Indo American Journal of Pharmaceutical Research, 2013
Journal home page: http://www.iajpr.com/index.php/en/
ISSN NO: 2231-6876
INDO AMERICAN JOURNAL OF PHARMACEUTICAL RESEARCH
Development and Validation of a Stability Indicating RP-HPLC Method for the Determination of Nilotinib (A Tyrosine Kinase Inhibitor) *G. Sowjanya, M. Mathrusri Annapurna and A. Venkata Sriram Department of Pharmaceutical Analysis & Quality Assurance, GITAM Institute of Pharmacy, GITAM University, Visakhapatnam, India-530045
ARTICLE INFO Article history Received 25/06/2013 Available online 30/06/2013
Keywords Nilotinib; RP-HPLC; stability-indicating; validation, ICH.
ABSTRACT Nilotinib is a tyrosine kinase inhibitor used for the treatment of chronic myelogenous leukemia. A stability indicating liquid chromatographic method (Isocratic mode) was developed and validated for the determination of Nilotinib. Chromatographic separation was achieved on a C18 column (250 mm × 4.6 mm i.d., 5 µm particle size) using water: acetonitrile: acetic acid (20:80: 0.03, v/v) with flow rate 1.0 mL/min (UV detection at 254 nm). Linearity was observed in the concentration range of 0.4–150 μg/mL (R2 = 0.9997) with linear regression equation y = 169118x - 11095. The LOQ was found to be 0.2384 μg/mL and the LOD was found to be 0.0785 μg/mL. Nilotinib was subjected to stress conditions and it was found that the drug is slightly sensitive towards oxidation in comparison to acidic, alkaline, thermal and photolytic degradation.
Corresponding author G. Sowjanya Department of Pharmaceutical Analysis, GITAM Institute of Pharmacy, GITAM University, Visakhapatnam, India-530045; Fax: 91-891-2795315 E-mail:-
[email protected] Mobile no: 91-9989640339.
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Copy right © 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Please cite this article in press as G. Sowjanya et.al. Development and validation of a stability indicating RP-HPLC method for the determination of Nilotinib (a tyrosine kinase inhibitor). Indo American Journal of Pharm Research.2013:3(6).
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INTRODUCTION Nilotinib (Figure 1) (NLT) chemically 4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5(trifluoromethyl)phenyl]-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-benzamide,mono hydro chloride, monohydrate is a white to slightly yellowish to slightly greenish yellow powder with molecular formula C28H22F3N7O•HCl• H2O and molecular weight 584 [1]. Rational design of novel inhibitors exhibiting effectiveness against imatinib-resistant mutants of BCR-ABL protein was carried out by researchers based upon the crystal structure of the imatinib-ABL complex and Nilotinib is a novel, selective BCR-ABL inhibitor so designed to fit into the ATP-binding site of the BCR-ABL protein with higher affinity than imatinib [2]. Literature survey revealed that Nilotinib was determined in pharmaceutical dosage forms by RP-HPLC [3-4] as well as in biological fluids using liquid chromatography [5-10] and liquid chromatography-mass spectrometric [11-12] methods. In the present work the authors have developed a simple, rapid, precise, accurate and robust stability indicating liquid chromatographic method for the determination of NLT in capsules and tablets as per ICH guidelines.
Figure 1: Chemical structure of Nilotinib MATERIALS AND METHODS Reagents and solutions: Nilotinib standard (purity 99.0 %) was obtained from Hetero Drugs, India). Acetonitrile (HPLC grade), sodium hydroxide (NaOH) and hydrochloric acid (HCl) and Hydrogen peroxide (H2O2) were obtained from Merck (India). Nilotinib is available (Label claim: 200 mg) with brand names TASIGNA as capsules and tablets. All chemicals were of analytical grade and used as received.
Twenty capsules and tablets were procured from local market and the contents were finely powdered. Powder equivalent to 25 mg Nilotinib was accurately weighed and transferred into a 25 mL volumetric flask and volume was made up with mobile phase. The volumetric flask was sonicated for 30 min to enable complete dissolution of Nilotinib and the solution was filtered through 0.45 mm nylon filter before injection. The filtrate was diluted with mobile phase to yield a concentration of 20 mg/mL. www.iajpr.com
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Assay:
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Instrumentation and chromatographic conditions: Chromatographic separation was achieved by using a Shimadzu Model CBM-20A/20 Alite HPLC system, equipped with SPD M20A prominence photodiode array detector with C18 (250mm × 4.6mm i.d., 5 µm particle size) column maintained at 25 ºC. Isocratic elution was performed using water: acetonitrile: glacial acetic acid (20: 80: 0.03, v/v). The overall run time was 10 min. and the flow rate was 1.0 mL/min. 20 µL of sample was injected into the HPLC system. The mobile phase was prepared by accurately mixing HPLC grade water: acetonitrile: glacial acetic acid (20: 80: 0.03, v/v) in a 1000 mL volumetric flask, sonicated and filtered through membrane filter. Nilotinib stock solution (1000 μg/mL) was prepared by accurately weighing 25 mg of NLT in a 25 mL volumetric flask with mobile phase. Working standard solutions were prepared on a daily basis from the stock solution with mobile phase and the solutions were filtered through a 0.45 μm membrane filter prior to injection.
