INTRODUCTION:

Imatinib Mesylate is a cancer medication prescribed to treat leukemia and gastrointestinal tumors. It operates by inhibiting proteins associated with cancer cell growth in order to relieve symptoms, prevent the spread of cancer cells, and aid other treatments[1]. Imatinib Mesylate is one of the newest anticancer drugs in the market and was one of the first drugs to be pushed through Food and Drug Administration's (FDA) fast track designation for approval. The drug is designed to inhibit tyrosine kinases such as Bcr-Ab and is used in the treatment of Chronic Myeloid Leukemia (CML) and gastrointestinal stroma tumors.

The Chemical name of Imatinib Mesylate is 4-4[(4-methyl-1- piperazinyl) methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl] amino] phenyl] – benzamide mono methane sulfonate. Fig 1 It has a molecular formula of C29H31N7O.CH4O3S and a molecular weight of 589.71. Imatinib Mesylate is a white crystalline powder which in freely soluble is distilled water, 0.1 N Hcl, methanol and sparingly soluble in dimethyl ether. Literature Survey revealed that the drug has been estimated by Liquid chromatography[2-9] and Spectrophotometry[10] methods in biological fluids and in pharmaceutical formulations[11-18]. The proposed Method was validated as per ICH guidelines Q2A.[19] Advantage of the present work is using acetonitrile and water as solvents for both the methods. Keeping this point into consideration, an attempt was made to develop a simple, accurate and validate UV spectroscopic and RP-HPLC method for the estimation of Imatinib in bulk and tablet dosage form.

Figure No.1: Structure of Imatinib Mesylate

MATERIAL AND METHOD:

Chemical reagents :

Imatinib Mesylate was gift sample from HETERO labs, Hyderabad, India. Acetonitrile (HPLC grade) were purchased from Qualigens, acetonitrile, water are purchased from Sd fine-Chem ltd; Mumbai.

Instrumentation:

Double beam UV-Visible Spectrophotometer (Shimadzu-1800) connected to a computer loaded with Shimadzu UV Probe 2.33 software was used for all the spectrophotometric measurements in all proposed spectrophotometric methods.

Reverse phase-High performance liquid chromatography (Agilent) equipped with VW detector. The software used is EZ Chrome and the column employed is Eclipse C₁₈ (100 mm×4.6 mm, 3.5 μm particle size)

Selection of solvent of Imatinib:

From the drug profile of Imatinib initially water was selected as a solvent, precipitate was occurred. Later acetonitrile was found to be a solvent to produce a clear solution. But to developed the method Acetonitrile:water (50:50) to produce a common solvent for drug. So, it was selected as solvent.

Preparation of standard stock solution of Imatinib for UV and HPLC:

Standard stock solution of Imatinib (10 µg /ml) was prepared by transferring 10 mg of Imatinib into a 10 ml volumetric flask separately and add 4mL of acetonitrile:water (50:50). It was then sonicated for 15 minutes and solution was diluted up to the volume by a solvent. From these, further dilutions were made using acetonitrile:water (50:50) to produce solution of Imatinib (10µg/ml).

Selection of wavelength of Imatinib:

10µg/ml of Imatinib were prepared in acetonitrile:water(50:50) solvent. The resulting solutions were scanned from 190 to 400 nm in UV-Visible spectrophotometer. The optimal response for drug was obtained at 242 nm. Hence the complete method was processed at the wavelength of 242 nm.

Preparation of sample solution of Imatinib for UV and HPLC:

20 Tablets of contents were weighed and the average weight was determined. They were crushed in to fine powder with glass mortar. The quantity of powder equivalent to 10 mg of active ingredient present in Imatinib into a 10 ml clean dry volumetric flask separately and solvent acetonitrile:water (50:50) was added to it and was shaken by mechanical stirrer and sonicated for about 30 minutes by shaking at intervals of five minutes and it was diluted up to the mark with diluent to give a concentration of 1000 µg/ml for Imatinib the solution was filtered through the whatman filter paper. The filtrate contains 10µg/ml of Imatinib to give the respective concentrations as per with standard solution

Selection of analytical concentration ranges of Imatinib:

From the standard stock solution of Imatinib (10µg/ml), appropriate aliquots of 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml and 3.0ml was pipette out and transferred into a 10ml volumetric flasks and dilutions were made with acetonitrile:water(50:50) to obtain working standard solutions with concentration ranging from 0.5 to 3µg/ml.

