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TRANSCRIPT
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8.0 Introduction
Nilotinib Hydrochloride, 4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-
(trifluoromethyl)phenyl]-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]benzamidesalt) in the form
of the hydrochloride monohydrate salt with trade name Tasigna, is a tyrosine kinase
inhibitor approved for the treatment of chronic myelogenous leukemia [272-276]. Nilotinib
hydrochloride monohydrate is used to treat chronic myeloid leukaemia (CML) in people who
have tested positive for Philadelphia chromosome.Nilotinib Hydrochloride is slightly yellow to
slightly greenish yellow powder.It is slightly soluble in methanol and in Dimethyl
sulphoxide.The empirical formula of Nilotinib Hydrochloride is C28H22F3N7O.HCl. The
molecular weight of Nilotinib Hydrochloride is 565.98.
8.0.F1: Chemical structures of Nilotinib Hydrochloride
NH
CH3
N
N
N
O
N
N
CH3
F
F
FHN
Hcl
8.1 Pharmacology of Nilotinib Hydrochloride
Nilotinib Hydrochloride is a tyrosine kinase inhibitor approved for the treatment of
chronic myelogenous leukemia.[274].It is used to treat chronic myeloid leukaemia (CML) in
people who have tested positive for Philadelphia Chromosome. Philadelphia Chromosome
is a genetic abnormality which is commonly found in people who have CML.Chronic
myelogenous (or myeloid) leukemia (CML), also known as chronic granulocytic leukemia
(CGL), is a cancer of the white blood cells. It is a form of leukemia characterized by the
increased and unregulated growth of predominantly myeloid cells in the bone marrow and
the accumulation of these cells in the blood.CML is a clonal bone marrow stem cell
273
disorder in which proliferation of mature granulocytes (neutrophils, eosinophils, and
basophils) and their precursors is the main finding.It is a type of myeloproliferative disease
associated with a characteristic chromosomal translocation called the Philadelphia
chromosome.CML is now largely treated with tyrosine kinase inhibitors (TKIs), such as
imatinib,dasatinib,or Nilotinib ,which have led to dramatically improved survival rates since
their introduction in the last decade.[275-277].
8.1.T1 Chemical structures, anticancer activity and adverse effects of some of
the common epidermal growth factor receptors (EGFR).
Drug Chemical structure
Mode of action Side effects
Nilotinib
NN
HNON
O
O
F
Cl
For certain type of lung cancer (non-small cell lung cancer or NSCLC)
Diarrhea, Skin reaction (rash, acne), Nausea Vomiting , Itching, Poor apetite Eye irritation.
Crizotinib
HN
NN
N
O
Cl
ClF
NH2
To treat advanced lung cancer
vision problems, constipation, and swelling due to fluid retention. fainting, fever, or breathing problems.
Erlotinib HCl
OH3C
H3CO O
O
NN
HN CCH
To treat lung cancer, pancreatic cancer and several other types of cancer.
Rashes occurs in the majority of patients. Rarely, ingrown hairs, such as eyelashes.
Cetuximab
HNO
S
N
O
OO
F
OH
F
OO
OH
For treatment of metastatic colorectal cancer and head and neck cancer
The incidence of acne-like rash. fevers, chills, rigors, urticaria, pruritis, rash, hypotension.
8.2 Synthesis of Nilotinib Hydrochloride
The synthesis of Nilotinib Hcl(III) involves three stages.In the first stage 3-(4-
Methyl-1h-Imidazole-1-yl)-5-(trifluro methyl) benzenamine and 4-methyl -3-((4-(pyridine-3-
yl) pyrimidin-2-ylamino) benzoic acid are condensed in presence of Diethyl cyano
phoshphonate and triethylamine to form Nilotinib base (NTH-I) through the elimination of
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water.Nilotinib base is further reacted with ethyl acetate Hcl to give Nilotinib Hydrochloride
and the wet material is dried.
8.2.1.F1 Chemical synthesis of Nilotinib HCl in the laboratory
N
NH2N
CF3
CH3
NH
CH3
N
N
NHO
O
Diethyl cyano phosphonate+
3-(4-Methyl-1H-imidazole-1-yl)-5-(tri fluro methyl) benzenamine
4-methyl-3-((4-(Pyridin-3-yl)pyrimidin-2-ylamino)benzoic acid
Solvents
I II
NH
CH3
N
N
N
O
N
N
CH3
F
F
F HN
NH
CH3
N
N
N
O
N
N
CH3
F
F
F HN
Hcl
Nilotinib Base Nilotinib Hydrochloride
Purification
HCl
III IV
8.2.2. Impurities of Nilotinib HCl (IV)- Structures
8.2.2. F1 Impurity-1:3-bromo-5-(trifluoro methyl) aniline.
NH2
BrCF3
Molecular weight : 227
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8.2.2.F2 Impurity-2:4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)
5-(trifluoromethyl)phenyl]-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-
benzamide.
NH
O
N
NCH3
HNN
N
N
CH3
CF3
Molecular weight : 529.52
8.2.2. F3 Impurity-3: 4-Methyl-3-(4-Pyridine -3-yl) pyrimidin-2-ylamino)benzoic acid
N
N
N
NH
CH3
CH3
O
Molecular weight : 306.32
8.2.2.F4 Impurity-4: 3-(4-methyl-1H imidazole-1-yl-5-(Trifluoromethyl)
benzene amine.
H2N
CF3
N
N
CH3
Molecular weight : 241.21
276
8.3 Availability of Nilotinib HCl analytical methods
Literature survey revelas that there are some mass detection methods reported for
determination of Nilotinib Hydrochloride. As far as we are aware there is no stability-
indicating UPLC method for determinationof related compounds (impurities) of Nilotinib
Hydrochloride.The purpose of the present research work is to develop a single stability-
indicating UP LC method for the determination of Nilotinib Hydrochloride and its related
impurities and to establish the degradation summary for Nilotinib HCl along with its four
potential impurities followed with method validation as per ICH recommended conditions
[278-280].
8.4. Objectives of the present work.
8.4.1 To optimize the Ultra Perfromance chromatographic conditions for separation of four
process related impurities of Nilotinib Hydrochloride.
8.4.2 Tostudy the forced degradation of Nilotinib Hydrochloride under thermal, photo,
acedic, basic,peroxide conditions.
8.4.3 Tostudy the degradation behaviour of Nilotinib Hydrochloride using UPLC having
PDA detector.
8.4.4 To identify the impurities that considerably undergone degradation with time using
LC-MS.
8.4.5 To study the suitability of the developed method for analysis of bulk drug samples.
8.5 Experimental
8.5.1 Materials and reagents
Methanol HPLC grade and acetonitrile HPLC grade were purchased from Rankem.
Formic acid, sodium hydroxide, hydrochloric acid, and hydrogen peroxide was purchased
from Merck. HPLC grade water was obtained from Milli-Q water purification system
(Millipore,Milford,USA). All impurities and the Nilotinib Hydrochloride standards are more
than 96% purity and individually which contain purity as, Nilotinib Hydrochloride (99.8%),
imp-1 (96.8%), imp-2 (99.1%), imp-3 (97.2%) and imp-4 (98.8%).
