Assessing imatinib mesylate in two different solid dosage forms

Imatinib mesylate (IM), is a tyrosine kinase inhibitor (TKI), which is used to treat chronic myeloid leukaemia (CML) and other malignant diseases related to over expression of tyrosine kinases protein (Savage and Antman, 2002). Imatinib is one of the first molecular therapies for clinical use that has shown impressive efficacy for CML, advanced gastrointestinal stromal tumours (GISTs), and myeloproliferative disorders associated with PDGFR. It is approved as a first-line therapy for Ph+ ALL (Iqbal and Iqbal, 2014). Several RP-HPLC methods can be found in the literature (Rele and Patil, 2019; Rosasco et al., 2005) for its determination in various pharmaceutical forms. The purpose of this work was to evaluate imatinib mesylate in two different solid dosage forms with a new RP-HPLC method developed in our laboratory.


Introduction
Imatinib mesylate (IM), is a tyrosine kinase inhibitor (TKI), which is used to treat chronic myeloid leukaemia (CML) and other malignant diseases related to over expression of tyrosine kinases protein (Savage and Antman, 2002). Imatinib is one of the first molecular therapies for clinical use that has shown impressive efficacy for CML, advanced gastrointestinal stromal tumours (GISTs), and myeloproliferative disorders associated with PDGFR. It is approved as a first-line therapy for Ph+ ALL (Iqbal and Iqbal, 2014).
Several RP-HPLC methods can be found in the literature (Rele and Patil, 2019;Rosasco et al., 2005) for its determination in various pharmaceutical forms. The purpose of this work was to evaluate imatinib mesylate in two different solid dosage forms with a new RP-HPLC method developed in our laboratory.
Chromatographic data were obtained using an Agilent 1200 reversed phase chromatographic system consisting of a High Pressure Mixing Binary Pump, DAD detector and equipped with a ChemStation PC-controlled. The pH measurements were made with a pH meter (Denver Instruments). Centrifuges, mixers and ultrasonic baths were also used. Nylon membrane filters were used for mobile phase filtration (type: Hydrophilic Nylon membrane filter) (EMD Millipore) (47 mm; 0.45 µm pore).

Preparation of standard solutions
100 mg of standard Imatinib was dissolved in methanol in a 100 mL volume flask and was made up to the volume mark with methanol. The obtained solution, at a concentration of 1 mg/mL, was filtered through 0.45mm porous filters, and stored in suitable containers, refrigerated at 4-8 °С. Solutions with concentrations of 5, 10, 20, 30 and 40 µg/mL, were prepared daily for the calibration curves by diluting the stock solution with distilled water. All the solutions obtained were filtered through 0.45 µm pore filters, prior to injection.

Chromatographic conditions
The flow rate was adjusted at 1.0 mL/min and the detection was carried out at 276 nm. The volume of injection was 20 µL.

Method validation parameters
Pharmaceutical Analysis/Quality Assurance/Regulatory Affairs Maced. pharm. bull., 68 (Suppl 1) 93 -94 (2022) The method was validated according to the ICH validation parameters and was assured to meet the conditions of linearity, accuracy and precision.

Preparation of the samples for the uniformity content
Each tablet is poured into a pre-filled Erlenmeyer flask with 100 mL of distilled water and placed in an ultrasound bath to disintegrate for 45 minutes. A 3 mL sample from each container is transferred in a test tube, which is centrifuged for 5 minutes. An aliquot of 2 mL from the supernatant liquid is further diluted in a 1 to 5 ratio with distilled water until the theoretical concentration reaches 20 µg/mL. The same procedure was applied for 10 tablets of each of the pharmaceutical products, which contain 100 mg of Imatinib. Samples were injected into the apparatus and the data obtained were plotted in graphs to compare the different products.

Results and Discussions
Literature review on the characteristics of Imatinib, revealed various evaluations with HPLC (Rele and Patil, 2019;Rosasco et al., 2005). Unfortunately, none of the published methods could be carried out in our laboratory, hence to evaluate imatinib in solid dosage forms a new method was developed. The tests revealed that the LiChroCART® 250-4 LiChrospher® 60 RP-select B C8 (5μm) column resulted the most suitable one. The chosen mobile phase consisted of Acetonitrile / H 2 O adjusted to pH 2.2 with orthophosphoric acid in the ratio (32.5: 65.5 v/v). Tests were carried out at room temperature. The analysis time was 6.5 minutes with imatinib eluting at 4.8 minutes. The method proved sensitive to the pH changes of the mobile phase.
The data obtained showed a consistent and practical method. DAD was used to confirm peak purity, and the result showed a peak with no interference with excipients, degradation products, or impurities.

Method validation
The linearity of the method was estimated through the correlation coefficient of the linear regression line, and regression equation was y = 68.83x + 17.18 with r 2 = 0.999. Repeated injections (n = 5) of each standard solution of 10 µg/mL, 20 µg/mL and 30 µg/mL were performed daily to determine accuracy and precision within day and n = 18 between days. All values of RSD for within day and between day tests were below 2.5%.

Uniformity content of Imatinib tablets
10 tablets from each pharmaceutical product were analysed as defined in European Pharmacopeia 10. The method demonstrates that all the tablets tested are within the specified range respectively between 85% and 115% of the average content.

Conclusions
The new method used to evaluate imatinib mesylate was accurate and precise. The results of the uniformity content for the two pharmaceutical products were within the specified range according to ICH and Ph. Eur. Determination of Imatinib in pharmaceutical dosage forms with this method enables the opportunity for further studies regarding the dissolution test.