23  

Chapter II A

Simultaneous estimation of erlotinib (Tarceva®) and its

process-related impurities formed in 6,7-bis-(2-methoxy

ethoxy)-4-quinazolinone route: Titrimetry v/s HPLC

method  

 

 

 

 

 

 

 

24  

2.1.1 INTRODUCTION

Erlotinib·HCl (E) and gefitinib (G) are two important anticancer drugs containing

quinazoline skeleton. In nature, the quinazoline skeleton is widely found in alkaloids and

in many biologically active compounds.1 This class of compounds have known to

possess wide range of biological activities such as fungicidal (albaconazole),2

antihypertensive (prazosin, quinethazone, fenquizone),3–5,6 anti-inflammatory

(praquazone)7 and anticancer (erlotinib, gefitinib, vandetanib, raltitrexed).8–10 Some of

the biologically active quinazolines are shown below.

25  

Cancer is a disease of cells characterized by a reduction or loss of effectiveness in normal

cellular control and maturation mechanisms, which regulate multiplication. The main

features of cancer are excessive cell proliferation, loss of tissue-specific characteristics,

invasiveness and metastasis.11 The erlotinib belongs to quinazoline class of anticancer

drugs. Erlotinib hydro chloride, 6,7-bis(2-methoxyethoxy)-N-(3-ethynylphenyl)

quinazoline-4-amine hydrochloride [Tarceva®] is a drug used to treat non-small cell

lung cancer (NSCLC), pancreatic cancer and several other types of cancer. Erlotinib has

been approved by US FDA in year 2004 for the treatment of patients with NSCLC and

pancreatic cancer. Erlotinib has the molecular formula C22H23N3O4, molecular weight of

393.4 g/mol and has a pKa value of 5.42. It is very slightly soluble in water, methanol

and practically insoluble in acetone and hexane.12 It is a tyrosine kinase inhibitor, which

acts on the epidermal growth factor receptor (EGFR). The target protein (EGFR) is a

family of receptors which includes Her1 (erb-B1), Her2 (erb-B2), and Her3 (erb-B3).

EGFR is over expressed in the cells of certain types of human carcinomas, for example

in lung and breast cancers. This leads to inappropriate activation of the anti-apoptotic Ras

signaling cascade, eventually leading to uncontrolled cell proliferation.13

Erlotinib.HCl bulk drug synthesized from different synthetic routes involves different

process intermediates.8,14 These process intermediates may be present in bulk drug as

impurities. In this study we have used bulk drug made by quinazolinone route.14

26  

Erlotinib synthesized by this method may contain un-reacted starting material and

process intermediates. Any compound other than the drug will be considered as an

impurity, according to the US FDA guidelines (Department of Health and Human

Services Food and Drug Administration, Center for Drug Evaluation and Research

(CDER) November 2010 OGD. http://www.fda.gov/Drugs/GuidanceCompliance

RegulatoryInformation/Guidances/ default.htm U.S.), such impurities present at the levels

greater than 0.1% must be identified and quantified using validated analytical procedures.

The quality of erlotinib not only depends on the adopted procedure, but also on the

synthetic precursors, side reaction products, un-reacted raw materials and process-

intermediates. Since they may possess unwanted toxicological effects, thorough

monitoring of process-intermediates is of higher importance for controlling the quality of

erlotinib in the final product. Since the starting material for synthesis of erlotinib is 3,4-

dihydroxybenzaldehyde with final molecule containing quinazoline ring, HPLC with UV-

Visible detection is the technique of choice for separation and estimation of process-

related impurities. Thorough literature survey has indicated some spectrophotometric

methods and few HPLC methods with different detection techniques for determination of

erlotinib in pharmaceuticals formulation as well as in biological fluids.

Fouad Chiadmi. et al. developed and validated an isocratic HPLC method for estimation

of erlotinib in human plasma using quinine as internal standard. Separation was done

using a reversed-phase Symmetry C18 (250 x 4.6 mm, 5 µm) column, mobile phase

consisted of 0.05 M potassium dihydrogen phosphate and acetonitrile (60:40 v/v), (pH

4.8).The flow rate of the method was 1.0 mL/min and detection was done at 348 nm.

Calibration curves of erlotinib in human plasma are linear in the concentration range of

50-1,000 ng/mL. Limits of detection and quantification in plasma are 6.3 and 21 ng/mL,

respectively.15

Lepper, E. R. et al. developed HPLC assay method with UV detection of erlotinib in

human plasma. Quantitative extraction was achieved by a single-solvent extraction

involving a mixture of acetonitrile and n-butyl chloride. Erlotinib was separated on Nova-

Pak C18 column and the mobile phase composed of acetonitrile and water (60:40, v/v),

pH 2.0. The column effluent was monitored with UV detection at 348 nm. The

27  

calibration graph was linear in the range of 100-4500 ng/mL, the limit of detection and

limit of quantification 10 ng/mL and 30 ng/mL respectively.16

Pan, J. et al. developed a bioanalytical HPLC method for the quantitative determination

of erlotinib in human plasma. The quantitative extraction was done using supported

liquid extraction (SLE) sample cleanup technique using erlotinib-d6 as internal standard.

Erlotinib was separated on a hydrophilic interaction liquid chromatography (HILIAC)

column and mobile phase composed of 0.1% formic acid in water-acetonitrile (15:85 v/v)

with flow rate of 1.0 mL/min. The column effluent was monitored using tandem mass

spectrometer (MS/MS). The calibration curve was linear over the range of 2 - 2,000

ng/mL, with a linear correlation coefficient > 0.999 and the limit of detection and limit of

quantification 0.7 ng/mL and 2 ng/mL respectively .17

Zho, M. et al. developed and validated HPLC methods for determination of OSI-774

(Erlotinib) and its metabolite, OSI-420, in human plasma. Sample pretreatment involved

a single protein precipitation step with acetonitrile. The analytes were separated on

Waters X-Terra C18 (50 x 2.1 mm, 3.5 μm) analytical column and eluted with

acetonitrile-water mobile phase (70:30, v/v) containing 0.1% formic acid. The analytes of

interest were monitored by tandem mass spectrometry with electron spray positive

ionization.18

Pujeri. et al. developed stability-indicating HPLC method for the assay of erlotinib in

presence of degradation products. Erlotinib and its forced degradation compounds were

eluted on C18 column using 0.01 M ammonium formate - acetonitrile containing formic

acid with flow rate of 1.0 mL/min. The linearity range and values for limits of detection

and quantification were found to be 1-198, 0.33, and 1.1 µg /mL, respectively.19

Literature survey indicated that not a single titrimetry method was reported for assay of

erlotinib. Also few HPLC methods and spectrophotometric methods were reported for

estimation of erlotinib in bulk drug, formulation and biological fluids. But none of the

methods explains simultaneous estimation of erlotinib and its process-related impurities.

28  

2.1.2 IMPORTANCE OF PRESENT WORK

We have developed simple, fast and cost effective non-aqueous titrimetric methods using

visual titration and potentiometric titration for the first time for estimation of erlotinib.

The identification and quantification of process-related impurities is of greater

importance, since they may have some toxicological effects.20 The impurities present in

erlotinib bulk drug were isolated using preparative HPLC and identified by using

spectroscopic techniques. The available literature doesn’t contain HPLC method for

simultaneous estimation of erlotinib and its process-related impurities. In this present

study, we have also developed RP-HPLC methods for separation and estimation of

erlotinib and process-related impurities formed during synthesis of erlotinib by

quinazolinone route. The developed methods were validated for specificity, linearity,

precision, limit of detection, limit of quantification and ruggedness as per ICH

guidelines.21

2.1.3 RESULTS AND DISCUSSION

2.1.3.1 Titrimetric method for estimation of erlotinib.HCl (E)

The reaction between erlotinib in non-aqueous medium and acetic acid is an acid-base

reaction, where the strong acid can donate a proton to nitrogen of the amino group of the

drug molecule.

In the presence of perchloric acid, acetic acid will accept a proton and forms

CH3COOH2+. It can be readily give up its proton to react with a base, hence the basic

property of a base is enhanced, which makes the titration between weak base(E) and

perchloric acid accurately carried out using acetic acid as solvent.

Since, E is a hydrochloride, which is too weakly basic to react quantitatively with acetous

perchloric acid. Addition of mercuric acetate to a halide salt replaces the halide ion by an

equivalent quantity of acetate ion, which is a strong base in acetic acid. (Figure 1)

29  

CH3COOH + HClO 4 CH3COOH 2+ + ClO 4

-

(CH 3COO) 2Hg +

O

O

H3CO

H3CO

N

N

NH

CH

.HCl 2CH 3COOH + HgCl 2

+

O

O

H3CO

H3CO

N

N

NH

CH

O

O

H3CO

H3CO

N

N

NH

CH

O

O

H3CO

H3CO

N

N

NH2+

CH

CH3COOH 2+ + CH3COOH +

Figure 1 Probable scheme of acid-base reaction

The enhanced basicity of E in acetic acid medium is due to non-levelling effect of acetic

acid and the determination of E is very easier. The analysis procedures involve the

titration of E with perchloric acid with visual and potentiometric end point detection.

Crystal violet gave satisfactory end point for the concentrations of analyte and titrant

employed. A steep rise in the potential was observed at the equivalence point with

potentiometric end point detection (figure 2). With both methods of equivalence point

detection and by Gran plot (figure 3), a reaction stoichiometry of 1:1 (drug : titrant) was

obtained which served as the basis for calculation. From this it is implied that the reaction

between E and perchloric acid proceeds stoichiometrically in the ratio 1:1 in the range

studied.

Method optimization

In both the methods, the optimum amount of mercuric acetate required to precipitate the

chloride ion was studied by varying its amount and keeping the drug amount constant

followed by the measurement of the stoichometric amount of drug found in each case. It

was found that, 2 mL of 1% mercuric acetate (20 mg) was sufficient for complete

replacement of chloride in drug (10-100 mg) by mercuric acetate and the same amount

30  

was fixed throughout the investigation. A contact time of 2 min was essential after the

addition of mercuric acetate before proceeding for titration.

Figure 2 Potentiometric titration curves of 50 mg E Vs 0.01 N HClO4

Figure 3 Gran plot

2.1.3.1.1 Estimation of erlotinib.HCl by visual titration method

The non-aqueous titration method by visual titration using crystal violet as indicator was

developed and optimized. The method was validated for linearity, accuracy, precision and

recovery using ICH guidelines.