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Forced degradation studies: The study was intended to ensure the effective separation of NLT and its degradation peaks of formulation ingredients at the retention time of NLT. Separate portions of drug product and ingredients were exposed to the following stress conditions to induce degradation. Forced degradation studies were performed to evaluate the stability indicating properties and specificity of the method. All solutions for use in stress studies were prepared at an initial concentration of 1 mg/mL of NLT and refluxed for 30 min at 80 ºC. All samples were then diluted in mobile phase to give a final concentration of 8 μg/mL and filtered before injection. Acidic and alkaline degradation studies: Acid decomposition was carried out in 0.1 M HCl at a concentration of 1.0 mg/mL NLT and after refluxation for 30 min at 80 ºC the stressed sample was cooled, neutralized and diluted with mobile phase. Similarly stress studies in alkaline conditions were conducted using a concentration of 1.0 mg /mL in 0.1 M NaOH and refluxed for 30 min at 80 ºC. After cooling the solution was neutralized and diluted with mobile phase as per the requirement before injection. Oxidation degradation studies: Solutions for oxidative stress studies were prepared using 0.3% H2O2 at a concentration of 1 mg/mL of NLT and after refluxation for 30 min at 80 ºC on the thermostat the sample solution was cooled and diluted with the mobile phase as per the requirement before injection. Thermal degradation studies: For thermal stress testing, the drug solution (1 mg/ mL) was heated in thermostat at 80 ºC for 30 min, cooled and diluted with the mobile phase as per the requirement before injection. Photolytic degradation studies: The drug solution (1 mg/ mL) for photo stability testing was exposed to UV light for 4 h UV light (365 nm) chamber and analyzed. METHOD VALIDATION The method was validated for the following parameters: system suitability, linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, selectivity and robustness.
Accuracy: The accuracy of the assay method was evaluated in triplicate at three concentration levels (80, 100 and 120%), and the percentage recoveries were calculated. Standard addition and recovery experiments were
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Precision: The intra-day precision of the assay method was evaluated by carrying out 9 independent assays of a test sample of NLT at three concentration levels (20, 40 and 80 µg/mL) (n=3) against a qualified reference standard. The % RSD of three obtained assay values at three different concentration levels was calculated. The inter-day precision study was performed on three different days i.e. day 1, day 2 and day 3 at three different concentration levels (20, 40 and 80 μg/mL) and each value is the average of three determinations (n=3). The % RSD of three obtained assay values on three different days was calculated.
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Linearity: Linearity test solutions for the assay method were prepared from a stock solution at different concentration levels (0.4–150 μg/mL) of the assay analyte concentration and 20 µL of each solution was injected in to the HPLC system and the peak area of the chromatogram obtained was noted. The calibration curve was plotted by taking the concentration on the x-axis and the corresponding peak area on the y-axis. The data was treated with linear regression analysis method [13].
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conducted to determine the accuracy of the method for the quantification of NLT in the drug product. The study was carried out in triplicate at 18, 20 and 22 µg/mL. The percentage recovery in each case was calculated. Sensitivity/Limit of quantification (LOQ) and limit of detection (LOD): The limit of quantification (LOQ) and limit of detection (LOD) were based on the standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in International Conference on Harmonization guidelines Q2 (R1) [13]. Sensitivity of the method was established with respect to limit of detection (LOD) and LOQ for NLT. LOD and LOQ were established by slope method as mentioned below. LOD and LOQ were experimentally verified by injecting six replicate injections of each impurity at the concentration obtained from the above formula. 3.3 × standard deviation of y-intercept LOD = Slope of the calibration curve 10 × standard deviation of y-intercept LOQ= Slope of the calibration curve Robustness: The robustness of the assay method was established by introducing small changes in the HPLC conditions which included wavelength (252 and 256 nm), percentage of acetonitrile in the mobile phase (58 and 62%) and flow rate (1.1 and 0.9 mL/min). Robustness of the method was studied using six replicates at a concentration level of 20 μg/mL of NLT. Solution stability and mobile phase stability: The solution stability of NLT in the assay method was carried out by leaving both the sample and reference standard solutions in tightly capped volumetric flasks at room temperature for 48 h. The same sample solutions were assayed at 12 h intervals over the study period. The mobile phase stability was also assessed by assaying the freshly prepared sample solutions against freshly prepared reference standard solutions at 12 h intervals up to 48 h. The prepared mobile phase remained constant during the study period. The % RSD of the NLT assay was calculated for the mobile phase and solution stability experiments. An additional study was carried out using the stock solution by storing it in a tightly capped volumetric flask at 4 ºC. RESULTS AND DISCUSSION A reversed-phase liquid chromatographic technique was developed to determine Nilotinib quantitatively pharmaceutical dosage forms. A detailed comparative study of the previously published methods with the present method was discussed in Table 1. Satisfactory resolution was achieved with use of a mixture of water: acetonitrile: glacial acetic acid (20:80:0.03%, v/v) (Figure 2A-2B). UV detection was carried out at 254 nm (PDA detector).
