Development and Validation of UPLC method for the
determination of Lenvatinib in Capsule formulation
Akula Srinivas1, Tayyaba Mahtab2, Sayeeda Tabasum2,
Abrar3, K. Jyothi4
1Department
of Pharmacology, St. Mary’s College of
Pharmacy, St. Francis Street,
Secunderabad, Telangana, India – 500025
2Department
of Quality Assurance, Bhaskar Pharmacy College, Yenkapally,
Moinabad, Telangana, India – 500075
3Department
of Quality Assurance, St. Mary’s College of Pharmacy, St. Francis Street,
Secunderabad, Telangana, India – 500025
4Department
of Pharmaceutical Chemistry, St. Mary’s College of Pharmacy, St. Francis Street,
Secunderabad, Telangana, India – 500025
*Corresponding Author E-mail: akulasrinivas305@gmail.com
ABSTRACT:
A
new, simple and selective method was developed to estimate Lenvatinib pharmaceutical dosage
form by UPLC. Ideal Chromatographic
peak of separation was attained on a Acquity BEH C18 (50*3.0mm. 1.7µm) using
mobile phase consisting 0.1% Orthophosphoric acid: ACN (60:40) v/v with
detection of 248 nm. Linearity of the drug was observed in the concentration
range 60-140 µg /ml (r2 =0.994). From the results, the developed
method was simple, sensitive, precise and accurate and it can successfully be applied
for the determination of API in the commercial formulations of Lenvatinib in quality control
laboratories.
KEYWORDS: Lenvatinib, development, validation, ICH guidelines,
UPLC method.
INTRODUCTION:
Lenvatinib
is chemically 4-[3-chloro-4-(cyclopropylcarbamoylamino)phenoxy]-7-methoxyquinoline-6-carboxamide
as shown in figure 1 with the molecular formula C21H19clN4O4
and molecular weight of 426.857g/mol[1]. It is an anticancer agent
which acts by inhibiting VEGFR (vascular endothelial growth factor) as well as FGFR
(fibroblast growth factor receptors) and also platelet derived growth factor
receptor (PDGFR)[2]. Hence, used in the treatment of thyroid cancer[3][4][5]
Hepatocellular cancer (HCC)[6][7] and renal carcinoma.[8]
Literature study shows very few validation
methods for determining Lenvatinib such as spectroscopy and HPLC method[9],
LC-MS/MS method[10][11] as well as RP-HPLC method[12][13].
However, in the present research study, a new and precise UPLC method was
established for determination of Lenvatinib in capsule formulation and validated
as per ICH guidelines[14].
Figure
1: Structure of Lenvatinib
MATERIALS
AND METHODOLOGY:
Chemicals:
Lenvatinib
was obtained as gift sample from Madras pharmaceuticals, Chennai, India.
Acetonitrile, water and methanol used were of HPLC grade and were purchased
from Merck whereas Potassium Dihydrogen phosphate, Dipotassium hydrogen
orthophosphate and Orthophosphoric acid of AR grade were purchased from Rankem,
India. Lenvima capsule formulation was purchased from nearby local pharmacy.
Instruments
and chromatographic conditions:
Determination
of Lenvatinib was done on the UPLC chromatographic system of Agilent 1290
Infinity equipped with diode array detection using Acquity BEH C18 (50*3.0mm.
1.7µm) analytical column. Mobile phase used was 0.1% of ortho phosphoric acid (H3PO4)
maintained at a flow rate of 0.5mL/min with injection volume of 10µL. Detection
was done at 248nm.
Preparation
of Diluent:
Taken
1mL of Orthophosphoric acid and transferred in to a 1000mL of water and filtered through 0.45µm filters to remove all fine
particles and gases.
Preparation
of mobile phase:
600mL of
0.1% Orthophosphoric acid was taken and mixed with 400mL of Acetonitrile. It
was mixed well and degassed. The final solution was filtered through 0.45µm filters to remove any particles if
present.
Preparation
of Standard solution:
10mg
of the pure Lenvatinib drug was weighed and transferred to 10mL volumetric
flask and mixed with 7mL of the diluent which was then subjected for sonication
for few minutes and final volume was made up with diluent. Further, 1mL was
taken and diluted to 10mL with the solvent to get final concentration of 100µg/mL.
Preparation
of Sample solution:
Twenty
capsules were taken and the granules were crushed to obtain fine powder from
which the amount of powder equivalent to 100mg of Lenvatinib was transferred to
100mL volumetric flask and dissolved in 70mL of the diluent which was then
subjected for sonication for few minutes and final volume was made up to 100mL.
Further 5mL of the solution was taken and dissolved in the 50mL volumetric
flask with the diluent and made up to the mark. Final solution was then
filtered through 0.45µm PVDF Syringe filter and assay was performed using the
following formula:
Where:
WT:
Weight of drug in assay sample
DT:
Dilution of the assay preparation
DS:
Dilution of the std preparation
P:
Purity of Lenvatinib
LC:
Labelled claim of Lenvatinib in capsules
RESULTS
AND DISCUSSION:
Method
optimization:
Several
trials were performed to optimize the method using various columns and
different composition of mobile phase. Upon screening the results, a clear peak
was obtained using 0.1% H3PO4 and Acetonitrile
(60:40%v/v) as mobile phase on Acquity BEH C18 (50*3.0mm. 1.7µm) column. The
peak obtained was symmetrical obtained at flow rate of 0.5mL/min and detection
at 248nm. Chromatogram of the optimized trial is shown in figure 2.
