Article Images (0)
×![]()
featured_play_listSave article to folder
X
Create new folder
Full Text
PDF
Table of Contents
Abstract
Keywords
Experimental
Chemicals
Equipment
Chromatographic Conditions
Preparation Of Solutions
Preparation Of Standard Solutions
Preparation Of Sample Solutions
Specificity
Analytical Method Validation
Precision
Sensitivity
Linearity And Range
Accuracy
Robustness
Solution Stability And Mobile Phase Stability
Results And Discussion
Method Development And Optimization
Results Of Forced Degradation Studies
Degradation In Acidic Solution
Precision
Sensitivity
Accuracy
Robustness
Solution Stability And Mobile Phase Stability
Assay Analysis
Conclusion
Acknowledgments
Abstract
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. 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 (Rs) 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 for a 10 lL injection volume. The interand 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.
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 (Rs) 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 lL 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.
Keywords
Column liquid chromatography Forced degradation Pharmaceutical dosage forms Deferasirox Deferasirox is an active iron chelating agent, chemically as 4-[3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]-benzoic acid and its structural formula is C21H15N3O4. Desirox is the generic name for deferasirox, is an orally active chelator that is selective for iron (Fe3+). Deferasirox is a tridentate ligand that binds iron with high affinity in a 2:1 ratio from the soluble iron pool in the plasma. Deferasirox has been developed to reduce iron-related morbidity and mortality. It is indicated for the treatment of chronic iron overload due to blood transfusions such as b-thalassemia and other chronic anemias (transfusional hemosiderosis). Clinical b-thalassemia is hereditary disorder characterized by defective production of hemoglobin, which leads to decreased production and increased destruction of red blood cells. It is the first oral medication approved in the USA for this purpose [1–6]. Few LC methods were reported in the literature for the analysis of deferasirox in biological samples [7] and the relative bioavailability of deferasirox tablets administered without dispersion and dispersed in various drinks, stability and compatibility tests were performed to identify beverages suitable for the dispersion of tablets for further testing in DOI: 10.1365/s10337-009-1023-1 0009-5893/10/09 2009 Vieweg+Teubner | GWV Fachverlage GmbH Original Chromatographia 2010, 72, September (No. 5/6) 441 man [8]. No LCmethods were reported in major pharmacopeias like USP, EP, JP and BP. Extensive literature survey reveals there is no stability-indicating LC method for determination of related substances and for quantitative estimation of deferasirox in bulk drugs and pharmaceutical dosage forms. An ideal stability indicating chromatographic method should estimate the drug to be able to resolve from its potential impurities and degradation products. The present drug stability test guideline Q1A (R2) issued by International Conference on Harmonization (ICH) suggested that stress studies should be carried out on a drug to establish its inherent stability characteristics, leading to separation of degradation products and hence supporting the suitability of the proposed analytical procedures. It also required that analytical test procedures for stability samples should be stability indicating and they should be fully validated [9–12]. Hence, an attempt has been made to develop an accurate, rapid, specific and reproducible method for the determination of deferasirox and all four impurities in bulk drug samples and in pharmaceutical dosage forms along with method validation as per ICH norms. The stability tests were also performed on both drug substances and drug products as per ICH norms.
Experimental
Chemicals
Samples of deferasirox and its related impurities were received from United States Pharmacopeia-India (Fig. 1). Commercially available 400 mg of deferasirox tablets (Desirox) were purchased from Cipla Pharmaceuticals, Mumbai, India. LC grade methanol and acetonitrile, analytical reagent grade potassium dihydrogen orthophosphate monohydrate and orthophosphoric acid were purchased from Merck, Darmstadt, Germany. High purity water was prepared by using Millipore Milli-Q plus water purification system. All samples and impurities used in this study were greater than 99.0% purity.
