Development And Validation Of Stability Indicating RP-HPLC Method For Determination Of Ceritinib

Abstract

A new, simple, specific, accurate and precise RP-HPLC method was developed for determination of Ceritinib. In the present study, stress testing of Ceritinib was carried out according to ICH guidelines Q1A (R2). Ceritinib was subjected to stress conditions of hydrolysis, oxidation, photolysis and neutral decomposition. Extensive degradation was found to occur in acidic, condition. Mild degradation was observed in basic and at thermal conditions. Successful separation of drug from degradation products formed under stress conditions was achieved on a Hypersil BDS C18 column (250 mm × 4.6 mm, 5.0 μ particle size) using acetonitrile: acetate buffer (pH 3.7 ± 0.05) (50:50 v/v), at a flow rate of 1.0 mL/min and column was maintained at 40˚C. Quantification and linearity was achieved at 272 nm over the concentration range of 5 - 100 μg/mL for Ceritinib. The method was validated for specificity, linearity, accuracy, precision, LOD, LOQ and robustness.

Full text

Research Article
Volume 28 Issue 4 (2017) 241
Indonesian J. Pharm. Vol. 28 No. 4 : 241 – 248
ISSN-p : 2338-9427
DOI: 10.14499/indonesianjpharm28iss4pp241
DEVELOPMENT AND VALIDATION OF STABILITY
INDICATING RP-HPLC METHOD FOR DETERMINATION
OF CERITINIB
Vaibhav Suresh Adhao1*, J. Sharma2, M. Thakare2
1DR. Rajendra Gode College
Of Pharmacy, Malkapur.
Maharashtra, India – 443101
2Dr. B.R. Ambedkar
University, Agra, Uttar
Pradesh, India - 282004
Submitted: 12-09-2017
Revised: 28-11-2017
Accepted: 03-12-2017
*Corresponding author
Vaibhav Suresh Adha
Email:
adhao.vaibhav@gmail.com
ABSTRACT
The purpose of the present work was to develop new,
simple, specific, accurate and precise stability indicating RP-HPLC
method for determination of ceritinib. In the present study,
stress testing of ceritinib was carried out according to ICH
guidelines Q1A (R2). ceritinib was subjected to stress conditions
of hydrolysis, oxidation, photolysis and neutral decomposition.
Extensive degradation was found to occur in acidic condition. Mild
degradation was observed in basic and at thermal conditions.
Successful separation of drug from degradation products formed
under stress conditions was achieved on a Hypersil BDS C18
column (250×4.6mm, 5.0μ particle size) using acetonitrile:
acetate buffer (pH 3.7±0.05) (50:50 v/v), at a flow rate of
1.0mL/min and column was maintained at 40°C. Quantification
and linearity were achieved at 272nm over the concentration
range of 5-100μg/mL for ceritinib. The Correlation Coefficient
was found to be 0.9960. The method was validated for
specificity, linearity, accuracy, precision, LOD, LOQ and
robustness. The developed method will be useful for routine
analysis for samples of stability studies in the formulation and
development.
Keywords: Stability-indicating, HPLC, ceritinib, validation, stress testing.
INTRODUCTION
Ceritinib, 5-Chloro-N4-[2-[(1-
methylethyl)sulfonyl]phenyl]-N2-[5-methyl-2-
(1-methyl-ethoxy)-4-(4-piperidinyl)phenyl] 2,4-
pyrimidine diamine, is an anaplastic lymphoma
kinase (ALK) inhibitor which induces complete
tumor regression in a xenograft model of
EML4-ALK-positive lung cancer. The
alternative names of ceritinib are LDK 378,
NVP-LDK 378, ZykadiaTM. Ceritinib is a
highly selective inhibitor of an important cancer
target, ALK (Heudi, et. al., 2014). Ceritinib, a
recently approved drug by Food and Drug
Administration, is used for the treatment of
late-stage (metastatic) non-small cell lung
cancer (Waters, 2014). The recommended
dosage of ceritinib is 750 mg administered
orally once daily on an empty stomach (Shaw, et
al., 2014). The chemical structure of ceritinib
(Figure 1).
An ultrafast, sensitive, selective, and
robust LDTD-APCI-MS/MS method was
developed for the quantification of ceritinib
in human plasma (Lanshoeft, 2015). A stability
Figure 1. Structure of Ceritinib
indicating reversed-phase high-performance
liquid chromatographic (RP-HPLC) method for
estimation of ceritinib was reported (Kumar.,
2014). Since, there are only two HPLC method
reported in the literature for the estimation of
ceritinib in pharmaceutical dosage forms

