A new analytical novel RP-HPLC method development and validation for the quantitative determination of Dasatinib in pure form and marketed pharmaceutical dosage form

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Introduction

Dasatinib Anhydrous is an orally bioavailable synthetic small molecule-inhibitor of SRC-family protein-tyrosine kinases. Dasatinib1 binds to and inhibits the growth-promoting activities of these kinases. Apparently because of its less stringent binding affinity for the BCR-ABL kinase, Dasatinib has been shown to overcome the resistance to imatinib of chronic myeloid leukemia (CML) cells harboring BCR-ABL kinase domain point mutations. SRC-family protein-tyrosine kinases interact with variety of cell-surface receptors and participate in intracellular signal transduction pathways; tumorigenic forms can occur through altered regulation or expression of the endogenous protein and by way of virally-encoded kinase genes. Dasatinib [2] is indicated for the treatment of newly diagnosed adults with Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) in chronic phase, as well as adults with chronic, accelerated, or myeloid or lymphoid blast phase Ph+ CML with resistance or intolerance to prior therapy including imatinib, and adults with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) with resistance or intolerance to prior therapy. Dasatinib is also indicated for the treatment of paediatric patients 1 year of age and older with Ph+ CML in chronic phase or newly diagnosed Ph+ ALL in combination with chemotherapy. Dasatinib is an orally available small-molecule multikinase inhibitor. During clinical trials, less than 1% of patients treated with Dasatinib [3] had QTc prolongation as an adverse reaction, and 1% experienced a QTcF higher than 500 ms. The use of Dasatinib is also associated with myelosuppression, bleeding-related events, fluid retention, cardiovascular toxicity, pulmonary arterial hypertension, severe dermatologic reactions, tumor lysis syndrome and hepatotoxicity. It may also cause embryo-fetal toxicity and lead to adverse reactions associated with bone growth and development in paediatric patients.

The IUPAC Name of Dasatinib is N-(2-chloro-6-methyl phenyl)-2-[[6-[4-(2-hydroxy ethyl) piperazin-1-yl]-2-methyl pyrimidin-4-yl] amino]-1, 3-thiazole-5-carboxamide. The Chemical Structure of Dasatinib is following.

Fig 1: Chemical Structure of Dasatinib Several analytical methods 33-36 have been devised for the determination of Dasatinib. These methods include titrimetric method, HPLC methods, HPTLC methods, LC methods, Uv- visible spectrophotometric methods, Amperometric method, fluorimetric method, Chemilumniscence method and voltametric methods. These methods are required expensive or sophisticated instruments and not simple for routine analysis. High performance liquid chromatography [4, 5] (HPLC) can be used for determination of drugs and for purposes of control throughout the entire manufacturing process of drugs, as well as quality control of the finished product. It has the advantages of being accurate, sensitive, rapid, selective, and reproducible. The present paper reports the development of a new high performance liquid chromatography (HPLC) method for determination of Dasatinib in different type of pharmaceutical formulations.

Materials and Methods

Instruments Used Table 1: List of Instrument used S. No. Instruments/Equipments/Apparatus 1. HPLC with Empower2 Software with Isocratic with UV-Visible Detector (Waters).

2. ELICO SL-159 UV – Vis spectrophotometer 3. Electronic Balance (SHIMADZU ATY224)

8. Vacuum filtration kit (BOROSIL)

Chemicals / Reagents Used Table 2: List of Chemicals used S.No.