0

50

100

150

200

250

0 2 4 6 8 10 12 14 16 18

de/dv (V/m

l)

Volume of HClO4 (ml)

0

5000

10000

15000

20000

0 5 10 15 20

V.10

‐pH

Volume of HClO4 (ml)

Gran plot

300

350

400

450

500

550

600

0 5 10 15 20

emf (mV)

Volume of HClO4 (ml)

31  

Linearity

Linearity of the method was checked at seven concentration levels ranging from 10 -100

mg using E standard solution by titration with 0.01 N perchloric acid (Table 1). The

correlation graph was plotted by taking volume of perchloric acid consumed against

quantity of E taken. The correlation coefficient (R2) was 0.999 (figure 4) indicated that

there was good correlation between concentration of E and volume of perchloric acid

consumed.

Table 1 Linearity data

Figure 4 Correlation graph

Accuracy and precision

Accuracy and precision of the method was checked by analyzing the E standard solution

at three concentration levels (10, 20 and 50 mg). The intra-day and inter-day, accuracy

and precision were checked by analyzing samples on two days. The relative error (RE)

was calculated at each level and the intra-day and inter-day RE values were < 1.5%

indicating the good accuracy of the method. The relative standard deviations (RSD) was

estimated at three concentration levels on two days and the RSD values were ≤1.5%

indicating the good precision of the method(Table 2).

RE, %Amount E taken Amount E found x100

Amount E taken

RSD, %SD x 100

Average value

y = 0.216x ‐ 0.022R² = 0.999

0

5

10

15

20

25

0 50 100 150

Volum

e of HClO

4consum

edWeight of E (mg) 

Sl.no Weight of E (mg) Volume of HClO4 Consumed (mL)

1 10 2.25 

2 20 4.3 

3 30 6.45 

4 40 8.5 

5 50 10.8 

6 70 15.15 

7 100 21.7   

32  

Table 2 Accuracy and precision data

a average of five determinations b average of eight determinations

Recovery

Recovery of the method was checked by spiking the erlotinib bulk drug with standard E

at three concentration levels (10, 20, & 30 mg).The study was done in triplicate and the

recovery values at each level falls between 99.37 – 100.04% indicating the good recovery

of the method. (Table 3)

Recovery, %Amount of E taken Amount of E found x 100

Amount of E taken

Table 3 Recovery dataa

Amount of E from bulk drug (mg)

Amount of E standard added(mg)

Total E, (mg)

Total amount of E found (mg)

Recovery,%

20.3 10 30.3 30.15 99.50

20.2 20 40.2 40.22 100.04

19.9 30 49.9 49.59 99.37 a average of three determinations

Application

The developed visual titration method was used for estimation of assay of erlotinib bulk

drug. Six bulk drug samples analysed in triplicate and assay values were in the range of

99.39- 99.68% and RSD values were <1.0%. (Table 4)

Amount of E taken(mg)

Intra-daya Inter-dayb Amount of E found(mg)

RE, % RSD, % Amount of E found(mg)

RE, % RSD, %

10 9.98 0.19 1.29 9.93 0.72 1.22 20 20.10 0.51 0.98 20.09 0.45 0.82 50 50.19 0.37 0.39 50.11 0.22 0.39

33  

Table 4 Sample analysis dataa

Sample name Assay, %w/w %,RSD

Erl-1 99.51 0.73 Erl-2 99.39 0.55 Erl-3 99.50 0.24 Erl-4 99.59 0.22 Erl-5 99.68 0.54 Erl-6 99.57 0.24 a average of three determinations

2.1.3.1.2 Estimation of erlotinib.HCl by potentiometric titration method

The nonaqueous titration method by potentiometric end point detection was developed

and optimized. The method was validated for linearity, accuracy, precision and recovery

using ICH guidelines.

Linearity

Linearity of the method was checked at seven concentration levels ranging from 10-100

mg using E standard solution by titration with 0.01 N perchloric acid. The correlation

graph was plotted by taking volume of perchloric acid consumed against quantity of E

taken. The correlation coefficient value 0.999 (figure 5) indicated that there was good

correlation between concentration of E and volume of perchloric acid consumed.

( Table 5)

Table 5 Linearity data

Figure 5 Correlation graph

y = 0.217x ‐ 0.061R² = 0.999

0

5

10

15

20

25

0 50 100 150

Volum

e of HClO

4

consum

ed

Weight of E  (mg) 

Sl.No Weight of E ( mg)

Volume of HClO4 Consumed (mL)

1 10 2.2 2 20 4.3 3 30 6.4 4 40 8.4 5 50 10.8 6 70 15.2 7 100 21.65

34  

Accuracy and precision

Accuracy and precision of the method was checked by analyzing the E standard solution

at three concentration levels (10, 20 & 50 mg). The intra-day and inter-day, accuracy and

precision were checked by analyzing samples on two days. The relative error (RE) was

calculated at each level and the intra-day and inter-day RE values were <1.0% indicating

the good accuracy of the method. The relative standard deviations (RSD) was estimated

at three concentration levels on two days and the RSD values were ≤2.0% indicating the

good precision of the method.(Table 6)

Table 6 Accuracy and precision data

a average of five determinations, b average of eight determinations

Recovery studies

Recovery of the potentiometric method was checked by spiking the bulk drug with

standard E at three concentration levels (10, 20, & 30 mg).The study was done in

triplicate and the recovery values at each level falls between 99.14 – 100.15% indicating

the good recovery of the method. (Table 7)

Table 7 Recovery dataa

Amount of E from bulk drug taken, mg

Amount of E standard added, mg

Total E, mg

Total amount of E found, mg

Recovery,%

20.1 10 30.1 30.07 99.90

20.0 20 40.0 40.06 100.15

20.1 30 50.1 49.67 99.14 a average of three determinations

Amount taken, mg

Intra-daya Inter-dayb Amount

found, mg RE, % RSD, % Amount

found, mg RE, % RSD, %

10 10.07 0.75 1.64 10.02 0.16 1.65

20 20.01 0.04 0.64 20.03 0.16 0.63

50 50.09 0.18 0.39 50.11 0.22 0.39

35  

Application

Accurately weighed 50 mg of erlotinib bulk drug into a dry 100 beaker and dissolved in

25 mL of glacial acetic acid. About 2 mL of mercuric acid was added and stirred for 2

min and titrated with 0.01 N HClO4 potentiometrically and assay of erlotinib was

calculated. Six bulk drug samples analysed in triplicate and assay values were in the

range of 99.27- 99.75% and RSD values were < 1.0%.( Table 8)

Table 8 Sample analysis dataa

a average of three determinations

2.1.3.2 Isolation and identification impurities and development HPLC method for

simultaneous estimation of erlotinib and its process related impurities

2.1.3.2.1 Isolation and characterization of impurities

The impurities present in bulk drug (figure 6) were isolated using preparative HPLC and

purified by extraction and concentration. All compounds were identified by recording

mass spectra, IR spectra, UV spectra, melting points and comparing with the standard

samples (figure 7).

Sample name Assay, % w/w %,RSD

Erl-1 99.73 0.81

Erl-2 99.35 0.46

Erl-3 99.27 0.38

Erl-4 99.65 0.49

Erl-5 99.63 0.31

Erl-6 99.75 0.51

36  

Figure 6 HPLC chromatogram of erlotinib.HCl bulk drug made by quinazolinone route

OH

OH

CHO O

O

CHOH 3CO

H 3CO

O

O

CNH 3CO

H 3CO

O

O

CNH 3CO

H 3CO NO 2

O

O

H 3CO

H 3CO

NH

N

O

E 0 E 1

E 2

E 3

E 4

O

O

H 3CO

H 3CO

N

N

NH

CH

.HCl

E (Erlotinib HCl) Figure 7 Structure of erlotinib and impurities

Impurity E0

The peak eluted at 4.5 min yielded off-white powder. The GC-MS analysis exhibited

molecular ion peak at m/z 138(M+). The IR spectrum showed strong band at 1643 cm-1

indicating the presence of carbonyl group and bands at 2850 and 3350 cm-1 indicated the

presence of phenolic hydroxyl groups. Further, the IR and UV-spectra and melting point

of isolated compound matched with the standard 3,4-dihydroxy benzaldehyde (E0).

37  

Impurity E1

The peak eluted at 7.0 min yielded brown colored liquid. The GC-MS analysis exhibited

molecular ion peak at m/z 254(M+). The IR spectrum showed strong band at 1690

indicating presence of carbonyl group. Further, the IR and UV-spectra of isolated

compound matched with the standard 3,4-bis(2-methoxyethoxy)benzaldehyde (E1).

Impurity E2

The peak eluted at 9.3 min yielded brown colored liquid. The GC-MS analysis exhibited

molecular ion peak at m/z 251(M+). The IR spectrum showed band at 2250 cm-1 indicated

the presence of cyano group (-CN).Further, the IR and UV-spectra of isolated compound

matched with the standard 3,4-bis(2-methoxyethoxy)benzonitrile (E2).

Impurity E3

The peak eluted at 12.5 min yielded yellow colored powder. The GC-MS analysis

exhibited molecular ion peak at m/z 296(M+). The IR spectrum, showed strong band at

1576 cm-1 indicated the presence of nitro group and band at 2258 cm-1 indicated cyano

group. Further, the IR and UV-spectra and melting point of isolated compound matched

with the standard 4,5-bis(2-methoxyethoxy)-2-nitrobenzonitrile (E3).

Impurity E4

The peak eluted at 3.1 min in HPLC chromatogram yielded yellowish brown colored

powder. The GC-MS analysis exhibited molecular ion peak at m/z 294(M+). The IR

spectra showed strong band at 1656 cm-1 indicated the presence of carbonyl group.

Further, the IR and UV-spectra and melting point of isolated compound matched with the

standard 6,7-bis (2-methoxyethoxy)quinazoline-4(3H)-one (E4).

38  

2.1.3.2.2 Development HPLC method for simultaneous estimation of erlotinib and

its process related impurities

2.1.3.2.2.1 Optimization of chromatographic conditions

All the impurities and erlotinib (figure 6) were subjected to separation by reverse-phase

HPLC using different columns and mobile phase. The separation and peak shapes were

good on Inertsil ODS-3V (250 x 4.6 mm, 5 µm) column using 1% ammonium acetate and

acetonitrile (55:45, v/v). A typical chromatogram of erlotinib spiked with 25 ppm of each

impurity was shown in figure 8. It is evident from figure 8 that all the compounds were

eluted and well separated with good peak shapes and resolutions. UV at 254 nm was

chosen for detection and quantification, since erlotinib and its impurities have good

absorption at that wavelength. The peaks were identified by injecting and comparing

retention times of individual compounds and studying absorption spectra using PDA

detector. The developed method was validated with respect to accuracy, precision,

linearity and robustness.