Selection of mobile phase:

Proper selection of the method depends on the nature of the sample (ionic or ionisable or neutral molecules), its molecular weight, pka value and stability. The drug selected in the present study is polar and so reverse phase or ion exchange chromatography can be used. The reversed phase HPLC was selected for the initial separation because of its simplicity and suitability. The trails are mentioned in the below Table 1

Out of all trials made in the lab, the 5^th trial of Imatinib was selected for further studies because when compared to other trials 5^th trial was found less in retention time, with good peak symmetry, and plate count within acceptable limits.

From the literature survey and with the knowledge of properties of the selected drug, Eclipse C18- 100mm x 2.5mm column was chosen as stationary phase and mobile phase with different compositions such as Acetonitrile and acetate buffer were used.

Optimized conditions:

The following optimized parameters were used as a final method in estimation of Imatinib in bulk and pharmaceutical formulations. The instrument used was Agilent model 1220 and Symmetry C₁₈ (4.6 x 150mm, 5mm), flow rate was 0.8ml/min and ambient column temperature, the capacity of injection sample volume is 20ml by using mobile phase of Acetonitrile: Water(50:50 v/v) with runtime of 5mins.

System Suitability studies of Imatinib:

According to the USP, system suitability tests are an integral part of chromatographic methods. These tests are used to verify that the resolution and reproducibility of the system are adequate for the analysis to be performed. System suitability tests are based on the concept that the equipment, electronics, analytical operations, and samples constitute an integral system that can be evaluated as a whole. The purpose of the system suitability test is to ensure that the complete testing system (including instrument, reagents, columns, analysts) is suitable for the intended application.

System suitability test parameters are:

· Plate number or number of theoretical plates (n)

· Capacity factor (capacity ratio) k

· The selectivity or Separation Factor (relative retention) α

· Peak Resolution R_s

· Peak asymmetry factor (As).

These are measured on a peak or peaks of known retention time and peak width.

Validation of UV spectroscopic and HPLC method:

Specificity of Imatinib:

The term specific generally refers to a method that produces a response for a single analyte only while the term selectivity refers to a method that provides responses for a number of chemical entities that may or may not be distinguished from each other. The analyte was assessed in the presence of components and it was found that there was no interaction with the analyte.

Selectivity studies of Imatinib:

The solutions were prepared and analysed with change in the analytical conditions like different instrument and different analyst. Standard concentrations of Imatinib/Capecitabine 1µg/ml were carried used to carry out the ruggedness studies.

Linearity of Imatinib:

The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in the sample within the range. The range of the analytical method is the interval between the upper and lower levels that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity.

For UV, appropriate aliquots from standard Imatinib stock solutions were transferred into different volumetric flasks of 10ml capacity. The volume was adjusted to the mark to obtain concentrations of 0.5, 1, 1.5, 2, 2.5, 3µg/ml. Absorbance spectra of each solution against acetonitrile: water (50:50) as blank were measured at 242nm and the graph of absorbance against concentration were plotted and the regression equation and correlation coefficient were determined.

For HPLC, the linearity of the method was demonstrated over the concentration range of Imatinib 0.25-1.5µg / ml. Aliquots of six levels were prepared from sample solution.

Precision of Imatinib:

The precision of an analytical method is the degree of agreement among individual test results, when the method is applied repeatedly to multiple sampling of homogeneous samples. It provides an indication of random error results and is expressed as relative standard deviation (%RSD).

Precision of the method was demonstrated by Repeatability, Intraday precision, Inter day precision.

Method precision:

Repeatability:

Repeatability expresses the analytical variability under the same operating conditions over a short interval of time. For UV, at least six determinations at 100% test concentration should be performed. The repeatability studies were carried out by taking 2µg/ml for as the test concentration and repeating it for six times.

For HPLC, the repeatability studies were carried out by taking 1µg/ml for Imatinib as 100% test concentration and repeating it for six times.

