INTRODUCTION:

Dasatinib is N-(2-chloro-6-methyl phenyl)-2-[[6-[4-(2-hydroxy Ethyl)-1- piperazinyl]-2-methyl-4-pyrimidinyl] amino] 5-thiazole carboxamide monohydrate. It is insoluble in water and slightly soluble in ethanol and methanol⁶. It is used in treatment of imatinib resistant chronic myeloid leukaemia.

Fig-1: Structure of Dasatinib monohydrate.

Dasatinib inhibits multiple tyrosine kinases, including BCR-ABL, the fusion protein created by the abnormal Philadelphia chromosome (Ph) which characterizes chronic myeloid leukemia. Competitive inhibition at the enzyme's ATP-binding site leads to inhibition of tyrosine phosphorylation of proteins involved in BCR-ABL signal transduction. Inhibition is not completely selective, as Dasatinib also inhibits other kinases including the Src family. Dasatinib may overcome imatinib resistance that results from BCR-ABL kinase domain mutations, activation of alternate signaling pathways involving the Src family kinases and multi-drug resistance gene over expression.

Literature survey reveals that very few colorimetric methods¹, HPTLC method², spectroscopic-LC method³, LC–MS/MS^4-10 methods are available for the determination of Dasatinib monohydrate in biological fluids. The aim of the present study is to develop a simple, precise, rapid and accurate RP-HPLC method for the determination of Dasatinib in tablets.

EXPERIMENTAL:

Materials and Reagents:

The instrument used was Aliance Waters (2487) HPLC with dual absorbance HPLC detector. A Shimadzu UV-visible spectrophotometer – 1601 with empower-2 software and a Cosmicsil BDS C₁₈ column (4.6 X 250, 5µ) were used. A 20 µl Hamilton injection syringe was used for sample injection. GR grade reagents were used for the preparation of buffer (pH 6.5 ± 0.05). Reagents were obtained from E Merck (India). Milli Q Water (Millipore (USA)) was used throughout the procedure. A freshly prepared triethylamine buffer solution (pH6.5 ±0.05) and solvent mixture (Methanol, Acetonitrile) in 50:50v/v was used as a mobile phase. The solvents were filtered through 0.45 µ membrane filter and sonicated before use. The flow rate of mobile phase was maintained at 1.0ml/min. The column temperature was maintained at 35^oC and the detection was carried out at 315 nm.

Selection of column:

Column trials were performed using Thermosil C₁₈ column, 5µ and Perfectsil C₁₈ column, phenomenex Gemini C₁₈ column, Cosmicsil BDS C₁₈ column. Better peak resolution with less tailing was observed with Cosmicsil BDS C18 column.

Selection of mobile phase:

The method development for the determination of Dasatinib monohydrate tablets was tried with different solvent systems. Different mobile phases containing methanol, Acetonitrile, water and triethylamine buffer (pH 6.5 ± 0.05) in various compositions were tried and finally triethylamine buffer (pH 6.5 ± 0.05) and solvent mixture of methanol: Acetonitrile of 50:50v/v was selected as good chromatograms were obtained with that composition. 100% Purified Milli Q Water was selected as diluent.

Preparation of mobile phase:

4ml of triethylamine was diluted to 1000 ml with Milli-Q water and pH was adjusted to 6.5 ± 0.05 with Orthophosphoric acid. Then a mixture of methanol and Acetonitrile in 50: 50 v/v was prepared.

A filtered (0.45µ) and degassed mixture of buffer and solvent mixture was prepared in the ratio of 50: 50 v/v respectively. Mobile phase was filtered through a 0.45 µ membrane filter, degassed with a helium purge for 20 min.

Selection of analytical wavelength:

From the standard stock solution, different dilutions were prepared using mobile phase and scanned over the range of 200-400 nm and the spectrum was overlain. It was observed that 315nm is the λ_maxfor Dasatinib monohydrate and it was selected as a suitable wavelength for detection. Eluents were monitored at 315nm.

