77

4.0 Introduction

Gefitinib is a Epidermal growth factor receptors (EGFR) tyrosine kinases

domain were a class of anticancer drugs used for certain breast, lung and other cancers. It

is chemically designated as N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-

ylpropoxy) quinazolin-4-amine. Gefitinib is off-white to white colour powder and is soluble

in glacial acetic acid. Gefitinib melts at approximately 194°C-196°C.The empirical formula

of Gefitinib is C22H24ClFN4O3 the molecular weight of Gefitinib is 446.90.

4.0.F1 Chemical structure of Gefitinib

O

'65'

N

3'

'2

7'

'8

9'

O8

910

76

5N

3

N

NH

'23'

Cl

6'

5'

F

OCH3

1'

4'

1'

4

Name: N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy) quinazolin-4-

amine

Molecular weight: 446.90

Chemical structures, anticancer activity and adverse effects of some of the common

epidermal growth factor receptors (EGFR) have been illustrated in 4.0.T1.

78

4.0.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

Gefitinib

NN

HNON

O

O

F

Cl

For the treatment of cancer Diarrhea Skin reaction

(rash, acne), Nausea

Vomiting , Itching, Poor

appetite etc.

Crizotinib

HN

NN

N

O

Cl

ClF

NH2

For the treatment of cancer Vision problems,

constipation, and swelling

due to fluid retention.

Fainting, fever, or

breathing problems.

Erlotinib HCl

OH3C

H3CO O

O

NN

HN CCH

For the treatment of cancer Rashes occurs in the

majority of patients.

Rarely, ingrown hairs,

eyelashes, hearing loss.

Partial hair loss. Loss of

appetite.

Cetuximab HNO

S

N

O

OO

F

OH

F

OO

OH

For the treatment of cancer The incidence of acne-like

rash. fevers, chills, rigors,

urticaria, pruritis, rash,

hypotension,.

4.1 Pharmacology of Gefitinib

Gefitinib,N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)

quinazolin-4-amine (4.1.F1) is a novel, non-small cell lung cancer or (NSCLC) first

introduced by Astra Zeneca, Sweden in 1998. It was one among the quinazoline class of

chemicals which include aliphatic and aryl Amines, Phenols and derivatives, ethers Halo

benzenes, Pyrimidine and Derivatives, Heterocyclic compounds, Anisoles, Morpholines,

79

Phenyl esters and anilines. Gefitinib is similar in efficacy to Erlotinib. In the last decade, two

small molecules orally active, selective and reversible EGFR-tyrosine kinase inhibitors have

been extensively developed in NSCLC i.e. Gefitinib and Erlotinib [180-184].

The comparison of Gefitinib with the combined groups of EFGR indicated superiority

of Gefitinib [185-189]. Gefitinib is one of the most widely used anticancer drugs in the world.

Gefitinib inhibits receptor tyrosine kinases (TKs) including the epidermal growth factor

receptor (EGRF)-TK. Gefitinib also inhibits ATP-binding cassette transporter-mediated drug

efflux, which in turn strongly increases the intracellular concentrations of co-administrated

drug molecules that are transporter substrates. After oral administration, Gefitinib is widely

distributed throughout the body. Gefitinib is metabolized in the liver by cytochromes

[190-194].

80

4.1. F1 Chemical synthesis of Gefitinib in the laboratory

ON O

NH

N

O

MeO

PoCl3

Toluene

Triethyl amine

ON O

NH

N

O

MeO

Cl

F

NH2

3-Chloro-4-Fluoro Aniline

Methanol

ON O

N

NMeO

HN

ClF

Gefitinib Hydrochloride

7-Methoxy-6-3-(morpholinopropoxy)-3,4-dihydro-4-quinazolinone

4-chloro-6-(3-morpholinopropoxy)-7-methoxy quinazoline

C16H20ClN3O4

Mol.Wt.319.36

Mol.Wt.337.8

Mol.Wt.145.56

C22H25Cl2FN4O3

Mol.Wt.483.36

ON O

N

NMeO

HN

ClF

Gefitinib

C22H24ClFN4O3

Mol.Wt.446.9

Ammonia

Ethanol

Methanol

I

III

C6H5ClFN

V

HCl

4.2 Synthesis

The synthesis of Gefitinib (III) involves three stages. In the first stage 4-chloro-6-(3-

morpholinopropoxy)-7-methoxyquinazoline (I) was reacted with POCl3 in presence of

triethylamine and toluene to give 4-chloro-6-(3-morpholinopropoxy)-7-methoxy quinazoline.

Further condensation reaction of 4-chloro-6-(3-morpholinopropoxy)-7-methoxy quinazoline

with 3-chloro-4-flouro-anilne in presence of methanol gives Gefitinib hydrochloride. Gefitinib

hydrochloride on basification with ammonia, methanol gives crude Gefitinib which is finally

81

purified in presence of ethanol to give Gefitinib (III). The chemical structures of related

substances and degradation products of Gefitinib are shown in 4.2.F1.

4.2. F1 Process–related impurities and degradation products of Gefitinib

ON O

NH

N

O

MeO

1

O

OON

O

N+O

O-O

II

O

N ON

NMeO

HN

ClF

O

IV

O

N ON

NMeO

HN

ClF

III

Several methods for determination of Gefitinib in human plasma, mouse plasma and

tissues using high performance liquid chromatography coupled to tandem mass

spectrometry are reported. Sample preparation involved a single protein precipitation

step by the addition of plasma or tissue homogenate diluted in human plasma. Separation

of the compounds of interest, including the internal standard Gefitinib. One method and

estimation of Gefitinib and its impurities by RP-HPLC. Rapid and accurate reverse phase

RRLC method developed for the estimation of Gefitinib drug substance.

4.3 Availability of analytical methods

From the available literature surveyed there are no ultra performance or rapid

resolution methods for determination of process related impurities, degradation products

and estimation of bulk pharmaceutical along with its impurities of Gefitinib in the available

literature. The present study is aimed at developing a fast liquid chromatographic related

compounds method for fast, accurate quantification of Gefitinib two process related

impurities originating from the starting materials and intermediates of Gefitinib bulk drug.

Forced degradation studies of Gefitinib were carried out under thermal, photo, acidic, basic,

82

peroxide conditions. The developed RRLC method was applied for the analysis of different

laboratory batches of Gefitinib. Process impurity whose area percentage ranged from 0.05

to 0.06% was detected consistently in almost all the batches.

A comprehensive study was undertaken to found the considerable Degradant that

was found to occur in oxidative stress condition and the drug gradually undergone

degradation with time and degraded into unknown (~14.0%), in this condition the

Degradant impurity was identified as Gefitinib N-oxide impurity by spectroscopic technique

i.e. LC-MS. The stress samples were assayed with validated RRLC method against a

qualified reference standard and the mass balance was found close to 99.6 %. The positive

electro spray ionization (ESI) spectrum of the Degradant impurity showed the N-Oxide of

Gefitinib.

