A simple, precise,
accurate and robust RP-HPLC method was developed and validated for the
determination of dasatinib in bulk and its Tablets, Nano-suspension drug
entrapment efficiency and serum. The software used is EZ Chrome and the column
employed is Syncronis C18 (250 mm × 4.6 mm, 5 ?m particle size). Acetonitrile:
Methanol: water 45:35:20 (v/v) was used as mobile phase at a flow rate of 1 mL
min-1 with UV detection at 325 nm. Linearity was observed in the concentration
range of 1–6 ?g mL-1 with regression equation y =40966x-6534(R2 = 0.999). The
Nano-suspension prepared by novel precipitation-combined ultra-sonic
homogenization technique and was then characterized by using particle size
analysis, zeta potential measurement. Liquid- liquid extraction is performed
for isolation of the drug and elimination of serum interferences samples of
serum was extracted with 50µL of ortho phosphoric acid and 3ml of methanol was
added and spiked with dasatinib. The method was validated as per ICH
guidelines. The RSD for intra-day (99.2) and inter-day (98.3) precision were
found to be less than 2 %.The developed method is simple, precise and robust
for the determination of dasatinib in bulk and applied to tablets,
Nano-suspension, drug entrapment efficiency and serum.
Abstract
Introduction
Dasatinib (DAS), a small molecule tyrosine kinase
inhibitor, can effectively fight against chronic myelogenous leukemic (CML) and
acute lymphoblastic leukaemia (ALL) by inhibiting the activity of both Src and
BCR-ABL tyrosine kinases in leukaemia cells [1-5]. However, DAS treatment has
been reported to cause serious hematologic and non-hematologic adverse effects
due to its interaction with non-disease-related processes and cells, which
often leads to a dose reduction or treatment discontinuation in clinic [6].
Peripheral edema and pleural effusion are the common non-hematologic side
effects occurred during DAS treatment, which is likely caused by endothelial
hyper permeability [7-9]. Dasatinib is official in I.P and B.P. A thorough
literature survey has revealed that UV spectroscopy [4-6], HPLC [7-9] method
for Dasatinib with combination of other drugs, UPLC [10], LC-MS [11,12], GC-MS
[13] for its estimation in bulk, pharmaceutical dosage forms and biological
samples 14. Till now there is no reported method for dasatinib preparation of
Nano-suspension and estimation drug entrapment.
Experimental
Materials
and reagents
Dasatinib was gift sample from NATCO Pharm.
(Hyderabad, A.P, INDIA). Acetonitrile and methanol was purchased from Rankem
Chemical Company (India). The 0.45 ?m pump Nylon filter was obtained from
Advanced Micro Devices (Ambala Cantt, India) & whatman no 5 filter paper
was obtained from Modern Science lab, (Nashik, India). Other chemicals used
were analytical or HPLC-grade and glassware’s used were Class A grade.
Instrumentation
Reverse phase - High performance liquid chromatography
(Agilent) equipped with UV detector. The software used is EZ Chrome and the
column employed is Syncronis C18 (250 mm × 4.6 mm, 5 ?m particle
size).
Selection
of wavelength
Selectivity of HPLC method that uses UV detector
depends on proper selection of wavelength. A wavelength which gives good
response for the drugs to be detected is to be selected. From the UV spectra
obtained for drugs, 325 nm was selected as the wavelength for study.
Selection
of mobile phase
Solvent selectivity (solvent type), solvent strength
(percentage of organic solvent in the mobile phase), strength and pH of buffer,
flow rate etc. were varied to determine the chromatographic conditions that
gave the best separation.
HPLC
method validation
As per the International Conference on Harmonization
(ICH) guidelines, the method validation parameters like linearity, precision,
accuracy, limit of detection, limit of quantification, specificity and robustness
were experimentally determined and the method validated.
Specificity
Specificity for an assay ensures that the signal
measured comes from the substance of interest and there is no interference from
excipient and/or degradation products and/or impurities. Specificity of the
method was done by comparing the chromatogram of drug with the chromatogram of
blank (mobile phase).
Linearity
Linearity was performed by taking from stock solution
(100µg/ml) aliquots of 1, 2, 3, 4, 5 and 6µg/ mL were taken in 10mL volumetric
flasks and diluted up to the mark with diluents such that the final
concentrations are in the range of 1-6µg/ml. Each of these drug solutions
(10?L) was injected into the chromatographic system for three times.