Preparation of standard solution:

50mg of Dasatinib working standard solution was accurately weighed and transferred into a 100ml clean dry volumetric flask and 60 ml of the solvent mixture was added. The solution was sonicated and cooled to room temperature and was further diluted with solvent mixture up to the mark. Then 2.0 ml of the above solution was transferred into 50 ml volumetric flask and was diluted with mobile phase.

Linearity and construction of calibration curve:

Aliquots of 10-30ppm solutions were prepared from standard solution to determine the linearity range. Each of these drug solutions (10 µl) was injected 5 times into the column by maintaining a flow rate of 1.0 ml/min. The detection was carried out at 315 nm. Chromatograms were recorded and peak area was recorded for all injections. A calibration plot of concentration over the peak area was constructed.

Estimation of Dasatinib monohydrate in tablet:

20 tablets were accurately weighed and finely powdered. The powder equivalent to 100 mg of Dasatinib was transferred into 250 ml clean, dry, volumetric flask. About 160 ml of solvent mixture was added, shaked on an orbital shaker for 15 min and sonicated for 30 min with occasionally shaking. The solution was cooled to room temperature and the volume was diluted with solvent mixture. The solution was centrifuged at 3000 rpm for 15 min. Then 5.0 ml of the above solution was transferred into 100 ml volumetric flask and was diluted with mobile phase

10µl of sample solution was then injected in to the column. The sample was injected for five times. The mean peak area of the five determinations was calculated and the drug content in the sample was quantified using the regression equation obtained from the calibration curve.

RESULTS AND DISCUSSION:

Method development:

In the present investigation we have developed a simple, precise and accurate reverse phase high performance liquid chromatographic method for the determination of Dasatinib monohydrate. The method was developed using mobile phase, a mixture of triethylamine buffer solution pH (6.5 ±0.05) and solvent mixture methanol and acetonitrile in 50:50v/v was used as mobile phase the flow rate of mobile phase was maintained at 1.0ml/min. The column temperature was maintained at 35^oC and the detection was carried out at 315 nm. The developed method was found to be appropriate for the determination of dasatinib monohydrate in tablet dosage form. A typical chromatogram obtained by using the above parameters was shown in fig-2.

Validation:

Precision:

Intra-day and inter-day precision studies were performed by injecting each concentration for six times and the percent RSD was calculated and it was found to be within limits. The results of precision studies were shown in table-1and 2.

Linearity:

The linearity of the method was found to be in the range of 10-30ppm. The values of linearity studies were shown in table-1. The regression coefficient was found to be 0.9996% from the calibration curve. The linearity plot was shown in fig-3.

Accuracy:

The percentage recovery studies were carried out at three different concentration levels. The percentage recovery and the percentage RSD values were found within the limits. The results of accuracy studies were shown in table-4.

Specificity:

Chromatogram of blank did not show any peak at the retention time of analyte peak. There is no interference due to blank at the retention time of analyte. No interference was observed from the excipients and degradation products of degradation studies. Hence the method was found to be specific and stable.

Robustness:

The robustness studies were performed by changing the organic phase proportion of the mobile phase and buffer pH. The results of robustness were shown in table-5.

Limit of detection (LOD):

The Detection Limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value

The results of LOD were shown in fig-4.

Limit of quantification (LOQ):

The Quantification limit of an analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The results of LOQ were shown in fig-5.

System suitability:

The system suitability studies were done with the 50mg of standard drug. The % of RSD values are below 2%, theoretical plate count is above 2000 and tailing factor is less than 2, indicating that the method is suitable.The results of system suitability were shown in table-6.

Fig-2: A typical chromatogram of dasatinib monohydrate

CONCLUSION:

The proposed HPLC method was found to be simple, rapid, precise, accurate and sensitive for the determination of dasatinib monohydrate in pharmaceutical dosage forms. Hence this method can be easily and conveniently adopted for routine analysis of dasatinib monohydrate in pure and its pharmaceutical formulations.