4.4 Objectives of the present work.

4.4.1. To optimize the rapid resolution chromatographic conditions for separation of

process related impurities of Gefitinib and to monitor the synthetic reactions by RRLC.

4.4.2 To study the forced degradation of Gefitinib under thermal, photo, acidic, basic,

peroxide conditions.

4.4.3 To study the degradation behaviour of Gefitinib using HPLC having PDA detector.

4.4.4 To identify the impurities that considerably undergone degradation with time using

LC-MS.

4.4.5 To study the suitability of the developed method for analysis of Gefitinib bulk drug.

4.5 Experimental

4.5.1 Materials and reagents

All the reagents were of analytical reagent grade unless stated otherwise. Milli-Q

water from the purification system. HPLC grade acetic acid, Sodium hydroxide, hydrochloric

acid, hydrogen peroxide and orthophosphoric acid (S.D.Fine chem. Mumbai India) were

purchased and used. Ammonium acetate was purchased from Merck, Darmstadt, Germany.

Chromatographic reagent grade acetic acid and acetonitrile was purchased from Merck,

Darmstadt, Germany were used. Samples of Gefitinib N-(3-chloro-4-fluoro-phenyl)-7

methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine(III), its process related substances

viz.,(I)4-chloro-6-(3-morpholinopropoxy)-7-methoxy quinazoline, (II) Ethyl-4-methoxy-6-nitro-

3-[3-(4-morpholinyl)propoxy] benzoate were collected during the process development work

in our laboratory and degradation product N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3

83

morpholin-4-ylpropoxy) quinazolin-4-amine N oxide (IV) was isolated and used. The Gefitinib

(reference standard) and bulk drug Samples were a kind gift from Hetero Labs Limited

Hyderabad, Andhra Pradesh India.

4.5.2 Apparatus

4.5.2.1 Ultra performance (Rapid resolution) liquid chromatograph

The analysis were performed on an Agilent 1200 series Rapid resolution LC system

(1200 RRLC system, Agilent technologies, Hachioji-shi 1-9,Tokyo Japan, equipped with

online degasser, high pressure binary pump, auto sampler with automatic temperature

controlled sample compartment, thermostatted column compartment and photodiode array

detector. For data processing and requisition, chemstation 01.03 (Agilent technologies) was

used.1200 RRLC system was performed with a normal instrument plumbing configuration

which has 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. Components installed

included 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 (13 L).

1. Agilent make XDB-C18, 50 x 4.6 mm with 1.8 µm particles

2. Agilent XDB-C8 50X4.6 mm with 1.8 µm particles

3. Waters BEH C18 50X4.6 mm with 1.8 µm particles

4. BEH C18 50X4.6 mm with 1.8 µm

4.5.2.2 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.

84

4.5.2.3 FTIR Spectrophotometer

The IR spectras necessary were recorded on a Fourier–Transform Infrared

Spectrophotometer (Perkin Elmer-Spectrum one). FTIR is equipped with Horizontal

attenuated total reflectance (HATR) accessory. The numbers of scans were 16 with 0.25

cm-1. The spectra were recorded in the solid state using an automatic KBR press pellet.

Spectrum is scanned between 400 and 4000 cm-1.

4.5.2.4 FT-NMR spectrometer

Bruker advance NMR spectrometer 300 MHz was used to record the 1HNMR

spectra. The operating conditions were, proton resonance frequency, 300 MHz, Spectral

width, 2929 Hz: Pulse width, 18 s: Data points, 8192; Spectral resolution, 0.2 Hz; probe

temperature, 270C. Chemical shifts were referenced to tetramethyl silane (TMS) at δ = 0.0

ppm.

4.5.2.5 Photo stability chamber

The Atlas Suntest CPS/CPS+ Photo stability chamber is used for photo stability

studies. Thermal stability studies are carried out in a dry hot air oven (Cintex precision hot

air oven).

4.5.2.6 Mass spectrometry

The experiments were performed using Agilent EV series liquid chromatography

system triple quadrupole mass spectrometer equipped with an Electro Spray Ion source. The

data was acquired suing the chemstation software. The typical source conditions were:

spray voltage, 5 Kv: capillary voltage, 4.5 Kv. The source and dissolvation temperatures are

250°C and 200°C, respectively.

The LC-MS system (Agilent 2010 EV series liquid chromatography system

triple quadrupole mass spectrometer) is used for the identification of unknown compounds

formed during forced degradation. A symmetry shield RP 18 100 x 4.6 mm, 3.5->m column

was used as the stationary phase. Acetonitrile is used as mobile phase for gradient

mode.0.01M ammonium formate and the pH is adjusted to 3.0 using formic acid and are

used as buffer. The flow rate was 0.6 ml/min. The injection volume is 20 >l. The analysis is

performed in positive and negative electrospray ionization modes. The capillary and cone

voltages are 4.5 kV and 5 V, respectively. The source and dissolvation temperatures are

250°C and 200°C, respectively, and the dissolvation gas flow is 1.2 min-1.

85

4.5.3 Chromatographic conditions

4.5.3.1 Analytical

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.

Column: Agilent make XDB-C18, 50 x 4.6 mm with 1.8 µm particles.

Flow rate : 0.5 mL min-1.

Injection volume : 4 μL

Detector : Photo diode array (PDA)

Wavelength (Max) : 250nm

Temperature : 400C

4.5.4 Analytical procedures

4.5.4.1 Preparation of mobile phase

About 0.01 Molar of ammonium acetate 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 6.5 with dilute ortho phosphoric acid. The resultant buffer (solvent A) was

thoroughly shaken and filtered through a PTFE membrane filter of 0.45um pore size using

a vacuum pump. Acetonitrile: Buffer (40:60 v/v) (solvent-B) was used as an organic

modifier in a gradient elution mode.

4.5.4.1 Preparation of standard solutions

Standards of Gefitinib and its process impurities (50mg) were accurately weighed,

transferred in to 50ml volumetric flasks, dissolved in acetonitrile and made up to the mark

with the diluent.

86

4.5.4.2 Preparation of sample solutions

Samples of bulk drugs of Gefitinib (1.0mg/ml) were prepared as described in section

4.5.4.2.Synthetic mixtures containing the Gefitinib and its process related substances

(10ug/ml each) were prepared by using respective stock solutions and a 10 l volume was

injected and chromatographed under the optimized chromatographic conditions. Impurities

stock solution (mixture of Gefitinib, impurity-1 & impurity-2) at a concentration of 0.1 mg mL-1

is also prepared in diluent. Working solutions of 1.5 μg mL-1 were prepared from above stock

solution for related compounds determination respectively by dissolving in diluents. The

stock solutions were adequately taken to study accuracy, precision, linearity, limits of

detection and Quantitation. The specification concentration of Gefitinib was taken as

0.1mg/ml.

4.6 Results and discussion

During the synthesis of Gefitinib, the intermediates were collected at each step of

the reactions and characterized. The data is given in 4.6.T1.

87

4.6.T1 UV, FT-IR and 1H NMR characterization of process related substances collected

during the synthesis of Gefitinib.