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HPLC method development and optimization: Initially the stressed samples were analyzed using a mobile phase consisting of water: acetonitrile (50:50, v/v) at a flow rate of 1.0 mL/min. Under these conditions, the resolution and peak symmetry were not satisfactory and therefore the mobile phase composition was changed to water: acetonitrile (20:80, v/v) with a flow rate of 1.0 mL /min under which a sharp peak was observed with good symmetry with slight tailing. Therefore, a slight amount of acetic acid was added to avoid tailing and therefore water: acetonitrile: glacial acetic acid (20:80:0.03%, v/v) was chosen as the best chromatographic response for the entire study.
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[B] Figure 2: Representative chromatograms of [A] Nilotinib (8 μg/mL) [B] Tasigna ® Capsules (Label claim: 200 mg) METHOD VALIDATION
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Table 1: Comparison of the performance characteristics of the present HPLC method with the published methods Linearity λ Re S. No. Mobile phase Remarks (nm) ( g/mL) f. Phosphate buffer: Acetonitrile Not stability 1 266 80-240 [3] (60:40, v/v) (pH 2.5) indicating Water: Acetonitrile Not stability 2 254 5-250 [4] (50:50, v/v) indicating Water: Acetonitrile: Glacial Stability indicating Present 3 254 0.4-150 acetic acid (20:80:0.03%, v/v) (PDA detector) method
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Linearity: The typical chromatogram for NLT obtained from the extracted marketed formulation (8 μg/mL) was shown in Figure 2B-2C. The calibration curve for NLT was linear over the concentration range of 0.4–150 μg/mL (Table 2). The data for the peak area of the drug for various concentrations was treated by linear regression analysis and the regression equation for the calibration curve (Figure 3) was found to be y = 169118x -11095 with correlation coefficient of 0.9997 which is nearly equals to unity. The LOD was found to be 0.0785 μg/mL and the LOQ was found to be 0.02384 μg/mL.
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Table 2: Linearity of Nilotinib Conc. (µg/mL) *Mean peak area ± SD 0.4 67425 ± 352.6 0.8 144145 ± 792.8 4 712517 ± 4346.4 8 1427763 ±10133.6 16 2826130 ± 18087.2 20 3369943 ± 23926.6 40 6543874 ± 35336.9 80 13449098 ± 88764.1 100 16619047 ± 121319.1 150 25617756 ± 174200.8 *Mean of three replicates
% RSD 0.52 0.55 0.61 0.71 0.64 0.71 0.54 0.66 0.73 0.68
30000000 y = 169118x - 11095 R2 = 0.9997
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Figure 3: Calibration curve of Nilotinib Precision: The precision of the method was determined by repeatability (intra-day precision) and intermediate precision (inter-day precision) of the NLT standard solutions. Repeatability was calculated by assaying three samples of each at three different concentration levels (20, 40 and 80 µg/mL) on the same day. The inter-day precision was calculated by assaying three samples of each at three different concentration levels (20, 40 and 80 µg/mL) on three different days. The % RSD range was obtained as 0.58-0.62 and 0.67-0.76 for intra-day and inter-day precision studies respectively (Table 3).
Intra-day precision Inter-day precision *Mean peak area ± SD % RSD *Mean peak area ± SD 3381251 ± 21061.06 0.62 3258969 ± 24721.91 6544854.3 ± 9403.16 0.61 6549822 ± 45825.76 13466108 ± 7704.46 0.58 13471747 ± 89875.29 *Mean of three replicates
% RSD 0.76 0.71 0.67
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Conc. (µg/mL) 20 40 80
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Table 3: Precision study of Nilotinib
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Accuracy: The method accuracy was proven by the recovery test. A known amount of NLT standard (10 μg/mL) was added to aliquots of samples solutions and then diluted to yield total concentrations as 18, 20 and 22 μg/mL as described in Table 4. The assay was repeated over three consecutive days. The resultant % RSD was in the range 0.344-0.707 (