Results of intra-day and inter-day precision:

Table 1:Intra-day precision:

S.No	Peak Name	Area
1	Dasatinib	775269
2	Dasatinib	774215
3	Dasatinib	776152
4	Dasatinib	774952
5	Dasatinib	775962
6	Dasatinib	776119
Mean		775445
SD		775.65
%RSD		0.1002

Table-2: Inter-day precision:

S.No	Peak Name	Area
1	Dasatinib	774823
2	Dasatinib	775219
3	Dasatinib	774518
4	Dasatinib	776251
5	Dasatinib	777021
6	Dasatinib	774505
Mean		775389
SD		1029.15
%RSD		0.132

Table-3: Results of the linearity studies:

S.No	Linearity Level	Concentration	Area
1	I (50%)	10 ppm	395495
2	II (75%)	15 ppm	603421
3	III (100%)	20 ppm	798654
4	IV (125 %)	25 ppm	984321
5	V (150%)	30 ppm	1186479
Correlation Coefficient			0.9996

Table-4: Results of Accuracy studies:

Fig-3: Standard calibration curve of the proposed method

Fig -4: Chromatogram showing limit of detection

Fig -5: Chromatogram showing limit of Quantification

Table-6: System suitability parameters

Drug

RT (min)

Peak

area

Theoretical plates

Tailing factor

Dasatinib monohydrate

6.4 min

792091

7610

1.24

REFERENCES:

1) Nasir Vadia and Sadhana Rajput. Development of colorimetric method for determination of Dasatinib in bulk and in tablet formulation. International Journal of Pharmacy and Pharmaceutical Sciences. 3 (2); 2011: 975-1491.

2) D. V. Mhaske and Dhaneshwar. Stability Indicating HPTLC and LC Determination of Dasatinib in Pharmaceutical Dosage Form. Chromatographia. 66 (1-2); 2007: 95-102.

3) Elisa Pirro, Silvia Racca, Francesco Di Carlo, Carmen Fava. A New HPLC–UV Validated Method for Therapeutic Drug Monitoring of Tyrosine Kinase Inhibitors in Leukemic Patients. Journal of Chromatographic Science. 49; 2011:753-757.

4) Sandra Rochea, Lorena Baietto, Jessica Cusato. Development of a high performance liquid chromatographic-mass spectrometric method for the determination of cellular levels of the tyrosine kinase inhibitors lapatinib and dasatinib. Journal of Chromatography B. 877 (18-19); 2009: 3982-3990.

5) Silvia De Francia, Antonio D’Avolio, Francesca De Martino, Development of a HPLC–MS method for the simultaneous quantification of antileukemia drugs imatinib, dasatinib, and nilotinib in human plasma. Journal of Chromatography B. 87(18-19); 2011:1721-1726.

6) Antonio D’Avolioa, Marco Simiele, Alessandra Ariaudo. Development of a HPLC–MS method for the simultaneous quantification of antileukemia drugs imatinib, dasatinib and nilotinib in human peripheral blood mononuclear cell. Journal of Pharmaceutical and Biomedical Analysis.59 (5); 2011:109-116.

7) http://www.ncbi.nlm.nih.gov/pubmed/22987603

8) http://www.ncbi.nlm.nih.gov/pubmed/23138519

9) http://www.sciencedirect.com/science/article/pii/ S1570023209007119

10) http://pubs.acs.org/doi/abs/10.1021/cg300152p

Received on 20.07.2013 Modified on 30.07.2013

Asian J. Research Chem. 6(9): September 2013; Page 859-862

Parameter	Organic phase composition		Variation in temperature(⁰C)		Variation in flow rate(ml)
Parameter	47:53	53:47	32	35	0.9	1.1
Injection no	1	2	3	4	5	6
Peak area	775493	775907	775777	776363	775705	775069
%RSD	0.11	0.43	0.433	0.31	0.10	0.13