Compound code

Chemical structure

UV

(nm)

FT-IR (cm-1

) Mass (amu)

[M+H]+

1H NMR(ppm)

Gefitinib (III)

NN

HNON

O

O

F

Cl

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)

Impurity-1 O

N O

N

N

Cl

CH3O

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)

Impurity-2

O

O

NO

OC2H

5

NO2

CH3O

204.29

224.04

249.01

334.16

3386,3078,1644,1620,1583,1517,1501,1470,1431,1364,1288,1271,1251,1207,1134,1071,1001,860,800,773

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)

4.6.1 Method development

For development of a suitable chromatographic system, it is necessary to know the

physicochemical characteristics such as molecular weight, polarity, basicity, liophilic or

hydrophilic character of the compounds to be separated. Gefitinib and its intermediates are

polar as well as basic in nature. Adsorption or normal phase chromatography although offers

good selectivity, but rarely used for the analysis of basic compounds. The reason for this is

that the interaction between the amino groups and the surface hydroxyls on the Silica used

as the stationary phase could be so strong as to make the elution difficult. The major

88

advantages of high throughput RRLC are the increase in throughput and the reduction in the

analysis cost. The shortening of analysis time is due to the use of a shorter column length.

Therefore, a rapid resolution liquid chromatographic system was selected for analysis of

these compounds. Separation, resolution reduction of analysis time has continuously

improved in high performance liquid chromatography.

The potential of high speed analysis by rapid resolution liquid chromatography

(RRLC) on 1.8 m porous particles packed into short columns operated at high flow rate was

investigated and compared to the performance of 5m porous particles packed into

conventional columns. Using similar chemistries the case of conversion from conventional

HPLC to RRLC method was demonstrated. In order to display the practicality of RRLC

separations the gradient analysis of Gefitinib was selected. The fastest separation was

achieved by RRLC on Agilent make XDB-C18, 50 x 4.6 mm RRLC column packed 1.8 µm

particles. The analysis time was reduced by a factor 15 compared to the conventional

method.

4.6.2 Optimization of chromatographic conditions

Preliminary experiments were carried out by reverse phase HPLC to separate

Gefitinib (III) and related impurities on different commercial C18 columns. Since the

compounds i, ii and iii were basic compounds with similar polarities their separation became

critical. Initially buffers such as 0.01M ammonium acetate (PH adjusted to 4.0 with acetic

acid) and 0.01M Potassium dihydrogen ortho-Phosphate (PH adjusted to 3.0 with

orthophosphoric acid) and Acetonitrile were tried. However, the separation of compounds I, II

and III was poor and peaks exhibited a characteristic tailing on all the columns. It could be

due to the ability of these compounds to form strong hydrogen bonds with the residual

silanols of the C18 materials.

4.6.2.1 Column selectivity

Four different C18 columns as described in 4.6.2.1.T1 were used for method

development. The stationary phase was found to have a great influence on the retention

time, resolution and peak shape. The experiments showed that BEH C18, 50X4.6 mm with

1.8 µm particles were not suitable as Principal peak retention time is late and impurities 1

and 2 are not resolved properly and on Agilent XDB, C8 50X4.6 mm with 1.8 µm particles,

with the 250 mm the tailing factor is observed more than 3(broad shape).

89

The best results were obtained with, Agilent XDB C18 column with the dimensions 50X4.6

with 1.8 µm particles. It provided a good peak symmetry and selectivity. It can be seen from

the Table 3 that on XDB C18 column with the dimensions 50X4.6 with 1.8 µm all the

compounds were well separated and the tailing was minimum. So it was chosen for further

development. Rapid resolution high throughput, (RRHT) 1.8 μm LC columns allows to

increase the speed of your separations without compromising the quality of your resolution

and results. Rapid Resolution 3.5 μm particles provide 60% greater efficiency than 5 μm

particles while Rapid Resolution HT 1.8 μm particles provide 200% greater efficiency.

Shorter columns can be used to significantly reduce analysis time of small molecules.

4.6.2.2 Effect of organic modifier

The separation of Gefitinib, Impurity-1 and Impurity-2 became critical as they eluted

very close to each other. With methanol broad peaks were observed and separation was

poor. When acetonitrile was used as an organic modifier, peaks became sharp and

resolution was improved for Gefitinib (III).

4.6.2.1.T1 Selectivity of C8 and C18 columns of different manufacturers.

Column Compound k1 Rs As

Agilent XDBC8 I 1.14 1.184 - 1.23

(50X4.6 mm) II 1.35 1.17 2.35 1.24

1.8 m III 1.58 2.50 15.09 1.99

Waters BEHC18 I 0.93 1.204 - 1.26

(50X4.6 mm) II 1.12 1.161 1.40 1.22

1.8 m III 1.30 2.823 16.34 1.46

Supelco DB-5C18 I 1.28 1.227 - 1.28

(50X4.6 mm) II 1.57 1.192 3.0 1.25

1.8 m III 1.87 2.262 17.69 1.52

Agilent XDBC18 I 1.14 1.184 - 1.20

(50X4.6 mm) II 1.35 1.17 10.84 1.40

1.8 m III 1.58 2.50 6.440 1.20

Impurity-1(I) and Impurity-2(II).For further improvement, a mixture of

acetonitrile and methanol was tried. Use of a mixed organic modifier such as

Acetonitrile and ammonium acetate buffer (40:60 v/v) resulted in good separation

90

(Resolution >2.0). The initial concentration of organic modifier was kept at 33% in the

gradient program to get optimum separation of Gefitinib (III), Impurity-1(I) and Impurity-

2(II).For further improvement, a mixture of acetonitrile and buffer was tried. Use of a mixed

organic modifier such as acetonitrile: ammonium acetate buffer (40:60 v/v) resulted in good

separation (Rs >2.0). The initial concentration of organic modifier was kept at 0.01 Molar in

the gradient program to get optimum separation of Gefitinib(III), Impurity-1(I) and Impurity-

2(II).

4.6.2.3 Effect of buffer concentration

The effect of concentration of phosphate and acetate buffer on the separation was

studied by varying its concentration from 0.01% to 0.1%.the pH of the buffer was adjusted to

3.0 with diluted ortho-phosphoric acid. The XDB, C8 50X4.6 mm with 1.8 µm column 400C.

The concentration of phosphate and acetate buffer had no effect on the retention of the test

compounds. But the resolution was increased tailing was reduced with an increase in

concentration of phosphate and acetate buffer. At 0.01 Molar of ammonium acetate buffer,

sharp symmetrical peaks with good resolution were obtained. As the desired symmetry

(1.33) and resolution (>2.0) were obtained with 0.01 M of phosphate and acetate buffer, it

was used for further optimization of other variables.