Stability Indicating RP-HPLC Method
242 Volume 28 Issue 4 (2017)
(Chintala, et al., 2015), (Reddy, et al., 2016 ),
therefore, there is a need to develop
quantitative methods under different conditions
to achieve improvement in sensitivity,
selectivity, as per ICH guidelines (2005). The
developed method will be novel for
Stability studies and use in the routine analysis
for samples in stability study which was very
important part of formulation and development.
MATERIAL AND METHODS
Reagents and materials
Ceritinib (purity > 99%) procured from
Spectrum lab, Hyderabad, India was used for
the study. All the chemicals used of HPLC
Grade (MERCK. Chem. Ltd., Mumbai) and
HPLC grade water was used for mobile phase
preparation. Nylon membrane filter 0.45µ
syringe filter. Hydrogen peroxide, sodium
hydroxide and hydrochloric acid are used of
AR grade.
Chromatographic conditions:
The chromatographic separation was
achieved on Perkin Elmer (USA) HPLC system
(series 200) equipped with Perkin Elmer series
200 pump system having back pressure 5000
psi, manual injector of 20μL loop, UV-Visible
detector and Hypersil BDS C18 column
(250mm x 4.6mm i.d., 5µm); Sartorious,
analytical balance; An ultra-sonicator; A
Shimadzu model 1800 double beam UV/Vis.
spectrophotometer with a pair of 10mm matched
quartz cells. The mobile phase comprised of
acetonitrile: acetate buffer (pH 3.7±0.05)
(60:40% v/v), at a flow rate of 1.0mL/min, and
column was maintained at 40°C. The mobile
phase was filtered through nylon 0.45 µm
membrane filter and was degassed before being
used for analysis. The UV-Visible detector was
set at 272nm. The injection volume was 20µL
and total run time was 10min.
Preparation of the mobile phase
The mobile phase was prepared by
mixing 60mL acetonitrile and 40mL acetate
buffer (pH 3.7±0.05) previously filtered
through 0.45µm nylon membrane filter. The
mobile phase was degassed for 15min by
sonicating the solution before use.
Preparation of Diluent
Acetonitrile: Water (60:40% v/v) was
used as diluent.
Preparation of standard solution
Accurately weighed ceritinib (25mg) was
transferred to a 25mL volumetric flask,
dissolved in and diluted to the mark with
diluent to obtain a standard stock solution
(1mg/mL).
Preparation of working standard
solution (10µg/mL)
Standard solution (0.1mL) was
transferred in a 10 mL volumetric flask and
diluted up to the mark with mobile phase.
Analysis of tablet dosage form
Twenty tablets (ZYKADIA) were
weighed and average weight was calculated.
The tablets were finely powdered, and a quantity
of powder equivalent to 25mg ceritinib was
weighed accurately and transferred to a 25mL
volumetric flask containing 15mL diluent, and
sonicated for 15min. The solution was allowed
to stand at room temperature for 5min and the
volume was made up to the mark with
diluent to obtain the sample stock solution
(1mg/mL). The solution was filtered through
0.45µ membrane filter. Aliquot (1.0mL) was
taken and transferred to 10mL volumetric flask
and volume was made up to the mark
with diluent to give a solution containing
100µg/mL ceritinib. The solution (2.0mL) was
transferred to 10 mL volumetric flask and
diluted up to the mark with mobile phase
to give a solution containing 20µg/mL
ceritinib. An aliquot (20µL) was injected
and the chromatogram was recorded. The
peak area was noted and the amount of
ceritinib was calculated from the regression
equation.
Forced degradation study
Ceritinib was subjected to various forced
degradation conditions to affect partial
degradation of the drug preferably in 20-80%
range. The study provided information about
the conditions in which the drug was unstable.
This Information was useful for development
of formulation.

