INTRODUCTION:

Imatinib mesylate is an antineoplastic agent which has its use in treating chronic myelogenous leukemia. It is chemically 4-[(4-methyl-1-piperazinyl) methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl] amino]-phenyl] benzamidemethanesulfonate¹. It is available in white crystalline powder form.

It is Soluble in Water and Methanol. Imatinib is a protein tyrosine kinase inhibitor that inhibits Bcr-Abl tyrosine kinase. It inhibits proliferation induces apoptosis in Bcr-Abl positive cell lines as well as fresh leukemic cells from Philadelphia chromosome-positive chronic myeloid leukemia. Imatinib is well absorbed after oral administration with Cmax achieved within 2-4 hours post-dose. Mean absolute bioavailability is 98%². Following oral administration in healthy volunteers, the elimination half-lives of Imatinib and its major active metabolite, the N-dimethyl derivative (CGP74588), are approximately 18 and 40 hours, respectively. Mean Imatinib AUC increases proportionally with increasing doses ranging from 25mg - 1,000mg. At clinically relevant concentrations of Imatinib, binding to plasma proteins in in-vitro experiments is approximately 95%, mostly to albumin and α1-acid glycoprotein³. CYP3A4 is the major enzyme responsible for the metabolism of Imatinib.

Other cytochrome P450 enzymes, such as CYP1A2, CYP2D6, CYP2C9, and CYP2C19, play a minor role in its metabolism⁴. The main circulating active metabolite in humans is the N- demethylated piperazine derivative, formed predominantly by CYP3A4. It shows invitropotency similar to the parent Imatinib. The plasma AUC for this metabolite is about 15% of the AUC for Imatinib⁵. The plasma protein binding of N-demethylated metabolite CGP74588 is similar to that of the parent compound. Human liver microsome studies demonstrated that Gleevec is a potent competitive inhibitor of CYP2C9; CYP2D6and CYP3A4/5 with Ki values of 27, 7.5, and 8μM respectively⁶. Imatinib elimination is predominately in the feces, mostly as metabolites. Based on the recovery of compound (s) after an oral 14C-labeled dose of Imatinib, approximately 81% of the dose was eliminated within 7 days, in feces (68% of dose) and urine (13% of dose). Unchanged Imatinib accounted for 25% of the dose (5% urine, 20% feces)⁷, the remainder being metabolites. Typically, clearance of Imatinib in a 50-year old patient weighing 50 kg is expected to be 8L/h, while for a 50-year old patient weighing 100kg the clearance will increase to 14 L/h⁸. The inter-patient variability of 40% in clearance does not warrant initial dose adjustment based on body weight and or age but indicates the need for close monitoring for treatment-related toxicity⁹.

Fig. 1: Chemical structure of Imatinib Mesylate Molecular formula: C₂₉H₃₁N₇O Molecular weight: 589.7gm

MATERIALS AND METHOD:

Instrument:

ELICO SL 210 UV and visible recording spectrophotometer with two 10-20mm. matched quartz cells were employed for all absorbance measurements used for experiments. All weights were taken on Analytical balance.

Reagents and Standards:

Imatinib mesylate reference standard was obtained from Hetero labs Hyderabad. Veenat brand (IMT) tablets having a label claim of 400mg were purchased from nearby stores. The pure form of IMT has shown good solubility in distilled water which has been used as a diluent.

Experimental:

Standard stock I:

10.0mg of IMT was accurately weighed and transferred to a 10ml volumetric flask. A few ml of diluent was added to dissolve with occasional shaking manually for 10 min and was made up to the mark with the same (1000µg/ml).

Standard stock II:

1.0ml from standard stock I was pipetted to a 10ml volumetric flask and was made up to the mark with the same (100µg/ml).^10,11

λ max establishment:

1.0ml of IMT was pipetted to a 10ml volumetric flask and the volume was made up to the mark with the same (10µg/ml). This solution was scanned in the entire UV range of 200-400nm. (Figure 2)

Fig. 2: UV spectrum of Imatinib Mesylate in distilled water

Validation of the developed method:

The developed UV method was validated for precision, linearity, range, accuracy, robustness, Ruggedness, LOD, and LOQ. Methods, as described in USP <1225>, were followed.

1. Precision:

The study was performed as method and intermediate precision. The absorbance of six replicate solutions of ITM (10μg/ml) were taken and the results were tabulated in Table 1.

Intermediate precision:

Intermediate precision was performed to demonstrate the inter-day precision study. Repeated the method precision study using same sample preparation ITM (10μg/ml). The absorbance of six replicate solutions was taken but under different conditions like a different day, different analyst, different instrument. Calculated the mean and relative standard deviation of six sets of test results and the results were tabulated in Table 1a.

2. Linearity:

A series of linear concentrations (1-12μg/ml) were prepared from standard stock II solutions. Appropriate dilutions were made from the aliquots into separate 10 ml volumetric flasks and made up to the volume with diluent. Noted down the absorbance for each of the concentrations (Table 2) and a plot of the calibration curve was constructed (shown in Fig. 3).