4.6.2.4 Effect of buffer pH

Further studies were carried out to find out to find out the effect of buffer pH on

resolution and retention. The pH had no affect on retention of compounds I,II,III. While for I

and II. Retentions were increased as the pH of the buffer increased from 2.0 to 8.0 and at

pH 8.0, Imp-3 eluted very close to Imp-2 (4.6.2.3.F1).But, with a decrease in pH, the

resolution was increased and tailing was reduced (4.6.2.3.F2) for all the compounds. At pH

6.5, symmetrical peaks with good resolution were obtained.

91

4.6.2.3. F1 The effect of buffer concentration A) on resolution (Rs) and B) tailing

factors (As)

(A) (B)

4.6.2.3.F2. The effect of buffer concentration A) on retention factor (Rs) and B)

Resolution (Rs)

(A) (B)

0

2

4

6

8

10

12

0.0.1

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

Resolution(R

s)

Ammonium Acetate

Impurity-1

Impurity-2

Gefitinib

0.75

0.95

1.15

1.35

1.55

1.75

0.0.1

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

Tailing factor(As)

Ammonium Acetate(Molar)

Impurity-1

Impurity-2

Gefitinib

0

1

2

3

4

5

6

7

8

9

10

2.003.004.005.005.506.006.507.008.00

Re

ten

tio

n f

acto

r(K

')

pH

Impurity-1

Impurity-2

Gefitinib

0

2

4

6

8

10

12

14

2.00 3.00 4.00 5.00 5.50 6.00 6.50 7.00 8.00

Re

solu

tio

n(R

s)

pH

Impurity-2

Gefitinib

92

4.6.2.5 Effect of column temperature

The column was maintained at different temperatures ranging from 25-400C in a

thermostatted oven. Retentions were decreased for compounds I, II and Gefitinib

(4.6.2.5.F1). The tailing was reduced with increasing temperature for all the compounds

(4.6.2.5.F1), and at 400C it was minimum.

4.6.2.5. F1 Effect of temperature on A) resolution (Rs) and B) tailing factors (As) of

Gefitinib and related substances.

(A) (B)

4.6.2.6 Selection of wavelength

The UV absorption spectra of Gefitinib and its related substances were recorded

using PDA detector in the range of 190-400 nm and shown in 4.6.2.6.F1. The UV spectra of

all the compounds were similar. From 4.6.2.6.F1, it could be seen that Gefitinib has three

well defined absorption bands at 204 (E1 B and of aromatic ring), 226(K Band of aromatic

ring), 250(B Band of aromatic ring) and 331(B Band of aromatic ring) nm. All the

compounds have max at 200nm, but the UV cut of acetonitrile and buffer was 250 nm and

also a secondary max 226nm was chosen for detection and quantitation throughout the

study.

0

5

10

15

20

25

30

35

20.00 25.00 30.00 35.00 40.00

Res

olu

tio

n(R

s)

Temperature (0C)

Impurity-2

Gefitinib

0.8

1

1.2

1.4

1.6

1.8

2

20.00 25.00 30.00 35.00 40.00

Taili

ng

faco

tr(A

s)

Temperature (0C)

Gefitinib

Impurity-2

Impurity-1

93

4.6.2.6.F1 UV-Spectra of Gefitinib (III)

4.6.2.6.F2 UV-Spectra of Impurity-1(I)

94

4.6.2.6.F3 UV-Spectra of Impurity-1(II)

Finally, the separation was carried out on a XDB-C18, 50 × 4.6 mm with 1.8 μm

maintained at 400C with gradient elution using 0.01 Molar Ammonium acetate (pH=6.5) as a

buffer and acetonitrile (40:60 V/V) as a organic modifier with PDA detector set at 250nm.

Synthetic mixtures containing Gefitinib (III) and its impurities were subjected to separation

under the optimum conditions developed in the present study.4.6.2.6.F4 shows the

chromatogram of a synthetic mixture of Gefitinib(III)an its process-related impurities.

Reproducible peak shapes were obtained under the above conditions. The peaks were

identified by injecting and comparing with the retention times of the individual compounds

and the absorption spectra recorded using the online PDA detector.

95

4.6.2.6.F4 A typical chromatogram of Gefitinib (III) (100ug/ml) spiked with 10%

(w/w) of each of impurities (I and II)

4.6.2.6.F5 Specimen Chromatograms-Method development

96

4.6.3 Specimen Chromatograms-Optimized Conditions

4.6.3.F1 Blank

4.6.3.F2 Impurity 1

97

4.6.3.F3 Impurity 2

4.6.3.F4 Gefitinib

98

4.6.3.F5 Impurity Spiked Sample:

4.6.3.F6 System suitability for standard solution

99

Similarly, different batches of Gefitinib (III) were analyzed by RRLC. Two impurities

having > 0.1% area at retention times 2.978 min (0.82 RRT), 4.575 min. (0.91RRT), were

detected and their retention times and the on-line PDA absorption spectra were perfectly

matched with their standards [4.6.3.F5]. One of the impurities. Eluted at 0.91RRT (i.e.,

marked as II) did not match with any of the process intermediates. It has shown similar

absorption Spectra like Gefitinib (III) (4.6.3.F4). A comprehensive study has been undertaken

to identify the impurities by Spectroscopic techniques (4.9).

4.7. Forced degradation studies of Gefitinib

Forced degradation studies provide information on drug degradation pathways,

potential identification and structural characterization of degration products. The degradation

products often possess unwanted Pharmacological-toxicological effects by which any benefit

from the administration of drug may be outweighed. According to ICH guidelines, forced

degradation is necessary to elucidate the inherent stability of drug products. Typical

conditions for forced degradation include strong acid and base, oxidative, photo stability and

thermal conditions [226]. Stress conditions that result in approximately 20-30% degradation

of drug product are generally utilized. For some drug products severe conditions may be

required for degradation. As the stability in gastrointestinal pH range 1 7.5 is important for

drug absorption, the stability of drug solution in acidic and basic medium is assessed. In the

long run, solution-state stability is important for overall drug product stability and possible

stabilization strategy. Drug degradation in solution typically involves hydrolysis, oxidation,

racemization and photo and thermal degradation. Forced degradation studies were carried

out by stressing the Gefitinib solution (100ug/ml) under degradation study includes photolytic

(carried out as per ICH Q1B), thermal (100°C), acid hydrolysis (1M HCl), base hydrolysis (2

M NaoH) and oxidation (6% H2O2). Under UV light, thermal and alkaline conditions and in the

presence of peroxide, no change in the purity of Gefitinib was observed, Gefitinib is gradually

undergone degradation with time in 6% H2O2 upon heating for 2 h and prominent

degradation is observed as formed and well separated from Gefitinib as shown in 4.7.3.F2.

100

4.7.1 Acid hydrolysis

Acid Hydrolysis is one of the major degradation processes employed to evaluate the

stability of the chemical entity. Gefitinib drug substance was exposed with 1 M HCl at room

temperature for 6 hours. No significant degradation noticed during oxidation process with

Gefitinib.