Limit of Detection and Limit of Quantification
The limit of detection (LOD) and the limit of
quantification (LOQ) of the drug were derived by calculating the
signal-to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the
following equations designated by International Conference on Harmonization
(ICH) guidelines.
LOD= 3.3 × ?/S
LOQ= 10 × ?/S
Where, ?= the standard deviation of the response and
S= slope of the calibration curve
Assay
Twenty tablets were weighed accurately and finely
powdered. A powder equivalent to 100 mg of Dasatinib was transferred carefully
to 100 mL volumetric flask and about 15mL diluent was added. The mixture was
sonicated for 10minutes. The volume was made up to 100mL with diluent, filtered
through Whatman no. 5 filter paper. The final solution was injected in HPLC,
chromatogram was recorded and area was measured.
Accuracy
Accuracy of the proposed method was determined using
recovery studies. The recovery studies were carried out by adding different
amounts (50%, 100%, and 150%) of the pure drug to the pre-analysed formulation.
The analysis was conducted in triplicate. Percentage recovery was calculated.
Robustness
It is carried by calculated by comparing the area
before and after the addition of working standard. Changing the flow rate of
mobile phase from 0.95-1.05 mL/min, and wavelength from 323 to 327 nm.
Dasatinib made in triplicates and were analysed.
Precision
Precision studies were carried out to ascertain the
reproducibility of the proposed method.
Repeatability
Repeatability was determined by preparing six
replicates of 20 µg/ml dasatinib separately inject equal volumes (20 µl) of
each solution. Record the chromatograms and measure the peak response of drug.
The results were reported as %RSD.
Intermediate
precision
Interday
precision: Interday precision study was carried out
by preparing drug solution of concentration (20 µg/ml) and analysing it at
three different days to determine interday precision. Record the chromatograms
and measure the peak response of Dasatinib. It is shown in Table 4. The results
were reported as %RSD.
Robustness
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 by changing the flow rate of mobile phase from 0.95 to 1.05
mL/min and by changing in wavelength from 323 to327 nm. 2 µg/ml dasatinib was
analysed and the %RSD is determined.
System
suitability parameters
System suitability tests are an integral part of
chromatographic method. To ascertain its effectiveness, system suitability
tests were carried out by injecting freshly prepared standard stock solution of
10µg/ml dasatinib six replications and the parameters like retention time, peak
area, plate number (N), and peak asymmetry of samples were calculated.
Formulation
of dasatinib nano-suspension
Dasatinib Nano-suspensions were prepared by
combination method of precipitation –ultrasonic homogenization.40mg of
dasatinib is dissolved in 10ml of DMSO. Eduragit RS 100 and poloxamer188 and
sonicated for about 10min. 1ml of tween80 is added to the above solution. Small
amount of HPMC K100 is dissolved in 10ml of water. With the help of a syringe
the polymer and surfactant mixture is added drop by drop to the drug and
stirred vigorously. The final suspension is kept in ultrasonic homogenizer
where the suspension was homogenized at 40 pulse and 50 power for about 30 min.
The temperature should be maintained at 8oC in refrigerator in order to avoid
particle aggregation. 0.1ml of Nano-suspension was dissolved in 100ml of
volumetric flask and make up the volume with mobile phase.
Particle
size
Particle size growth is mainly responsible for
agglomeration. Precise sizing techniques can give useful information about the
particle size distribution in Nano-suspension.18 The particle size was measured
using particle size analyser HORIBA scientific Nano particle SZ-10 appropriate
scattering intensity at 25°C and sample was placed in disposable
sizing cuvette at a count rate of 372.0 (kcps) for 20 s.
The
proposed method applied to dasatinib nano-suspension entrapment efficiency
Entrapment efficiency is the % of drug that is
successfully entrapped or absorbed into the Nano-suspension. Samples from each
dasatinib Nano-suspensions were centrifuged at 10,000 rpm for 30 min using
centrifuge. The amount of untrapped drug in the supernatant obtained after
centrifugation was determined using HPLC. The encapsulation efficiency was
determined after the reading of the filtered samples in the HPLC, performed in
triplicate and calculated [14,15].
The percentage entrapment efficiency was calculated according to the following equation.
Methods
Dasatinib
spiked in serum extraction process
Trail
1: 0.2ml of serum samples, 50µL of ortho
phosphoric acid and 3ml of n-hexane was added. The sample were mixed in a
mechanical shaker for 20 min and centrifuged at 1000 rpm for 10 min. After
centrifuged, supernant layer was separated and make up to 10ml with mobile
phase. To the above solution, 2 ug/ml concentration of drug solution was spiked
and solution was injected.
