American Journal of Analyt ical Chemistry, 2011, 2, 75-83
doi:10.4236/ajac.2011.21008 Published Online February 2011 (
Copyright © 2011 SciRes. AJAC
Identification of Degradant Impurity in Gefitinib by Using
Validated RRLC Method
Madireddy Venkataramanna1,3, Indukuri Venkata Somaraju2,3, Kondra Sudhakar Babu3
1Hetero Labs Lt d., Hetero House, Santhnagar, India
2Invagen Pharmaceuticals INC, Hauppaug e, New York, USA
3Department of Che mistry, Sri Krishna Devaraya University, Anantapur, India
E-mail: {venky75, ivsraju}
Received August 4, 2010; revised Sept em ber 30, 2010; accepted October 5, 2010
Degradation pathway for gefitinib is established as per ICH recommendations by validated and stability in-
dicating reverse phase liquid chromatographic method. Gefitinib is subjected to stress conditions of acid,
base, oxidation, thermal and photolysis. Significant degradation is observed in acid and base stress condi-
tions. Two impurities are studied among which one impurity is found prominent degradant. The stress sam-
ples are assayed against a qualified reference standard and the mass balance is found close to 99.5%. Effi-
cient chromatographic separation is achieved on a Agilent make XDB-C18, 50 × 4.6 mm with 1.8 µm parti-
cles stationary phase with simple mobile phase combination delivered in gradient mode and quantification is
carried at 250 nm at a flow rate of 0.5 mL·min–1. In the developed RPLC method the resolution between ge-
fitinib and the potential impurities is found to be greater than 5.0. Regression analysis shows an r value (cor-
relation coefficient) of greater than 0.998 for gefitinib and the two potential impurities. This method is capa-
ble to detect the impurities of gefitinib at a level of 0.01% with respect to test concentration of 0.5 mg·mL–1
for a 4-µL injection volume. The developed RRLC method is validated with respect to specificity, linearity
& range, accuracy, precision and robustness for impurities determination and assay determination.
Keywords: Column Liquid Chromatography, Gefitinib, Forced Degradation, Validation, Stability Indicating
1. Introduction
Gefitinib: N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-
morpholin-4-yl propoxy) quinazolin-4-amine (Figure 1)
is an anticancer. The generic name of gefitinib is iressa;
gefitinib is a drug that is used to treat several types of
cancer. It works by preventing lung cancer cells from
growing and multiplying. Gefitinib is used alone (mono-
therapy) for the treatment of patients with a certain type
of lung cancer (non-small cell lung cancer or NSCLC)
that has not responded to chemotherapy [1-4]. Literature
survey reveals an analytical method is reported for the
determination of gefitinib in human plasma, mouse plas-
ma and tissues using high performance liquid chroma-
tography coupled to tandem mass spectrometry and a
method is reported for estimation of gefitinib in tablet
dosage forms by RP-HPLC [5-6]. As far as we are aware
there is no stability-indicating rapid resolution liquid
chromatography method for determination of related
substances and assay determination of gefitinib. In this
paper we describe validation of related substances and
assay method for accurate quantification of two potential
process impurities in gefitinib samples as per ICH rec-
ommendations. Intensive stress studies are carried out on
gefitinib; accordingly a stability-indicating method is
developed, which could separate various degradants.
The present active pharmaceutical ingredient (API)
stability test guideline Q1A (R2) issued by international
conference on harmonization (ICH) [7] suggests that
stress studies should be carried out on active pharmaceu-
tical ingredient (API) to establish its inherent stability
characteristics, leading to separation of degradation pro-
ducts and hence supporting the suitability of the pro-
posed analytical procedures. It also requires that analyti-
cal test procedures for stability samples should be stabil-
ity indicating and they should be fully validated. Ac-
cordingly, the aim of present study is to establish degra-
dation pathway of gefitinib through stress studies under a
Gefitinib: N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-
ylpropoxy) quinazolin-4-amine.
Impurity-1: 4-chloro-6-(3-morpholinopropoxy)-7-methoxyquinazoline.
Impurity-2: Ethyl-4-methoxy-6-nitro-3-[3-(4-morpholinyl) propoxy]
N-Oxide impurity: N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-mor-
morpholin-4-ylpropoxy) quinazolin-4-amine N oxide.
Figure 1. Chemical structures and labels of gefitinib and its
variety of ICH recommended test conditions [7-9].
2. Experimental Design
2.1. Chemicals
Samples of gefitinib and its impurities are received from
Hetero Laboratories Ltd, a research foundation of the
firm Hetero drugs Ltd, Hyderabad, India. HPLC grade
ammonium acetate and acetonitrile are purchased from
Merck, Darmstadt, Germany. Chromatographic reagent
grade hydrogen peroxide, hydrochloric acid, sodium hy-
droxides are purchased from Merck, Darmstadt, Ger-
many. High purity water is prepared by using Milli-Q
water purification system. All samples and impurities
used in this study are of having greater than 99.8% pu-
2.2. Procedure
2.2.1. Equipment
The RRLC system, used for method development and
method validation is Agilent 1200 RRLC. The output
signal is monitored and processed using chemstation
software on Pentium computer (Digital equipment Co).