4.7.1.F1 Acid-Blank

101

4.7.1.F2 Acid-Hydrolysis

4.7.1.F3 Acid hydrolysis-Peak purity plot

Purity Angle Purity Threshold Purity Flag Peak Purity

0.062 0.283 No Pass

102

4.7.2 Base hydrolysis

No notable degradation was noticed when the Gefitinib drug substance was

exposed to base hydrolysis at room temperature for 6 hours. Drug substance was found to

be very stable under base hydrolysis and no degradation is observed.

4.7.2.F1 Base-Blank

4.7.2.F2 Base –Hydrolysis

103

4.7.2.F3 Base hydrolysis-Peak purity plot

Purity Angle Purity Threshold Purity Flag Peak Purity

0.062 0.283 No Pass

4.7.3 Oxidation

Gefitinib is gradually undergone degradation with time in 6% H2O2 upon heating for 2

h and prominent degradation is observed as Gefitinib N-Oxide. Gefitinib is sensitive to

oxidative condition and is degraded into unknown impurities by oxidation using 6% H2O2.

Gefitinib has shown significant sensitivity towards oxidative treatment. Oxidation is one of

the major degradation processes employed to evaluate the stability of the chemical entity.

Gefitinib was exposed with 6% hydrogen peroxide at room temperature for 2 hours.

Gefitinib is sensitive to oxidative condition and is degraded into unknown impurities by

oxidation using 6% H2O2. The drug gradually undergone degradation with time and

degraded into unknown (~14.0%). The positive electro spray ionization (ESI) spectrum of

the RT~20.0 min impurity showed peaks at m/z 463.11(M+1) which indicate the N-Oxide of

Gefitinib. The chemical name of the Degradant at RT ~ 20.0 min could be N-(3-chloro-4-

fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy) quinazolin-4-amine N oxide typical

chromatograms and spectrums are added in this section.

104

The peak purity report also presented which explains, no co-elution is associated

with Gefitinib peaks during oxidation. Efforts were made to characterize this major

degradation impurity using LC-MS, IR and NMR techniques. This is detailed in the section

4.9 under the title “identification of degradation product”.

4.7.3 F1 Oxidation-Blank

4.7.3 F2 Oxidation-Test Sample

105

4.7.3.F3 Oxidation -Peak purity plot

Peak Purity Report

Purity Angle Purity Threshold Purity Flag Peak Purity

0.062 0.283 No Pass

4.7.4 Water hydrolysis (Neutral condition)

No notable degradation was noticed when the Gefitinib drug substance were

exposed to water at room temperature for 48 hours. The drug candidate was found to be

very stable under water (neutral) hydrolysis.

106

4.7.4.F1 Water hydrolysis-Blank

4.7.4.F2 Water hydrolysis-Test Sample

Purity Angle Purity Threshold Purity Flag Peak Purity

0.062 0.283 No Pass

4.7.5. 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. Gefitinib 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/m hr) (in photo stability chamber), photo stability chamber.

107

4.7.5.F1 Photo degradation-Test Sample

4.7.6 Thermal Degradation

Heat is most important component, which induce change in the drug candidate.

Generally it is one of the key environmental factors that affect the regular storage, shipping

and stability of drug candidate. Hence thermal degradation is typically prescribed by ICH to

evaluate the change associated with the chemical entity when exposed to heat. Gefitinib was

exposed to dry heat for 10 days and 48 hours respectively. No significant degradation

observed in both Gefitinib drug substance.

4.7.6.F1 Thermal degradation-Test Sample.

108

Peak Purity Report

Forced degradation studies conducted above concludes that Gefitinib is stability

indicative towards different kinds of stress such as heat, light, water hydrolysis and

oxidation. Gefitinib found sensitive under Oxidation environment. An impurity at retention

time ~20 formed under oxidation condition significantly. A detailed study of the identification

of this impurity was discussed in the section 4.9. Assay of all stressed samples were

calculated using qualified reference standard of Gefitinib. 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 Gefitinib in all

stressed samples were close to 99.0 %. This clearly demonstrates that the developed

HPLC method was found to be specific for Gefitinib in presence of its degradation products.

Peak purity test results derived from PDA detector, confirmed that the Gefitinib peak is

homogeneous and pure in all the analyzed stress samples.

Purity Angle Purity threshold Purity Flag Peak Purity

0.056 0.272 No Pass

109

4.7.6. T1: Forced degradation-Summary

Stress condition

Time

% Assay of active

substance

Total Impurities

(%)

Mass balance (%Assay+

%impurities + % Degradation

products)

Oxidation (6% H2O2 at 80°C)

2hrs 86.2 14.0 100.2

Acid hydrolysis (1M HCl at 80°

for 6 hrs )

6 hrs 99.4 0.15 99.6

Base hydrolysis (2M NaoH at 80° for 6

hrs )

6 hrs 99.5 0.14 99.6

Humidity(100%RH) 48 hrs 99.4 0.15 99.6

Thermal (100° C )

168 hrs 99.5 0.16 99.7

Light (photolytic degradation)

200 watts/hr/sq.

m 1200 KLUX

99.7 0.15 99.9

4.8 Identification and characterization of oxidation degradation impurity.

The attempt was made to isolate, identify and characterize the major degradation

product formed during the oxidation degradation of the drug substance. Major degradation

impurity enhanced all through oxidation degradation at relative retention time 0.51. The

degradant was identified using the conditions mentioned in 4.9.

4.8.1 Optimized Conditions for LCMS

4.8.1.1: LC/MS Conditions

Mobile phase for Solvent Buffer (0.1% Ammonia in water and pH adjusted to 3.0 Gradient

Mode with formic acid and Solvent B is methanol.

Column : Zorbax SB C18, 150 X 4.6 mm, 5 m

Flow rate : 0.6 ml/min

Wavelength (Max) : 245nm

Injection volume : 20 μL

Temperature : 350C

110

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

Disolvation Temp [ºC] : 350 ºC

Cone Gas Flow [L/Hr] : 100 L/Hr

Disolvation Gas Flow [L/Hr]: 650 L/Hr

LM 1 Resolution : 15.0

HM 1 Resolution : 15.0

Ion Energy 1 : 0.5

Mass range : 80-1200 m/z

Column : Waters Symmetry C-18, 250mm x 4.6mm,5Particle size

Mobile phase : Water: Acetonitrile: Acetic acid 95:5:0.1(v/v)

Elution : Gradient 0/0,5.5/55,8.5/45,9.5/0,10.5/0

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

Dissolvation Temperature : 350° C

Gas flow : 500 L/Hr

Gefitinib (100 mg) dissolved in 6% H2O2 was subjected to Oxidation degration at

250C for 2 hrs. About 30% of Gefitinib was degraded and the degradation product was

identified by LC/MS. The retention times of Gefitinib and the degradation product was at ~ 4

min (Fig. 2.7.3 F2). The isolated compounds were characterized by ESI-MS-MS, 1H NMR

and FT-IR spectroscopy.