Figure 8 Chromatogram of five impurities spiked to erlotinib

39  

2.1.3.2.2.2 Method validation

Specificity

To demonstrate specificity of the method, erlotinib bulk drug was spiked with known

amount of impurities and chromatographed. All the impurities were well separated from

erlotinib, the chromatographic peak purity and homogeneity was evaluated with PDA

detector. The peaks with flat-top indicated that erlotinib has homogeneous peak with no

impurities embedded in it. Also specificity was checked by stressing pure erlotinib under

UV light at 254nm, and under extreme conditions such as 0.1N NaOH, 0.1N HCl and 3%

H2O2 at 40°C for 24 hours. Under UV and acidic condition there was no change in

purity, but in alkaline and oxidative conditions, the degradation products were formed,

but they are well separated from erlotinib and the process impurities, indicating that the

method is specific for the separation and estimation of erlotinib and its process

impurities(figure 9).

Figure 9 Chromatogram of forced degradation study

System suitability

The system suitability was conducted by making five replicate injections of the erlotinib

(100 µg/mL) solution spiked with 1% of each impurity. System suitability parameters

retention time (tR), theoretical plate (TP) and tailing factors (TF) were evaluated and

values are recorded in Table 9. The % RSD values calculated for retention time was in

the range 0.06 – 0.19, TF was in the range of 0.97 – 1.78 and theoretical plates in the

40  

range 0.30 – 1.92 respectively. It is evident from results that, the parameters evaluated

were within the acceptable range (RSD < 2%), indicating, the system was suitable for

estimation of erlotinib and its process-related impurities.

Table 9 System suitability dataa

Compound name Abbrevi

ation

tR/ min Tailing

factor

Theoretical plate

3,4-Dihydroxy benzaldehyde E0 4.47 ±0.14 1.16 ± 1.19 8218 ± 1.47

3,4-Di-(methoxyethoxy)

benzaldehyde

E1 6.75 ± 0.09 1.26 ± 1.78 22246 ± 1.85

3,4- Di-(methoxyethoxy)

benzonitrile

E2 8.94 ± 0.06 1.2 ± 1.38 14376 ± 0.79

4,5- Di-(methoxyethoxy)-2-

nitro benzonitrile

E3 11.90 ± 0.09 1.08 ± 1.09 14980 ± 0.66

6,7-bis-(2-methoxyethoxy)

quinazolin -4-(3H)-one

E4 2.99 ± 0.19 1.13 ± 1.13 5301 ± 1.92

N-(3-ethynylphenyl)-6,7-bis(2-

methoxyethoxy)-4-quinazolin

amine (Erlotinib)

E 15.09 ± 0.07 1.03 ± 0.97 14038 ± 0.30

a average of five determination ± RSD,%

Linearity

Linearity of detector response to different concentrations of impurities was studied by

analyzing erlotinib spiked with each impurity at eight levels ranging from 0.1-2.0 µg/mL;

similarly, the linearity of erlotinib was studied by preparing standard solutions at seven

different levels ranging from 25-500 µg/mL (Table 10). The data’s were subjected to

statistical analysis using linear-regression model and the correlation coefficients of the

impurities and erlotinib were >0.995, indicating the good linearity between the detector

response and the concentration in all compounds.(Figure 10 & 11)

41  

Table 10 Linearity data

Conc. µg/mL

Area Conc. µg/mL

Area

E0 E1 E2 E3 E4

E

0.1 4982 1522 9350 14676 8177 25 2448349

0.2 10901 2888 16158 30712 14251 50 4879988

0.3 16174 4826 24109 45155 21433 75 7356985

0.5 20711 5044 32571 58838 27552 100 9506751

0.8 33137 8071 52113 94140 44083 150 14289220

1 50513 14336 74857 142584 64451 200 19308948

1.5 77877 19668 112669 215843 96069 250 24258460

2 97117 25967 147384 274586 124504 300 29620660

Figure 10 Correlation graphs of erlotinib and impurities

42  

Figure 11 HPLC chromatograms of impurities (0.1 – 2.0 µg/mL)

Accuracy

The accuracy of the method for impurities was checked by spiking each impurity at four

different concentration levels ranging from 0.1-2.0 µg /mL (0.1, 0.5 1.0 & 2.0 µg /mL) to

the erlotinib at specified level (100 µg /mL). All estimation were done in triplicate (n=3),

recovery and RSD for all five impurities were found to be 92.86 – 106.23% and 0.39 –

2.83% respectively. The accuracy for determination of assay of erlotinib was checked at

four different levels: i.e. 50, 100,200 and 300 µg /mL each in triplicate. The recovery and

RSD of erlotinib were found to be 98.48-99.57% and 0.20-0.57% respectively. The

percentage recoveries and RSD values are recorded in table 11.

43  

Table 11 Accuracy dataa

Compound Spiked quantity, µg /mL

Found quantity ± SD, µg /mL

Recovery (%)

%,RSD

E0 0.1 0.102 ± 0.002 102.42 2.83

0.5 0.491± 0.004 97.89 0.99 1 0.940 ± 0.006 94.05 0.63 2 1.986 ± 0.010 99.30 0.65

E1

0.1

0.106 ± 0.001

106.23

1.14 0.5 0.498 ± 0.002 99.57 0.6 1 0.974 ± 0.005 97.42 0.55 2 1.997 ± 0.009 99.85 0.53

E2

0.1

0.099 ± 0.001

99.37

1.63 0.5 0.499 ± 0.004 99.80 1.01 1 0.990 ± 0.007 99.08 1.21 2 1.986 ± 0.013 98.80 0.96

E3

0.1

0.093 ± 0.002

92.86

1.95 0.5 0.478 ± 0.002 95.60 0.4 1 0.960 ± 0.006 96.09 0.59 2 1.956 ± 0.013 97.80 0.49

E4

0.1

0.099 ± 0.002

99.79

1.64 0.5 0.503 ± 0.002 100.60 0.39 1 1.000 ± 0.007 100.04 0.74 2 1.996 ± 0.010 99.80 0.56

E

25

24.62 ± 0.27

98.48

0.51 100 99.04 ± 0.27 99.04 0.27 200 199.81 ± 0.40 99.90 0.20 300 298.55 ± 0.59 99.51 0.25

a n=3, average of three determination. RSD: relative standard deviation SD: standard deviation

Precision

The precision of the method was estimated by repeatability and intermediate precision.

Repeatability is the intra day variation obtained at three different concentration levels and

is expressed in terms of RSD, % calculated for each day. The RSD values for all impurity

were in the range of 0.49-2.83% indicating good repeatability. The intermediate precision

is the inter-day variation at the same concentration levels determined on three successive

44  

days. The inter-day variations calculated for three concentration levels were expressed in

terms of RSD, %. At each concentration level, the RSD, % values for all impurity were in

the range 0.53-2.92%, thus indicating a good intermediate precision.Similarly the

repeatability of the method for assay of erlotinib, was expressed interms of RSD values

was in the range of 0.20-0.51% and intermediate precision was in the range of 0.23-

0.53% respectively. Precision values for impurities was <3% and for assay of erlotinib

was <1%, indicates that method has got good presion for analysis of impurities and bulk

drug.Results are recorded in table 12.

Table 12 Precision data

Compound Spiked quantity,

µg /mL Intraday precision,

% RSD (n=3) Interday precision,

% RSD (n=9) E0 0.1 2.83 2.92

1 0.63 0.85 2 0.65 0.96

E1

0.1

1.14 1.25

1 0.55 0.85 2 0.53 0.76

E2

0.1

1.63 1.70

1 1.21 1.06 2 0.96 0.97

E3

0.1

1.95 2.05

1 0.59 0.56 2 0.49 0.53

E4

0.1

1.64 1.85

1 0.74 0.81 2 0.56 0.60

E

25

0.51 0.53

100 0.27 0.25 200 0.20 0.23

300 0.25 0.27

Limit of detection and quantification

Limit of detection (LOD) and quantification (LOQ) represent concentrations of the

analytes that would yield signal-to-noise ratio of 3 for LOD and 10 for LOQ,

45  

respectively. LOD and LOQ were determined by measuring the magnitude of analytical

background by injecting blank samples and calculating the signal-to-noise ratio for each

compound by injecting series of solutions until the S/N ratio is 3 for LOD and 10 for

LOQ. LOD and LOQ of erlotinib and impurities in the range of 0.016 - 0.029 µg /mL and

0.045-0.095 µg /mL respectively.(Table 13)

Table 13 LOD and LOQ data

Compound E0 E1 E2 E3 E4 E

LOD, µg /mL 0.016 0.025 0.015 0.022 0.025 0.029

LOQ, µg /mL 0.047 0.076 0.045 0.065 0.076 0.095

Robustness

Small but deliberate variations in the HPLC parameters were made to verify the

robustness of the analytical method. Robustness was studied by varying ±0.2 mL of flow

rate, ±2mL of acetonitrile composition in mobile phase and ±2°C in column temperature

to the actual method parameters. In all the above variations, test samples were injected in

triplicate and system suitability parameters were evaluated. The results were recorded in

table 14. There was slight change in relative retention time (RRT) and asymmetry (Asy)

of erlotinib and its impurities on changing mobile phase concentration, but all peaks are

well separated without affecting accuracy of quantitative estimation of erlotinib and

impurities. There was no significant change in RRT and Asy by changing the flow rate

and temperature. The result indicates that method is suitable for separation and estimation

of erlotinib and its synthetic impurities.

Stability of the solutions

Erlotinib and impurities solutions kept in room temperature and under refrigeration were

analyzed after 1, 3, and 5 days. The RSD of assay values of erlotinib and all procecess

related impurities were less than 2% during five days of the solution stability

experiments. The results from these experiments confirm that sample solutions used

during analysis were stable up to the study period of five days.

