272

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

274

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

275

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.