111

4.9 Identification of the degradation product (IV)

The studies on the stability of Gefitinib indicated that the acidic degradation product

would be N-Oxide (m/z 463) hypothetically. The mass spectrum of IV showed a Protonated

molecular ion at m/z 462 and the difference of mass between Gefitinib and IV was 18 amu. It

suggests the addition of a water molecule from Gefitinib to form IV. The addition of H2O may

be possible in oxidation using H2O2 gives two possible structures for the impurity IV. The MS-

MS studies gave same fragmentation as that of Gefitinib [4.9.F1].

The fragmentation pathways are shown in 4.9.F2. The 1H NMR Spectrum of

Gefitinib had multiple peaks between 2.0-9.0 ppm with integration equals to 10 protons.

Whereas, the 1H NMR spectrum of degradation product showed three signals in the aliphatic

region between 1.4-2.2 ppm, accounts for eight protons. The FT-IR data (4.9.F1) of Gefitinib

N-Oxide exhibited characteristic stretching absorption band at 3350 cm-1, indicating the

presence of OH group. This band was absent in the FT-IR spectrum of the degradation

product. Further an additional strong C-C double bond in the degradation product. This

confirms the structure of degradation product as N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-

(3-mor-morpholin-4-ylpropoxy) Uinazolin-4-amine N-oxide (IV). The degradation pathway of

Gefitinib is shown in 4.9.F4.

4.9.F1 IR Spectrum of Gefitinib N-Oxide

112

4.9.F2 ESI-MS-MS fragmentation patterns of [M+H]+ ions of A) Gefitinib(III), m/z 447

4.9.F3 ESI-MS-MS fragmentation patterns of [M+H]+ ions of A) Gefitinib-N-Oxide, m/z

463

113

4.9.T1 Spectral data of degradation product

Compound Code

Chemical structure

UV(nm) FT-IR (CM-1

) 1H NMR(ppm)

IV

ON

N

HN

O N+

O-

FCl

C22H24ClFN4O4

462.90

203.63

226.37

249.45

3413,2933,1629,1580,1530,1501,1474,1431,1339,1253,1218, 1146,1115,1071,1053,1008,959,930&857

8.46(s,1H,H-3),8.00-8.03(dd,2H, H-2),7.78 (s, 1H,H-6),7.67-.71(m,1H,H-5),7.27-7.30(t,1H,H-),7.19 (s,1H,H-9), 4.33-4.37 (t,2H,H-2) ,4.19-4.27(t,2H,H-),4.00 (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,H-7’), 2.46-2.54(m,2H,H-8’)

4.9. F4 Degradation Pathway of Gefitinib by Oxidation hydrolysis (H2O2)

ON O

N

NMeO

HN

ClF

ON O

N

NMeO

HN

ClF

O

H2O2

(III) (IV)

114

4.9.F5 Mass Fragmentation of Gefitinib N-Oxide by Oxidation hydrolysis H2O2

ON O

N

NMeO

HN

ClF

O

+

ON O N

NMeO

HN

ClF

ON O

N

NMeO

HN

ClF

O

H2O2 Oxidation degradation

ON O

N

NMeO

HN

ClF

O

465.20(M+2)463.20(M+2)

C22H24ClFN4O4

C22H24ClFN4O4

C22H24ClFN4O4

C18H16ClFN3O2

ON

NO

HN

ClF

H3C+

ONH

OH

+

C4H10NO2

360.20(M+2) 103.98(M+2)

115

4.9.1 Identification and Confirmation of the degradation impurity by LC/MS

The impurity at relative retention time 0.70, was present at a significant level in all

the analyzed samples. The degradation impurity is significantly present in some of the

samples of Gefitinib, the elution volumes from the HPLC at that retention time were

collected using a volatile mobile phase. An attempt was made to identify this impurity by

LC-MS using those volumes. Mass spectrum was generated for this impurity in the positive

electron spray ionization (ESI) mode. The positive ion ESI-MS spectrum of (IV) showed a

Protonated molecular ion at m/z 463.11 and the difference of mass between Gefitinib and

IV was 16 amu. This indicated the addition of O2 group in IV compared to Gefitinib. Mass

fragmentation path ways are shown IV to get structural information and the mass

fragmentation path ways are shown in 4.9.1.F1. Like Gefitinib the MS-MS fragmentation

pattern of IV has same fragment ions corresponding to m/z 447.07.20, 463.11, 336.16.But

additional fragment ions m/z 272.23 and m/z 180.95 were also observed.

The fragment ions at m/z 463.11 corresponds to the loss of H2O from M+[H]+ions

(m/z 264) and m/z 44 corresponds to N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-mor-

morpholin-4-ylpropoxy)quinazolin-4-amine-N-oxide (IV). This data indicates that the IV has

O- group in addition to Gefitinib. Further, the FT-IR spectrum of degradation impurity IV

exhibited an additional O functionality in IV.

The 1H NMR spectral data of Gefitinib N-Oxide, indicating the presence of an

additional O functionality in IV. The 1H NMR spectral data of Gefitinib and IV have same

number of aromatic protons, OCH3 protons and the aliphatic protons of cyclohexane ring

and indicated of Gefitinib and is N-Oxide. From the above spectral data, the structure of IV

was confirmed N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)

quinazolin-4-amine-N-oxide. This was formed due to single Oxidation of NH group of

Gefitinib during the degradation study of Gefitinib (4.9.1.F1.)

116

4.9.1.F1 Mass spectrum for imp at RRT 2.6 [+ve mode]

117

4.9.1.F2 Proposed Structure for impurity at RRT ~ 0.70

O

N

N

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

Name: N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-mor-morpholin- 4-ylpropoxy)

quinazolin-4-amine N oxide.(C22H24ClFN404)

Molecular weight: 462.90

4.10 Method validation

Analytical method validation is a process that demonstrates the proposed

procedures are suitable for the intended purpose. More specifically, it is a matter of

establishing documented evidence that provides a high degree of assurance that the

method consistently provides accurate results to evaluate the product against defined

specifications.The RRLC 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.

4.10.1 System suitability

This is an integral part of development of a chromatographic method to verify that

the resolution and reproducibility of the system are adequate enough for the analysis to be

performed. 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 (4.10.1.T1).

These parameters were measured during the analysis of a “sample” containing the main

118

components and related substance. System suitability terms were determined and

compared with the recommended limits 1>As <2 and Rs>1.5. The specificity of the

developed LC method for Gefitinib is determined in the presence of its impurities namely

impurity-1 and impurity-2 at a concentration of 1.5 μg·mL-1 and Degradants. For specificity

determination, all the known impurities were added to Gefitinib and the response of each

analyte in the mixture was compared with that of Gefitinib. The assay of Gefitinib for three

determinations was found to be 99.91% 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.