Chemical Name Specifications Manufacturer/Supplier Purity Grade 1. Doubled distilled water 99.9% HPLC Sd fine-Chem ltd; Mumbai 2. HPLC Grade Water 99.9% HPLC Sd fine-Chem ltd; Mumbai 3. Methanol 99.9% HPLC Loba Chem; Mumbai. 4. Hydrochloric Acid 99.9 A.R. Sd fine-Chem ltd; Mumbai 5. Acetonitrile 99.9% HPLC Loba Chem; Mumbai. 6. Sodium Hydroxide 99.9 A.R. Sd fine-Chem ltd; Mumbai 7. Ethanol 99.9 A.R. Sd fine-Chem ltd; Mumbai 8. Octanol 99.9 A.R. Sd fine-Chem ltd; Mumbai Method Development Selection of Wavelength:

The standard & sample stock solutions were prepared separately by dissolving standard & sample in a solvent in mobile phase diluting with the same solvent [6]. (After optimization of all conditions) for UV analysis. It scanned in the UV spectrum in the range of 200 to 400nm. This has been performed to know the maxima of Dasatinib, so that the same wave number can be utilized in HPLC UV detector for estimating the Dasatinib. The scanned UV spectrum is attached in the following page, Sample & Standard Preparation for the UV-Spectrophotometer Analysis: 25 mg of Dasatinib standard was transferred into 25 ml volumetric flask, dissolved & make up to volume with mobile phase [7]. Further dilution was done by transferring 1ml of the above solution into a 10ml volumetric flask and make up to volume with mobile phase. Fig 2: UV spectrum for Dasatinib Observation: While scanning the Dasatinib solution we observed the maxima at 286nm. The UV spectrum [8] has been recorded on ELICO SL-159 make UV – Vis spectrophotometer model UV-2450.

Optimization of Chromatographic Conditions The chromatographic conditions [9] were optimized by different means. (Using different column, different mobile phase, different flow rate, different detection wavelength & different diluents for sample preparation etc.

Table 3: Summary of Process Optimization Column Used Mobile Phase Flow Rate Wave length Observation Result Xterra ODS (C18) RP Column, 150 mm x 4.6 mm, 5µm Methanol : Acetonitrile: Water = 40:30:30 0.8ml/min 286nm Very Low response Method rejected Zorbax ODS (C18) RP Column, 250 mm x 4.6 mm, 5µm Methanol : Acetonitrile = 70 : 30 0.9ml/min 286nm Low response Method rejected Develosil ODS (C18) RP Column, 150 mm x 4.6 mm, 5µm Acetonitrile: Methanol = 60 : 40 1.0ml/min 286nm Tailing peaks Method rejected Methanol = 80:20 1.0ml/min 286nm Resolution was not good Method rejected Methanol = 55:45 1.0ml/min 286nm Tailing peak Method rejected Methanol = 65:35 1.0ml/min 286nm Nice peak Method accepted Summary of Optimized Chromatographic Conditions: The Optimum Chromatographic conditions obtained from experiments [10] can be summarized as below:

Table 4: Summary of Optimized Chromatographic Conditions Mobile phase Phosphate Buffer (pH-4.6) : Methanol = 65:35% Column Phenomenex Luna (C18) RP Column, 250 mm x 4.6 mm, 5µm Column Temperature Ambient Detection Wavelength 286 nm Flow rate 1.0 ml/ min.

Temperature of Auto sampler Ambient Diluent Mobile Phase Injection Volume 20µl Type of Elution Isocratic Retention time 4.778 minutes Fig 3: Chromatogram for Blank Solution Fig 4: Chromatogram of Dasatinib in Optimized Condition Preparation of Mobile Phase: 650ml of prepared phosphate buffer and 350ml of HPLC Grade Methanol were mixed well and degassed [11] in ultrasonic water bath for 15 minutes. The solution was filtered through 0.45 µm filter under vacuum filtration.

Observation: The selected and optimized mobile phase was Phosphate Buffer (pH-4.6): Methanol = 65:35% and conditions optimized were flow rate (1.0 ml/minute), wavelength (286nm), Run time [12] was 10 mins. Here the peaks were separated and showed better resolution, theoretical plate count and symmetry. The proposed chromatographic conditions were found appropriate for the quantitative determination of the drug.

1. System Suitability Test

System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analysed constitute an integral system that can be evaluated as such. Following system suitability test parameters [13-16] were established. The data are shown in Table-5.

Table 5: Data of System Suitability Test S.No.

