeatable, selective and accurate for quantification of methyltestosterone and can be employed for routine analysis.

Development and Validation of a Stability-Indicating Method for Perillyl Alcohol Incorporated in Poly(lactide-co-glycolide) Nanoparticles and Stress Degradation Studies

Perillyl alcohol has been studied in the treatment of cancer disease. However, its high toxicity is a drawback, which can be overcome by its incorporation in nanostructured systems. The aim of this work was to develop and validate a chromatographic method for determination of perillyl alcohol encapsulation efficiency in a polymeric nanoparticles formulation and evaluation of the presence of related degradation products. Perillyl alcohol was subjected to forced conditions of hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as suggested in the International Conference of Harmonization (ICH) guidelines. The drug showed significant degradation under acidic conditions. The degradation products could be adequately separated on an XBridge C18 column (100 × 2.1 mm, 3.5 µm) using isocratic elution (350 µL min−1) of water/acetonitrile (65 : 35, v/v) at 210 nm. Data from validation studies demonstrated that the method is selective, linear (coefficient of determination (R2) > 0.999) over the range of 20.0-80.0 µg mL−1, precise (relative standard deviation (RSD) < 2.0%), accurate (98.07 to 101.99%) and robust for minor changes. The method was successfully applied to determine the encapsulation efficiency of perillyl alcohol in polymeric nanoparticles, both for product development and for quality control purposes. After nanoparticles production, the presence of degradation products was not observed indicating that the single-emulsion solvent-evaporation technique used does not favor chemical degradation of the drug.

Stress Degradation Studies and Development of Validated SpectrometricAssay-Method For Determination of Tofacitinib In Pure and Physical Admixtures

Aim:The aim is to develop simple validated analytical method for analysis of Tofacitinib by UV Spectroscopy and to study the forced degradation and stress conditions have been used to detect the stability of Tofacitinib. Method: Tofacitinib was estimated at 285.9nm. Linearity range was found to be 10-50 mcg/ml. The correlation coefficient was found to be 0.9996. The molar absorptivity was found to be 12468.77mol/cm. The proposed method Sandell’s sensitivity was found to be 0.040410 µg cm2/0.001AU. The limit of detection and quantification were found to be 0.8169 and 2.4755 µg/ ml respectively. The degradation behavior of Tofacitinib was carried out as per the standard procedures and guidelines. Forced acid hydrolytic degradation, alkali degradation and oxidative degradation of was performed in bulk Tofacitinib and laboratory prepared admixtures using 1M Hydrochloric acid up to 48 hrs, in 10 % Hydrogen peroxide up to 48 hrs and for 1.0 M Sodium hydroxide up to 10 min at room temperature. The resulting solutions were analyzed for content by UV spectrophotometry at the maximum absorption of 285.9 n. The assay value of Tofacitinib in bulk and physical admixture was calculated at different time intervals for intraday and interday experiments. Results and Conclusions: The proposed method was successfully applied for the determination of tofacitinib in pure and laboratory prepared physical mixtures. The % RSD value of Tofacitinib in bulk and physical admixture was calculated at different time intervals for recovery , precision (Iintraday and Interday experiments) and quantification studies were found to be less than 2 %.

Development and validation of RP-UPLC method for simultaneous estimation of Cobicistat and Darunavir

A new, simple, precise and stability-indicating UPLC (Ultra Performance Liquid Chromatography) method was developed and validated for the simultaneous determination of anti-viral drugs Cobicistat and Darunavir. The method was developed using Acquity UPLC HSS C18 x 1.8 μ.with isocratic elution. 0.1% orthophosphoric acid buffer and acetonitrile (35: 65 v/v) were used as mobile phase with 0.4 ml/min flow rate at 30°C temperature. The detection wavelength was fixed at 250 nm, the run time was within 2 min. The Retention time of Cobicistat and Darunavir were 1.06 and 0.79 min respectively. The method was validated in terms of linearity, precision, accuracy and robustness. Calibration plots were linear over the range of 7.5–45 μg/ml for Cobicistat and 40–240 μg/ml for Darunavir. Recovery was in the range of 98–102% with the relative standard deviation of less than 2% for both drugs. The limit of detection and the limit of quantification for the Cobicistat were found to be 0.161 and 0.488 μg/ml, respectively, and for Darunavir 0.55 and 1.67 μg/ml respectively. The specificity of the method was analyzed by the stress degradation study in acidic, alkaline, oxidative, thermal, UV and hydrolytic conditions. The suggested method was suitable for determination of Cobicistat in bulk and pharmaceutical dosage forms.