4.10.1.T1 System suitability results

Compound tR(min) (+S.D)

USP resolution (RS )

USP tailing factor (As)

No. of theoretical plates (USP

tangent method)

Impurity-1

5.923+0.05 --- 1.2 9998

Impurity-2

2.978+0.08 10.842 1.4 12115

Gefitinib

4.575+0.12 6.440 1.2 9099

4.10.2 Precision

The precision in determination of assay was studied by repeatability, intermediate

precision and reproducibility (ruggedness).Repeatability is the intra-day variation of results

at different concentration levels for impurities and Gefitinib respectively, indicating a good

repeatability (4.10.2.T1). The inter-day variations calculated for five concentration levels

from the above data of 3 days, the % R.S.D. values were <4.02% (for impurities) and 1.0%

(Gefitinib), 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

119

consecutive assays are 9.5%, 99.4%, 99.1%, 98.9%, 99.2% and 99.4% were within 1.0%

R.S.D.

4.10.2.T1 Precision Results-Impurities in Gefitinib

S.No. Results in %a %R.S.D

Impurity-1 0.01 0.76

Impurity-2 0.02 0.91

4.10.2. T2 Intermediate Precision Results-Impurities

S.No

Parameter

Variation

Assay Related Substances

1 Different System System-1

System-2

99.97%

99.48%

< 0.10%

< 0.10%

2 Different Column Column-1

Column-2

99.91%

99.62%

< 0.10%

< 0.10%

3

Different Analyst

Analyst-1

Analyst-2

99.59%

99.82%

< 0.10%

< 0.10%

4.10.3 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 have indicated good linearity. 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. Different dilutions of

120

Gefitinib and its impurities were injected to establish the limit of detection and limit of

quantification respectively.The results are recorded in 4.10.3.T1.

4.10.3.T1 Gefitinib Limit of quantification (LOQ) & detection (LOD)

Compound Concentration of LOQ Solution in %

Signal to noise ratio

Gefitinib 0.03 19.5

Impurity-1 0.03 10.2

Impurity-2 0.03 21.8

4.10.4 Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement

between the value, which is accepted either as a conventional true value or an accepted

reference value and the value found. Standard addition and recovery experiments were

conducted to determine accuracy of the present method for the quantification of impurities

in Gefitinib test samples at LOQ level. The recovery studies for both the impurities were

carried out in triplicate preparations at LOQ level of the analyte concentration. The

percentage recovery of the impurities is calculated.

The recoveries of I and II were determined by spiking a known amount of the

impurity stock solutions are spiked to the previously analyzed samples at LOQ (100%

sample + 0.03% impurities), 100 (100% sample + 0.15% impurities) and 150% (100%

sample + 0.225% impurities) of the analyte concentration (0.5 mg·mL-1).The percentage of

recoveries for impurity-1, impurity-2 are calculated The percentage recovery of impurity-1

and impurity-2 in bulk drug samples ranged from 97.35-101.91(4.10.4.T1). A known

amount of Gefitinib impurity stock solutions spiked to the sucrose at 50%, 100% and 150%

of the analyte concentration (0.5 mg·mL-1). Each concentration level is prepared for three

times. The percentage of recoveries is calculated. The criteria for % recovery between 85

and 115 percent at each level have met. The obtained absolute recoveries were normally

distributed around the mean with standardized RSD values.

121

4.10.4. T1 Recovery data

LOQ Level Gefitinib Impurity 1 %RSD Impurity 2 %RSD

50% 0.5(µg/mL)

99.08 99.80 0.89 101.91 1.07

100% 1.0(µg/mL)

99.59 99.18 1.12 101.77 1.17

150% 1.50 (µg/mL)

99.26 101.22 0.84 97.30 0.68

4.10.5 Linearity and limits of detection and quantitation

Linearity corresponds to the ability of the analytical procedure within a given range to

obtain the response directly proportional to the concentration of the analyte in the sample

system.The linearity of detector response to different concentrations of impurities was

studied in the Range from 50% to 150% with respect to test concentration at five different

levels. Similarly, the linearity of Gefitinib was also studied by preparing standard solutions

at ten different levels ranging from 25 to 300 g/ml. The data were subjected to statistical

analysis using a linear-regression model; the regression equations and coefficients (r2) are

given in 4.10.5.T1.The results have indicated good linearity. The limit of detection of

Gefitinib, impurity-1 and impu-rity-2 is 0.01 and 0.01% (of analyte concentration, i.e.0.50

mg·mL-1) respectively for 4 L injection volume. The limit of quantification of Gefitinib,

impurity-1 and impurity-2 is 0.03 and 0.03% (of analyte concentration, i.e. 0.50 mg·mL-1)

respectively for 4 L injection volume. The % RSD for area of impurity-1 and impurity-2 are

less than 5.0 for precision at LOQ level.

122

4.10.5.T1 Linearity results

Sample

Trend Line Equation

Range Regression coefficient

(r2

)

Slope Intercept % Intercept (100% Con.response)

Residual sum of

squares

I

Y = 221.31x − 4758

0.03-0.15%

0.999 221.31 −0.4758 −1.84 0.5688

II

Y = 147.38x − 0.3698

0.03-0.15%

0.998 147.38 −0.3698 −2.22 0.556

III

Y =

307.72x − 1.0334

0.03-0.15%

0.999 307.72 −1.0334 -2.29 2.7990

4.10.5.T2 Linearity Results (A)– Impurity-1,Impurity-2 and Gefitinib

Level Conc. (%)

Meana area Impurity-1

Meana area Impurity-2

Meana area Gefitinib

Level-1 0.03 6.502 4.003 8.258

%RSD -- 2.81 2.83 4.42

Level-2 0.05 10.198 7.143 14.705

%RSD -- 0.28 1.89 2.80

Level-3 0.10 21.937 14.571 29.681

%RSD -- 2.02 1.93 1.81

Level-4 0.12 25.779 16.694 34.574

%RSD -- 0.54 2.45 1.01

Level-5 0.15 33.730 22.062 46.088

%RSD -- 2.61 3.50 2.05

a: Mean area of three replicates

123

4.10.5. T3 Linearity Results (B) – Impurity-1, Impurity-2 and Gefitinib

Parameter Impurity-1 Impurity-2 Gefitinib

Regression coefficient 0.99939 0.99870 0.99849

Y Intercept -0.47592 -0.36978 -1.03327

%Y Intercept -1.84 -2.22 -2.99

Slope(m) 221.30952 147.38224 307.71565

4.10.5. F1 Linearity plot –Impurity-1, Impurity-2 and Gefitinib

y = 221.3x - 0.475y = 147.3x - 0.369

y = 307.7x - 1.033

0

5

10

15

20

25

30

35

40

45

50

0.00 0.05 0.10 0.15 0.20

Linearity chart for Gefitinib and its impurities

Impurity-1 Impurity-2 Gefitinib

Parameter Trend line equation 10% of 100%

concentration response

Impurity-1 y = 221.3095x-0.4759 2.194

Impurity-2 y = 147.38224x-0.36978 1.457

Gefitinib y = 307.71565x-1.03327 2.968

124

4.10.5.T4 Residual Summary (A) –Impurity-1, Impurity-2 and Gefitinib

Residual summary of Impurity-1, Impurity-2 and Gefitinib

Conc. (%) (with respect to test conc)

Mean Area Response achieved

Response calculated thru Trend line equation

Residual (Response practical -Response theoretical)