Equipment
The LC system, used for method development, forced degradation studies and method validation was a Waters 2695 binary pump plus auto sampler and a 2996 photo diode array detector (MA, USA). The output signal was monitored and processed using Empower software on Pentium computer (Digital Co). Photo stability studies were carried out in a photo stability chamber (Sanyo, Leicestershire, UK). Thermal stability studies were performed in a dry air oven (Mack Pharmatech, Hyderabad, India).
Chromatographic Conditions
The chromatographic column used was a Waters Inertsil ODS 3V (150 9 4.6) mm with 5 lm particles. The mobile phase consisted of a mixture of buffer, acetonitrile and methanol in the ratio (50:45:5, v/v). Buffer consists of 10 mM potassium dihydrogen orthophosphate monohydrate, pH adjusted to 3.0 using orthophosphoric acid. The column temperature was maintained at 35 C and the detection was monitored at a wavelength of 245 nm. The injection volume was 10 lL. Methanol was used as diluent.
Preparation of Solutions
Preparation of Standard Solutions
A stock solution of deferasirox (2.0 mg mL-1) was prepared by dissolving an appropriate amount in methanol. Working solutions were prepared from above stock solution for related substances determination and assay determination, respectively. A stock solution of impurities (mixture of imp-1, imp-2 imp-3 and imp-4) at a concentration of 0.5 mg mL-1 was also prepared in methanol.
Preparation of Sample Solutions
Desirox tablets contained 400 mg of deferasirox. The inactive ingredients present in Desirox were lactose monohydrate, crospovidone, povidone, sodium lauryl sulphate, microcrystalline cellulose, silicon dioxide, and magnesium stearate. Twenty Desirox tablets (400 mg) were weighed and the average weight was calculated. The tablets were powdered in a mortar and a sample of the powder equivalent to 50 mg of the active pharmaceutical ingredient (deferasirox) was transferred to a 100 mL volumetric flask. Approximately 75 mL methanol were added and the flask was placed on rotatory shaker for 10 min and sonicated for 10 min to dissolve the material completely. The solution was then diluted to 100 mL and centrifuged at 3,000 rpm for 10 min. The supernatant was collected and filtered through a 0.45 lm pore size Nylon 66-membrane filter. The filtrate was used as sample solution.
Specificity
is the ability of the method to measure the analyte response in the presence of its potential impurities. Stress testing of the drug substance can help to identify the likely degradation products, which can in turn help to establish the degradation pathways and the intrinsic stability of the molecule and validate the stability indicating power of the analytical procedures used. The specificity of the deferasirox in the presence of its impurities namely imp-1, imp-2, imp-3, imp-4 and degradation products was determined by the developed LC method. Forced degradation studies were also performed on deferasirox to provide an indication of the stability indicating property and specificity of the proposed method. The stress conditions employed for degradation study included light (carried out as per ICH Q1B), heat (60 C), acid hydrolysis (1 N HCl), base hydrolysis (0.1 NaOH), water hydrolysis (room temperature at 48 h) and oxidation (10% H2O2). For heat and light studies, the study period was 10 days where as for acid, base, peroxide and water hydrolysis the test period was 48 h. Peak purity of stressed samples of deferasirox was checked by using a 2996 photo diode array detector of Waters Corporation, MA, USA. 442 Chromatographia 2010, 72, September (No. 5/6) Original
Analytical Method Validation
The developed chromatographic method was validated for linearity, precision, accuracy, sensitivity, robustness and system suitability.
Precision
The precision of the related substance method was checked by injecting six individual preparations of (500 lg mL-1) deferasirox spikedwith 0.15% each imp-1, imp-2, imp-3 and imp-4. Each %RSD area of imp-1, imp-2, imp-3 and imp-4 was calculated. study was also determined by performing the same procedures on a different day (inter-day precision). The intermediate precision (ruggedness) of the method was also evaluated by a different analyst, different column and different instrument in the same laboratory. Assay method precision was evaluated by carrying out six independent assays of test sample of deferasirox against qualified reference standard. The %RSD of six assay values obtained was calculated. The intermediate precision of the assay method was evaluated by different analyst and by using different instrument from the same laboratory.