277
In addition, HPLC grade acetonitrile and orthophosphoric acid were purchased from
Merck, Darmstadt, Germany. Samples of Nilotinib Hydrochloride (E)-7-[2-cyclopropyl-4-(4-
fluorophenyl)quinolin-3-yl]-3,5-dihydroxy-hept-6-enoic acid, its process related substances
viz 3-bromo-5-(trifluoro methyl) anilineImp-1),4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-
trifluoromethyl) phenyl]-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-benzamide(Imp-2),4-Methyl-
3-(4-Pyridine -3-yl) pyrimidin-2-ylamino) benzoic acid (Imp-3) and 3-(4-methyl-1H imidazole-
1-yl-5-(Trifluoromethyl) benzene amine (Imp-4) were collected during the process
development work in our laboratory and degradation products 4-Methyl-3-(4-Pyridine-3-
yl)pyrimidin-2-ylamino)benzoic acid 3-(4-methyl-1H imidazole-1-yl-5-(Trifluoromethyl)
benzene amine were isolated and used.The Nilotinib (reference standard)and
bulk drug samples were a kind gift from Hetero Labs Limited Hyderabad,Andhra Pradesh
India.
8.5.2 Apparatus
8.5.2.1 Ultra performance liquid chromatograph
The analysis were performed on an Shimadzu UPLC system Hachioji-shi 1-
9,Tokyo Japan, equipped with online degasser, high pressure binay pump, autosampler with
automatic temperature controlled sample compartment, thermostatted column
compartmenta and photodiode array detector. For data processing and equisition,
chemstation 01.03(Agilent technologies)was used. 1200 UPLC system was performed with
a normal instrument plumbing configuration which was an internal system volume of
approximately 600-800 L down to 120 μL, flow rates up to 5 mL/min and 600 bar
pressure provide universal applicability in narrow and standard bore HPLC and RRLC with
column IDs from 1 to 4.6 mm. Componants installedincluded internal tubing connections with
0.17 mm i.d capillary tubing, a 400 L static mixture, damper (pressure dependant volume
ranging from 80-280 L, and 10 mm path length DAD flow cell(13L).
8.5.2.2 pH meter
The pH measurements were carried out with Elico,model LI 120, pH meter
equipped with a combined glass calomel electrode calibrated using standard buffer solutions
of pH 4.0,7.0 and 9.2.
8.5.2.3 FTIR Spectrophotometer
The IR spectras necessary were recorded on a fourieir–Transform Infrared
Spectrophotometer (Perkin Elmer-Spectrum one ).FTIR is equipped with Harizontal
278
attenuated total reflectance (HATR) accessory.The number of scans were 16 with 0.25
cm-1. The spectra were recorded in the solid state using a automatic KBR press
pellet.Spectrum is scanned between 450 and 4000cm-1.
8.5.2.5. Photo stability chamber :
The Atlas Suntest CPS/CPS+ Photo stability chamber is used for photostability
studies.Thermal stability studies are carried out in a dry hot air oven (Cintex precision hot air
oven).
8.5.2.6. Mass spectrometry
The LC-MS system (Waters–Micro mass, Quattro micro-API-ESCI) was used for
the identification of unknown compounds formed during forced degradation.
A symmetry Shield 100 x 4.6 mm, 2.2-µm column is used as the stationary phase with a
mobile phase containing a gradient of solvent A 0.01M ammonium formate in water (0.063 g
of ammonium formate in 1000mL of water and adjusted to pH 3.0 with formic acid solution)
and solvent B (acetonitrile). The flow rate of the mobile phase was kept at 0.8 mL min-1 with
a gradient program of 0/0, 5.5/55, 8.5/45, 9.5/0 and 10.5/0 (time (min) / %B). The
injection volume is 6 µL.The analysis is performed in positive and negative electrospray
ionization modes.The capillary and cone voltages are 3.50 KV and 25.00 V, respectively.The
extractor and RF lens voltages are 3.0 V and 0.3 V, respectively.The source and desolvation
temperatures are 120°C and 350°C, respectively, and the cone gas flow and desolvation gas
flow are 100 Lhr-1 650 Lhr-1respectively.
8.6. Analytical procedures
8.6.1 Preparation of mobile phase
About 0.01 Molar of ammonium formate was taken with the help of a calibrated
balance,dissolved in 1000ml of de-ionized water in a 1 liter-measuring cylinder and the PH
was adjusted to 3.0 with dilute formic acid.The resultant buffer (solvent A, 1.0ml of formic
acid in 1000ml of water)was thoroughly shaken and filtered through a PTFE membrane filter
of 0.45m pore size using a vacuum pump.Buffer:Acetonitrile (40:60 v/v)(solvent-B)was
used as an organic modifier in a gradient elution mode.
279
8.6.2 Preparation of standard and sample solutions
A stock solution of Nilotinib (1.0 mg/ml) was prepared by dissolving appropriate
amount in the diluent.Working solutions of 0.10 % and for impurities 0.15% are prepared
from the stock solution for the determinations of related substances respectively. A stock
solution of impurities (mixture of impurity-1, impurity-2, impurity-3 and impurity-4) at 0.03
mg/ml is also prepared in the diluent. The drug substance powder equivalent to 100 mg of
sample is transferred into a 100-ml volumetric flask, and 70 ml of diluent is added.The flask
is attached to a rotary shaker and shaken for 2 min to disperse the powder completely.The
mixture is sonicated for 2 min and then diluted to the appropriate volume with diluent to
make a solution containing 1.0 mg/ml.The solution is then filtered through a 0.45-μ Nylon 66
membrane filter.
8.6.3 Specificity and generation of stress samples
The specificity of the developed LC method for Nilotinib was determined in the
presence of its impurities (namely imp-1, imp-2, imp-3, and imp-4) and degradation
products.Forced degradation studies (stress studies)under various conditions are
recommended by the international conference on harmonization (ICH) during
pharmaceutical development of drug candidate.These guidelines are designed in order to
assist in the understanding of the stability and degradation behavior of the drug candidate.
The details of the stress conditions applied are as follows.
In Acid hydrolysis, drug substance was exposed at room temperature for 2 hours in
1N HCl.In base hydrolysis,drug was exposed at 800C temperature for 2hours in 0.1M
NaOH.During Oxidative stress,drug was exposed at 800C temperature for 4hours in 5%
H2O2.In water hydrolysis,drug was exposed at room temperature for 24 hours in
water.Thermal stress, drug was subjected to dry heat at 100°C for 24 hours.The photo
degradation [16] was carried out by carried out as per ICH Q1B exposing the Nilotinib
samples For heat, humidity and light studies, the samples were exposed for 5 days and 1.2
million LUX hrs, 200 Watt-Hours/Sq.mts respectively [17].
The above stress conditions were also applied on all the drug candidate samples to
check the applicability of the developed method for the drug substance.The peak purity of
the Nilotinib stressed samples was checked by using a Waters 2695 binary pump with an
280
auto sampler and a 2996 photodiode array detector (PDA).The mass balance (% assay + %
of impurities + % of degradation products) was calculated for all of the samples.
8.7 Method Development
Method development was initiated using the impurity-spiked samples and placebo
samples used for the preparation of pharmaceutical drug substance. Forced degradation
studies were performed to develop a stability-indicating HPLC method for the quantitative
determination and purity evaluation of Nilotinib drug substance. Stressed samples
obtained during forced degradation studies were also utilized in the optimization of HPLC
method.The method was optimized to separate all the process related impurities and major
degradation products formed under various stress conditions.It was also ensured that there
is no interference with the blank (inactive substances) used in the preparation of sample
solution.