Imp-1 Imp-2 GTB Imp-1 Imp-2 GTB Imp-1 Imp-2 GTB

0.03 6.626 4.003 8.258 6.1634 4.052 8.198 0.3386 0.0487 0.0598

0.05 10.210 7.143 14.705 10.5896 6.999 14.353 -0.3916 -0.1437 0.3525

0.10 21.443 14.571 29.681 21.6551 14.368 29.738 0.2819 0.2026 -

0.0573

0.12 25.850 16.694 34.574 26.0812 17.316 35.893 -0.3022 -0.6221 -

1.3186

0.15

33.081 22.062 46.088 32.7205 21.738 45.124 0.3605 0.3244 0.9639

125

4.10.5. F2 Residual summary (B) – Impurity-1, Impurity-2 and Gefitinib

4.10.5. T5 Sensitivity Summary (A) – Impurity-1, Impurity-2 and Gefitinib

0.463

-0.380

-0.212

-0.231

0.360

-0.049

0.1440.203

-0.622

0.324

0.512

-0.524-0.415

0.391

0.036

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2

Residuals plot

Impurity-1 Impurity-2 Gefitinib

Residual summary of Impurity-1,Impurity-2 and Gefitinib

Conc. (%) (with respect to test conc)

Mean area Response achieved Sensitivity (Response per unit

concentration)

Imp-1 Imp-2 Gefitinib Imp-1 Imp-2 GTB

0.03 6.502 4.003 8.258 216.733 133.433 275.267

0.05 10.198 7.143 14.705 203.960 142.860 294.100

0.10 21.937 14.571 29.681 219.370 145.710 296.810

0.12 25.779 16.694 34.574 214.825 139.117 288.117

0.15 33.081 22.062 46.088 220.540 147.080 307.253

126

4.10.5. T6 Sensitivity Summary – Impurity-1, Impurity-2 and Gefitinib

Parameter 90% of 100% concentration

sensitivity 110% of 100% concentration sensitivity

Impurity-1 197.433 241.307

Impurity-2 131.139 160.281

Gefitinib 267.129 326.491

4.10.5.F3 Sensitivity Plot –Impurity-1,Impurity-2 and Gefitinib

4.10.6 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.

Gefitinib and Impurity-2 was calculated and found greater than 10.0, illustrating the

robustness of the developed method. The results obtained are captured in the table

4.10.6.T1.

220.867 204.200 214.430 215.417 220.540

133.433 142.860 145.710 139.117 147.080

275.267294.100

296.810288.117

307.253

120

170

220

270

320

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16

Sensitivity plot (Response per uint concentration)

Impurity-1 Impurity-2 Gefitinib

127

4.10.6.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 7.5

0.6 ml/min 7.9

3 pH [±0.1Units] 6.0 7.3

7.0 7.4

4.10.7 Solution Stability

No significant changes are observed in the content of impurity-1 and impurity-2

during solution stability and mobile phase stability experiments.Bench-top stability of the

test solutions at room temperature was studied for two days for the drug substance of

Gefitinib 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 Gefitinib 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. The data obtained in both the

above experiments proves that sample solutions during assay and related substance

determination were stable up to 48 hours. Analysis is performed for different samples of

Gefitinib (n = 3). The two impurities in these samples are less than 0.1%.The results are

reported in table 4.10.7.T1.The solution stability and mobile phase stability experiments

data confirms that sample solutions and mobile phase used related sub-stance

determination are stable up to the study period of 48 h.

128

4.10.7.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%

4.10.8 Stability Samples Analysis

Three lots of Gefitinib were placed for stability study in suitable stability chambers

maintained at ICH set conditions. 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 4.10.8.T1. The developed UPLC method showed acceptable

performance for the quantitative evaluation of stability samples. The results show Gefitinib

is stable drug substance.

4.10.8.T1 Accelerated stability data [40°C/75% RH)

Parameter Specification Lot No# 001

Initial 3rd Month 6th Month

Description an off-white to white colour powder

Complies Complies Complies

IR Should match to the spectrum of working standard

Complies Complies Complies

Chromatographic purity by RRLC

Impurity-1 Not more than 0.15% Not detected Not detected Not detected

Impurity-2 Not more than 0.15%

MSUI(+S.D) Not more than 0.10% 0.06 0.07 0.08

Total Impurities (+S.D)

Not more than 1.0% 0.09 0.12 0.16

%Assay 99.0%w/w-101.0%w/w 99.56 99.37

99.18

129

4.10.8. T2 Long-Term stability data [25°C/60% RH)

Parameter Specification Lot No# 001

Initial 3rd Month 6th Month

Chromatographic purity by RRLC

Impurity-1 Not more than 0.15% Not detected Not detected Not detected

Impurity-2 Not more than 0.15% Not detected Not detected Not detected

MSUI(+S.D) Not more than 0.10% 0.06 0.05 0.06

Total Impurities (+S.D)

Not more than 1.0% 0.09 0.10 0.12

%Assay 99.0%w/w-101.0%w/w 99.56 99.37 99.42

4.10.9 Analysis of bulk drug samples

A rapid resolution chromatographic technique was employed for detecting trace level

impurities present in bulk drugs and capsule formulations of Gefitinib. Accordingly a very

high concentration (1000ug/ml) bulk drug sample solutions were prepared and analyzed by

the developed method and the results are recorded in table 4.10.9.T1.The unknown

impurities represent different synthetic route for the Gefitinib, but still they were separated

using the developed RRLC conditions.

4.10.9 T1 Levels of impurities in bulk drug samples of Gefitinib

% Impurities (+S.D)a (w/w)

Sample I(+S.D)a II(+S.D)a MSUI(+S.D)a Total Impurities (+S.D)a

Bulk-00I Not detected Not detected 0.06+0.0012 0.09+0.0025

Bulk-002 Not detected Not detected 0.04+0.0005 0.06+0.0015

Bulk-003 Not detected Not detected 0.05+0.0021 0.06+0.0018

S.D. Standard deviation; a; average of three determinations;

130

4.11 Conclusions

To monitor the synthetic process of Gefitinib, a Rapid resolution (Ultra performance)

liquid chromatographic method was developed. In this chapter a sensitive specific, accurate

validated and well-defined stability indicating RRLC method for the determination of

Gefitinib in the presence of degradation products, its process related impurities was

described. The separation of Gefitinib and its process related substances was achieved on

XDB-C18, 50 x 4.6 mm with 1.8 µm particles. The forced degradation of Gefitinib was

studied under thermal, photo, acidic, basic and peroxide conditions. One degradation

product (IV) was formed at RRT 0.70 under Oxidation conditions. The degradation product

(IV) was identified by LC/MS.

The method was validated with respect to accuracy, linearity, For Gefitinib and 0.5-

5.0l with r2> 0.9942 for impurities), limit of detection (LOD) and limit of quantitation (LOQ)

and specificity. The developed method was found to be selective, sensitive, and precise

and stability indicating. The method was applied to determine Gefitinib and its process-

related substances in Gefitinib bulk drug samples.