Vaibhav Suresh Adhao
Volume 28 Issue 4 (2017) 243
Effect of acid, alkaline and neutral hydrolysis
Accurately weighed ceritinib 25mg was
transferred to three different 100mL volumetric
flasks and dissolved in diluent (20mL).
Hydrochloric acid (0.1N, 10mL), sodium
hydroxide (0.1N, 10mL) and water (10mL)
were added to separate flasks containing drug
samples and mixed properly for acidic, alkaline
and neutral degradation respectively and stored
at room temperature for 72h.
The samples were neutralized with base
or acid as appropriate and diluted up to the
marks with diluent to obtain stock solutions
(250µg/mL). Dilutions were made with mobile
phase to obtain the degraded ceritinib solutions
(25µg/mL).
Effect of oxidation
Accurately weighed ceritinib 25 mg was
transferred to a 100mL volumetric flask and
dissolved in diluent 20mL. Hydrogen peroxide
solution (3%) 10mL was added, mixed, and
stored at room temperature for 72h. The
sample was diluted up to the mark with
acetonitrile to obtain stock solution (250µg/mL).
Dilution was made with mobile phase to obtain
the degraded Ceritinib solution (25µg/mL).
Effect of heat
Ceritinib 25mg was distributed over a
glass plate and kept in an oven at 60°C for 72h,
then ceritinib was transferred in a 100mL
volumetric flask, and dilutions were made with
mobile phase to obtain the degraded ceritinib
solution (25µg/mL).
Effect of light
Ceritinib solution (prepared by
dissolving 25mg ceritinib in 20mL acetonitrile
in 100mL volumetric flask) was exposed to sun
light for 48h, while ceritinib powder 25mg was
exposed to UV light for 48h. After exposure,
dilutions were made to obtain the degraded
ceritinib solutions (25µg/mL). Aliquots
(20µL) of the stressed samples were injected
into the HPLC system as described under
chromatographic conditions and the chromate-
grams were recorded.
Method validation
As per the ICH guideline Q2 (R1)
(2005), the method validation parameters
namely specificity, linearity, accuracy, precision,
limit of detection, limit of quantitation and
robustness were studied.
Solution stability
Sample solutions were kept at 25±2°C
(24h) and 2-8°C (3 days), respectively.
Assay percentage of initial time period
was compared with these two time periods.
The change in the assay percentage was
calculated. The difference between assay results
should not be more than 2% for formulation,
and 0.5% for active pharmaceutical ingredient
(API).
Specificity
Chromatograms of ceritinib solutions
and degraded samples were studied in order to
provide an indication of the stability indicating
properties and specificity of the method. The
stress conditions employed were acidic,
alkaline, neutral, oxidative, thermal and
photolytic degradation. The degraded samples
were analyzed against freshly prepared sample
solutions.
Linearity (Calibration Curve)
Standard solutions (0.05, 0.1, 0.15, 0.2,
0.25 and 0.3mL equivalent to 5.0, 10.0, 15.0,
20.0, 25.0 and 30.0µg/mL of ceritinib) were
transferred in a series of 10mL volumetric
flasks and diluted to the mark with mobile
phase. An aliquot (20µL) of each solution was
injected under the operating chromatographic
conditions as described earlier. Calibration
curve was constructed by plotting peak areas
versus concentrations, and the regression
equation was calculated. Each response was
average of three determinations.
Accuracy (% recovery)
Accuracy of the method was determined
by calculating percentage recovery of ceritinib
by the standard addition method. Known
amount of standard solutions of ceritinib (0, 5,
10 and 15µg/mL) were added to a pre-analyzed
sample solution of ceritinib (10µg/mL). Each
solution was injected in triplicate, and the
percentage recovery was calculated by
measuring the peak areas and fitting these
values into the regression equation of the
calibration curve.