Intermediate precision:

Intra-day and Inter-day precision:

For UV and HPLC, variation of results within the same day (intra-day), variation of results between days (inter-day) were analysed. Intra-day precision was determined by analyzing Imatinib for 6 times on the same day at 242nm. Inter-day precision was determined by analyzing 3 concentrations of Imatinib on the preceding day at 242nm and %RSD was calculated.

Accuracy studies of Imatinib:

Accuracy is the closeness of the results obtained by the method to the true value. Recovery studies for UV were carried out at 50%, 100% and 150% by adding known amount of standard drug solution of Imatinib i.e. (1.5, 2, 2.5 µg/ml), to the sample solution whose concentration is maintained constant for Imatinib i.e. 1µg/ml.The %recovery was calculated.

Recovery studies for HPLC were carried out at 50%, 100% and 150% by adding known amount of standard drug solution of Imatinib i.e. (0.75, 1, 1.25 µg/ml) to the sample solution whose concentration is maintained constant for Imatinib i.e. 0.5µg/ml. The %recovery was calculated and reported.

Limit of detection (LOD) and Limit of quantification (LOQ) of Imatinib:

The sensitivity of the proposed method for the measurement of Imatinib was estimated in terms of Limit of detection (LOD) and Limit of quantification (LOQ). The LOD and LOQ were calculated by using the slope and SD of response (intercept). The mean slope value and the SD of response were obtained from the calibration curve. The LOD and LOQ calculations were done and reported.

Ruggedness of Imatinib:

The solutions were prepared and analysed with change in the analytical conditions like different instrument and different analyst. Standard concentrations of Imatinib 2µg/ml for UV and 30µg/ml for HPLC were used to carry out the ruggedness studies.

Robustness of Imatinib:

Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It is carried out by changing the flow rate of mobile phase from 0.8 to 1.2 mLmin^-1, changing the detection wavelength from 240 to 242nm.

The flow rate was varied for Imatinib at 0.9 mLmin^-1 to 0.7 mLmin^-1

Standard solution of Imatinib was analysed at 0.9 and 0.7mL min-1 i.e. at ± 0.8unit of optimized flow rate (1mL/min).

The detection wavelength was varied at 240 to 244nm

Standard solution of Imatinib was analysed at 240 and 244nm i.e. at ± 1unit of optimized wavelength (242nm).

System suitability testing (SST) of Imatinib:

System suitability tests are an integral part of chromatographic method. They were used to verify that the reproducibility of the chromatographic system is adequate for the analysis. It is defined as tests to measure that the method can generate result of acceptable accuracy and precision.

Assay studies of Imatinib:

Assay studies were carried out by weighing twenty tablets of Imatinib formulation and powdered. The powder equivalent to 10mg was taken and the solution equivalent to 1000µg/ml was prepared and was used for further dilutions.

RESULTS AND DISCUSSION:

The objective of the proposed work was to develop new analytical methods for the determination of Imatinib to validate the methods according to the ICH guidelines and applying the same for its estimation in marketed formulations. The developed UV spectrophotometric and RP-HPLC method was found to be rapid, simple, precise, accurate and economic for routine estimation of Imatinib in commercial dosage forms. in pure and pharmaceutical dosage forms by UV spectrophotometric method, and RP-HPLC method by using same solvent(50:50) and same wavelength(242nm). The work extends to validate for the developed methods as per ICH guidelines

Selection of wavelength of Imatinib for UV:

The standard stock solution of imatinib of 10µg/ml concentration was scanned from 200-400nm and the absorption spectras were recorded at 242nm wavelength in UV spectrophotometer which is presented in Fig.2

Selection of mobile phase and flow rate of Imatinib for HPLC:

Initially various mobile phase compositions were tried, to separate title ingredients. Mobile phase composition and flow rate selection was based on peak parameters (height, tailing, theoritical plates, capacity or symmetry factor) and run time. The system with acetonitrile:water 0.1% ortho phosphoric acid(50:50) of 0.8ml/min flowrate was found to be quite robust. The optimum wavelength for detection was 242nm at which better detector response for Imatinib was obtained. The retention time was found to be 1.0min for imatinib and the total runtime for this method along with the elution of and indicates that the developed method is economical and quite fast and the results are shown in Fig.3

Fig 2 Absorption spectrum of Imatinib

Fig. 3 Trial – 1 Acetonitrile : Water (70:30) V/V as mobile phase.