46  

Table 14 Robustness data

Parameters E0 E1 E2 E3 E4 E RRT Asy RRT Asy RRT Asy RRT Asy RRT Asy RRT Asy

Mobile phase composition (acetonitrile %) 43 0.276 1.23 0.423 1.03 0.567 0.97 0.764 1.06 0.185 0.97 1 1.05 45 0.298 1.42 0.448 1.35 0.592 1.23 0.785 1.17 0.202 1.05 1 1.12 47 0.302 1.25 0.450 1.19 0.589 1.10 0.773 1.10 0.208 1.01 1 1.03 Flow rate (mL/min) 0.8 0.301 1.33 0.449 1.19 0.596 1.09 0.791 1.15 0.203 0.93 1 1.08 1.0 0.298 1.42 0.448 1.35 0.592 1.23 0.785 1.17 0.202 1.05 1 1.12 1.2 0.296 1.44 0.446 1.40 0.589 1.23 0.780 1.15 0.201 1.13 1 1.14 Temperature (°C) 28 0.299 1.40 0.448 1.30 0.593 1.09 0.788 1.16 0.201 1.04 1 1.09 30 0.298 1.42 0.448 1.35 0.592 1.23 0.785 1.17 0.202 1.05 1 1.12 32 0.296 1.42 0.448 1.35 0.590 1.23 0.776 1.17 0.203 1.05 1 1.12

Analysis of samples

Accurately weighed 200 mg of erlotinib bulk drug sample into 100mL volumetric flask

and dissolved in mobile phase. This solution was used for estimation of impurities. The

results are recorded in table 15. Almost all impurities are found in different quantities in

all studied samples.Erl-2 has the highest amount of impurity (0.14%) of which impurity

E4 alone was 0.05%. The impurities are present in very small quantity, hence their

presence was confirmed by comparing the UV spectra of impurity peaks in sample with

that of standard impurities (figure 12) using PDA detector. The assay of erlotinib was

carried out by diluting the above solution to 200 ppm with mobile phase and the assay

values were ≥99.39%.

Table 15 Sample analysis resultsa

Impurities/assay, % w/w ± SD

Sample E0 E1 E2 E3 E4 E

Erl-1 - - 0.01 ± 0.001 - 0.03 ± 0.002 99.86 ± 0.12

Erl-2 0.01 ± 0.002 0.03 ± 0.002 0.04 ± 0.003 0.01 ± 0.001 0.05 ± 0.003 99.56 ± 0.23

Erl-3 0.02 ± 0.001 0.03 ± 0.002 0.02 ± 0.001 0.02 ± 0.001 0.03 ± 0.002 99.39 ± 0.35 a average of three determinations

47  

Figure 12Comparitive UV-pattern of standard impurities and sample

48  

Calculation

. , % /Asm x Wst x Ps

Ast x Wsm

, % /Asi x Wst x Pi

Ai x Wsm

Where, Asm- area of sample peak

Ast- area of standard erlotinib peak

Asi- area of impurity peak in sample

Ai- area of impurity standard peak

Wst- weight of standard in mg

Wsm- weight of sample in mg

Ps- purity of erlotinib standard

Pi- purity of impurity standard

2.1.4 CONCLUSION

Simple, fast and cost effective nonaqueous titration methods using visual and

potentiometric end point detection were developed for assay of erlotinib in erlotinib.HCl

bulk drug. The method was validated for linearity, precision and accuracy and

successfully applied for the assay of erlotinib bulk drug. The impurities present in

erlotinib bulk drug was isolated by using preparative HPLC and characterized by

recording IR spectra, mass spectra and UV-visible spectra and comparing with that of

authentic standards. Also, a simple, fast and sensitive RP-HPLC method was developed

for simultaneous estimation of erlotinib and its process related impurities formed in

erlotinib synthesis by quinazolinone route. All impurities are separated with good peak

shape and symmetry indicating the selectivity of the method. The LOD and LOQ of the

method is <0.03 and <0.10 μg/mL for all impurities and erlotinib indicating the good

sensitivity of the method. The recovery of the method within accepted range of 90-110%

and precision values are <4 indicating the good accuracy and precision of the method.

49  

The assay values are unaffected by the minor variation in the instrumental parameters

indicating the robustness of the method. Developed HPLC method is successfully used

for estimation of process related impurities and assay of erlotinib bulk drug.

2.1.5 MATERIALS AND METHODS

2.1.5.1 Titrimetric method for estimation of erlotinib

2.1.5.1.1 Reagents and chemicals

All reagents were of analytical grade unless stated otherwise. Analytical grade acetic

acid(99%), mercuric acetate, perchloric acid(70%) and crystal violet dye were procured

from MERCK India Ltd (India). Erlotinib.HCl (99.9%) (E) was obtained from in-house

research facilities of Vittal Mallya Scientific Research Foundation, Bangalore.

2.1.5.1.2 Apparatus

Potentiometric titration was performed with an Elico 120 digital pH meter provided with

a combined glass-standard calomel electrode system. The KCl of the salt bridge was

replaced with 0.1 M lithium perchlorate in glacial acetic acid.

2.1.5.1.3 Preparation of solutions

Preparation of 0.1 N perchloric acid

About 8.5 mL of perchloric acid (70%, w/w) was transferred in to 1000 mL volumetric

flask containing 250 mL of acetic acid, to this 50 mL of acetic anhydride was added.

Allowed to cool for 30 min and make up to volume using glacial acetic acid. Allow the

solution to stand for 24 h before use.

The 0.01 N perchloric acid was prepared by diluting 100 mL of 0.1 N solution to 1000

mL by acetic acid. This solution was standardized using potassium hydrogen phthalate

before use.

50  

Preparation of crystal violet indicator and mercuric acetate solution

The crystal violet Indicator was prepared by dissolving 0.1 g of dye in 100 mL glacial

acetic acid. 1% mercuric acetate solution was prepared by dissolving 1 g of mercuric

acetate in 100 mL of glacial acetic acid.

Standardization of 0.01 N perchloric acid

Accurately weighed 25 mg of potassium hydrogen phthalate (dried at 110 °C for 2 hour)

in to a dried 100 mL conical flask and dissolved in 50 mL of acetic acid by sonication.

About 2-3 drops of crystal violet indicator was added and titrated to emerald green end

point.

N W

MW x V

N - normality of perchloric acid

W- weight in mg of potassium hydrogen phthalate

V -volume of perchloric acid consumed (mL)

MW- molecular weight of potassium hydrogen phthalate

Preparation of erlotinib.HCl (E) standard solution:

Accurately weighed 500 mg of E into a 100 mL volumetric flask, added 1000 mg of

mercuric acetate and dissolved in acetic acid. This 5 mg/mL E solution was used for

validation study.

2.1.5.1.4 General procedures

Estimation of erlotinib.HCl by visual titration method

Accurately weighed 10- 100 mg of bulk drug and transferred into a clean and dry 100 mL

beaker and dissolved in 25 mL of glacial acetic acid Then, 2 mL of 1% mercuric acetate

was added, the contents were mixed, after 2 min, two drops of crystal violet indicator was

51  

added and titrated with 0.01 N perchloric acid until the color changes from blue to

emerald green.

A blank titration was performed in the same manner without E, and the necessary volume

corrections were made. Each mL of 0.01 N perchloric acid equivalents to 4.298 mg

erlotinib.HCl.

Assay, % w/wV x N x Mw x 100

W x 0.01

V - volume of perchloric acid consumed in mL Mw - relative molecular mass of the compound N - normality of the perchloric acid W – weight of sample in mg

The method was validated for linearity, accuracy, precision and robustness as per ICH

guidelines.

Estimation of erlotinib.HCl by potentiometric titration method

Accurately weighed 10 - 100 mg of bulk drug and transferred into a clean and dry 100

mL beaker and dissolved in 25 mL of glacial acetic acid followed by the addition of 2 mL

of 1% mercuric acetate. The combined glass-SCE (modified) system was dipped in the

solution. The contents were stirred magnetically and the titrant (0.01 N HClO4) was

added from a micro burette. Near the equivalence point, titrant was added in 0.05 mL

increments. After each addition of titrant, the solution was stirred magnetically for 30 s

and the steady potential was noted. The addition of titrant was continued until there was

no significant change in potential on further addition of titrant. The equivalence point was

determined by applying the graphical methods.

A blank titration was performed in the same manner without E, and the necessary volume

corrections were made. Each mL of 0.01 N perchloric acid equivalent to 4.29896 mg

erlotinib.HCl.

Assay, %w/wV X N X Mw X 100

W X 0.01

Where, V - volume of perchloric acid consumed in mL, Mw - relative molecular mass of the drug

52  

N - normality of the perchloric acid W – weight of sample in mg The method was validated for linearity, accuracy, precision and robustness as per ICH

guidelines.

2.1.5.2 Isolation and identification of impurities and HPLC method development for

simultaneous estimation of erlotinib and its process related impurities

2.1.5.2.1 Reagents and chemicals

All reagents were of analytical grade unless stated otherwise. HPLC-grade water was

provided by a Milli-Q® water purification system, Millipore Corporation (USA), HPLC-

grade acetonitrile and ammonium acetate procured from MERCK India Ltd

(India).Standard erlotinib. HCl (E) and process-impurities (E0, E1, E2, E3, & E4) were

obtained from in-house research facilities of Vittal Mallya Scientific Research

Foundation, Bangalore.

2.1.5.2.2 Apparatus

High performance liquid chromatography (Analytical)

An Shimadzu HPLC equipped with two LC-10AT VP pumps, an SPD-M10A VP photo

diode array detector (PDA),CTO-10 AS VP oven and SCL-10A VP controller was used

for analysis of samples. The compounds were separated on reverse-phase Inertsil ODS-

3V column (250 x 4.6 mm i.d.; 5 μm) (GL Sciences Inc, Japan) was used for separation

and chromatograms were integrated using Class vp software. The mobile phase consisted

of 1% ammonium acetate and acetonitrile (55:45, v/v); before delivering into the column

it was filtered through 0.45 μm nylon filter and degassed. The analysis was carried out

under isocratic condition using a flow rate of 1.0 mL/min at 30°C with 20 μl injection

volume. Chromatograms were recorded at 254 nm using PDA.

53  

High performance liquid chromatography (Preparative)

The WatersTM prep LC 4000 system with single pump, system controller and tunable

absorbance detector was used for isolation of impurities from erlotinib bulk drug. The

compounds were separated on an Inertsil ODS-3V column (250 x 19 mm i.d.; 5 μm) (GL

Sciences Inc, Japan) with mobile phase consisting of 1% ammonium acetate and

acetonitrile (55:45 v/v) with flow rate 17 mL/min.