Stability Indicating RP-HPLC Method
244 Volume 28 Issue 4 (2017)
Precision
Repeatability was checked by repeatedly
(n = 6) injecting ceritinib solution (10 µg/mL)
and recording the chromatogram. Intra-day and
inter-day precisions of the developed method
was determined by measuring the
corresponding responses 3 times on the same
day and on 3 different days over a period of 1
week for 3 different concentration of ceritinib
(10.0, 20.0 and 30.0µg/mL). The results were
reported in terms of relative standard deviation.
Limit of detection and limit of quantification
Limit of detection (LOD) and the limit
of quantification (LOQ) were calculated using
the standard deviation of response (σ) and
slope (S) of the calibration curve.
LOD = 3.3 x σ/S
LOQ =10 x σ/S
Robustness
Robustness was studied by analyzing the
samples of ceritinib by deliberate variation in
the method parameters. The change in the
response of ceritinib was noted. Robustness of
the method was studied by changing the
extraction time of ceritinib from tablet dosage
form by ±2 min, composition of mobile phase
by ±2% of organic solvent, wavelength by ±2
nm, flow rate by ±0.2 mL/min and column
oven temperature by 2ºC. The changes in the
response of ceritinib were noted and compared
with the original one.
System-suitability test
System suitability tests were used to
verify that the resolution and repeatability of
the system were adequate for the analysis
intended. The parameters used in this test were
retention time, tailing factor and theoretical
plates of chromatographic peak as RSD of peak
area for replicate injections.
RESULTS AND DISCUSSION
Selection of column and mobile phase
As per the published literature and
knowledge of the molecule, reverse phase liquid
chromatography (RP-HPLC) is suitable for
analysis of ceritinib. In case of RP-HPLC
various columns are available, but as the
main aim of the method was to resolve the
compound from degraded products, C18
column (250x4.6mm i.d., 5µm particle size) was
preferred over the other columns. Resolution is
the most important criteria for the method, it is
imperative to achieve good resolution among
the compound and degraded products. As per
the value of pKa and solubility of compound
various composition of mobile phase were
tried.
The chromatographic conditions were
optimized with a view to develop a stability
indicating assay method, which can separate the
drug from its degradation products with good
resolution. Mobile phase consisting of
acetonitrile: acetate buffer (pH 3.7±0.05)
(50:50% v/v) at a flow rate of 1.0mL/min, was
found to be satisfactory to obtain well-resolved
peaks with better reproducibility and
repeatability for ceritinib (Figure 2).
Method validation
The change in assay results after storage
at 25°C (24h) and 2-8°C (3 days) was evaluated.
It was found that the difference in assay results
was not more than 2% for formulation, and
0.5% for API, indicating stability of ceritinib
solution.
The developed analytical method was
found to be specific as there was no inference
of any related impurities after the stress
degradation study (Figure 3). It was shown that
the ceritinib peaks were free from excipients
and co-eluting impurities.
The linear correlation was obtained
between peak area and concentration of
ceritinib in the range of 5-30µg/mL, the
linearity of the calibration curve was validated
by the value of correlation coefficient of the
regression (r) and the Y intercept were found to
be 0.9960 and Y =38217x+72833 respectively.
The regression analysis of the calibration curves
(Table I).
The accuracy study was carried out by
the standard addition method at level of ±50 %
of standard concentration. The percent
recoveries were found in the range of 98.86-
101.12%, which indicated accuracy of the
method (Table II). The accuracy study had
shown results within the limit of % RSD less
than 2%.

Vaibhav Suresh Adhao
Volume 28 Issue 4 (2017) 245
The % RSD for repeatability (Table III)
of ceritinib was found to be 1.25. The value of
% RSD for intra-day precision was found to be
in the range of 0.93 - 1.15% and inter-day
precision was found to be in the range of 1.07 -
1.22 %, which indicated that the method was
precise (Table IV). The Precision study showed
results within the limit of % RSD less than 2%.
The Limit of detection (LOD) for ceritinib was
found to be 0.062µg/mL, while the Limit of
quantification (LOQ) was 0.187µg/mL. The
method was found to be robust as the results
were not significantly affected by slight
variation in extraction time, composition of
mobile phase, wavelength and flow rate of the
mobile phase.
System-suitability test
The % RSD of retention time,
tailing factor, and theoretical plates were
found to be 1.26%, 1.08% and 0.46%
respectively. The% RSD of system-suitability
test parameters was found satisfactory as
% RSD should not be more than 2%
(Table V).
Analysis of tablet dosage form
The proposed RP-HPLC method
was successfully applied for determination
of ceritinib from tablet dosage form.
The percentage of ceritinib was found to be
99.07%; which was comparable with the
corresponding label claim (Table VI).
Figure 2. Chromatogram of ceritinib with retention time of 4.41min
Figure 3. Calibration curve of ceritinib