Trail
2: 0.2ml of serum samples, 50µL of ortho
phosphoric acid and 3ml of methanol was added. The sample were mixed in a
mechanical shaker for 20 min and centrifuged at 1000 rpm for 10 min. After
centrifuged, supernant layer was separated and make up to 10ml with mobile
phase. To the above solution, 2 ug/ml concentration of drug solution was spiked
and solution was injected.
Results and Discussion
Different mobile phase systems in different proportions were tried. From this a mixture of methanol: acetonitrile: water (4.5:3.5:2.0 v/v) produced symmetric peak shape for the drugs. A wavelength of 325nm was selected based on UV studies. Chromatographic conditions used are stationary phase Systronis C18 (250mm x 4.6 mm), 5m. The flow rate was maintained at 1ml/min, column temperature was set to 30oC and diluents were mobile phase conditions were finalized as optimized method. Out of 6 trials performed, the 6th trail was selected because when compared to other trails, the 6th trial had the least in retention time, with good peak symmetry as mention in (Table 1 and Figure 1).
Figure 1: Different
trails of mobile phases.
Specificity
Specificity of the method was done by comparing the
chromatogram of drug with the chromatogram of blank (mobile phase) (Figure 2).
Table 1: Selection of mobile
phase.
|
Trials |
Mobile phase |
Observation |
Remarks |
|
1 |
Methanol:
Water (80:20) v/v |
Broad peak
appearance |
Not satisfactory |
|
2 |
Acetonitrile :
Water (50:50 v/v) |
Negative Extra peak
with tailing |
Not satisfactory |
|
3 |
Methanol :
Acetonitrile: water (50:40: 10 v/v/v) |
Sharp peak with
tailing |
Not satisfactory |
|
4 |
Methanol :
Acetonitrile: water (40:45: 15 v/v/v) |
Sharp peak with
small fronting |
Not satisfactory |
|
5 |
Methanol:Water
(45:55 v/v) |
Sharp peak with extra
peak |
Not satisfactory |
|
6 |
Acetonitrile: methanol:Water (45:35:20 v/v) |
Sharp peak appear |
Satisfactory |
Figure 2: Typical Chromatogram
Blank (Mobile Phase).
Linearity
Standard solutions of dasatinib in the concentration range of 1- 6µg/ml were injected into chromatographic system and peak areas were measured. A graph of peak areas (on Y-axis) versus concentration (on X-axis) was plotted and calibration graph was shown in Figure 3. The regression equation was found to be y = 40966x + 6534. The correlation coefficient should not be less than 0.999 for dasatinib. The results were reported as %RSD. The precision result showed a good reproducibility shown with percentage relative standard deviation less than 2.
Figure 3: Calibration curve of
Dasatinib.
Limit
of detection and limit of quantification
The parameters LOD and LOQ were determined on the
basis of response and slope of the regression equation. LOD = 0.046µg/ml and
LOQ = 0.1400µg/ml.
Assay
Estimation of dasatinib tablet dosage forms were carried out 100.00 as assay value. The % purity for Dasatinib should be 98-102 %. The result of assay obtained was found to be in good agreement with the labelled claim, indicating the absence of interference of the excipients and results are shown in (Table 2) and chromatogram was recorded and area was measured which was shown in (Figure 4).
Figure 4: Typical
Chromatogram of Dasatinib flow rate a.1.1 b. 0.9 mL/min.
Table 2: Assay results of
Dasatinib.
|
Formulation |
Label claim |
Amount found |
% Purity |
|
SPRYCEL (100mg) |
100mg |
98.98mg |
99.83% |
Accuracy
The % RSD for the individual recoveries of each level
and mean recovery should not be more than 2%. The % recovery at each level and
mean recovery should be between 98.0-102% and results are shown in (Table 3).
Robustness
In the case of the robustness study, the values
obtained for the statistical parameters of the chromatographic responses for
all variations (detection wavelength and flow rate) at the target concentration
level of 2 ?g/ml. The small magnitude of % RSD obtained as a result of
introducing small calculated variations in the eluent composition, detection
wavelength and flow rate suggested the robustness of the developed system. The
results were presented in the (Table 4). The chromatograms of flow rate and
wave length are shown in (Figure 5).