8.7.1 Selection of wavelength:
Nilotinib and its all-potential process related impurities were prepared in diluent at a
concentration of 0.1% and injected individually.They were scanned through the photo diode
array (PDA) detector.UV spectrums of and also its potential impurities have shown
common UV maxima at around 260 nm. Hence detection at 260 nm was selected for the
initiation of method development. Further, the major degradation impurities found during
acid and basedegradation also shown the UV maxima at around 260nm.Hence 260nm
detection was confirmed for further optimization of LC method.
281
8.7.1.F1: Nilotinib Hydrochloride UV spectrum
8.7.1.F2 Impurity-1 UV spectrum
282
8.7.1.F3 Impurity-2 UV spectrum
8.7.1.F4 Impurity-3 UV spectrum
8.7.1.F5 Impurity-4 UV spectrum
283
8.7.2 Selection of Mobile phase and Column
The main target of the chromatographic method is to get the separation of Imp-1,
Imp-2, Imp-3, Imp-4,Nilotinib Hydrochloride and the degradation products generated
during stress studies from the analyte peak. As a part of method development initiation
available literature was searched and found no methods for admittance. USP monographs
and EP monograph was also pursued and found that no monographs are specified for
Nilotinib HCl.
The main objective of the chromatographic method was to separate imp-1, imp-2,
imp-3 and imp-4, Nilotinib Hydrochloride and the generated degradation products from the
analyte peak during stress studies.Impurities and degradation products are co-eluted by
using different stationary phases,such as C18 with various mobile phases and organic
modifiers, including acetonitrile and methanol,in the mobile phase. Impurities and Nilotinib
HCl solutions are prepared in diluent at a concentration of 0.01% and scanned in UV-visible
spectrometer; all the four impurities and Nilotinib Hydrochloride are having UV maxima at
around 260 nm which is selected for method development purpose. In order to separate
Nilotinib Hydrochloride and impurities,optimization started with acetonitrile–buffer (adjusted
to pH 2 using orthophosphoric acid) in proportion of 20:80 v/v on Shim-pack XR-ODSII
75mmx3.0mm, on which Nilotinib hydrochloride is retained with poor peak shape and no
separation is observed between impurities, later on by changing ratio of acetonitrile-buffer
(adjusted to pH 3 using orthophosphoric acid) in proportion of 30:70 v/v on Shimpack XR-
ODSII 75mmx3.0 mm, Nilotinib hydrochloride is retained with peak tailing, no separation
between impurities.
Further development is continued acetonitrile–buffer (adjusted to pH 3 using
orthophosphoric acid) in proportion of 20:80 v/v on Hypersil BDS C8 100 x 4.6mm, 3.0μ
Column Nilotinib Hydrochloride is tailed with asymmetry of 3.5. Different gradient programs
are investigated and satisfactory results are obtained when a gradient program with a flow
rate of the mobile phase at 0.6 ml/min and a gradient program of 0/0, 5.5/55, 8.5/45, 9.5/0
and 10.5/0 (time (min)/%B) is used. The column used with the said satisfactory conditions is
Shim-pack ODS-II 75mm x 3.0 mm, 1.8 microns column.
284
8.7.2.1 T1 Selectivity of 3 micron and 1.8 micron columns of different manufacturers
Column Compound K’ Rs As
Shim-pack XR-ODS II 75mm x 3.0 mm
Imp-1 1.498 1.126 8.665 1.00
Imp-2 1.705 1.348 2.112 1.01
Imp-3 2.636 1.639 8.405 1.12
Imp-4 2.635 1.218 --- 1.10
Nilotinib HCl 3.733 1.211 6.914 1.39
Hypersil BDS C8 100 x 4.6mm, 3.0μ
Imp-1 1.733 1.181 1.518 1.214
Imp-2 2.045 1.505 2.084 1.508
Imp-3 3.077 1.297 -- 1.513
Imp-4 3.992 1.358 5.204 1.485
Nilotinib HCl 6.103 1.156 9.927 1.469
Shim-pack ODS-II 75mm x 3.0 mm, 1.8 microns column
Imp-1 1.732 1.181 8.665 1.00
Imp-2 2.045 1.505 2.112 1.01
Imp-3 3.077 1.297 8.405 1.12
Imp-4 3.992 1.358 --- 1.10
Nilotinib HCl 4.312 1.156 6.914 1.39
8.7.3 Specimen Chromatograms-Method development
8.7.3.F1 Isocratic Pursue-Symmetry C-18 150mm Column
285
8.7.4 Effect of organic modifier
The separation of impurities i.e Imp-3 and Imp-4 and Nilotinib became critical as they
eluted very close to each other. With acetonitrile–buffer (adjusted to pH 2 using
orthophosphoric acid) in proportion of 20:80 v/v Nilotinib hydrochloride is retained with
poor peak shape and no separation is observed between impurities,later on by changing
ratio of acetonitrile-buffer (adjusted to pH 3 using orthophosphoric acid) in proportion of
30:70 v/v. Nilotinib hydrochloride is retained with peak tailing, no separation
betweenimpurities.Further developmentis continued acetonitrile–buffer (adjustedto pH 3
using orthophosphoric acid) in proportion of 20:80 v/v.
Nilotinib Hydrochloride is tailed with asymmetry of 3.5.To optimize the resolution
between the impurities and the retention time of the impurities, trails are carried out with
different mobile phase ratios using buffer and acetonitrile (buffer:acetonitrile: 70 : 30, 80 :
20, 60:40 v/v). Isocratic trials are not successful in achieving a favorable resolution
between impurities Imp-3 and Imp-4 and analyte peaks and the elution of the process
impurities. Therefore, a gradient method is selected usingbuffer and acetonitrile in a
ratio of 0/0, 5.5/55, 8.5/45, 9.5/0 and 10.5/0 (time (min) / %B) is used as mobile phase A and
water and acetonitrile in a ratio of 30 : 70 (v/v) as mobile phase B. Different gradient
programs were investigated and satisfactory results are obtained when a gradient program
of 0/10, 10/50, 20/50, 25/10, and 30/10 (time (min)/%B) is used.
8.7.4.1 Effect of buffer concentration
The effect of concentration of phosphate, sulphate and acetate buffers on the
separation was studied by varying its concentration from 0.05 M to 0.3 M. The pH of the
buffer was adjusted to 3.0 with orthophosphoric acid. The retention of impurities 3,2,1
was effected more wehre as retention of Imp 4 and Nilotinib was slightly changed with
buffer concentration. As the concentration of the buffer was kept at 0.3 M retention
times were increased for II and IV and decreased for remaining compounds.By keeping
the buffer concentration at 0.1 M resolution was increased for (Imp1,Imp3),( Imp3, Imp4)(
Imp 3, Imp 2) and decreased for (Imp2,Imp3).
286
Buffer concentration had significant effect on peak shapes and symmetry. Peaks
become Symmetrical and tailing fronting was minimized as the buffer concentration was
decreased with 0.1 M ammonium acetate buffer, good resolution and peak shapes were
obtained. So it was optimized for further studies.
8.7.4.1.T1 The effect of Buffer Concentration on retention factors, resolution and
tailing factors of Nilotinib(V) and its related substances.