Stability Indicating RP-HPLC Method
246 Volume 28 Issue 4 (2017)
Degradation study
Forced degradation study of ceritinib
was carried out under various stress conditions
as follows
Effect of acid, alkaline and neutral
hydrolysis
Ceritinib was found to undergo 64.67%
decomposition under acidic stress condition
with a major degradation product at retention
time of about 3.88 min and minor degradation
product at retention time of about 2.30min
and minute decomposition about 3.7% under
basic stress condition with a degradation product
at retention time of about 2.30min. Under
neutral degradation condition, no degradation
was observed. Hence, ceritinib was found to be
highly degradable in basic condition, and very
minute degradable in acidic condition but not
degradable in neutral condition.
Effect of oxidation
In oxidation stress condition, almost
9.5% of ceritinib was degraded and degradation
peak appeared in chromatogram at 2.30min
retention time.
Effect of heat
Under dry thermal stress condition,
ceritinib was degraded about 12.23% with
degradation product at retention time of about
2.58 and 6.83min.
Effect of light
When ceritinib in solution state was
exposed to sun light; and ceritinib in powder
state was exposed to UV light, no degradation
was observed, respectively.
Table I. Optical and regression characteristics (n=3)
Parameter
Ceritinib
Linearity range (µg/mL)
5-30
Linearity equation
y = 38217x+72833
LOD (µg/mL)
0.062
LOQ (µg/mL)
0.187
Correlation coefficient (r)
0.9960
Table II. Results of recovery study (n=3)
Amount
Taken (µg/mL)
Amount
added
(µg/mL)
Amount
Found
(µg/mL)
Recovery
± S.D, %
%
RSD
10
0
10.05
100.50±0.92
0.92
10
5
14.83
98.86±1.65
1.65
10
10
19.90
99.50±1.37
1.37
10
15
25.28
101.12±0.77
0.77
Table III. Results of repeatability (n=6)
Drug
1
2
3
4
5
6
Mean
SD
% RSD
Ceritinib
443614.0
454953.1
443275.4
445228.7
448743.8
438564.2
445729.9
5590.19
1.25
Peak area
Table IV. Results of Intra-day and Inter-day precision (n=3)
Ceritinib
(µg/mL)
Intra-day precision
Inter-day precision
Mean peak area ± SD
% RSD
Mean peak area ± SD
% RSD
10
443614.0±4152.12
0.91
448745.3±4836.18
1.04
20
875777.0±9638.35
1.08
878418.6±9858.21
1.07
30
1200410.0±13864.16
1.13
1201059.6±14728.26
1.12

Vaibhav Suresh Adhao
Volume 28 Issue 4 (2017) 247
The samples exposed to acidic, alkaline,
neutral, oxidative, thermal and photolytic
conditions were colorless. In Photolytic
stability, ceritinib was found to be stable
showing no degradation. All degradates were
resolved from ceritinib peak and the percentage
degradation for each condition indicated that
there was no interference from degradates in
determination of the ceritinib in tablet dosage
form. Thus, the proposed, method was found
to be "Stability Indicating".
CONCLUSION
An isocratic stability indicating reverse
phase liquid chromatographic method has been
developed and validated for the estimation of
ceritinib in tablet dosage form, the method was
found to be specific as there was no
interference of any co-eluting impurities after
stress degradation study. The proposed method
was found to be simple, accurate, precise,
sensitive and robust. Hence, it can be used
successfully for the routine analysis of ceritinib
in pharmaceutical dosage forms, and for
analysis of stability samples obtained during
accelerated stability study
ACKNOWLEDGEMENT
We are grateful to Dr. Rajendra Gode
College of Pharmacy, for supporting this
research.
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Table V. System suitability test parameters (n = 6)
No.
Retention time,
Min.
Tailing factor
Theoretical plates
1
4.41
1.49
9282.12
2
4.41
1.49
9254.23
3
4.35
1.48
9237.48
4
4.27
1.49
9187.75
5
4.38
1.47
9265.58
6
4.41
1.45
9176.38
Mean
4.37
1.48
9233.92
SD
0.055
0.016
42.88
% RSD
1.26
1.08
0.46
Table VI. Analysis results of tablet dosage form (n=3)
Formulation
Drug
No. of
Injection
Amount
Taken
Amount
found
Label
Claimed
Amount Found
per Tablet
% Label
Claim
ZYKADIA
Ceritinib
6
30 mg
29.72 mg
15 mg
14.86 mg
99.07
Table VII. Results of stress degradation study
Stress conditions/duration
% Degradation
Acidic/0.1N HCl
64.67
Alkaline/ 0.1N NaOH
3.70
Oxidative/ 3% H
2
0
2
9.50
Thermal 60°C
12.23

Stability Indicating RP-HPLC Method
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