Table 3: Accuracy studies of
Dasatinib.
|
Spiked level (%) |
Formulation Conc (µg/ml) |
Pure Drug Conc (µg/ml) |
Amount recovered (µg/ml) |
% Recovery |
% Mean recovery ±SD |
%RSD |
|
50 |
1 |
2 |
1.91 |
95.93 |
99.77±0.76 |
0.9 |
|
1 |
2 |
2.02 |
101.4 | |||
|
1 |
2 |
2.05 |
102 | |||
|
100 |
1 |
4 |
3.68 |
98.6 |
99.53±0.520 |
1.09 |
|
1 |
4 |
3.76 |
99.5 | |||
|
1 |
4 |
4.05 |
100.5 | |||
|
150 |
1 |
6 |
5.71 |
95.2 |
98.6±0.501 |
0.89 |
|
1 |
6 |
6.01 |
100.2 | |||
|
1 |
6 |
6.02 |
100.4 |
Table 4: Repeatability studies of
Dasatinib.
|
Concentration [µg/ml] |
Area |
RT |
Area Mean ±SD |
%RSD |
|
2 |
487534 |
1.800 |
479218 ± 9025
|
0.5 |
|
2 |
468667 |
1.660 | ||
|
2 |
476756 |
1.753 | ||
|
2 |
486003 |
1.623 | ||
|
2 |
468887 |
1.702 | ||
|
2 |
487462 |
1.698 |
Table 5: Inter-day precision of
Dasatinib.
|
|
Inter day |
Intra day | ||||||
|
Concentration[µg/ml] |
AUC |
RT |
AUC Mean ±SD |
%RSD |
AUC |
RT |
AUC Mean ±SD |
%RSD |
|
2 |
469387 |
1.643 |
449473± 31877
|
0.28
|
486097 |
1.73 |
487595±1879 |
0.38 |
|
2 |
410015 |
1.713 |
489743 |
1.80 | ||||
|
2 |
477304 |
1.801 |
488528 |
1.78 | ||||
|
2 |
407318 |
1.670 |
484678 |
1.77 | ||||
|
2 |
465382 |
1.5048 |
487754 |
1.79 | ||||
|
2 |
467434 |
1.5043 |
488775 |
1.81 | ||||
Precision
Repeatability of the method was determined by
analysing six samples of same concentrations of drug i.e. 2?g/mL.
Chromatographs were recorded and area of each chromatograph was measured and
the values are represented in the Table IV. A method is said to be precise if
the % RSD is < 2 %, the results show % RSD for repeatability studies was
1.06 which indicates the results meet the acceptance criteria and hence the
method is said to be precise. The inter-day and intra-day precision study was
performed at 10?g/mL concentration levels and each value is the average of six
determinations. The results were reported as %RSD and the results are presented
in the (Table 5).
Table 6: System Suitability
Parameters.
|
Parameters |
Results |
|
Retention time
(min) |
1.743 |
|
Theoretical plates |
4708 |
|
Asymmetry |
0.23 |
System
suitability parameters
System suitability tests are an integral part of
chromatographic method. System suitability tests like theoretical plates,
tailing factor and HETP results of Rilpiverine hydrochloride are shown in
(Table 6).
Figure 7:
Zeta potential report.
Dasatinib
nano-suspension particle size and zeta potential
The Nano-suspension prepared by
precipitation-ultrasonic homogenization method are discrete, uniform, nearly
spherical Nano-Meteric particle in the size of 345 nm are mentioned in (Figure
6 and 7). The zeta potential of the prepared dasatinib Nano-suspension
formulations was found to be -25.4 [16-18].
Drug
entrapment efficiency
Dastanib Nano-suspension formulation the drug particles
were reduced to Nano sized. During the formulation process there was not any
drug loss step involved, so theoretically the formulation was considered as
being 100% drug content. The formulations, formulation drug loading efficiency
of 84.2%.
Dasatinib
spiked in serum
Out of 2 trial performed, the 2nd trial was
selected for further studies because when compared to other trials 2nd
trial was found good separation of saliva, with good peak symmetry. The
proposed method is applied to dasatinib spiked with serum and done in high
performance liquid chromatography with ultraviolet detection and the peak
obtained was good and same retention time to bulk sample.
Declaration of Interest
The author has no relevant affiliations or financial
involvement with a financial interest in or financial with the subject matter
or materials discussed in the manuscript.
Conflicts of Interest
There
is no conflict of interest.
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