Resolution (Rs) Tailing factor (As)
Impurities
Buffer
Conc. I II III IV
Niloti
nib I II III IV
Niloti
nib
0.05 0.895 6.380 --- 13.423 7.851 1.562 1.691 1.469 1.873 0.827
0.10 1.738 2.426 --- 5.361 9.872 1.068 1.224 1.232 1.342 1.443
0.20 1.176 4.978 --- 3.574 7.462 1.276 1.013 1.117 1.199 0.991
0.30 1.258 2.885 --- 2.658 6.717 1.051 1.009 1.033 1.156 1.079
8.7.4.2 Effect of buffer pH
Further studies were carried out on the effect of buffer pH on resolution and
retention. pH had little effect on retention of compounds 3,4 and III where as retentions of
remaining compounds were decreased with decreasing the pH of the buffer
(8.7.4.2.T1).Beacuse of the drastic change in the retention of compound III with pH, elution
order and resolution were effected. At pH=2.4 the resolution between the impurities and
analyte is poor, Imp-1(I) and imp-3(III) are almost co-eluted with the analyte. At pH = 4, II
was coeluted.I,II,III coeluted close to one another and V was nearer to IV at pH=5. But at
pH=3.5 all the compounds were separated with good resolution.Peak shapes were improved
on decreasing the pH and at pH=3.0 symmetrical peaks with minimum tailing were
observed. So the buffer pH of 3.0 was optimised.
287
8.7.4.2.T1. Effect of pH on tailing factors of Nilotinib and related substances.
8.7.4.3 Effect of column temperature
The column was maintained at different temperatures ranging from 25- 400C Nin a
thermostatted oven. Retentions were decreased for compounds 3,4 and
Nilotinib(8.7.4.3.T1). The tailing was reduced keeping the moderate temperatureMfor all the
compounds and at 400C it was minimum.
8.7.4.3.T1 Effect of Column temperature on tailing factors of Nilotinib and related
substances.
Retention (K1) Tailing factor (As)
Impurities
Temp0
C I II III IV NTB I II III IV NTB
25 1.95 1.52 2.64 2.61 5.67 1.84 1.73 2.08 1.92 1.81
35 2.57 3.15 3.95 4.35 7.48 1.87 1.92 2.41 2.15 2.37
40 1.74 2.46 3.18 5.14 4.62 1.08 1.12 1.21 1.24 1.34
42 2.62 3.67 4.84 4.83 6.39 1.14 1.26 1.58 1.93 1.86
8.8 Optimized chromatographic conditions.
Mobile phase :Solvent A: The mobile phase is prepared by mixing buffer and
acetonitrile in the ratio of 40:60 (v/v). Buffer is prepared by dissolving 0.77 g of ammonium
acetate dissolved in 1000 mL of water .Mobile phase was pumped at a flow rate of 0.5 mL
min-1. according to the isocratic elution program.
pH Tailing factor
I II III IV Nilotinib HCl
2.5 2.45 2.13 3.12 1.98 1.84
3.0 1.05 1.12 1.04 1.42 1.29
4.0 3.25 3.05 3.71 3.29 3.14
288
Column : Agilent make XDB-C18, 50 x 4.6 mm with
1.8 µm particles.
Flow rate : 0.6 mL min-1.
Injection volume : 4 μL
Detector : Photo diode array(PDA)
Wavelength (Max ) : 260nm
Temperature : 400C
Time in (min)/%B 0 5.5 8.5 9.5 10.5
Mobile Phase 0 55 45 0 0
In the optimized conditions Nilotinib, Imp-1, Imp-2, Imp-3 and Imp- 4 were well
separated with a resolution of greater than 2 and the typical retention times of Imp-1,
Nilotinib, Imp-2, Imp-3 and Imp-4 were about 1.18,4.162, 3.99, 1.182 and 6.91 min,
respectively.
8.8.1 Charactarisation of Impurities and Nilotinib
During the method developement the impurities and Nilotinib samples were
collected and characterized was performed. The characterization data is given in Table
8.8.1.T1.
289
8.8.1.T1 Charactarisation details of Impurities and Nilotinib HCl
Compound code
UV(nm) FT-IR (cm-1) Mass (amu)
[M+H]+
1H NMR(ppm)
Nilotinib
HCl
204.23
226.19
250.11
331.62
3400,3099,3043,2956,2941,2808,1625,1578,1532,1501,1471,1428,1399,1356,1301,1248,1219,1137,1110,1069,1037,1013,930,872 &848
446.90
9.57(s,1H,H-12),8.50 (s,IH,H-3),8.10-8.14 (dd,1H,H-6),7.76-7.80(m,2H,H-6,9),7.42-7.48(t,1H,H-5’),7.20 (s,1H,H-2),4.16-4.20 (t,2H,H-9’)9.394(s,3H,H-9),3.57-3.60(t,4H,H-2,6),2.46(m,2H,H-7’)2.39(m,4H, H-3,5’)19.6-2.04(m,2H,H-8)
Imp-1
204.59
235.90
300.46
3438,3322,3064,1626,1602,1501,
1300,1260,1214,1128,1054,1045,908,848,810
145.56
10.38(s,1H, H-10), 10.27
(s,1H,H-7) 8.72(s,1H,H-2),8.07-8.10(2d,1H,H-
6),7.87(s,1H,H-8),7.70-7.76(m,IH,H-2’),7.47-7.53(t,1H,H-5),7.24(s,1H,H-
5),4.01(s,3H,H-9)
Imp-2
204.29
224.04
249.01
334.16
3386,3078,1644,1
620,1583,1517,1501,1470,1431,1364,1288,1271,1251
,1207,1134,1071,1001,860,800,773
368.39
8.46(s,1H, H-3), 8.00-8.03
(dd,1H,H-2’) 8.78(s,1H,H-6),7.67-7.71(m,1H,H-5’),7.24-
7.30(t,1H,H-6), 7.19 (s,1H,H-6),4.33-4.37 (t,2H,H-9),4.19-4.27 (t,2H,H-2),4.0 (s,3H,H-
11),3.81-3.85(m,2H,H-6),3.50-3.61(m,4H,H-
3’,5’),3.14- 3.18 (m,2H,H7), 2.46-2.54(m,2H,H-8)
290
Compound
code
UV
(nm) FT-IR (cm-1)
Mass
(amu) [M+H]+
1H NMR(ppm)
Imp-3
203.04
240.29
332.04
3280,3072,2959,2927,2815,1682,1593,1574,1451,
1423,1336,1303,1264,1235,1198,
1129,1023,1000,961,787,770,759
306.32
12.83(s,1H,H-9),9.28(s,1H, H-2),9.07 (s, 1H,H-
7),8.69(d,1H,H-6),8.53-8.55(d,1H,H-6),8.46 (d,1H,H-4), 8.30 (s,1H,H-6)
,7.65(d,1H,H-5),7.54 (m,2H,H-2,3),7.37 (d,1H,H-
5’), 2.33 (s,3H,H-7)
Imp-4
209.19
221.99
311.48
3356,3212,3105,2931,1652,162
5,1509,1484,1407,1383,1327
241.21
7.98-7.99(m,1H,H-
2),7.24(s,1H,H-5),6.88-
6.95(m,3H,H-
2,4,6),2.24(d,3H,H-6)
8.8.2 Specimen Chromatograms-Optimized Conditions
8.8.2.F1 Blank
291
8.8.2.F2 Standard solution
8.8.2.F3 Impurity Spiked Sample
292
8.9 Forced/stress degradation
As stated in the initial discussion, forced degradation (stress studies) studies
under various degradation conditions are recommended by international conference on
harmonization (ICH) guidelines. These guidelines are designed to assist the development of
pharmaceutical drug substances and also to support the long-term stability and storage
conditions to be associated with drug samples. The analysis of these stress samples was
done using HPLC-PDA (photo diode array detector)as per the method conditions mentioned
in this section. The formed impurity during acid and base hydrolysis were identified using
LC-MS.The chromatographic conditions used for both hese studies are detailed in section
8.10.
