INTRODUCTION:

Nilotinib¹ (Tasigna® 200mg ) is an orally available signal transduction inhibitor of the Bcr-Abl kinase, c-kit and Platelet Derived Growth Factor (PDGF), all of which play a role in cell proliferation, cell migration, and angiogenesis. Chemically it is 4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl) - 5-(trifluoromethyl)phenyl]-3- [(4-pyridin-3-ylpyrimidin-2-yl) amino]benzamide. Its molecular weight is 529.52 and molecular formula is C₂₈H₂₂F₃N₇O^2,3_. Literature survey reveals no chromatographic methods for the estimation of Nilotinib from pharmaceutical dosage forms. The availability of an HPLC method with high sensitivity and selectivity will be very useful for the determination of Nilotinib in pharmaceutical formulations. The aim of the study was to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of Nilotinib in bulk drug samples and in pharmaceutical dosage form.

Structure of Nilotinib

EXPERIMENTAL:

Materials and Methods:

Nilotinib was obtained as a gift sample from Hetero Drugs Ltd, Hyderabad. Acetonitrile and water used were of HPLC grade (Qualigens). Commercially available Nilotinib capsules (Tasigna® 300 mg, Novartis pharma) were procured from local market.

Instrument:

Quantitative HPLC was performed on liquid Chromatograph, Waters separation 2996, PDA detector module equipped with automatic injector with injection volume 10 µl, and 2693 pump. A RP Inertsil ODS-3V C-18 column (250x4.6 mm i.d; particle size 5 μm) was used. The HPLC system was equipped with Empower Software.

HPLC Conditions:

The contents of the mobile phase were 0.02M Dipotassium hydrogen orthophosphate in water pH-2.5 with orthophosphoric acid and Acetonitrile in the ratio of 60:40 v/v. They were filtered before use through a 0.45μm membrane filter, and pumped from the respective solvent reservoirs to the column at a flow rate of 1.0 ml/min. The run time was set at 16.0 min and the column temperature was ambient. Prior to the injection of the drug solution, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The eluents were monitored at 266 nm.

Preparation of Standard Stock solution:

A standard stock solution of the drug was prepared by dissolving 200 mg of Nilotinib in 100 ml volumetric flask containing 30 ml of water, sonicated for about 15 min and then made up to 100 ml with water to get 2000µg/ml standard stock solution.

Working Standard solution:

5ml of the above stock solution was taken in 50 ml volumetric flask and thereafter made up to 50 ml with mobile phase to get a concentration of 200µg/ml.

Preparation of Sample solution:

Twenty capsules (Tasigna® 300mg, Novartis Pharma) were weighed, and then powdered. A sample of the powdered capsules, equivalent to 200mg of the active ingredient, was mixed with 30 ml of water in 100 ml volumetric flask. The mixture was allowed to stand for 1 hr with intermittent sonication to ensure complete solubility of the drug, and then filtered through a 0.45μm membrane filter, followed by adding water up 100 ml to obtain a stock solution of 2000µg/ml. Transfer for 5ml of this solution to a 50 ml of volumetric flask and made upto sufficient volume with mobile phase to give an concentration of 200µg/ml.

Linearity:

Aliquots of standard Nilotinib stock solution were taken in different 10 ml volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of Nilotinib are in the range of 80-240μg/ml. Each of these drug solutions (10µl) was injected three times into the column, and the peak areas and retention times were recorded. Evaluation was performed with PDA detector at 266 nm and a Calibration graph was obtained by plotting peak area versus concentration of Nilotinib (Fig 2).

The plot of peak area of each sample against respective concentration of Nilotinib was found to be linear in the range of 80–240µg/ml with correlation coefficient of 0.999. Linear regression least square fit data obtained from the measurements are given in table I. The respective linear regression equation being Y= 81268.215X+174323.3.The regression characteristics, such as slope, intercept, and %RSD were calculated for this method and given in Table I.

Table I: Linear Regression Data for Calibration curves.

Parameters

Results of proposed HPLC Method

Concentration range (µg/ml)

Slope (m)

Intercept (c)

Correlation coefficient

% RSD

Standard error of estimate

80-240

81268.215

174323.3

0.999

0.5

150312.7

Assay:

10µl of sample solution was injected into the injector of liquid chromatograph. The retention time was found to be 8.508 minutes. The amount of drug present per capsule was calculated by comparing the peak area of the sample solution with that of the standard solution. The data are presented in Table II.

Table II: Results of HPLC Assay and Recovery studies

Sample

Amount claim

(mg/capsule)

% Found by the proposed method

% Recovery*

200

99.55

99.95

99.35

108.05

108.65

108.22

*Average of three different concentration levels.

Recovery Studies:

Accuracy was determined by recovery studies of Nilotinib, known amount of standard was added to the preanalysed sample and subjected to the proposed HPLC analysis. Results of recovery study are shown in Table II. The study was done at three different concentration levels.

RESULTS AND DISCUSSION:

The system suitability tests were carried out on freshly prepared standard stock solution of Nilotinib. Parameters that were studied to evaluate the suitability of the system are given in Table III.

Table III Validation Summary

Validation Parameter

Results

System Suitability

Theoretical Plates (N)

Tailing factor

Retention time in minutes

Resolution

% Area

9706.63

1.17

8.508

7.91

99.97

LOD (µg/ml)

LOQ (µg/ml)

0.1

0.3

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The limit of detection (LOD) and limit of quantification (LOQ) for Nilotinib were found to be 0.1µg/ml and 0.3µg/ml respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ.

From the typical chromatogram of Nilotinib as shown in fig 1, it was found that the retention time was 8.508 min. A mixture of 0.02M Dipotassium hydrogen orthophosphate in water pH-2.5 with orthophosphoric acid and Acetonitrile in the ratio of 60:40 v/v was found to be most suitable to obtain a peak well defined and free from tailing. In the present developed HPLC method, the standard and sample preparation required less time and no tedious extraction were involved. A good linear relationship (r=0.999) was observed between the concentration range of 80-240µg/ml. Low values of standard deviation are indicative of the high precision of the method. The assay of Nilotinib capsules was found to be 99.61%. From the recovery studies it was found that about 108.30% of Nilotinib was recovered which indicates high accuracy of the method. The absence of additional peaks in the chromatogram indicates non-interference of the common excipients used in the capsules. This demonstrates that the developed HPLC method is simple, linear, accurate, sensitive and reproducible.

Thus, the developed method can be easily used for the routine quality control of bulk and capsule dosage forms of Nilotinib within a short analysis time.

ACKNOWLEDGEMENTS:

The authors are grateful to M/s Hetero Drugs, Hyderabad for the supply of as a gift sample Nilotinib and to the Management, Omega college of Pharmacy, Hyderabad, for providing the necessary facilities to carry out the research work.

REFERENCES:

1. Kantarjian H et al. "Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL". N Engl J Med, Volume 354, Issues 24, Pages 2542–51, (2006).

2. Silvia De Francia et al, New HPLC–MS method for the simultaneous quantification of the antileukemia drugs imatinib, dasatinib, and nilotinib in human plasma; Journal of Chromatography B ,Volume 877, Issues 18-19, Pages 1721-1726, (2009).

3. Andrea Davies, Alison K. Hayes, et al, Simultaneous determination of nilotinib, imatinib and its main metabolite (CGP-74588) in human plasma by ultra-violet high performance liquid chromatography; Leukemia Research, Volume 34, Issue 6, Pages 702-707, (2010).

Received on 29.08.2011 Accepted on 19.10.2011

Asian J. Pharm. Ana. 1(4): Oct. - Dec. 2011; Page 100-102