Gas chromatography mass spectrometer (GC-MS)

The mass spectra of samples were recorded on a Shimadzu GCMS-QP2010S with

electron impact ionization (ionization energy 70 eV), direct probe sample injection mode

and data analysed using GC solutions software. The compounds were dissolved in

acetonitrile and 1µl was injected.

Infrared spectrophotometer

IR spectra of samples were recorded using Nicolet Avatar 320 FT-IR spectrometer. The

solid samples were analysed by making potassium bromide (KBr) pellets and liquid

sample were analysed by making thin film on KBr disc.

Melting point was recorded using melting point apparatus Acro Steel Pvt Ltd. A photo

stability chamber Caron Model 6540-2 (Caron, Marietta, OH) was used for stressing the

sample. An Ultrasonic cleaner model 8890 (Cole Parmer, India) was used for preparation

of sample and standard. For evaporation of solvents Buchii Rotavapour Model R124 was

used.

2.1.5.2.5 Preparation of standard and Sample solution

Solutions of (1000 μg/mL) erlotinib (E) and its process intermediates E0, E1, E2, E3 and

E4 were prepared by dissolving known amounts of compounds in acetonitrile. These

solutions were further diluted with mobile phase to determine the accuracy, precision,

linearity, LOD and LOQ.

Sample solution of erlotinib bulk drug (2000 μg/mL) was prepared by dissolving an

appropriate amount in mobile phase.

54  

2.1.5.2.6 HPLC Method validation

Validation is the means to assure a minimum level of accepted quality that a method will

generate according to its intended purpose. It is supposed to guarantee evidence that the

interpreted quantitative conclusion is correct and that the results are obtained accordingly

with in advance set performance criteria. According to ICH guidelines method should be

validated for specificity, linearity, limit of detection, limit of quantification, accuracy,

precision, ruggedness and sample stability.

Specificity

The specificity of the method was demonstrated by spiking the 25 ppm of each impurity

to erlotinib and chromatographed. Chromatographic peak purity and homogeneity was

evaluated with PDA detector. Also, the specificity is demonstrated by forced degradation

studies.

Forced degradation studies

A stock solution of erlotinib (1000 μg/mL) was prepared using mobile phase for

degradation studies.

(a) Acid induced degradation

Erlotinib stock solution was treated with 10 mL of 0.1 N HCl and kept at 40 °C for 24 h.

The above solution was neutralized to pH 7 using sodium hydroxide and diluted to 25

μg/mL concentration using mobile phase.

(b) Base induced degradation

Erlotinib stock solution was treated with 10 mL of 0.1 N NaOH and kept at 40 °C for 24

h. The above solution was neutralized to pH 7 using HCl and diluted to 25 μg/mL

concentration using mobile phase.

(c) H2O2 induced degradation

Erlotinib stock solution was treated with 10 mL of 3% v/v H2O2 and kept at 40 °C for 24

h and diluted to 25 μg/mL concentration using mobile phase.

55  

(d) UV induced degradation

Erlotinib stock solution was diluted to 25 μg/mL concentration using mobile phase and

exposed to UV light for 6 hrs, resulting in an overall illumination of ≥ 210 Wh/m2 in a

photo stability chamber.

System suitability

System suitability test parameters and acceptance criteria are based on the concept that

the equipment, electronics, analytical operations, and samples to be analyzed constitute

an integrated system. System suitability testing ensures that the system is working

properly at the time of analysis.21 System suitability of chromatographic method was

evaluated by performing five replicate injection of solution containing 1.0% of

impurities in erlotinib (100 μg/mL). The system suitability parameters like retention time

(tR), relative retention time (RRT), tailing factor, capacity factor (CF) and theoretical

plates were evaluated. To satisfy the system suitability test RSD values should be less

than 2%.

Accuracy

Accuracy of the analytical method is defined as the similarity of the results obtained by

the analytical method to the true value. Accuracy of the method for impurities was

studied at four concentration levels ranging from LOQ to maximum analysis limit (0.1,

0.5, 1.0 & 2.0 µg/mL) by spiking the impurities to erlotinib at its specification level (100

µg/mL). Analysis was done in triplicate in all specified levels. The accuracy of the

method for assay of erlotinib is checked at four different levels ranging from 25 – 300

µg/mL (25,100,200 & 300 µg/mL) in triplicate. The acceptance limit for accuracy is

given in table 16.

Table 16 Accuracy limits

Active ingredient/impurities content Acceptance limit

≥ 10% 98-102%

≥ 1% 90-110%

0.1-1.0% 80-120%

< 0.1% 75-125%

56  

Precision

Precision of the method was established by spiking the impurities at three concentration

levels i.e. at LOQ, mid range and maximum range (0.1, 1.0 & 2.0 µg/mL) to erlotinib in

specified level (100 µg/mL) and the recoveries of each impurity was calculated. The

analysis was done in triplicate in each level to find the intra-day precision (repeatability)

and %RSD values are recorded. Same study was done on different days to find the inter-

day precision. (Intermediate precision)

Precision for assay of erlotinib was checked at four concentrations 25,100,200 & 300

µg/mL, analysis done in triplicate and values are recorded in %RSD to find the

repeatability or intra-day precision. Same study was done on different days to find the

inter-day precision. The acceptance limit for accuracy is given in table 17.

Table 17 Precision limits

Active ingredient/impurities content Acceptance limit

≥ 10% ≤ 2%

1-10% ≤ 5%

0.1-1.0% ≤ 10%

< 0.1% ≤ 20%

Linearity

The linearity of the detector response to different concentrations of impurities was

studied by analyzing erlotinib solution containing impurities at eight different

concentration levels ranging from 0.1 – 2.0 µg/mL. Similarly, the linearity of erlotinib

was studied at eight different levels ranging from 25 – 300 µg/mL. Linearity of the

method was established by plotting the peak area against the concentration and using the

statistical analysis.

57  

Limit of detection (LOD) and Limit of quantification (LOQ)

The LOD and LOQ of the method, is the minimum quantity that can be detected and

minimum quantity that can be quantified with proper precision. The LOD and LOQ of

the method for erlotinib and its impurities was calculated by using the signal-to-noise

(S/N) ratio. The S/N ratio of 3 and 10 were used to calculate the LOD and LOQ.

Robustness

Robustness of a method is defined as a measure of its capacity to remain unaffected by

small, but deliberate changes in method parameters and provides an indication of its

reliability during normal usage.22 The robustness of the method was studied by varying

the flow rate by ± 0.2 mL, the acetonitrile composition in the mobile phase by ± 2 mL,

and the column temperature by ±2°C to the actual method parameters. The test solution

was injected in triplicate and system suitability parameters were evaluated.

Stability of the solutions

Drug stability in pharmaceutical formulations is a function of storage conditions and

chemical properties of the drug and its impurities. Conditions used in stability

experiments should reflect situations likely to be encountered during actual sample

handling and analysis. Stability data is required to show that the concentration and purity

of analyte in the sample at the time of analysis corresponds to the concentration and

purity of analyte at the time of sampling. The stability study of the analyte in

pharmaceutical formulation should be conducted at the room temperature and refrigerator

conditions (i.e., 2°C – 8°C) that will be experienced over the period needed to process a

batch of study samples.23 About 2 µg/mL solutions of impurities and erlotinib were

prepared using mobile phase and analyzed immediately (0 day) to find the assay. The

aliquots were stored at room temperature and refrigerator conditions and analyzed after 1,

3 and 5 days. The stability of the erlotinib and its process-related impurities in solutions

were ascertained by finding the assay of solutions by HPLC using freshly prepared

standard solutions.

58  

2.1.6 EXPERIMENTAL SECTION

2.1.6.1 Isolation of the impurities by preparative HPLC

The chromatographic conditions mentioned under preparative HPLC was used for

isolating the impurities. About 5 g of erlotinib.HCl bulk drug was dissolved in 50 mL of

acetonitrile and 0.2 mL of the solution was injected to preparative HPLC and fraction

corresponding to each impurity was collected separately. 100 injections were made and

fractions were collected each time and pooled (750 mL). The fractions collected were

concentrated to remove acetonitrile and aqueous layer (400 mL) containing impurities

were re-extracted with (100 x 3) mL of ethyl acetate. The ethyl acetate layer containing

impurities were dried over anhydrous sodium sulphate. The evaporation of ethyl acetate

yielded individual components in pure form (7 mg).

2.1.6.2 Characterization of impurities

The impurities isolated using preparative HPLC were characterized by recording IR

spectra, mass spectra, UV spectra and melting point.

Impurity E0

Off-white solid

mp: 151-156 °C (lit. 150-157 °C)14

UV: λ max 224, 285, 333 nm

IR(KBr): 852, 940, 1038,1130,1203,1308, 1436, 1501, 1556, 1693, 2905, 3402 cm-1

GC-MS (DI, m/z, Spectra 2.1.1): 138 (M+)

Impurity E1

Brown colored liquid

UV: λ max 230, 276, 308 nm

IR (Thin film): 706, 792, 1010, 1108, 1253, 1426, 1503,1578, 1690, 2900, 3010 cm-1.

GC-MS (DI, m/z, Spectra 2.1.2): 254 (M+)

59  

Impurity E2

Brown colored liquid

UV: λ max 220, 254, 285 nm

IR(Thin film): 786, 885, 1020, 1118, 1226, 1446, 1502,1580, 2250, 2908, 3015 cm-1.

GC-MS(DI, m/z, Spectra 2.1.3): 251( M+)

Impurity E3

Yellow colored solid

mp: 137-140 °C (lit. 136-140 °C) 14

UV: λ max 253, 300, 340 nm

IR (KBr): 780, 896, 1056, 1137, 1345, 1501, 1576, 2258, 2906, 3024 cm-1

GC-MS (DI, m/z, Spectra 2.1.4): 296 (M+)

Impurity E4

Yellowish brown solid

mp: 183-185 °C (lit. 182-184 °C) 14

UV: λ max 241, 308, 320 nm

IR(KBr): 790, 1055, 1163, 1289, 1365, 1405, 1490, 1590, 1656, 2902, 3400 cm-1

GC-MS (DI, m/z, Spectra 2.1.5): 294 (M+)

2.1.6 BIBLIOGRAPHY

 

1. Herz, W.; Falk, H.; Kirby, G. W. Progress in the Chemistry of Organic Natural

Products; Springer, 2000.

2. Carrillo, A. J.; Guarro, J. Antimicrob. Agents Chemother. 2001, 45, 2151–2153.

3. Yu, C. X.; Zhu, C. B.; Xu, S. F.; Cao, X. D.; Wu, G. C. Neurosci. Lett. 2000, 282,

161–164.