8.9.1 Acid hydrolysis
No significant degradation was observed when Nilotinib was subjected to stress with
1.0 M HCl at 800c for 24 hours.There observed an impurity formed up to 3-4% quantitatively
at relative retention time 0.29 with respect to Nilotinib.This indicates Nilotinib is sensitive
to acid hydrolysis. An attempt was made to isolate, identify and characterize the impurity,
which is further discussed in the section 8.10. Representative chromatograms and
spectrums are added in this section. The peak purity report also presented which
explains, no co-elution is associated with Nilotinib peaks during acid stress.
8.9.1.F1 Acid-Blank
293
8.9.1.F2 Acid hydrolysis
8.9.1.F3 Acid hydrolysis-Peak purity plot
Purity Angle Purity Threshold Purity Flag Peak Purity
0.678 0.999 No Pass
294
8.9.2 Base hydrolysis
Nilotibnib also found to be sensitive towards base and was degraded up to 13-14%
when subjected to 0.1 M base at 800C temperature for 1 hour. A major impurity formed at
relative retention time 1.2 and it matches with the same impurity formed during acid
hydrolysis with respect to relative retention time and UV spectrum. The quantum of impurity
found during base hydrolysis is slight more than that formed during acid hydrolysis. Typical
chromatograms and spectrums are added in this section. Interestingly, the Nilotinib
peak split when the base hydrolysis sample is injected as is without neutralizing with
acid. This can be attributed towards the non-compatibility of diluent (base medium) with
column.Hence, the experiment was repeated and the sample was reanalyzed after
neutralizing the sample with 1.0 M acid. The peak purity report also presented which
explains, no co-elution is associated with Nilotinib peaks during base hydrolysis. Efforts
were made to identify this major degradation impurity using LC-MS, IR and NMR
techniques. This is detailed in 8.10 under the title “identification of degradation impurity”.
8.9.2.F1 Base hydrolysis
295
8.9.2.F2 Base hydrolysis-Peak purity plot
8.9.3 Oxidation
Oxidation is one of the major degradation processes employed to evaluate the
stability of the chemical entity.Nilotinib was exposed with 5% hydrogen peroxide at 800C
temperature for 8 hours.No significant degradation noticed during oxidation process with
Nilotinib.The purity report presented converse the homogeneity of the Nilotinib impurity.
Purity Angle Purity Threshold Purity Flag Peak Purity
0.549 0.928 No Pass
296
8.9.3.F1 Oxidation-degradation sample
8.9.3.F2 Oxidation hydrolysis- Peak purity plot
8.9.4 Water hydrolysis (Neutral condition)
No notable degradation was noticed when the Nilotinib drug substance and drug products
were exposed to water at room temperature for 48 hours.The drug candidate was found to
be very stable under water (neutral) hydrolysis.
Purity Angle Purity Threshold Purity Flag Peak Purity
0.349 0.905 No Pass
297
8.9.4.F1 Water hydrolysis
8.9.4.F2 Water hydrolysis-Peak purity plot
8.9.5 Photo Degradation
Photo degradation (exposing the drug candidate under light) is one of the
environments prescribed by ICH to evaluate the change associated with the chemical
entity under light. Nilotinib drug substance was stable and no prominent degradation was
observed when exposed to light for an overall illumination of 1200 K lux/Hour and an
integrated near ultraviolet energy in an integrated near ultraviolet energy of 200-watt
hours/square meter (w/mhr) (in photo stability chamber), photo stability chamber.
298
8.9.5.F1 Photo Degradation
8.9.5.F2 Thermal degradation-Test Sample
Purity Angle Purity Threshold Purity Flag Peak Purity
0.753 0.999 No Pass
299
The above analysis concludes that Nilotinib are stable towards different kinds of
stress such as heat, light, water hydrolysis and oxidation.Nilotinib found sensitive under
Acidic and basic environment.An impurity at relative retention time ~1.2 formed under those
conditions significantly in Nilotinib HCl. A detailed study of the identification,characterization
and formation of this impurity discussed in the section 8.10. Assay of all stressed samples
were calculated using qualified reference standard of Nilotinib.
Considering the purities from the respective chromatograms of stressed samples, mass
balance (%assay + % degradants + % impurities) was calculated for each stressed
sample.The mass balance of Nilotinib in all stressed samples was close to 99.0 %.
This clearly demonstrates that the developed HPLC method was found to be specific for
Nilotinib in presence of its degradation products. Peak purity test results derived from
PDA detector,confirmed that the Nilotinib peak is homogeneous and pure in all the analyzed
stress samples.
8.10 Identification of major degradation product formed in Base degradation
using LC/MS.
An attempt was made to isolate, identify and characterize the major degradation
product formed during the base degradation of the drug substance. Major degradation
impurity enhanced all through oxidation degradation at relative retention time 1.2.Nilotinib
(100 mg) dissolved in 0.1 M NaoH was subjected to degration at 800C for 1 hr. About 13-
14%of Nilotinib was degraded
Purity Angle Purity Threshold Purity Flag Peak Purity
0.827 0.999 No Pass
300
8.9.6.T1 Forced degradation-Summary
and the degradation products was identified by LC-MS, using the conditions as described in
section 8.10.1. The retention times of Nilotinib and the degradation product was at at
RRT~1.2 and 1.3 min (5.7.6.F2).The chromatographic purity of the degradation product was
tested by analytical LC and found to be 80.0%.Total degradation impurities are observed
18.0% with Major unspecified impurities-RRT at about 1.2 and 1.3
The same impurity was also enhanced during acid hydrolysis confirming the reaction
of Nilotinib degraded under basic conditions.This is confirmed with relative retention time
and spectral match analysis.The chromatographic purity of the degradation product was
tested by analytical LC method and found to be more than 98.8%.
Stress condition % of
TI Imp-1 Imp-2 Imp-3 Imp-4 % Assay
of Nilotinib
Mass balance*
As it is sample 0.10 0.02 ND ND ND 99.7
99.8
Kept the flask at 80°C for 8Hours by adding the 10 mL of 1M HCl on
shaking water bath 1.40 0.03 ND 0.70 0.20 97.2 98.6
Kept the flask at 80°C for 1Hour by adding the 10 mL of 0.1M NaoH on
shaking water bath 18.30 0.02 ND 12.5 3.1 80.5 98.8
Kept the flask at 80°C for 8 hours by adding the 10 mL of 5% H2O2 on
shaking water bath 3.50 0.01 ND 0.03 0.01 95.6 99.1
Exposed for thermal degradation at 105°C for about 5 days
0.10 0.02 ND ND ND 99.6 99.7
Exposed to Photo light both for about 1.2 Million Lux hours and
200 Watt-Hours / Sq.mts in photo stability chamber
0.15 0.02 ND ND ND 99.5 99.7
Exposed to humidity at 25°C, 90%RH for about 5 days.
0.12 0.03 ND ND ND 99.7 99.8
301
8.10.1 Optimized Analytical Conditions for LCMS
Mobile phase for Solvent A Buffer (0.1% Ammonia in water and pH adjusted to 3.0
Gradient Mode with formic acid and Solvent B is methanol.