Fig. 3 Trial – 2 Methanol : water (70:30) V/V as mobile phase

Fig. 3 Trial – 3 Acetonitrile : water (80:20) V/V as mobile phase

Fig. 3 Trial – 4 Acetonitrile : water (60:40) V/V as mobile phase

Fig 3 Trial-5 Acetonitrile : water (50 :50) V/V as mobile phase

Validation of UV spectroscopic and HPLC method:

Specificity studies of Imatinib:

Fig. 4 chromatogram of specificity

Linearity studies of Imatinib for UV and HPLC:

The linearity was found in the concentration range of Imatinib i.e. 0.5-3 µg/ml for the developed UV spectrscopy method. The X-axis is concentration and the Y-axis is absorbance. The correlation coefficient was found to be 0.9991 and the regression equation was found to be Y=0.3076x + 0.0025 for Imatinib and the results are shown in Fig5.

Fig 5 Calibration curve for Imatinib at 242nm

The linearity was found in the concentration range of 0.25-1.5 µg/ml for the developed HPLC spectrscopy method. The X-axis is concentration and the Y-axis isarea under curve. The correlation coefficient was found to be 0.999 and and the regression equation was found to be Y = 6E+06x + 15314 for Imatinib and the results are shown in Fig 6.

Precision studies of Imatinibfor UV and HPLC:

Repeatability studies for UV were carried out by taking test concentration and repeating it six times. Interday and intraday precision were done by taking three concentrations and repeating it three times and the values for both system precision and method precision in terms of % RSDwere found to be <2.0% The results show that the %RSD value for repeatability, intraday and interday is 0.241, 0.123-1.968, 0.061-1.882 for Imatinib the results shown in table3

The results show that the %RSD value for repeatability, intraday and interday for HPLC is 0.43, 0.56, 0.55 for Imatinib the results shown in table 2, 3 which indicate that they meet the acceptance criteria and hence the method is said to be precise.

Fig 6 Calibration curve for Imatinib at 242nm

Table 2 Repeatability results for of Imatinib

UV Method			HPLC Method
Concentration (µg/ml)	Absorbance mean± S.D (n=6)	%RSD	Concentration (µg/ml)	Area mean± S.D (n=6)	%RSD
2	0.610±0.012	0.59	0.5	2641638±1121.51	0.49

Table 3: Intermediate Precision For Imatinib

Conc .(µg/ml)	UV Method				Conc. (µg/ml)	HPLC Method
	Intraday		Interday			Intraday		Interday
	Absorbance (mean± S.D) n=3	% RSD	Absorbance (mean± S.D) n=3	% RSD		Peak Area (mean± S.D) n=3	% RSD	Peak Area (mean± S.D) n=3	% RSD
1	0.293± 0.005	0.5	0.306±0.005	1.882	0.5	3243280 ±1212.5	1.67	3294280 ±4212.5	1.96
2	0.610± 0.001	0.42	0.610 ± 0.002	0.340	1	6177323± 1352.5	1.60	6147323± 1249.3	1.53
3	0.933± 0.001	0.36	0.933±0.0005	0.061	1.5	8916832± 1356.2	1.47	8891832± 1499.2	1.66

Table 6 Robustness results for Imatinib (flow rate and wavelength) for HPLC

Factor	Level	R_t	Area	Height	Platecount	Tailing
Flow Rate	Less Flow	1.2	411480	44852	2561.1	1.71
Flow Rate	More Flow	1.0	363858	42569	2463.2	1.58
Wavelength	244 nm	1.1	669994	40256	2870.4	1.60
Wavelength	240 nm	1.0	658779	49632	2583.5	1.64