Fig. 3: Calibration plot of IMT

3. Range:

The range of an analytical procedure is an interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy, and linearity. The specified range was 2-12µg/ml

4. Accuracy:

Recovery of the spiked standard solution (known concentration) was performed at three different levels (50%, 100%, and 150%) of the sample solution. The % recovery was then calculated from the recorded absorbance values. (Table 3)

5. LOD and LOQ:

The LOD and LOQ of Imatinib Mesylate were determined by using the standard deviation of response and slope approach as defined by ICH guidelines and the values of LOD and LOQ were found to be 0.2925 and 0.8977 respectively.

6. Robustness:

Six replicate solutions of 10ppm concentration were prepared and absorbance values at a wavelength of ±1nm of λmax were noted down. (Table 4)

Drug Stability Studies:

a) Acid and Base Degradation:

2.5ml from Standard stock II (100µg/ml) was pipetted to a 25ml, volumetric flask, and 5ml.of 0.1NHCl/0.1N NaOH was added. After 24hrs neutralize the solution with 5ml of 0.1N NaOH/0.1N HCl the remaining volume made up to the mark with water (10µg/ml). 5ml each of 0.1N HCl and 0.1N NaOH were taken into respective 25ml volumetric flasks and made up to the mark with water. These were used as blank. The absorbance of the acid/base degraded samples were noted applying the developed method.

b) Oxidation (Hydrogen peroxide):

2.5ml from Standard stock II (100µg/ml) was pipetted to a 25ml volumetric flask and 5ml of 0.3%H₂0₂was added. After 24hrs the solution is finally made up to the mark with distilled water. The absorbance was measured using the above solution against a blank containing 5ml of 0.3%H₂0₂ in a 25ml volumetric flask was kept as blank. The absorbance of the peroxide degraded samples was noted applying the developed method.

c) Photolytic Degradation:

2.5mg of pure IMT was placed in a UV chamber where the drug substance is exposed to UV-Visible light for 10hours.IMT was evaluated for the effect of light by noting the absorbance applying the developed method.^{12, 13,14}

d) Solution stability studies:

Benchtop and refrigerator: Benchtop stability of the solution was under observation for 20hrs at room temperature. The test solution is kept on the benchtop under normal laboratory conditions and was analyzed at suitable time intervals to establish benchtop solution stability up to 8 hrs. The solution stability was evaluated by storing in the refrigerator for the medium period (3days) and in the freezer (-20^o)

RESULTS AND DISCUSSIONS:

Table1: Method Precision results of IMT

S. No	Concentration(µg/ml)	Absorbance
1	10	0.8832
2	10	0.8795
3	10	0.8815
4	10	0.8627
5	10	0.8842
6	10	0.8785
% RSD		0.82%

Table1a: Intermediate Precision results of IMT

Day 1			Day2
Analyst 1		Analyst 2	Analyst 1	Analyst 2
Concentration	Abs.	Abs.	Abs.	Abs.
10(µg/ml)	0.8832	0.8568	0.8693	0.8742
10(µg/ml)	0.8795	0.8493	0.8548	0.8756
10(µg/ml)	0.8815	0.8536	0.8675	0.8845
10(µg/ml)	0.8627	0.8532	0.8656	0.8749
10(µg/ml)	0.8842	0.8546	0.8648	0.8723
10(µg/ml)	0.8785	0.8579	0.8653	0.8777
Mean	0.8783	0.8542	0.8646	0.8765
%RSD	0.82%	0.32%	0.53%	0.44%

Table2: Linearity results of IMT

Concentration	Absorbance
2	0.1689
4	0.3578
6	0.5215
8	0.6908
10	0.8791
12	1.0612

Table3: Accuracy results of IMT

Accuracy level	Sample (bulk + formulation)	Absorbance	% Recovery	% RSD
50%	2.5+5 2.5+5 2.5+5	0.5578 0.5499 0.5611	99.16 97.76 99.75	1.03%
100%	5+5 5+5 5+5	0.7834 0.7953 0.7962	97.05 98.52 98.63	0.903%
150%	7.5+5 7.5+5 7.5+5	1.0298 1.0268 1.0123	98.56 98.27 96.88	0.914%

Table4: Robustness results of IMT

S. No	concentration(µg/ml)	257nm	259nm
1	10	0.8712	0.8825
2	10	0.8793	0.8798
3	10	0.8715	0.8826
4	10	0.8759	0.8751
5	10	0.8708	0.8816
6	10	0.8542	0.8749
Mean		0.8705	0.8794
%RSD		0.90	0.37

CONCLUSION:

An accurate, precise, and reproducible UV – Spectrophotometric method has been developed for the quantitative estimation of Imatinib Mesylate. The method is validated and found to be linear, robust, and rugged. Hence it can be concluded that the developed Spectrophotometric methods are accurate, precise, and can be employed successfully for the quantification of Imatinib Mesylate in bulk and its tablet dosage form.

ACKNOWLEDGEMENTS:

The authors are very much thankful to the Hetero Labs Hyderabad, Telangana, India for facilitating and supporting the research study.

CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

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Received on 19.02.2022 Modified on 15.03.2022

Asian J. Pharm. Ana. 2022; 12(2):83-86.

DOI: 10.52711/2231-5675.2022.00015