INTRODUCTION:

Imatinib(IMA) mesylate (fig. 1), is a new chemotherapy drug indicated for the treatment of patients with chronic myeloid leukemia (CML) in blast crisis accelerated phase, or in chronic phase after failure of interferon-∝ therapy. It is the first of its kind developed to fight cancer by turning off an enzyme that causes cells to become cancerous and multiply. Food and Drug Administration (FDA) has approved IMA for treatment of patients at any of the three stages of CML. IMA designated chemically as 4-[(4-Methyl-1-piperazinyl) methyl]-n-[4-methyl-3-[[4-(3- pyridinyl)-2-pyrimidinyl] amino]-phenyl] benzamide methane sulfonate. IMA is a protein kinase inhibitor which potently inhibits the abelson tyrosine kinase in in-vitro and in-vivo studies. Oral IMA is indicated as first-line therapy in newly diagnosed patients with Philadelphia positive chronic myeloid leukemia in blast crisis^1-5. Literature survey reveals that the drug can be estimated by HPLC in human and monkey plasma^6-16. These methods involve the extraction of IMA by solid phase extraction and protein precipitation techniques. Different types of analytical techniques such as liquid chromatography coupled to tandem mass spectrometry (LC-MS)^17-25,

HPTLC²⁶ determination of IMA in bulk drug and pharmaceutical dosage form and capillary electrophoresis^27,
28 for estimation of IMA and its metabolite in biological fluids have been reported. These methods are sensitive and have low quantitation limits. However, these methods require sophisticated instrumentation and are expensive.

EXPERIMENTAL:

Instrument:

Elico double beam Ultraviolet-Visible double beam spectrophotometer SL-164 with 1 cm matched quartz cells were used for all spectral measurements. A UNICAM UV2 UV/Visible spectrometer was used for robustness study. Transonic Digital S (Sonicator) and Shimadzu electronic one pan balance were also used in this study.

Reagents:

a) Double Distilled water

b) 0.1N HCl

Procedure:

The solubility of IMA was determined in a variety of solvents using essentially a method of Schefter and Heguchi²⁹. From the solubility studies, distilled water and 0.1 N HCL were selected as solvents for UV spectroscopical studies of IMA in bulk drug and tablet dosage form. The λmax was determined in distilled water and 0.1 N HCL.

Table 1. Assay of imatinib in Tablets.

Sample (Tablet)	Labeled Amount (mg)	Amount Obtained (mg)* by proposed method		** % Recovery by the proposed method
		Method A.	Method B	Method A.	Method B
1	100	100.92	99.96	100.96	99.98
2	100	101.01	100.12	100.99	100.09
3	100	100.26	100.57	100.23	100.61

*Average of three determinations, ** After spiking the sample.

Table 2. Optical characteristics of proposed method

Parameters	Method A	Method B
λ _max (nm)	251	230
Beer’s law limit (μg mL^-1)	10 - 50	10 - 50
Sandell’s sensitivity (μg cm^-2/0.001 absorbance unit)	1.89 × 10^-5	1.63× 10^-5
Molar absorptivity (L mol^-1 cm^-1)	1.0767 × 10⁴	9.6121× 10³
Regression equation (Y = a + bc) Slope (b) Intercept(a)	0.0184 0.0061	0.0164 -0.0032
Correlation coefficient (r²)	0.9992	0.9997

able 3: Summary of validation parameters

Parameters	Method A	Method B
Linearity and range (μg/ml)	10 - 50	10 -50
LOD (μg/ml)	0.0111	0.0092
LOQ (μg/ml)	0.0334	0.0279
Accuracy (% Recovery) ( n = 9)	100.72%	100.23%
%RSD	0.8827	0.8899
Precision (%RSD)
Intra-day (n = 3)	0.748-0.891	0.799-0.915
Inter-day ( n = 3)	0.691-0.893	0.611-0.899
Repeatability( %RSD) (n = 6)	0.749	0.812
Ruggedness (%RSD) (n = 3)
Analyst I (% label claim)	0.471	0.364
Analyst II( % label claim)	0.281	0.386
Robustness( % RSD) (n = 3)
Laboratory-I	0.346	0.425
Laboratory-II	0.431	0.487

