INTRODUCTION:

It is a 4-[(4-methylpiperazin-1-yl) methyl]-N-(4-methyl-3-{[4-(pyridin-3-yl) pyrimidin-2-yl] amino} phenyl) benzamide, imatinib mesylate. It is known for chronic myelogenous leukemia and acute lymphocytic leukemia that are philadephia chromosome positive, certain types of gastrointestinal stromal tumors, hypersinoiphilic syndrome, chronic eosinophilic leukemia, systemic mastocytosis and myelodysplastic syndrome.

According to the literature review several methods has been developed for drug, like spectroscopy methods^1-4.HPLC^5-18, and HPTLC¹⁹. This UV–Area under curve method can be used for the routine analysis. In the proposed methods optimization and validation of this method are reported.

MATERIALS AND METHOD:

Instrument and reagents:

Spectral scan was made on a Shimadzu UV-spectrophotometer, model 1800 (Shimadzu, Japan) with spectral band width of 0.5 nm with automatic wavelength corrections by using a pair of 10 mm quartz cells. All spectral measurements were done by using UV-Probe 2.42 software.

Reference standard of imatinib mesylate was obtained from reputed firm with certificate of analysis.

Preparation of standard drug solutions:

10 mg standard imatinib mesylate was weighed accurately and transferred to a 10 ml volumetric flask and sonicated with 3 ml of distilled water for 5 minutes. The volume was made up to the mark with distilled water to give a stock solution of imatinib mesylate of concentration 1000 μg /ml. From this solution, 1 ml of solution was pipetted out and transferred into 10 ml volumetric flask. The volume was made up to mark with distilled water to give a working standard solution of concentration 100 μg/ml.

Estimation from tablets:

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 10 mg of imatinib mesylate was weighed and transferred in 100 ml of volumetric flask. A 30 ml of distilled water was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 100 μg /ml. Such solution was used for analysis.

Experimental:

Method: Area under curve (AUC) method:

Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ₁ and λ₂. The area under curve between λ₁ and λ₂were calculated by UV probe 2.42 software. In this method, 10 μg/ml solution of imatinib mesylate was scanned in the spectrum mode from 200 nm to 350 nm. From zero order spectrums the AUC calculation was done. The AUC spectrum was measured between 250 nm to 270 nm (Fig. 1).

Fig. 1. Area under curve spectrum of imatinib mesylate ( 10 μg/ml) showing area from 228 nm to 236 nm.

Into series of 10 ml graduated flask, varying amount of standard solutions of imatinib mesylate was pipette out and volume was adjusted with distilled water. Solutions were scanned between 350 nm to 200 nm in spectrum mode. The AUC calculations were done and the calibration curve for imatinib mesylate was plotted in the concentration range of 1 to 20 μg/ml (Fig. 2).

Fig. 2. Calibration curve for imatinib mesylate by area under curve spectroscopy

Results of analysis are given in table 1.

Table 1: Values of results of optical and regression of drug

Parameter	Area under curve (AUC) method
Detection Wavelength (nm)	250-270
Beer Law Limits (µg/ml)	1-20
Correlation coefficient(r²)	0.9999
Regression equation (y=b+ac)
Slope (a)	0.020
Intercept (b)	0.0001

Validation:

Accuracy:

Accuracy of the proposed methods was carried as on the basis of recovery studies. It is performed by the standard addition method. Recovery studies were performed by adding standard drug at different levels to the pre-analyzed tablets powder solution and the proposed method was followed. From the amount of the drug estimated, the percentage recovery was calculated. The results of the analysis are shown in table (2).

Precision:

The method precision was established by carrying out the analysis of homogenous powder blend of tablets. The assay was carried out of drug by using proposed analytical method in six replicates. The values of relative standard deviation lie well within the limits indicated the sample repeatability of the method. The results obtained are tabulated in table 3.