Sensitivity
was determined by establishing the limit of detection (LOD) and limit of quantitation (LOQ) for imp-1, imp-2, imp-3 and imp-4 estimated at a signal-tonoise ratio of 3:1 and 10:1 respectively, by injecting a series of dilute solutions with known concentrations. The precision study was also carried out at the LOQ level by injecting six individual preparations of imp-1, imp-2, imp-3 and imp-4, and the %RSD for the areas of each impurity was calculated.
Linearity and Range
Linearity test solutions for the assay method has prepared from stock solution at five concentration levels from 50 to 200% of assay analyte concentration (250, 375, 500, 750 and 1,000 lg mL-1). A linearity test solution for related substance method was prepared by diluting the impurity stock solution to the required concentrations. The solutions were prepared at seven concentration levels. From LOQ to 200% of the permitted maximum level of the impurity (i.e. LOQ 0.0375, 0.075, 0.1125, 0.15, 0.225 and 0.3%) was subjected to linear regression analysis with the least squares method. Calibration equation obtained from regression analysis was used to calculate the corresponding predicted responses. The residuals and sum of the residual squares were calculated from the corresponding predicted responses. Linearity was checked for three consecutive days in the same concentration range for both assay and related substance method and the %RSD value of the slope and Y-intercept of the calibration curve were calculated. Upper and lower levels of range were also established.
Accuracy
The accuracy of the assay method was evaluated in triplicate at five concentration levels, i.e. 250, 375, 500, 750 and Imp-2: Salicylic Acid Original Chromatographia 2010, 72, September (No. 5/6) 443 1,000 lg mL-1 in bulk drugs and pharmaceutical dosage forms. At each concentration, three sets were prepared and injected in triplicate. The percentage of recovery was calculated at each level. The bulk sample shows the presence of imp-4 at a level of the 0.05 and 0.05% of total impurities (limit: not more than 0.15% for a single unknown impurity, for total impurities the limit was 0.50%). The study was carried out in triplicate at 0.075, 0.125, 0.15, 0.225 and 0.3% of the analyte concentration (500 lg mL-1). The percentage of recoveries for imp-1, imp-2, imp-3 and imp-4 were calculated.
Robustness
To determine the robustness of the developed method, experimental conditions were deliberately changed and the resolution (Rs) between deferasirox, imp-1, imp-2, imp-3 and imp-4 were evaluated. The flow rate of the mobile phase was 1.0 mL min-1. To study the effect of flow rate on the developed method, 0.2 units of flow were changed (i.e. 0.8 and 1.2 mL min-1). The effect of column temperature on the developed method was studied at 30 and 40 C instead of 35 C. The effect of pH on resolution of impurities was studied by varying ±0.1 pH units (i.e. buffer pH altered from 3.0 to 2.9 and 3.1). In all the above varied conditions, the components of the mobile phase were held constant.
Solution Stability and Mobile Phase Stability
The solution stability of deferasirox in the assay method was carried out by leaving the test solutions of samples in tightly capped volumetric flasks at room temperature for 48 h. The same sample solutions were assayed at 6 h intervals up to the study period against freshly prepared standard solution. The mobile phase stability was also carried out by assaying the freshly prepared sample solutions against freshly prepared reference standard solutions at 6 h intervals up to 48 h. Mobile phase prepared was kept constant during the study period. The %RSD of assay of deferasirox was calculated for the study period during mobile phase and solution stability experiments. The solution stability of deferasirox and its related impurities was carried out by leaving both spiked and unspiked sample solution in tightly capped volumetric flasks at room temperature for 48 h. Content of imp-1, imp-2, imp-3 and imp-4 were determined at every 6 h interval, up to the study period. Mobile phase stability was also carried out for 48 h by injecting the freshly prepared sample solutions, for every 6 h interval. Content of imp-1, imp-2, imp-3 and imp-4 was checked in the test solutions. Mobile phase prepared was kept constant during the study period.