Method A:

Standard stock solution of IMA (1000 μg mL^-1) was prepared in distilled water. It was further diluted to obtain 10, 20, 30, 40 and 50 μg mL^-1 with distilled water. The absorbance was measured at 251 nm against distilled water as blank. The calibration curve was plotted in the concentration range of 10 to 50 μg mL^-1 of IMA in distilled water. The sample solution was also treated in the similar manner. The amount of drug in the sample was computed from Beer-Lambert plot.

Method B:

Standard stock solution of IMA (1000 μg mL^-1) was prepared in 0.1 N HCL. It was further diluted to obtain 10, 20, 30, 40 and 50 μg mL^-1 with 0.1 N HCL. The absorbance was measured at 230 nm against 0.1 N HCL as blank. The calibration curve was plotted in the concentration range of 10 to 50 μg mL^-1 of IMA in 0.1 N HCL. The amount of IMA present in the sample solution was computed from its calibration curve.

Figure 1. Chemical structure of Imatinib mesylate.

Figure 2. UV spectrum of Imatinib mesylate in water.

Figure 3. UV spectrum of Imatinib mesylate in 0.1 N HCL.

Preparation of sample solution:

Tablets containing IMA were successfully analyzed by the proposed methods: Twenty tablets of IMA were accurately weighed and powdered. Tablet powder equivalent to 100 mg of IMA was dissolved in 50 ml of distilled water and sonicated for 15 minutes, filtered and washed with distilled water, the filtrate and washings were combined and the final volume was made to 100 ml with distilled water. The solution was suitably diluted and analyzed as given under the assay procedure for bulk samples. Same procedure was followed by using 0.1 N HCL as solvent. The results are represented in Table 1. None of the excipients usually employed in the formulation of tablets interfered in the analysis of IMA, by the proposed methods.

Recovery Studies:

To ensure the accuracy and reproducibility of the results obtained, recovery experiments were performed by adding known amounts of pure drug to the previously analysed formulated samples and these samples were reanalyzed by the proposed method. The percentage recoveries thus obtained were given in Table 1.

RESULTS AND DISCUSSION:

The UV spectrum of IMA in distilled water and 0.1 N HCL (Fig 2 and 3) has showed maximum absorbance at 251 nm and 230 nm respectively. The methods were validated for accuracy, precision, ruggedness and robustness. The precision of the methods were studied as intra-day, interday and repeatability. The % RSD values less than 2 indicate the methods are accurate and precise. Ruggedness of the proposed methods was studied with the help of two analysts. Robustness of the methods was studied in two different laboratories using UV-visible spectrophotometer. The results did not show any statistical difference between operators and environmental conditions, suggesting that methods developed were rugged and robust. The results from validation studies are shown in table 3.

The optical characteristics such as absorption maxima, Beer’s law limits, molar absorptivity and Sandell’s sensitivity are presented in Table 2. The regression analysis using the method of least squares was made for slope (m), intercept (b) and correlation obtained from different concentrations and the results are summarized in Table 2.

CONCLUSION:

Both these methods are simple, rapid and accurate and precise. It can be used for routine analysis of IMA from tablet formulations.

ACKNOWLEDGEMENT:

The authors are thankful to Don Bosco College of Pharmacy for providing the facilities to carryout this study. The authors are also thankful to Dr. P. Srinivasababu, Principal, Vignan Pharmacy college to his valuable suggestion during this research work.

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Received on 15.05.2009 Modified on 02.06.2009

Research J. Pharm. and Tech.2 (3): July-Sept. 2009,;Page 578-581