Table: 7 Ruggedness results for Imatinib

Parameters	UV Method		HPLC Method
Parameters	Different instrument	Different analyst	Different instrument	Different analyst
Conc. (µg/ml)	2	2	-	1
Absorbance/ Area (242nm)	0.609	0.610	-	6243294
Absorbance / Area mean± S.D	0.609±0.001	0.610±0.001	-	6209408±35056.4
%RSD	0.163	0.250	-	0.163

System suitability studies of Imatinib for HPLC:

Procedure:

System suitability standard solution which contained 10µg/ml of Imatinib was prepared by appropriately diluting and mixing the corresponding stock standard solutions. System suitability was determined from six replicate injections of the system suitability standard before sample analysis. Parameters such as a number of theoretical plates (N), tailing factor and retention time, resolution were calculated and reported in the results shown Table 8

Table: 8 System suitability parameter for Imatinib

Parameter	Imatinib
Retention time	1.2
Asymmetric factor	1.1
Tailing factor	1.7
Number of Theoretical plates	2645

Assay of Imatinib for UV and HPLC:

The results show that the %purity was found to be 100.1 for UV and 99.5% for HPLC and reported in the results shown Table 9 and Fig7

Table 9 Assay results of Imatinib for UV

Formu

lation

Label claim

Method

Amount found

%Purity

Mean ± S.D

RSD

Imatinib

Glevec

100mg

100.1

100±

0.005

1.04

HPLC

99.8

100.8±0.65

0.54

Fig. 7 Assay of Imatinib

Optimum conditions, optical characteristics and Statistical data of the Regression equation in UV method:

The optical characteristics such as Beer’s law limits molar absorptivity, LOD and LOQ in each method were calculate and the results are displayed in below tables. Also the regression equation like slope(b), intercept(a) and correlation coefficient(R²) using the method of least squares were calculated. The results show that the methods are reasonably precise

Optimum conditions, optical characteristics and Statistical data of the Regression equation in HPLC method:

The optical characteristics such as Beer’s law limits molar absorptivity, LOD and LOQ in each method were calculate and the results are displayed in Table 10 andAlso the regression equation like slope(b), intercept(a) and correlation coefficient(R²) using the method of least squares were calculated. The results show that the methods are reasonably precise.

Table 10 Summary of HPLC validation parameters for Imatinib

Parameter	UV Method	HPLC Method
max (nm)	242nm	242nm
Linearity range µg/ml	0.5-3(µg/ml)	0.25 to 1.5µg/ml
Correlation coefficient ( r²)	0.999	0.999
Regression equation (y=mx+c)	y=0.307x+0.002	y=6E+06x+3576
Slope (m)	0.307	6E+06x
Intercept(c)	0.02	3576
Accuracy	99.6-100.8	99.24 – 100
Precision (%RSD)	0.165	0.43
LOD	0.103	0.0421
LOQ	0.312	0.1276

ACKNOWLEDGEMENTS:

The authors are grateful to Malla Reddy College of Pharmacy for providing necessary research facilities to carry out the research work and Herero labs, Hyderabad for providing the gift sample of the drug.

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Received on 15.03.2018 Modified on 04.04.2018

Asian J. Research Chem. 2018; 11(3):555-562.

DOI:10.5958/0974-4150.2018.00100.1

S.NO	Mobile phase	OBSERVATION	REMARKS
1	Aetonotrile : water (70 :30) v/v	Single peak was observed.	Not satisfactory
2	Methanol : water (70 :30) v/v	Single peak was observed	Not satisfactory
3	Acetonitrile : water (80 :20) v/v	Single peak was observed.	Not satisfactory
4	Acetonitrile : water (60 :40) v/v	Single peak was observed.	Not satisfactory
5	Acetonitrile : water (50 :50) v/v	Sharp peaks were observed	Satisfactory

Spiked level (%)	UV				HPLC
Spiked level (%)	Amount added	Amount found	% Mean recovery ±SD	%RSD	Amount added	Amount found	% Mean recovery ±SD	%RSD
50	1.5	1.49	100.4±0.404	0.404	1.25	1.24	100.8±0.797	0.78
100	2	2.03	100.3±0.568	0.566	1.5	1.49	100.60±0.567	0.52
150	2.5	2.47	100.1±0.305	0.304	1.75	1.76	99.5±0.267	0.26