4. Cohen, E.; Klarberg, B.; Vaughan, J. R. J. Am. Chem. Soc. 1960, 82, 2731–2735.

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5. Maggi, G. C.; Donati, C.; Gueli Alletti, D. Arzneimittelforschung. 1985, 35, 994–998.

6. Boyapati, S.; Kulandaivelu, U.; Sangu, S.; Vanga, M. R. Arch. Pharm. 2010, 343,

570–576.

7. Stephan Krause, P. A Guide for Testing Biopharmaceuticals Part 2: Acceptance

criteria and analytical method maintenance. BioPharm Int. 2006.

8. Chandregowda, V.; Rao, G. V.; Reddy, G. C. Synth. Commun. 2007, 37, 3409–3415.

9. Chahbouni, A.; den Burger, J. C. G.; Vos, R. M.; Sinjewel, A.; Wilhelm, A. J. Ther.

Drug Monit. 2009, 31, 683–687.

10. Widemann, B. C.; Balis, F. M.; Godwin, K. S.; McCully, C.; Adamson, P. C. Cancer

Chemother. Pharmacol. 1999, 44, 439–443.

11. Williams, H.; Smith, H. J. Smith and Williams’ introduction to the principles of drug

design; Wright: London; Toronto, 1988.

12. Dowell, J.; Minna, J. D.; Kirkpatrick, P. Nat. Rev. Drug Discov. 2005, 4, 13–14.

13. Pao, W.; Miller, V.; Zakowski, M.; Doherty, J.; Politi, K.; Sarkaria, Proc. Natl. Acad.

Sci. 2004, 101, 13306–13311.

14. Chandregowda, V.; Venkateswara Rao, G.; Chandrasekara Reddy, G. Heterocycles

2007, 71, 39-46.

15. Fouad Chiadmi,; Mathieu Duprez,; Joël Schlatter. EJHP Science. 2007, 13, 48-51

16. Lepper, E. R.; Swain, S. M.; Tan, A. R.; Figg, W. D.; Sparreboom, A. J. Chromatogr.

B Analyt. Technol. Biomed. Life. Sci. 2003, 796, 181–188.

17. Pan, J.; Jiang, X.; Chen, Y.-L. Pharmaceutics 2010, 2, 105–118.

18. Zhao, M.; Hartke, C.; Jimeno, A.; Li, J.; He, P.; Zabelina, Y. Chromatogr. B 2005,

819, 73–80.

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19. Pujeri, S. S.; Khader, A. M. A.; Seetharamappa, J. Anal. Lett. 2009, 42, 1855–1867.

20. Raman, N. V. V. S. S.; Reddy, K. R.; Prasad, A. V. S. S.; Ramakrishna, K.

Chromatographia 2008, 68, 481–484.

21. Guideline, I. H. T. IFPMA Geneva 2005.

22. Chow, S.-C. In Statistics in Drug Research; CRC Press, 2002; Vol. 10.

23. GUIDELINE FOR GOOD CLINICAL PRACTICE - Q1A (R2). http://www.ich.org/

fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1A_R2/Step4/Q1A_

R2__Guideline.pdf

 

 

 

 

 

 

 

 

 

 

 

 

62  

 

Spectra 2.1.1: Mass spectra of impurity E0 (molecular weight 138)

 

 

 

Spectra 2.1.2: Mass spectra of impurity E1(molecular weight 254)

63  

Spectra 2.1.3: Mass spectra of impurity E2 (molecular weight 251)

 

 

Spectra 2.1.4: Mass spectra of impurity E3 (molecular weight 296)

64  

 

Spectra 2.1.5: Mass spectra of impurity E4 (molecular weight 294)

65  

Chapter II B

HPLC method for simultaneous estimation of erlotinib

(Tarceva®) and its process-related impurities formed in

6,7-bis-(2-methoxyethoxy)-4-quinazoline-thione route:

Isolation and characterization of a new impurity

66  

2.2.1 INTRODUCTION

Herein we describe isolation and characterization of impurities present in the

erlotinib.HCl bulk drug made by quinazoline-thione route 1,2 and development HPLC

method for simultaneous estimation of erlotinib, its process-related impurities and

characterization of new impurity. Erlotinib synthesized by this method may contain un-

reacted starting material and process intermediates. Any compound other than the drug

will be considered as an impurity, according to the US FDA guidelines, such impurities

present at the levels greater than 0.1% must be identified and quantified using validated

analytical procedures as discussed under chapter IIA.

As described in chapter IIA, literature survey revealed many HPLC methods with UV,

photo diode array and mass detection for the estimation of erlotinib in bulk drugs, tablets

and in biological fluids.

2.2.2 IMPORTANCE OF THE PRESENT WORK

The identification and quantification of process-related impurities estimation is of greater

importance, since they may have some toxicological effects.3 The impurities present in

erlotinib.HCl bulk drug made from quinazoline-thione route were isolated using

preparative HPLC and characterized using spectroscopic techniques. The available

literature doesn’t contain HPLC method for simultaneous estimation of erlotinib and its

process-related impurities. In this present study, we have developed RP-HPLC methods

for separation and estimation of erlotinib and its seven process-related impurities formed

during erlotinib synthesis as mentioned above. The developed HPLC method was

validated for specificity, linearity, precision, limit of detection, limit of quantification and

ruggedness as per ICH guidelines.4

67  

2.2.3 RESULTS AND DISCUSSION

2.2.3.1 Isolation and characterization of impurities

The impurities present in erlotinib.HCl bulk drug were isolated using preparative HPLC

(figure 1) and purified by extraction and concentration. The mass spectra, IR spectra, UV

spectra and melting points were recorded for purified compounds. Further, 1H NMR and 13C NMR was recorded for structure elucidation of new impurity E-NO.

Figure 1 HPLC chromatogram of erlotinib.HCl bulk drug made by quinazoline-thione

route

The spectroscopic data of impurities eluted at 3.2, 4.5, 7.0, 9.3 and 12.4 min were

matching with compounds characterized as impurities E4, E0, E1, E2 and E3 in chapter

IIA. (Figure 2)

68  

OH

OH

CHO O

O

CHOH3CO

H3CO

O

O

CNH3CO

H3CO

O

O

CNH3CO

H3CO NO2

O

O

H3CO

H3CO

NH

N

O

O

O

H3CO

H3CO

N

N

NH

CH

.HCl

E0 E1 E2

E3 E4

E (Erlotinib.HCl)

O

O

H3CO

H3CO

NH

N

S

E5

O

O

H3CO

H3CO

N

N

S CH3

E6

Figure 2 Structure of erlotinib and process-related impurities

Impurity E5  

The peak eluting at 4.9 min in HPLC chromatogram yielded pale yellow colored powder.

The GC-MS analysis exhibited molecular ion peak at m/z 310(M+). The IR and UV-

spectra and melting point of isolated compound were matched with the standard

6,7-bis(2-methoxyethoxy)quinazoline-4(3H)-thione (E5).

Impurity E6  

The peak eluting at 10.3 min in HPLC chromatogram yielded pale yellow colored

powder. The GC-MS analysis exhibited molecular ion peak at m/z 324(M+). The IR and

UV-spectra and melting point of isolated compound were matched with the standard

6,7-bis(2-methoxyethoxy)quinazoline-4-(methylthio)quinazoline(E6).

Impurity E-NO

The new impurity(E-NO)(figure 3) which is eluting at 6.1 min has been isolated using

preparative HPLC. IR spectrum showed a peak at 1190 cm-1 indicating the probability of

N-oxide group. Further mass spectrum showed M+ peak at 409 which is 16 mass units

more than the parent molecule erlotinib. The 1H NMR spectrum showed two peaks at δ

69  

3.34 and 3.36 for 2 x -OCH3 protons and the 2 x –CH2-OCH3 protons were observed

as multiplets at δ 3.77. The acetylenic proton was seen as singlet at δ 4.18 and 2 x –CH2-

O-ф protons were observed as multiplets at δ 4.33. The aromatic protons on aniline ring

were observed as singlet, doblets and triplet between δ 7.20 and 7.79 and quinazoline

protons attached to C-5, C-8 and C-2 were observed at δ 7.88, 7.93 and 8.65 as three

singlets, another singlet at δ 9.57 was seen for –NH proton. Six peaks observed in 13C

NMR in the range of δ 58.83 to 70.45 indicated primary and secondary alkyl carbons,

two peaks at δ 81.13 and 83.39 for two acetylinic carbons. Apart from these 14 aromatic

carbon peaks were clearly seen confirming all structural features of quinazoline molecule

present in erlotinib. It is known that in 4-substituted quinazolines, 1 N-oxides are

favoured products over 3 N-oxides due to stability and steric factors. (Reference:

M.Uchida, T.Higashino and E.Hayashi; Mass Spectrometry Vol.21, 245-254 (1973).5

Thus it was characterized as [6,7-bis-(2-methoxyethoxy)-quinzolin-4-yl]-(3-ethynyl

phenyl) amine-1-N-oxide (E-NO). Further it was confirmed by synthesizing it from

erlotinib using meta chloroperbenzoic acid.

O

O

H3CO

H3CO

N

N+

NH

CH

O-

Figure 3 Structure of erlotinib N-oxide (E-NO)

2.2.4.2 Development of HPLC method for simultaneous estimation of erlotinib and

its impurities

2.2.4.2.1 Optimization of chromatographic conditions

Figure 2 shows the process related impurities present in erlotinib bulk drug made by

quinazoline-thione route. The present study was aimed at developing a chromatographic

70  

system capable of eluting and separating erlotinib and its intermediates/synthetic

impurities.

All the intermediates and erlotinib were subjected to separation by using different

columns and mobile phase in order to select the suitable column and solvent system for

accurate analysis and to achieve good resolution of all the intermediates. The suitability

of the mobile phase and the flow rate was decided on the basis of selectivity, resolution

and time required for analysis. The separation and peak shapes were good on Inertsil

ODS-3V (4.6 x 250 mm i.d, 5 µm) column, using 1% ammonium acetate and acetonitrile

(55:45 v/v). A typical chromatogram of erlotinib spiked with 25 ppm of each impurity is

shown in figure 4. It is evident from figure 4 that, all the compounds were eluted, and

well separated with good peak shapes and resolutions. The UV wavelength at 254 nm

was chosen for the detection and quantification, since erlotinib and its impurities have

good absorption at that wavelength. The peaks were identified by injecting and

comparing retention times of the individual compounds and studying absorption spectra

using PDA detector.

