INTRODUCTION:

Gefitinib is a novel antiemetic agent used in cancer chemotherapy; with a chemical name N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine. It’s molecular weight is 446.902363 g/mol with molecular formula C₂₂H₂₄ClFN₄O_3. Literature survey reveals only two chromatographic methods^1,2 for the determination of Gefitinib which are concerned with biological fluids. So far, no assay procedure has been reported for the estimation of Gefitinib from pharmaceutical dosage form. The availability of an HPLC method with high sensitivity and selectivity will be very useful for the determination of Gefitinib in pharmaceutical formulations. The aim of the study was to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of Gefitinib in bulk drug samples and in pharmaceutical dosage form.

Structure of Gefitinib

EXPERIMENTAL:

Gefitinib was obtained as a gift sample from Benzochem Organics, Mumbai. Dipotassium Hydrogen orthophosphate was of analytical grade, and supplied by M/s S.D.Fine Chem Limited, Mumbai. Methanol and water used were of HPLC grade (Qualigens). Commercially available Gefitinib tablets (Geftinat 250 mg, Natco) 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 20 µl, and 2693 pump. A RP C-18 Hypersil BDS column (250x4.6 mm i.d; particle size 5 μm) was used. The HPLC system was equipped with Empower Software.

Table I: Linear Regression Data for Calibration curves.

Drug

Gefitinib

Concentration range (µg/ml)

Slope (m)

Intercept (b)

Correlation coefficient

% RSD

25-300

94342263

77672.77

0.9999

0.25

HPLC Conditions:

The contents of the mobile phase were 0.02 M Dipotassium Hydrogen ortho phosphate and Methanol in the ratio of 10:90 v/v was used. They were filtered before use through a 0.45 μm 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 8 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 246 nm.

Table II: Results of HPLC assay and Recovery studies

Sample	Amount claim [mg/ tablet]	% found by the Proposed method.	% Recovery
1.	250	99.5	99.82
2.	250	99.24	99.92
3.	250	99.77	99.79

*Average of three different concentration levels.

Preparation of Standard Stock solution:

A standard stock solution of the drug was prepared by dissolving 250 mg of Gefitinib in 100 ml volumetric flask containing 30 ml of diluent [HPLC Grade Water: Methanol 5:95 v/v], sonicated for about 15 min and then made up to 100 ml with diluent to get 2.5 mg/ml standard stock solution.

Working Standard solution:

5 ml of the above stock solution was taken in 50 ml volumetric flask and thereafter made up to 50 ml with diluent to get a concentration of 250mcg/ml.

Preparation of Sample solution:

Twenty tablets (Geftinat 250 mg, Natco Pharma) were weighed, and then powdered. A sample of the capsule powder, equivalent to 250 mg of the active ingredient, was mixed with 25 ml of diluent [HPLC Grade water: Methanol 5: 95]. The mixture was allowed to stand for 1 hr with intermittent sonication to ensure complete solubility of the drug, and then filtered through a 0.45 μm membrane filter, followed by adding mobile phase to obtain a stock solution of 2.5 mg/ml. Transfer 5ml of this solution to a 50 ml volumetric flask and made up to sufficient volume with mobile phase to give an concentration of 250 μg/ml.

Linearity:

Aliquots of standard Gefitinib 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 Gefitinib are in the range of 25-300μg/ml. Each of these drug solutions (20 μL) was injected three times into the column, and the peak areas and retention times were recorded. Evaluation was performed with PDA detector at 246 nm and a Calibration graph was obtained by plotting peak area versus concentration of Gefitinib (Fig 2). The plot of peak area of each sample against respective concentration of Gefitinib was found to be linear in the range of 25–300µg/ml with correlation coefficient of 0.9999. Linear regression least square fit data obtained from the measurements are given in table I. The respective linear regression equation being Y=94342.26x+77672.7. The regression characteristics, such as slope, intercept, and %RSD were calculated for this method and given in Table I.

Fig 1: Typical Chromatogram of Gefitinib by HPLC:

Assay:

20 µl of sample solution was injected into the injector of liquid chromatograph. The retention time was found to be 3.7 minutes. The amount of drug present per tablet was calculated by comparing the peak area of the sample solution with that of the standard solution. The data are presented in Table II.

Recovery Studies:

Accuracy was determined by recovery studies of Gefitinib, known amount of standard was added to the preanalyzed 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.

Table III Validation Summary

Validation Parameter

Results

System Suitability

Theoretical Plates (N)

Tailing factor.

Retention time in minutes.

14234.45

1.22

3.667

LOD (µg/ml)

LOQ (µg/ml)

0.125

0.375

RESULTS AND DISCUSSION:

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

Fig 2: Calibration curve of Gefitinib by HPLC.

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

The limit of detection (LOD) and limit of quantification (LOQ) for Gefitinib were found to be 0.125µg/ml and 0.375µg/ml respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ.

From the typical chromatogram of Gefitinib as shown in fig 1, it was found that the retention time was 3.667 min. A mixture of 0.02 M Dipotassium Hydrogen ortho phosphate and Methanol in the ratio of 10:90 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=0.9999) was observed between the concentration range of 25-300 µg/ml. Low values of standard deviation are indicative of the high precision of the method. The assay of Gefitinib tablets was found to be 99.5%. From the recovery studies it was found that about 99.86 % of Gefitinib 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 tablets. 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 tablet dosage form of Gefitinib within a short analysis time.

ACKNOWLEDGEMENTS:

The authors are grateful to M/s Natco Pharma, Hyderabad for the supply of as a gift sample Gefitinib and to the Management, Sree Chaitanya Institute of Pharmaceutical Sciences, LMD Colony, Karimnagar-505527.AP, for providing the necessary facilities to carry out the research work.

REFERENCES:

1. Ming Zhao; Hartke Carol ; Jimeno Antonio ; Jing Li ; Ping He ; Zabelina Yelena ; Hidalgo Manuel⁽¹⁾ ; Baker Sharyn D. Journal of chromatography. B, 2005, vol. 819[1], pp. 73-80.

2. Guetens G., Prenen H.^, De Boeck G., Van Dongen W.,Esmans E, Lemiere F., Van Oosterom A. T., Schöffski P., De Bruijn E. A. Journal of chromatography, 2005, vol 1082[1], pp 2-5.

Received on 16.04.2009 Modified on 10.05.2009

Research J. Pharm. and Tech.2 (2): April.-June.2009; Page 341-343