2. Linearity

To evaluate the linearity, serial dilution of analyte were prepared from the stock solution was diluted with mobile phase to get a series of concentration [17] ranging from 60- 140μg/ml. The prepared solutions were sonicated. From these solutions, 10μl injections of each concentration were injected into the HPLC system and chromatographed under the optimized conditions. Calibration curve [17] was constructed by plotting the mean peak area (Y-axis) against the concentration (X-axis).

Fig 5: Calibration Curve of Dasatinib Table 7: Linearity Data for Dasatinib Conc. (μg/ml) Area 0 0 60 461404 80 606157 100 748506 120 891041 140 1032196 3. Accuracy: The accuracy of the method was determined by recovery studies18 and the percentage recovery [19] was calculated. The recoveries of Dasatinib were found to be in the range of 99-102%. The proposed Liquid Chromatographic method was applied to the determination of Dasatinib. The results for Dasatinib comparable with the corresponding labeled amounts.

Table 8: Shown Accuracy Observation of Dasatinib Accuracy Amount Added Amount Recovered Peak Area % Recovery Mean Recovery 80% 100.437% 100% 120%

4. Precision

Repeatability: The precision [20] of each method was ascertained separately from the peak areas & retention times obtained by actual determination of six replicates of a fixed amount of drug. Dasatinib (API). The percent relative standard deviation [21-23] was calculated for Dasatinib are presented in the table-9.

Table 9: Repeatability Data for Dasatinib S. No. Injection Peak Area 7 Average 744529.6667 8 SD 2440.4116 9 % RSD 0.32777 Intermediate Precision The Intermediate Precision [24] consists of two methods:- Intra Day: In Intra Day process, the 80%, 100% and 120% concentration are injected at different intervals of time in same day.

Inter Day: In Inter Day process, the 80%, 100% and 120% concentration are injected at same intervals of time in different days.

Table 10: Results of Intra-Assay & Inter-Assay Conc. of Dasatinib (API)

Observed Conc. of Dasatinib (µg/ml) by the proposed method Intra-Day Inter-Day Mean (n=6) % RSD Mean (n=6) % RSD Observations: The intra & inter day variation [25] of the method was carried out for standard deviation & % RSD (% RSD < 2%) within a day & day to day variations for Dasatinib revealed that the proposed method is precise.

5. Specificity

Specificity26 can be determined by comparing the chromatograms obtained from the drugs with the chromatogram obtained from the blank solution. Blank solution was prepared by mixing the excipients in the mobile phase without drug. Drug solutions were prepared individually and the sample containing one drug was also prepared. Now these mixtures were filtered by passing through 0.45 μ membrane filter before the analysis. In this observation no excipient peaks were obtained near the drug in the study run time. This indicates that the proposed method was specific.

The chromatograms representing the peaks of blank, Dasatinib and the sample containing the one drug was shown in following figures respectively.

Observation: In this test method blank, standard solutions were analyzed individually to examine the interference. The above chromatograms show that the active ingredient was well separated from blank and their excipients and there was no interference of blank with the principal peak. Hence the method is specific.

6. Limit of Detection (LOD) and Limit of Quantification

The LOD27 and LOQ parameter was evaluated by mistreatment the slope of line and variance obtained from accuracy studies.

The detection limit (LOD) and quantization limit [28] (LOQ) may be expressed as:

Where, SD = Standard deviation of the response S = Slope of the calibration curve The slope S may be estimated from the calibration curve of the analyte.

The Minimum concentration level at which the analyte can be reliable detected (LOD) & quantified (LOQ) were found to be 1.469 & 4.454µg/ml respectively.

7. Method Robustness: Influence of small changes in

chromatographic conditions such as change in flow rate 1.0 ml ( 0.1ml/min), Wavelength of detection 286 (2nm) & organic phase content in mobile phase (5%) studied to determine the robustness [29] of the method are also in favour of (Table-11, % RSD < 2%) the developed RP-HPLC method for the analysis of Dasatinib (API).