Figure 4 Typical chromatogram of erlotinib (E) spiked with 25 ppm of each impurity

(E0, E1, E2, E3, E4, E5 and E6).

71  

2.2.4.2.2 Method validation

The developed method was validated for accuracy, precision, linearity, LOD, LOQ,

specificity and robustness as per ICH guidelines.

System suitability

The system suitability test was performed to confirm that the LC system to be used was

suitable for the intended application. The test was conducted by using 1.0% of all

intermediates spiked to the erlotinib (100 µg/mL) and evaluated by making five replicate

injections. The system suitability parameters like retention time (tR), tailing factor and

theoretical plates were evaluated and the values were recorded in table 1. The system

suitability experimental results showed that the parameters evaluated were within the

acceptable range (RSD < 2.0%) indicating that the system is suitable for the estimation of

erlotinib and impurities.

Table 1 System suitability dataa Sl.No Compound Name  Abbrev

iation tR/ min  Tailing

factor Theoretical

plates 1 3,4-Dihydroxy

benzaldehyde E0 4.41 ± 0.11 1.24 ± 1.58 8951 ± 1.89

2 3,4-Di(methoxyethoxy) benzaldehyde

E1 6.68 ± 0.09 1.14 ± 0.98 22481 ± 1.13

3 3,4-Di(methoxyethoxy) benzonitrile

E2 8.78 ± 0.12 1.12 ± 1.59 15584 ± 1.28

4 4,5-Di(methoxyethoxy)-2-nitrobenzo nitrile

E3 11.57 ± 0.20 1.06 ± 1.85 16653 ± 0.42

5 6,7-Bis-(2-ethoxyethoxy) quinazolin-4-(3H)-one

E4 3.00 ± 0.26 1.20 ± 1.73 7278 ± 1.44

6 6,7-Bis-(2-ethoxyethoxy) quinazolin-4-(3H)-thione

E5 4.71 ± 0.10 1.15 ± 1.26 9819 ± 1.94

7 6,7-Bis-(2-ethoxyethoxy)-4-(methylthio)quinazoline

E6 9.50 ± 0.09 1.22 ± 1.79 11610 ± 0.80

8 N-(3-Ethynylphenyl)-6,7-bis(2-metho xyethoxy)-4-quinazolinamine

E 14.78 ± 0.15 0.99 ± 0.85 14612 ± 0.28

a average of five determinations ± RSD, %

72  

Specificity

To demonstrate specificity of the method, erlotinib bulk drug was spiked with 25 ppm of

impurities and chromatographed (figure 4). All the impurities were well separated from

erlotinib, the chromatographic peak purity and homogeneity was evaluated with PDA

detector. The peaks with flat-top indicated that all peaks are homogeneous with out

impurities embedded in it. Also the specificity was checked by forced degradation study.

Under UV and acidic condition there was no change in purity, but in alkaline and

oxidative conditions, the degradation products were formed, but they were well separated

from erlotinib and the process impurities, indicating that the method is specific for the

separation and estimation of erlotinib and its process impurities.

Linearity

Linearity of the detector response to different concentrations of impurities was studied

by analyzing the erlotinib spiked with each impurity at seven levels ranging from 0.1-2.0

µg/mL. Linearity of erlotinib was studied at seven different concentration levels ranging

from 25-300 µg/mL (table 2 & figure 5). The solutions were injected in triplicate and

data’s were subjected to statistical analysis using linear-regression model; the regression

equation and coefficients are shown in figure 6. The correlation coefficients of all

compounds >0.997 indicating the excellent relationship between the peak area and the

concentration of compounds.

Table 2 Different data points of linearity study Concen. µg/mL

Area Concen. µg/mL

Area

E0 E1 E2 E3 E4 E5 E6 E

0.1 5379 1180 7651 11270 7902 12918 18792 25 2508345

0.2 10081 2438 14065 20385 13554 15119 22568 50 4919782

0.3 17985 4930 25128 36539 23698 18192 32069 100 9707736

0.5 27145 7712 38032 55361 34815 30859 43394 150 14883327

1 51421 16064 71674 104529 63609 51745 68088 200 19808743

1.5 81948 26596 113459 165155 100253 77641 102653 250 24758529

2 106150 35651 148173 217002 130525 95443 130099 300 29871562

73  

Figure 5 HPLC chromatograms of impurities (0.1 – 2.0 µg/mL)

Figure 6 Regression curves of erlotinib and process-related impurities

74  

Accuracy

The accuracy of the method was checked by spiking each impurity at four different

concentrations within calibration range (0.1, 0.5, 1.0 and 2.0 µg/mL) to the erlotinib at

specified level (100 µg/mL) and estimating recovery and RSD,%. All estimations were

carried in triplicate (n=3), the recovery and RSD for all seven impurities were found to be

96.83 - 100.96% and 0.11 - 2.46% respectively. Similarly the accuracy of the method for

assay of erlotinib was studied at four concentration levels (25, 100, 200, & 300

µg/mL).The recovery values were in the range of 98.59-99.70% and RSD 0.25-0.81%

respectively. Results indicated that method has got very good recovery values, intern

having good accuracy. (Table 3)

Precision

The precision of the method for impurites and erlotinib assay was estimated by

repeatability and intermediate precision. Repeatability is the intra day variation obtained

at three different concentration levels (0.1, 1.0 & 2 µg/mL)and is expressed in terms of

RSD,% calculated for each day. The RSD values for all intermediates were in the range

of 0.11-2.25%, indicating good repeatability. The intermediate precision is the inter-day

variation at the same concentration levels determined on three successive days. The inter-

day variations calculated for three concentration levels are expressed in terms of RSD, %.

At each concentration level, the RSD, % values for all intermediates were in the range

0.29-3.76%, thus indicating a good intermediate precision. Similarly the intra-day and

interday precision of assay of erlotinib at four concentration levels (25, 100, 200 & 300

µg/mL) expressed as RSD were in the range of 0.25-0.81% and 0.24-0.85% respectively.

Results are recorded in table 4.

75  

Table 3 Accuracy dataa

Compound Spiked quantity,

µg/mL Found quantity ,

µg/mL ± SD Recovery

(%) %RSD E0 0.1 0.099 ± 0.001 99.06 1.13

0.5 0.494 ± 0.004 98.85 0.81 1 0.984 ± 0.010 98.91 0.11 2 1.990±0.019 99.5 1.01

E1

0.1

0.098 ± 0.001

98.36

1.10

0.5 0.495 ± 0.005 98.99 0.93 1 0.984 ± 0.002 98.78 0.22 2 2.014 ± 0.015 100.69 0.73

E2

0.1

0.099 ± 0.001

98.61

0.47

0.5 0.497 ± 0.004 99.37 0.88 1 0.980 ± 0.002 98.58 0.20 2 1.993 ± 0.012 99.65 0.62

E3

0.1

0.098 ± 0.002

98.11

2.0

0.5 0.492 ± 0.012 98.37 2.46 1 0.982 ± 0.002 98.62 0.20 2 1.999 ± 0.042 100.67 1.04

E4

0.1

0.099 ± 0.001

99.11

0.26

0.5 0.497 ± 0.004 99.44 0.79 1 1.010 ± 0.001 100.96 0.04 2 1.950 ± 0.011 97.5 0.54

E5

0.1

0.098 ± 0.002

98.11

2.01

0.5 0.492 ± 0.012 98.37 2.46 1 0.982 ± 0.002 98.62 0.20 2 1.999 ± 0.042 100.67 1.02

E6

0.1

0.099 ± 0.001

99.11

0.26

0.5 0.497 ± 0.004 99.44 0.79 1 1.010 ± 0.001 100.96 0.04 2 1.950 ± 0.011 97.5 0.54

E

25

24.65 ± 0.20

98.59

0.81

100 99.01 ± 0.36 99.01 0.36 200 199.44 ± 0.52 99.72 0.26 300 299.10 ± 0.75 99.70 0.25

a. n=3, average of three determinations RSD: relative standard deviation SD: standard deviation

76  

Table 4 Precision data

Compound Spiked quantity, µg /mL

Intra-day precision,

RSD%(n=3)

Inter-day precision, RSD%(n=9)

E0 0.1 1.13 1.64 1 0.11 0.69 2 1.0 1.18

E1

0.1

1.10

1.25

1 0.22 0.34 2 0.73 1.7

E2

0.1

1.15

2.79

1 0.14 0.29 2 0.93 1.25

E3

0.1

2.25

3.76

1 0.21 1.37 2 0.87 0.85

E4

0.1

0.47

1.61

1 0.20 0.91 2 0.62 0.78

E5

0.1

2.0

2.1

1 0.2 1.53 2 1.0 0.82

E6

0.1

0.26

2.56

1 0.14 0.41 2 0.54 0.43

E

25

0.81

0.85

100 0.36 0.35 200 0.26 0.27 300 0.25 0.24

Robustness

Small but deliberate variations in the HPLC parameters were made to verify the

robustness of the analytical method. Robustness was studied by varying ±0.2 mL of flow

rate, ±2mL of acetonitrile composition in mobile phase and ±2°C in column temperature

77  

to the actual method parameters. In all the above variations, test samples were injected in

triplicate and RRT, capacity factor and percentage recovery was evaluated. A slight

change in the retention time of erlotinib and its impurities was observed on changing

acetonitrile composition in mobile phase, but all peaks were well separated without

affecting the accuracy of quantitative estimation. There was no significant change

observed by changing the flow rate and temperature. The result indicated that the method

adopted is suitable for separation and estimation of erlotinib and its synthetic impurities.