Column : Symmetry shield , 100 X 4.6 mm,2.2 m
Flow rate : 0.8 ml/min
Wavelength (Max ) : 245nm
Injection volume : 6 μL
Temperature : 350C
Source :
Capillary [KV] : 3.50 [KV]
Cone [V] : 25.00 V
Extractor [V] : 3.0 V
RF Lens [V] : 0.3 V
Source Temp [ºC] : 120 ºC
Desolvation Temp [ºC] : 350 ºC
Cone Gas Flow [L/Hr] : 100 L/Hr
Desolvation Gas Flow [L/Hr] : 650 L/Hr
Analyzer :
LM 1 Resolution : 15.0
HM 1 Resolution : 15.0
Ion Energy 1 : 0.5
Mass range : 80-1200 m/z
Mobile phase : Water:Acetonitrile:formic acid 95:5:0.1(v/v)
Elution : Isocratic
Flow rate : 1.0 mL/min
Wavelength of detection : 254 nm
Diluent: Water : Acetonitrile (1:1, v/v)
Capillary Voltage : 3.5 (kV)
Cone Voltage : 25.0 (v)
Extractor : 2.00 (v)
Source Temperature : 120° C
302
Dissolvation Temperature : 350° C
Gas flow : 500 L/Hr
Nilotinib (100 mg) dissolved in 5% H2O2 was subjected to Oxidation degration at
800C for 1 hrs. About 18% of Nilotinib was degraded and the degradation products was
identified by LC/MS. The chromatographic purity of the degradation products was tested by
analytical LC and found to be 98.3%, indicating that the fractions were quite stable during
isolation.
8.10.2 Identification of degradation impurities.
During the degradation studies of Nilotinib sample using 0.1 M NaoH was subjected
to degration at 800C for 1 hr. The sample is collected and the analysis was carried out
using LC MS. The LC MS reporting showing the UV detector and Mass detector results
are as follows.LC-MS study was carried to determine m/z value of the major degradation
product formed under base hydrolysis.
Mass chromatogram in the positive electron spray ionization (ESI) mode for the
impurity at relative retention times of 1.2 and 1.3 was presented in figure 8.10.2.F1.The m/z
value obtained was corresponds to the molecular weights of 306.31 and 241.21. It was
further confirmed by the m/z value obtained in negative ESI mode [8.10.2.F1&F2]. The
m/z value obtained was 241.95,307.05[M-H] corresponds to the molecular weights of
306.31 and 241.21.The structures were further studied and confirmed by characterization
through FTIR, and 1H/13C NMR spectral analysis represented in below table 8.8.1.T1.
303
8.10.2.F1: Mass spectrum of impurity at RRT~1.2 [+ve mode]
8.10.2.F2: Mass spectrum of impurity at RRT~1.3 [-ve mode]
304
8.10.3 Confirmation of the degradation impurity by LC MS
The studies on the stability of Nilotinib indicated that the base degradation product
would be Impurity-3 and Impurity-4 (m/z 307 & 242) hypothetically [43]. It was observed
from the route of synthesis Nilotinib base is formed using the intermediates3-(4-Methyl- 1H-
imidazole-1-yl)-5-(trifluromethyl) benzenamine (306.32) and 4-methyl-3-((4-(Pyridin-3-yl)
pyrimidin-2-ylamino)benzoic acid (241.21). Upon the base degradation these two
compounds are formed because of the base hydrolysis.
The degradation reaction path ways are shown in 8.10.3.F1. The FT-IR spectrum of Nilotinib
exhibited characteristic stretching absorption band at 3350 cm-1, indicating the presence of
O group. This band was absent in the FT-IR pectrum of the degradation product.
The m/z value obtained for the degradation product resolving at 1.2 & 1.3
RRT in ESI positive mode was 307 (M+1) and 242 (M+1).Based on the mass number the
identified degradant is 3-(4-Methyl-1H-imidazole-1-yl)-5-(trifluron methyl) benzenamine and
4-methyl-3-((4-(Pyridin-3-yl)pyrimidin-2- ylamino)benzoic acid 8.10.3.F1).
8.10.3.F1 The degradation of Nilotinib HCl by Base hydrolysis
N
NH2N
CF3
CH3NH
CH3
N
N
NHO
O+
Nilotinib HCl
Hydrolysis
NaoHNH
CH3
N
N
N
O
N
N
CH3
F
F
F HN
Hcl
Impurity4 Impurity3
The LC-MS nalaysis confirms the structures of degradation products as N-(3-chloro-4-fluoro-
phenyl)-7-methoxy-6-(3-mor-orpholin-4-ylpropoxy) quinazoline -4-amine N oxide (IV).
305
The degraded impurities in the test sample are identified with m/z values and the same are
confirmed with pure impurities.
8.10.3.F1 Structure for impurity at RRT~1.2 i.e Impurity-3 and at RRT~1.3 i.e Impurity-3
and Impurity-4.
O
NN
HN
O N+
O-
F
Cl
3.67
2.37
3.67
2.37 2.36
1.39
1.62
2.55
3.73
7.01
6.72
4.06.32
6.45
6.66
O
NN
HN
O N+
O-
F
Cl
3.67
2.37
3.67
2.37 2.36
1.39
1.62
2.55
3.73
7.01
6.72
4.06.32
6.45
6.66
8.11 Validation of Analytical method
The UPLC method that was developed and optimized was taken up for
validation.The validation parameters viz., specificity, accuracy, precision, linearity, limit of
detection, limit of quantitation, robustness, system suitability have to be evaluated as per
the ICH guidelines are discussed.
8.11.1 System suitability
Parameters such as plate number (N), asymmetry or tailing factors (As), relative
retention time (RRT), resolution (Rs) and reproducibility (%R.S.D), retention time and area
were determined and compared against the specifications set for the method(8.11.1.T1).
The specificity [10-11] of the developed LC method for Nilotinib is determined in the
presence of its impurities namely impurity-1,impurity-2,impurity-3 and impurity-4 at a
concentration of 1.0 μg·mL-1 and degradants. For specificity determination, all the known
impurities were added to Nilotinib and the response of each analyte in the mixture was
compared with that of Nilotinib.The assay of Nilotinib for three determinitions was found to
be 99.73% with 0.025% R.S.D, while in the presence of impurities (0.5%w/w) it was 99.86%
with % R.S.D.0.04. It suggests that the assay results did not change in the presence of
impurities.
306
8.11.1.T1 System Suitability results.
8.11.2 Precision
The precision in determination of assay was studied by repeatability, intermediate
precision and reproducibility ruggedness). Repeatability is the intra- day variation in assay
obtained at different concentration levels for impurities and Nilotinib respectively, indicating a
good repeatability (8.11.2.T1). The inter-day variations calculated for five concentration
levels from the above data of 3 days, the % R.S.D.values were <2.0% (for impurities)and
1.0% (Nilotinib), indicating a good intermediate precision. The same samples were analyzed
by another instrument (RRLC system containing two pumps and a PDA detector) by a
different analyst with different lots of reagents and columns. The data obtained for six
consecutive assays are98.9%,98.6%,99.1%,98.9%,98.9%, and 99.1% were within 1.0%
R.S.D.
The precision of the related substance method was checked by injecting six
individual preparations of Nilotinib spiked with 0.15% of each Imp-1, Imp- 2 Imp-3 and Imp-
4 with respect to the analyte concentration. The %RSD of area of impurities for six
consecutive determinations is calculated and reported in table 8.12.2.T1.