Table 3: Precision- method precision

Experiment No.	Weight of imatinib mesylate taken in mg	Weight of imatinib mesylate found in mg
1	10	9.9951
2	10	10.004
3	10	9.9945
4	10	9.9955
5	10	10.000
6	10	10.004
	Standard deviation	0.00444
	%RSD	0.044407

Inter-day and intra-day precision

An accurately weighed quantity of tablets powder equivalent to 10 mg of imatinib mesylate was transferred to 10 ml of volumetric flask. A 3 ml of distilled water was added and sonicated for 5 minutes and filtered. The filtrate and washing were diluted up to the mark with distilled water to give concentration as 1000 μg /ml. A 1 ml of such solution further diluted to 10 ml to give concentration as 100 μg /ml Such solution was used for analysis.

Solution was scanned between 400 nm to 200 nm in spectrum mode. The area under curve of resulting solutions was measured at between 250 nm to 270 nm by using as blank as distilled water. The area under curve of final solutions was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell at 250 nm to 270 nm. Similarly area under curve of the same solution was read on 1^st, 2^nd and 5^th day. The amount of imatinib mesylate was estimated by comparison with standard at 250 nm to 270 nm, table 4.

Table 4: Summary of validation parameter for intra-day and inter-day

Sr. No.	Parameters	Area under curve (AUC) method values
(A)	Intra-day precision ( n=3) Amount found ± % RSD	100.35% 0.4012
(B)	Inter-day precision ( n=3) Amount found ± % RSD	100.12% 0.3256
(c)	Ruggedness Analyst to analyst( n= 3) % RSD	0.1248

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

The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from back-ground levels. In this study, LOD and LOQ were based on the standard deviation of the response and the slope of the corresponding curve using the following equations-

LOD=3.3 σ/S and

LOQ=10 σ/S

Where σ is the standard deviation of the signal to noise ratio of the sample and S is the slope of the related calibrations graphs.

The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with an acceptable accuracy, precision and variability .The values of LOD and LOQ are given in table 5.

Table 5: Values of results of LOD and LOQ

parameters

Values

Limit of Detection (μg/ml)

0.02835

Limit of Quantification (μg/ml)

0.08591

Ruggedness:

The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of imatinib mesylate sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of imatinib mesylate was conducted spectrophotometrically on one laboratory. It was again tested in another laboratory using different instrument by different analyst. The assays obtained in two different laboratories were well in agreement. It proved ruggedness of the proposed methods.

RESULT AND DISCUSSION:

The area under curve UV-spectroscopic method is useful for routine analysis of imatinib mesylate in bulk drug and formulation. The method was validated according to International Conference on Harmonization guidelines for validation of analytical procedures. Imatinib mesylate has the absorbance maxima in the areas were measured between 250 nm to 270 nm. The polynomial regression data for the calibration plots showed good linear relationship in the concentration range of 1 to 20 μg/ml and given in table1. Recovery studies were carried out by adding the pure drug to the previously analyzed tablet powder sample and shown in table 2. The percentage recovery value indicates non interference from excipients used in formulation. The reproducibility and accuracy of the method were found to be good, which was evidenced by low standard deviation.

The most striking features of method is its simplicity and rapidity, not requiring tedious sample solutions preparations which are needed for other instrumental methods. From the results obtained it can be concluded that the proposed methods are fully validated and found to be simple, sensitive, accurate, precise, reproducible, rugged and robust and relatively inexpensive. So, the developed methods can be easily applied for the routine quality control analysis of imatinib mesylate in pharmaceutical formulation.

ACKNOWLEDGMENT:

Authors express sincere thanks to the Principal, Dr. Tushar M. Desai of D. G. Ruparel college

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Received on 23.06.2019 Modified on 22.07.2019

Asian J. Research Chem. 2019; 12(4):213-216.

DOI: 10.5958/0974-4150.2019.00040.3

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery (%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
2	0	2.006969	100.3484	0.008382	0.41763
2	2	4.012544	100.3136	0.007375	0.183797
2	6	8.014634	100.1829	0.012906	0.161032
2	8	10.00906	100.0906	0.004389	0.04385
			Mean =100.2339	Mean =0.008263	0.201577 Mean =