Results and Discussion
Method Development and Optimization
The main target of the chromatographic method was to achieve the separation of closely eluting impurities namely imp-2, imp-3 and retention time of the deferasirox peak. Potassium dihydrogen orthophosphate monohydrate buffer (10 mM) with pH 6.5 and methanol (70:30, v/v) was chosen for initial trial on a C18 stationary phase with a 25 cm length, 4.6 mm ID and 5 lm particle size. Flow rate was 1.0 mL min-1. When deferasirox sample spiked with all the impurities was injected the resolution (Rs) between all impurities was <2.0 and the retention time (Rt) of deferasirox was very high (*90 min). Another trial was carried out with mobile phase consisting of buffer (10 mM potassium dihydrogen orthophosphate monohydrate, pH adjusted to 3.0 with orthophosphoric acid) and methanol. With Inertsil ODS 3 V (250 9 4.6 mm) with 5 lm particles column improvement in resolution (Rs) between impurties was observed and the retention time (Rt) of deferasirox was about 60 min. To reduce the retention time a Waters Inertsil ODS 3 V (150 9 4.6 mm) with 5 lm particles column was chosen and marginal improvement in terms of resolution (Rs) and the retention time of deferasirox peak (Rs * 2 and Rt * 45 min) was observed. To further improve the retention time (Rt) of deferasirox a, higher percentage of acetonitrile was added to the above mobile phase. With a mobile phase consisting of buffer (10 mM potassium dihydrogen orthophosphate monohydrate buffer with pH 3.0), acetonitrile and methanol (50:45:5, v/v) satisfactory results (retention time (Rt) of deferasirox at *17 min and the resolution (Rs) between all the impurities of >2) were obtained. At 35 C column temperature, the peak shape of deferasirox was found symmetrical. In the optimized isocratic conditions deferasirox, imp-1, imp-2, imp-3 and imp-4 were well separated with a resolution (Rs) greater than 2 and the typical retention times of imp-1, imp2, imp-3, imp-4 and deferasirox were about 2.5, 3.4, 4.3, 10.2, and 16.8 min respectively. The system suitability results are given in Table 1. Effect of buffer pH was also studied. When the pH of the buffer was adjusted to 7.5 the tailing factor of deferasirox was high (about 2.5) and imp-2 and imp3 were co-eluted. When pH decreased towards acidic side the symmetry of the deferasirox peak was improved and, the resolution (Rs) between all the impurities was also improved. Satisfactory resolution between impurities and symmetry of the deferasirox peak was observed at pH 3.0. 444 Chromatographia 2010, 72, September (No. 5/6) Original
Results of Forced Degradation Studies
Degradation in Acidic solution
The drug was exposed to 1 N HCl at 60 C for 1 h. Deferasirox has shown significant sensitivity towards the treatment of 1 N HCl. The drug gradually undergone degradation with time in 1 N HCl and prominent degradation was observed (*35%) at RRT 2.9 (Fig. 2a). No major degradation products were observed when the sample was stressed in base hydrolysis, oxidative conditions, neutral, photolytic and thermal conditions after 48 h. From the degradation studies, peak purity test results derived from PDA detector, confirmed that the deferasirox peak was homogeneous and pure in all the analyzed stress samples. The mass balance of stressed samples was close to 99.5%. No degradants were observed after 60 min in the extended runtime of 90 min of all the deferasirox samples. The developed LC method was found to be specific in the presence of imp-1, imp-2, imp-3, imp-4 and their degradation products confirm the stability indicating power of the developed method. Method Validation
Precision
The %RSD deferasirox during the assay method precision study and intermediate precision study was 0.3 and the %RSD of area of imp-1, imp-2, imp-3 and imp-4 in the related substance method precision study were within 2.0. Confirming the good precision of the developed analytical method.