Compound tR(min) (+S.D)a
USP resolution (RS )
USP tailing factor (As)
No. of theoretical plates (USP
tangent method)
Impurity-3 1.184+005 ---- 1.10 3592
Impurity-4 2.667+0.05 8.405 1.12 8325
Nilotinib
3.901+0.10
6.914 1.39
9124
Impurity-2
4.162+0.08 2.112
1.01
69168
Impurity-1
6.152+0.10 8.665 1.00 87339
307
8.11.2.T1 Precision Results-Impurities
Compound Nilotinib Drug substance (%RSD)
Imp-1 0.76%
Imp-2 0.59%
Imp-3 0.66%
Imp-4 0.74%
8.11.2.T2: Intermediate Precision Results-Impurities
8.11.3 Limit of quantification (LOQ) & detection (LOD)
Limits of detection (LOD) and quantitation (LOQ) represent the concentration of the
analyte that would yield a signal-to-noise Ratio of 3 for LOD and 10 for LOQ respectively.
LOD and LOQ were determined by measuring the magnitude of the analytical back ground
by injecting blank samples (mobile phase) and calculating the signal-to-noise ratio for each
compound by injecting a series of solutions until the S/n ratio 3 for LOD and 10 for LOQ
were obtained.The results are recorded in 8.12.3.T1.
Compound Parameter Variation Assay (%RSD)
Related substances
Imp-1 Different
System
System-1
System-2
0.63
0.25
< 1.0%
< 1.0%
Imp-2 Different
Column
Column-1
Column-2
0.32
0.27
<1.0%
< 1.0%
Imp-3 Different
Analyst
Analyst-1
Analyst-2
0.28
0.21
<1.0%
< 1.0%
Imp-4 Different
Analyst
Analyst-1
Analyst-2
0.19
0.23
<1.0%
< 1.0%
308
8.11.3.T1 Limit of detection and quantification
Compound Concentration of
LOQ Solution in % (With respect to sample Concentration)
Signal To noise ratio
Imp-1 0.002 0.005
Imp-2 0.004 0.01
Imp-3 0.01 0.02
Imp-4 0.004 0.01
Nilotinib 0.004 0.01
8.11.4 Accuracy
Standard addition and recovery experiments were conducted to determine
accuracy of the present method for the quantification of impurities in Nilotinib test samples
at LOQ level. The recovery studies for both the mpurities were carried out in triplicate
preparations at LOQ level of the analyte concentration. The percentage recovery of all the
impurities is calculated.
The recoveries of Imp-1,Imp-2,Imp-3 and Imp-4 were determined by spiking a known
amount of the impurity stock solutions are spiked to the previously analysed samples at LOQ
(100% sample + 0.03% impurities),100 (100% sample + 0.15% impurities) and 150%
(100% sample + 0.225% impuri-ties) of the analyte concentration (0.5 mg·mL-1).The
percentage recovery 97.35-101.91(8.11.4.T1).
309
8.11.4.T1 Accuracy results of Nilotinib and Impurities.
Amount Spiked Imp-1 Imp-2 Imp-3 Imp-4
LOQa 99.26 -101.29 99.00 -99.63 99.99 - 100.25 99.70 –100.45
%RSD 1.18 0.89 0.92 0.90
50%a 0.5(µg/mL)
99.85 –100.45 98.95 –99.11 99.51 -100.88 99.66–100.88
%RSD 1.07 1.08 0.94 0.82
100%a 1.50 µg/mL)
99.54-101.10 99.23-100.05 98.77– 100.56 98.11–100.58
%RSD 1.25 0.82 0.76 0.84
150%a 0.5(µg/mL)
98.11 -102.56 97.94 -102.34 99.18 -103.11 98.12 -99.99
%RSD 0.69 0.91 0.82 0.87
a: average of three determinations;R.S.D.:relative standard deviation.
8.11.5. Linearity
The linearity of Nilotinib was also studied by preparing standard solutions at
five different levels ranging from LOQ to 150%. The data is subjected to statistical analysis
using a linear-regression model; the regression equations and coefficients (r2) are given
in the below tables.The results have indicated good linearity. The limit of detection of
Nilotinib, Imp-1, Imp-2 ,Imp-3 and Imp-4 is 0.01 and 0.01% (of analyte concentration,
i.e.0.50 mg·mL-1) respectively for 8 L injection volume. The limit of quantification of
Nilotinib, Imp-1, Imp-2, Imp-3 and Imp-4 is 0.03 and 0.03% (of analyte concentration,
i.e.0.50 mg·mL-1) respectively for 8 L injection volume.The % RSD for area of Imp-1,Imp-2,
Imp-3 and Imp-4 are less than 5.0 for precision at LOQ level.
310
8.11.5.T1 Linearity results
8.11.5.T2: Linearity Results –Impurity-1
Level
Concentration (%)
Mean Area Imp-1
Mean Area Imp-2
Mean Area Imp-3
Mean Area Imp-4
Mean Area Nilotinib HCl
Level-1 0.050 11645 1356 2041 1184 1558
Level-2 0.075 115237 6532 5059 5893 7759
Level-3 0.120 185672 10596 8059 9364 12293
Level-4 0.150 231121 13620 10175 11640 15483
Level-5 0.180 275743 15965 12057 14341 18477
Level-6 0.225 349563 20186 15037 17518 23066
%RSD(Mean) 0.96 1.33 0.83 1.51 0.72
Sample
Trend Line equation
Range Regression coefficient
(r2)
Slope Intercept % Intercept
(100% Con.
response)
Residual sum of squares
Imp-1
Y = y =140280 x-113
0.01-0.15%
0.99993 2321199 −537.45 23112 6013757
Imp-2
y = 136181x - 83.01
0.01-0.15%
0.99977 134810 −83.01 1362 183514
Imp-3
y = 66793x +53
0.01-0.15%
0.99899 104175 −403.54 1018 238853
Imp-4
y = 90394 x + 89
0.01-0.15%
0.99976 117397 11.27 1164
72588
Nilotinib
HCl
y = 108677x +72
0.01-0.15%
0.99998 153653 45.46 1543 8793
311
8.11.5.T3 Residual summary(B) of Impurities and Nilotinib
8.11.5.T4 Residual summary of Impurities and Nilotinib Hydrochloride
8.1
Parameter Imp-1 Imp-2 Imp-3 Imp-4 Nilotinib
Trend line equation
y =2321199 x-537.45
y = 136181x -83.01
y = 104175x -403.54
y = 117397x + 11.27
y = 153653x+45.46
10% of 100% con. response
23112 1362 1018 1164 1543
Residual sum of squares
6013575 183514 238853 72588 8793
Residual summary of Impurity-1
Conc. (%) (with
respect to test conc)
Mean Area Response achieved
Response calculated thru
Trend line equation
Residual(Response practical -Response
theoretical)
Residual squares
0.005 11645 11069 576 332300
0.05 115237 115523 -286 81510
0.08 185672 185158 514 263713
0.10 231121 231582 -461 212936
0.12 275743 278006 -2263 5123115
0.15 349563 347642 1921 3688704
Residual summary of Impurity-2
0.005 1356 1279 77 5960
0.05 6532 6726 -194 37652
0.08 10596 10811 -215 46427
0.10 13620 13535 85 7210
0.12 15965 16259 -294 86266
0.15 20186 20344 -158 25008
Residual summary of Impurity-3
0.005 2041 1680 361 130350
0.05 5059 4805 254 64409
0.08 8059 7930 129 16523
0.10 10175 10014 161 25934
0.12 12057 12097 -40 1637
0.15 15037 15223 -186 34488
312
8.11.5.T5 Sensitivity summary Results(B) – Impurities and Nilotinib
Residual summary of Impurity-4
Conc. (%) (with
respect to test conc)
Mean Area Response achieved
Response calculated thru
Trend line equation
Residual(Response practical -
Response theoretical)
Residual squares
0.005 1184 1185 -1 2
0.05 5893 5881 12 141
0.08 9364 9403 -39 1523
0.10 11640 11751 -111 12314
0.12 14341 14099 242 58608
0.15 17518 17621 -103 10572
Residual summary of Nilotinib Hydrochloride
0.005 1558 1582 -24 576
0.05 7759 7728 31 954
0.08 12293 12338 -45 1998
0.10 15483 15411 72 5219
0.12 18477 18484 -7 47
0.15 23066 23093 -27 751
Sensitivity summary(A) of Impurities and Nilotinib HCl
Conc. (%)(with respect to test conc)
Mean area Response achieved
Imp-1 Imp-2 Imp-3 Imp-4 Nilotinib
0.030 2329000 135600 102050 118400 155800
0.075 2304740 130640 101180 117860 155180
0.120 2320900 132450 100738 117050 153663
0.150 2311210 136200 101750 116400 154830
0.180 2297858 133042 100475 119508 153975
0.225 2330420 134573 100247 116787 153773
90% of 100% con.Sen
2080089 122580 91575 104760 139347
110% of 100% con,sen
2542331 149820 111925 128040 170313
313
8.11.5.T6 Sensitivity summary Results(A)– Impurities.