The results are recorded in table 5

Table 5 Robustness data

Param eters

E0 E1 E2 E3 E4 E5 E6

RRT CF RRT CF RRT CF RRT CF RRT CF RRT CF RRT CF Mobile phase composition (acetonitrile %)

43 0.310 42 0.460 63 0.601 83 0.784 109 0.211 29 0.327 45 0.652 90 45 0.290 43 0.436 66 0.577 87 0.763 116 0.198 29 0.311 46 0.634 96 47 0.277 45 0.423 70 0.563 93 0.753 125 0.187 30 0.298 49 0.615 102

Temperature (°C)

28 0.299 44 0.445 65 0.588 73 0.781 116 0.199 29 0.315 46 0.632 93 30 0.290 43 0.436 66 0.577 87 0.763 116 0.198 29 0.311 46 0.634 96 32 0.298 43 0.445 65 0.587 86 0.771 113 0.201 29 0.316 46 0.638 94

Flow rate (mL / min)

0.8 0.301 54 0.449 82 0.596 90 0.78 117 0.203 36 0.318 57 0.64 108 1.0 0.290 43 0.436 66 0.577 87 0.763 116 0.198 29 0.311 46 0.634 96 1.2 0.288 36 0.435 54 0.574 61 0.758 72 0.197 24 0.309 38 0.63 79

Limit of detection and quantification

LOD and LOQ represent the concentration of the analytes that would yield signal-to-

noise ratio of 3 for LOD and 10 for LOQ, respectively. LOD and LOQ were determined

by measuring the magnitude of analytical background by injecting blank samples and

calculating the signal-to-noise ratio for each compound by injecting series of solutions

until the S/N ratio is 3 for LOD and 10 for LOQ. LOD and LOQ of all the compounds lie

in the range 0.015 - 0.030 µg/mL and 0.045-0.089 µg/mL respectively. The results are

given in table 6.

78  

Table 6 LOD and LOQ data

Compound E0 E1 E2 E3 E4 E5 E6 E

LOD, µg/mL 0.017 0.024 0.015 0.023 0.025 0.03 0.018 0.028

LOQ, µg/mL 0.05 0.075 0.045 0.067 0.075 0.089 0.055 0.091

Stability of solutions

The solution stability of erlotinib and process-related impurities were investigated by

keeping 2 µg/mL solution of all the impurities in room temperature, refrigeration (4°C)

for 5 days and estimating the purity of the solution by comparing with freshly prepared

standards. The RSD of assay values of erlotinib and all procecess related impurities were

less than 2% during five days of the solution stability experiments. The results from these

experiments confirm that sample solutions used during analysis were stable up to the

study period of five days.

Analysis of samples

Accurately weighed 200 mg of erlotinib bulk drug sample into a 100 mL volumetric

flask, and dissolve in mobile phase. The solution was filtered through 0.45 µm nylon

filter and used for estimation of impurities. Compounds were identified by their retention

time and comparing the UV spectra of sample peaks, with that of authentic standards

using PDA detector. Almost all impurities are found in different quantities in all studied

samples. Sample Erl-5 has the highest amount of impurity (0.28%) of which impurity E6

alone was 0.17%. Assay of erlotinib was carried out by diluting above solutions to 200

µg/mL, by using the mobile phase and the assay values were in the range of 99.49-

99.81%. The results are recorded in table 7.

79  

Table 7 Sample analysis dataa

Sam

ple

Impurities/assay, % w/w ± SD

E0 E1 E2 E3 E4 E5 E6 E

Erl-4 0.03 ± 0.002 0.02 ± 0.001 0.01 ±0.001 0.01 ±0.001 0.02 ±0.002 0.01 ± 0.001 0.02 ± 0.003 99.76 ± 0.21

Erl-5 0.02 ± 0.001 - - 0.01 ±0.001 0.06 ± 0.001 0.02 ± 0.001 0.17 ± 0.005 99.49 ± 0.30

Erl-6 0.01 ±0.000 0.06 ± 0.002 - - 0.01 ±0.001 - 0.06 ±0.002 99.81 ± 0.22

a (n=3) average of three determinations

2.2.4 CONCLUSION

Isolated impurities present in erlotinib.HCl made by quinazoline-thione route using

preparative HPLC. All process related impurities and one new degradation impurity [6,7-

bis-(2-methoxyethoxy)-quinzolin-4-yl]-(3-ethynyl phenyl) amine-1-N-oxide was

characterized using spectrophotometric techniques. Also, simple, fast and precise RP-

HPLC method was developed and validated for simultaneous estimation of erlotinib and

its process related impurities present in erlotinib.HCl bulk drug made quinazoline-thione

route. The method has good selectivity, the accuracy values were in the range of 94-107

and precision values were < 4%. The develolped HPLC method was used for estimation

of erlotinib and its process related impurities present in erlotinib bulk drug.

2.2.5 EXPERIMENTAL SECTION

2.2.5.1 Reagents and Chemicals

Except for intermediates E5 and E6, other reagents used are same as mentioned in

Chapter IIA.

2.2.5.2 Apparatus and methods

The analytical HPLC, preparative HPLC, GC-MS, IR spectrophotometer, photostability

chamber, pH meter and ultrasonic cleaner were used in analysis.(Detailed discussion on

80  

instruments is provided in chapter IIA). The chromatographic conditions were same as

mentioned in chapter IIA. The NMR spectra were recorded in DMSO-d6 with TMS(tetra

methyl silane) as internal standard at 400 Hz on a Bruker spectrometer.

2.2.5.3 Preparation of standard solution and sample solution

Known amounts of intermediates E5 and E6 standards were dissoved in acetonitrile to

get 1000 µg/mL solutions.Other standard and sample prepartion was same as discussed

in Chapter IIA.

2.2.5.4 Method validation

Method validation was carried out as per ICH guidelines as discussed in Chapter IIA.

2.2.5.5 Isolation of the impurities by preparative HPLC

The chromatographic conditions mentioned under preparative HPLC was used for

isolating the impurities. In this solvent system, the impurity E4, E0, E5, E-NO, E1, E2,

and E3 (figure 2) were eluting at 3.2, 4.5, 4.9, 6.1, 7.0, 9.3, and 12.5 min respectively.

About 5 g of erlotinib bulk drug was dissolved in 50 mL of acetonitrile and 0.2 mL of the

solution was injected to preparative HPLC and fraction corresponding to each impurity

was collected separately. 200 injections were made and fractions were collected each

time and pooled (1300 mL). The fractions collected were concentrated to remove

acetonitrile and aqueous layer (750 mL) containing impurities were re-extracted with

(100 x 3) mL of ethyl acetate. The ethyl acetate layer containing impurities were dried

over anhydrous sodium sulphate. The evaporation of ethyl acetate yielded individual

components in pure form (20 mg).

2.2.5.6 Characterization of impurities E5, E6 and E-NO

The impurities isolated using preparative HPLC were characterized by recording IR

spectra, mass spectra, UV spectra and melting point. Further, 1H NMR and 13C NMR was

recorded for structure elucidation of new impurity E-NO.

81  

Impurity E5

Pale yellow colored solid

mp: 191-194 °C (lit. 190-195 °C)1

UV: λ max 229, 253, 275, 361 nm

IR (KBr): 720, 806, 1006, 1115, 1275, 1448, 1507,1596, 1698, 2945, 3180 cm-1

GC-MS (DI, m/z, Spectra 2.2.1): 310 (M+)

Impurity E6

Yellow colored solid

mp: 84-89 °C (lit. 86-90 °C) 1

UV: λ max 243, 327, 339 nm

IR (KBr): 652, 798, 872, 1021, 1135, 1165, 1220, 1310, 1402, 1495, 1586,

2915, 3280 cm-1

GC-MS (DI, m/z, Spectra 2.2.2): 324 (M+)

Impurity E-NO

Brown colored solid

mp: 162-165 °C

UV: λ max 240, 280, 363 nm

IR (KBr): 675, 786, 860, 1005, 1102, 1190, 1232, 1438, 1505, 1575, 1603,

2955, 3350 cm-1 1H NMR (DMSO-d6, 400 MHz, Spectra 2.2.3): δ 3.34 (s, 3H), 3.36 (s, 3H), 3.75-3.80

(m, 4H), 4.18 (s, 1H), 4.33 (m, 4H), 7.20(d, J=8.0 Hz, 1H), 7.39 (t, J=8.0, 1H), 7.80

(d, J=8.0 Hz, 1H), 7.85 (s, 1H), 7.88 (s, 1H), 7.93 (s, 1H), 8.65 (s, 1H), 9.57 (s, 1H). 13C NMR (DMSO-d6, 400 MHz, Spectra 2.2.4): δ 58.83, 58.89, 69.01, 69.14, 70.33,

70.45, 81.13, 83.89, 100.01, 104.63, 110.25, 122.35, 122.64, 124.80, 126.94, 129.48,

139.39, 139.78, 140.01, 148.01, 149.99, 154.83.

GC-MS (DI, m/z, Spectra 2.2.5): 409 (M+), 392, 348, 334, 302, 276, 230, 101, 59.

82  

Preparation of Erlotinib N-oxide (E-NO)

About 250 mg of the erlotinib was dissolved in 25 mL of water and added 5 mL of 50%

w/v solution of sodium hydroxide. The precipitate formed was filtered and washed with

25 mL of 5% w/v sodium bicarbonate solution and dried to get 200 mg of erlotinib free

base. About 200 mg of erlotinib free base was dissolved in 10 mL of chloroform, 120 mg

of 3-chloroperoxybenzoic acid (mcPBA) was added under chilled condition. Solution

was stirred for about 12 h at room temperature and chloroform was evaporated under

vacuum. The residue was dissolved in 5% sodium bicarbonate and extracted with 25 mL

of chloroform. The chloroform layer evaporated under vacuum to get light yellow

colored solid. The crude material was recrystallized from ethyl acetate to afford 185 mg

of yellow crystalline compound (E-NO). The melting point and mixed melting point

(mmp) were measured for both synthesized compound and erlotinib N-oxide isolated.

The mp 163-165°C and mmp 162-164 °C of synthesized and isolated compounds

indicated that both were same.

2.2.7 BIBLIOGRAPHY

 1. Chandregowda, V.; Rao, G. V.; Reddy, G. C. Synth. Commun. 2007, 37, 3409–3415.

2. Chandregowda, V.; Venkateswara Rao, G.; Chandrasekara Reddy, G. Heterocycles

2007, 71, 39.

3. Raman, N. V. V. S. S.; Reddy, K. R.; Prasad, A. V. S. S.; Ramakrishna, K.

Chromatographia 2008, 68, 481–484.

4. Guideline, I. H. T. IFPMA Geneva 2005.

5. Uchida, M.; Higashino, T.; Hayashi, E. J. Mass Spectrom. Soc. Jpn. 1973, 21,

245–254.

 

  

83  

Spectra 2.2.1: Mass spectra of impurity E5

 

Spectra 2.2.2: Mass spectra of impurity E6

 

Spectr

Spectr

ra 2.2.3: 1H

ra 2.2.4: 13C

NMR spect

NMR spect

tra of impur

tra of impu

rity E-NO

rity E-NO

84 

85  

 

Spectra 2.2.5: Mass spectra of impurity E-NO