Sensitivity
The limit of detection of imp-1, imp-2, imp-3 and imp-4 were 0.003, 0.002, 0.007, and 0.008% (of analyte concentration, i.e. 500 lg mL-1) respectively for 10 lL injection volume. Under the same conditions, the LOQ were 0.009, 0.006, 0.021 and 0.024% (of analyte concentration, i.e. 500 lg mL-1) respec- tively. The precision at LOQ concentration for imp-1, imp-2, imp-3 and imp-4 were below 2%. Linearity and Range Linear calibration plot for the assay method was obtained over the calibration ranges tested, i.e. 250–1,000 lg mL-1 and the correlation coefficient obtained was greater than 0.999. The result showed an excellent correlationbetween thepeakarea and concentration of the analyte. Linear calibration plot for the related substance method was obtained over the calibration ranges tested, i.e. LOQto0.3% for imp-1, imp-2, imp-3 and imp-4. The correlation coefficient obtained was greater than 0.999 for all four impurities. The result showed an excellent correlation between the peak area and concentration of imp-1, imp-2, imp-3 and imp-4. The best-fit linear equation obtained was y = 8.3914x + 37.8876. At all concentration levels, standard deviation of peak area was significantly low and RSD was below 1.0%. Analysis of residuals indicated that they were scattered within ±2%with respect to 100% concentration response. Linearity was checked for related substances over the same concentration ranges on three consecutive days the %RSD of the slopes and Y-intercept of the calibration plots were with in 2.2 and 5 respectively. The range of the method had on LOQ to 0.3% of the analyte concentration (500 lg mL-1).
Accuracy
The percentage recovery of deferasirox in bulk drug samples ranged from 98.3 to 101.1% and in pharmaceutical dosage forms from 100.2 to 103.1% (Table 2). The percentage recovery of imp-1, imp-2, imp-3 and imp-4 in bulk drug samples ranged from 99.7 to 102.1% (Table 3). LC chromatograms of spiked sample with all four impurities in deferasirox bulk drug sampled are shown in Fig. 3a.
Robustness
Close observation of analysis results deliberately changed chromatographic conditions (flow rate, pH and column temperature) revealing that the resolution between closely eluting impurities, namely imp-1, imp-2, imp-3 and imp-4 were always greater than 2.0, illustrating the robustness of the method.
Solution Stability and Mobile phase Stability
The %RSD of assay of deferasirox during solution stability and mobile phase stability experiments was within 1.0. No significant changes were observed in the content of imp-1, imp-2, imp-3 and imp-4 during solution stability and mobile phase stability experimental. The solution stability and mobile phase stability experiments data confirmed that sample solutions and mobile phase used during assay and Original Chromatographia 2010, 72, September (No. 5/6) 445 related substance determinations were stable up to the study period of 48 h.
Assay Analysis
Analysis was performed for different batches of deferasirox in both bulk drug samples (n = 3) ranged from 99.95 to 99.96% and dosage forms (n = 3) ranged from 100.9 to 103.1%.
Conclusion
The RP-LC method developed for quantitative and related substance determinations of deferasirox in both bulk drugs and pharmaceutical dosage forms were precise, accurate and specific. Themethod was completely validated showing satisfactory data for all the method validation parameters tested. The developedmethod is stability indicating and can be used for the routine analysis of production samples and also to check the stability of deferasirox samples.
Acknowledgments
The authors wish to thank the management of United the States Pharmacopeia Laboratory-India for supporting this work.
Article Images (0)
×![]()
Bioz is the world’s most comprehensive AI search engine for scientific experimentation. The Bioz search engine offers researchers billions of data-driven product, technique, and protocol recommendations. Bioz taps into the latest advances in Artificial Intelligence (AI) – including Natural Language Processing (NLP), Machine Learning (ML) and Generative AI to mine and structure hundreds of millions of pages of complex and unstructured scientific papers. By harnessing the power of AI, Bioz provides researchers with an unprecedented amount of summarized scientific experimentation knowledge right at their fingertips, ultimately helping to speed up drug discovery and the rate of success in finding cures for diseases. Bioz is used by over 15 Million researchers from over 17,000 different universities and companies in 196 countries.