Sensitivity summary(B) of Impurities and Nilotinib HCl
Conc. (%)(with respect to test conc)
Sensitivity (Response per unit concentration)
Imp-1 Imp-2 Imp-3 Imp-4 Nilotinib HCl
0.030 11645 1356 1363 1184 1558
0.075 115237 6532 5059 5893 7759
0.120 185672 10596 8059 9364 12293
0.150 231121 13620 10175 11640 15483
0.180 275743 15965 12057 14341 18477
0.225 349563 20186 15037 17518 23066
314
8.11.5.F1: Linearity Results for impurities and Nilotinib HCl
8.11.5.F2: Residual summary Plot – Impurities and Nilotinib HCl
Linearity chart for Nilotinib and Impurities
0
5000
10000
15000
20000
25000
30000
35000
0.02 0.075 0.12 0.15 0.18 0.225
Imp-1
Imp-2
Imp-3
Imp-4
Nilotinib
Y =140280.24161 x-112.74212(Imp-1)
Y = 136181x -24.5868(Imp-2)
Y = 66792.95659x +53.12613(Imp-3)
Y = 90394x + 89.05988(Imp-4)
Y = 108676.55610 x+71.89752(Nilotinib)
-3000
-2000
-1000
0
1000
2000
3000
0 1 2 3 4 5 6 7Re
sid
ual
s
Order of residuals
Residual plot for impurities and Nilotinib HCl
Impurity-1
Impurity-2
Impurity-3
Impurity-4
Nilotinib HCl
315
8.11.5.F3: Sensitivity summary Plot Impurities and Nilotinib
8.11.6 Robustness Study
All the chromatographic conditions (Flow rate, pH of the buffer and Column temperature)
were altered deliberately. The resolution between critical pair of peaks i.e.Nilotinib,
Impurity-3 and impurity-4 was calculated and found greater than 10.0,illustrating the
robustness of the developed method. The results obtained were captured in the table
8.11.6.T1.
8.11.6.T1 Robustness Results
S.No. Parameter Variation Resolution
1 Temperature [+20C] 250C
290C
7.2
8.6
2 Flow Rate [±10%] 0.4 ml/min
0.6 ml/min
7.5
7.9
3 pH [±0.1Units] 3.1
2.9
7.5
7.8
8.11.7 Solution Stability
No significant changes are observed in the content of impurity-3 and impurity-4 during
solution stability and mobile phase stability experiments. Bench-top stability of the test
0
10000
20000
30000
40000
50000
1 2 3 4 5 6
Sensitivity plot(Response for unit concentration)
Impurity-1
Impurity-2
Impurity-4
Nilotinib HCl
Impurity-3
316
solutions at room temperature was studied for two days for the drug substance of Nilotinib
under related substances by UPLC method.The same sample solutions were assayed at
every 12 hours interval up to the study period against freshly prepared standard
solution.The % RSD of assay of Nilotinib during solution stability and mobile phase
stability experiments is within 1.0%.No significant change was observed in the content of
impurities during solution stability experiments up to the study period of 48 hours. Analysis
is performed for different samples of Nilotinib (n = 3).The results are reported in table
8.11.7.T1.
8.11.7.T1 Solution Stability Results
S.No. Parameter Variation Resolution
1 Initial (0Hrs) 98.6 1.28%
2 12Hrs 99.2 1.21%
3 24Hrs 99.3 1.26%
4 36Hrs 98.9 1.19%
5 48Hrs 99.4 1.12%
8.11.8 Stability Samples Analysis
The analysis of stability samples was carried up to 6 months period using the above
stability-indicating method.The results obtained are presented in Table 8.11.8.T1.The
developed UPLC method showed acceptable performance for the quantitative evaluation
of stability samples.The results show Nilotinib is stable drug substance.
317
8.11.8.T1 stability data [Accelearated(40°C/75% RH), Long term 25°C/60% RH]
Specification
Accelerated conditions 40°C/75% RH
Long term conditions 25°C/60% RH
1st month 3rd
month 6th month 1st month
3rd month
6th month
Description Complies Complies Complies Complies Complies Complies
Impurity-1 0.03 0.04 0.04 0.03 .0.02 0.04
Impurity-2 ND ND ND ND ND ND
Impurity-3 ND ND ND ND ND ND
Impurity-4 ND ND ND ND ND ND
MSUI 0.06 0.05 0.06 0.06 0.05 0.05
Total Impurities
0.17 0.20 0.19 0.21 0.20 0.19
Assay by HPLC
100.1 .
100.0 99.8 99.9 99.9 99.9
8.12 Analysis of bulk drugs
A Ultra performance chromatographic technique was employed for detecting trace
level impurities present in bulk drugs samples of Nilotinib. Accordingly a very high
concentration (1000ug/ml) of bulk drug sample solutions were prepared as described
in experimental section (8.5) to increase the concentration of the impurities above their
detection limits.The solutions were analyzed by the developed method and the results
are recorded in table 8.12.T1.
8.12.T1 Levels of % Impurities (+S.D)a (w/w) in bulk drug of Nilotinib.
Sample I(+S.D)a II(+S.D)a III(+S.D)a IV(+S.D)a MSUI(+S.D) Total
Impurities
(+S.D)a
Bulk-1 0.02 ND ND ND 0.06 0.10
Bulk-2 0.03 ND ND ND 0.05 0.12
Bulk-3 0.02 ND ND ND 0.05 0.16
S.D.Standard deviation;a;average of three determinations;Bulk drug. ND :Not detected
318
8.13 Conclusions
In this paper, a sensitive, specific, accurate, validated and well defined stability
indicating UPLC method for the determination of Nilotinib HCl in the presence of
degradation products and its process related impurities was described.
The behavior of Nilotinib HCl under various stress conditions was studied, and the
hydrolysis (acid and base) degradants were identified by LC-MS and other spectral
analysis presented. All of the degradation products and process impurities were well
separated from the drug substance demonstrates the stability- indicating power of the
method. The information presented in this study could be very useful for regular quality
monitoring of drug substance and its dosage forms and be used to check drug quality
during stability studies. This whole study will also help in the cost effective development or
improvement in the analysis of drug substance containing Nilotinib HCl.