Effect of Variation of Flow Conditions The sample was analyzed at 0.9ml/min and 1.1ml/min instead of 1ml/min, remaining conditions are same. 10µl of the above sample was injected and chromatograms were recorded.

Effect of Variation of Mobile Phase Organic Composition The sample was analyzed by variation of mobile phase [30] i.e. Methanol: Phosphate Buffer was taken in the ratio and 40:60, 30:70 instead of 35:65, remaining conditions are same. 20µl of the above sample was injected and chromatograms were recorded.

8. Estimation of Dasatinib in Pharmaceutical Dosage

Form Label Claim: 100mg Each tablet contains: 100mg Twenty Tablets were taken and the I.P. method was followed to determine the average weight. Above weighed tablets were finally powdered and triturated well. A quantity of powder equivalent to 25 mg of drugs were transferred to 25 ml volumetric flask, make and solution was sonicated for 15 minutes, there after volume was made up to 25 ml with same solvent. Then 10 ml of the above solution was diluted to 100 ml with mobile phase. The solution was filtered through a membrane filter (0.45 m) and sonicated to degas. The solution prepared was injected in five replicates into the HPLC system [31] and the observations were recorded. The Assay [32] data are shown in Table-12.

AT = Peak Area of drug obtained with test preparation AS = Peak Area of drug obtained with standard preparation WS = Weight of working standard taken in mg WT = Weight of sample taken in mg DS = Dilution of Standard solution DT = Dilution of sample solution P = Percentage purity of working standard Table 12: Recovery Data for estimation Dasatinib in Invista-100 Tablet Brand Names of Dasatinib Labelled Amount of Drug (mg)

Mean ( SD) Amount (mg) Found by the Proposed Method (n=6)

Invista-100 Tablet (Dr. Reddy’s Laboratories)

Result & Discussion: The amount of drug in Invista-100 Tablet was found to be 99.596 ( 0.638) mg/tab for Dasatinib & % Purity was 99.428%.

Summary To develop a precise, linear, specific & suitable stability indicating RP-HPLC method for analysis of Dasatinib, different chromatographic conditions were applied & the results observed are presented in previous chapters. Isocratic elution is simple, requires only one pump & flat baseline separation for easy and reproducible results. So, it was preferred for the current study over gradient elution. In case of RP-HPLC various columns are available, but here Phenomenex Luna (C18) RP Column, 250 mm x 4.6 mm, 5µm Column was preferred because using this column peak shape, resolution and absorbance were good. Detection wavelength was selected after scanning the standard solution of drug over 200 to 400nm. From the U.V spectrum of Dasatinib it is evident that most of the HPLC work can be accomplished in the wavelength range of 286 nm conveniently. Further, a flow rate of 1.0 ml/min & an injection volume of 20µl were found to be the best analysis. The result shows the developed method is yet another suitable method for assay and stability related impurity studies which can help in the analysis of Dasatinib in different formulations.

Conclusion

A sensitive & selective stability indicting RP-HPLC method has been developed & validated for the analysis of Dasatinib in bulk and pharmaceutical dosage form. Based on peak purity results, obtained from the analysis of samples using described method, it can be concluded that the absence of co-eluting peak along with the main peak of Dasatinib indicated that the developed method is specific for the simultaneous estimation of Dasatinib in the bulk and pharmaceutical dosage forms.

Further the proposed RP-HPLC method has excellent sensitivity, precision and reproducibility. The specific Retention time for Dasatinib are found to be 4.778min. The tailing factor was found to be 1.37 with theoretical plates 6859 for Dasatinib. The % Recoveries was determined as 100.437% for Dasatinib in Accuracy. The %RSD in Repeatability is 0.327 with Intermediate Precision (Intra & Inter Day) are 0.767 & 0.725 for Dasatinib in Precision respectively. In Linearity, the correlation coefficient was found to be 0.9995 for Dasatinib. The LOD for Dasatinib was 1.469 and LOQ for Dasatinib are 4.454 respectively.