Figure
2: Chromatogram of the optimized trial of Lenvatinib
System Suitability and
System precision:
100µg/mL
of Lenvatinib was injected six times into the chromatographic system and
results were recorded for the same to verify the system. The plate count and
tailing factor results were found to be within the limits and the % RSD was
found to be 0.1 which determines that the system was suitable and can produce
precise results. Results of system suitability studies are presented in table
1.
Table
1: Result of System Suitability Studies
|
Injection
|
RT
|
Peak area
|
Theoretical plates (TP)
|
Tailing Factor (TF)
|
|
1
|
1.192
|
44125114
|
2560
|
1.2
|
|
2
|
1.193
|
44176891
|
2421
|
1.2
|
|
3
|
1.193
|
44165637
|
2676
|
1.2
|
|
4
|
1.190
|
44147346
|
2706
|
1.2
|
|
5
|
1.187
|
44105682
|
2704
|
1.2
|
|
6
|
1.190
|
44102411
|
2786
|
1.2
|
|
Mean
|
1.191
|
44137180
|
-
|
-
|
|
SD
|
0.00
|
31102.11
|
-
|
-
|
|
%RSD
|
0.2
|
0.1
|
-
|
-
|
Method
validation:
A UPLC
method developed was validated as per the guidelines given by ICH for specificity,
linearity, accuracy and precision, limit of detection and quantitation and
robustness. Linearity results obtained were good with correlation coefficient
value > 0.99 within the range of 60 - 140µg/mL concentration. Assay for the
developed method was found to be 99.7% as 9.87mg of the drug was found from the
label claim (10mg) which is within the acceptance range. Drug solutions with
the concentrations of 50%, 100% and 150% were analyzed for accuracy study and
the mean recovery was found to be 98.98. Detection and quantitation limit were
calculated from calibration curve and robustness study was performed changing
various conditions.
Specificity:
A study to establish
and determine the interference
of blank as well as placebo was
conducted. The sample and placebo
were injected into the system in triplicates. Chromatograms
of blank as well as placebo solutions
shows no peaks at the retention
times
of Lenvatinib indicating that the excipients do not interfere with the drug.
Accuracy:
Accuracy of the developed method
was analyzed by submitting 50%, 100% and 150% of the drug solution in
triplicate to the chromatographic system and their recovery studies were
observed. The mean percentage recovery was 98.98. The results are shown in
table 2.
Table 2: Result of Accuracy
Studies
|
Sample
|
Amount added
|
Amount recovered
|
% Recovery
|
Mean
|
Average mean
|
|
50%
|
51.25
|
50.84
|
99.2
|
99.36
|
98.98
|
|
51.36
|
51.36
|
100.0
|
|
51.24
|
50.71
|
98.9
|
|
100%
|
97.21
|
94.96
|
97.7
|
97.5
|
|
97.25
|
94.81
|
97.5
|
|
97.36
|
94.81
|
97.4
|
|
150%
|
150.21
|
150.73
|
100.3
|
100.1
|
|
150.89
|
150.75
|
99.9
|
|
150.22
|
150.64
|
100.2
|
Precision:
The precision of the developed
method was investigated by submitting six individual sample preparations into
the chromatographic system and its %RSD was calculated which was found to be 0.3
and is within the acceptance range.
Linearity:
From the standard stock
solution, serial dilutions were made to get the concentrations of 60, 80,100,
120, 140µg/mL and injected into the chromatographic system and the area were
noted. A calibration plot was constructed taking concentration against peak
area. The linearity data is shown in table 3 and the graph is shown in figure
3.
Table 3: Linearity
Results
|
S. No
|
Concentration (µg/mL)
|
Area
|
|
1
|
60
|
21720461
|
|
2
|
80
|
34167231
|
|
3
|
100
|
44035624
|
|
4
|
120
|
52943892
|
|
5
|
140
|
67035271
|
|
Slope
|
547031.405
|
|
Intercept
|
-10722644.7
|
|
Correlation coefficient
|
0.994
|
Figure 3: Calibration
curve of Lenvatinib
Detection and
quantitation limit (LOD and LOQ):
LOD and LOQ of Lenvatinib
were calculated using the following formula based on response of the
calibration curve.
Where,
σ = Std dev of the
response,
S = Slope of the
calibration curve
LOD and LOQ values were
found to be 2.7 µg/mL and 8.185µg/mL respectively.
Robustness:
The Robustness of the method
was determined by changing the chromatographic conditions such as flow rate and
the temperature as a result, the tailing
factor was found to be within the limits on small variation of flow rate. The results
of robustness are depicted in table 4.
Table 4: Robustness Results
|
Chromatographic changes
|
Retention time (Rt)
|
Tailing factor
|
Theoretical plates
|
%RSD for standard areas
|
|
Flow rate (mL/min)
|
0.4
|
1.473
|
1.1
|
2942
|
0.2
|
|
0.6
|
0.933
|
1.2
|
2047
|
0.1
|
|
Temperature
|
25
|
1.143
|
1.2
|
2467
|
0.3
|
|
35
|
1.137
|
1.1
|
2496
|
0.2
|
CONCLUSION:
A
new precise, accurate, rapid method has been developed for the estimation of Lenvatinib in capsule formulation
by UPLC. The results obtained by the validation of the developed method produce
within the desired criteria. Hence, this method can be adopted for the analysis
of Lenvatinib myselate in quality control laboratories.
ACKNOWLEDGEMENT:
All
the authors are thankful to St. Mary’s college of pharmacy for providing the
facilities to undergo the research work.
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DOI: 10.5958/2231-5691.2019.00040.6