RRLC is equipped with Binary gradient pump, Auto
Sampler, thermostatted column compartment, variable
wavelength detector, Auto sampler thermostatted (G
1330B), Computer with windows based chemstation
software version B.03.02. Photo stability studies are car-
ried out in a photo stability chamber equipped with Ze-
non arc lamp (Atlas Suntest CPS+;). Thermal stability
studies are carried out in a dry hot air oven (Cintex pre-
cision hot air oven).
2.2.2. Chromatographic Conditions
The chromatographic column used is Agilent make
XDB-C18, 50 × 4.6 mm with 1.8 µm particles. The mo-
bile phase is prepared by mixing buffer and acetonitrile
in the ratio of 40:60 (v/v). Buffer is prepared by dissolv-
ing 0.77 g of ammonium acetate dissolved in 1000 mL of
water. The flow rate of the mobile phase is maintained
0.5 mL·min–1. The column temperature is maintained at
40˚C and the detection is monitored at a wavelength of
250 nm.The injection volume is 4 μL. Diluent is a mix-
ture of buffer and acetonitrile in the ratio of 60:40 (v/v).
2.2.3. Preparation of Solutions
Stock solution of gefitinib (0.1 mg·mL–1) is prepared by
dissolving appropriate amount in the diluent.Working
solutions of 1.5 μg·mL–1 were prepared from above stock
solution for related compounds determination and assay
determination, respectively by dissolving in diluents.
Impurities stock solution (mixture of gefitinib, impu-
rity-1 & impurity-2) at a concentration of 0.1 mg·mL–1 is
also prepared in diluent.
2.3. Method Development and Optimization
Impurities and gefitinib solutions are prepared in diluent
at a concentration of 100 ppm and scanned in UV-visible
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
Figure 2. Typical chromatograms of specification level impurities spiking in 100% sample and stress samples.
Copyright © 2011 SciRes. AJAC
are analysed for an extended run time of 10 min to check
.4.2. Precision
the related substance method is checked
e method
.4.3. Sensitivity
mined by establishing the limit of de-
.4.4. Linearity and Range
LOQ to 150% with respect
.4.5. Accuracy
of the impurity stock solutions are
.4.6. Robustness
obustness of the developed method,
experimental conditions are deliberately changed and the
spectrometer; the two impurities and gefitinib are having
UV maxima at 250 nm which is selected for method de-
velopment purpose. To achieve separation of gefitinib
from its impurities and degradation products chroma-
tographic method is developed using various stationary
phases like C8 and C18, different mobile phases con-
taining buffers like phosphate and acetate and using or-
ganic modifiers like acetonitrile and methanol in the mo-
bile phase across the pH 2 to 8. Mobile phase of ammo-
nium acetate and acetonitrile (60:40, v/v) is selected for
initial trial on a BEH C18, 50 × 4.6 mm with 1.8 µm
particles and flow rate at 1.0 mL·min–1. Spike sample
analysis revealed that principal peak retention time is late
and impurities 1 and 2 are not resolved properly. Similar
results are obtained with Agilent XDB, C8 50 × 4.6 mm
with 1.8 µm particles, with the 250 mm the tailing factor
is observed more than 3 (broad shape).
To increase the resolution between each component
Agilent XDB C18 column with the dimensions 50 × 4.6
with 1.8 µm particles is selected. After several other tri-
als satisfactory results (retention time of gefitinib is
~5.935 min and the resolution between all the impurities
is >5.0) are obtained. In the optimized conditions gefit-
inib, impurity-1, impurity-2 are well separated with a
resolution greater than 5.0 and the typical retention times
of gefitinib, impurity-1 and impurity-2, are about 5.923,
2.978 and 4.575 min respectively meeting the chroma-
tographic system suitability requirements. In XDB C18
column with the dimensions 50 × 4.6 mm with 1.8 µm
particles the components are separated with resolution
above 5. In the optimized conditions tailing factor for all
the components is observed between 0.8-1.5. Theorical
plates are observed above 8000.
2.4. Analytical Method Validation
The developed chromatographic method is validated for
specificity and stress studies, sensitivity, linearity &
range, precision, accuracy, robustness and system suit-
ability [10-15].
2.4.1. Specificity and Stress Studies
Specificity is the ability of the method to measure the
analyte response in the presence of its potential impuri-
ties. The specificity [10-11] of the developed LC method
for gefitinib is determined in the presence of its impuri-
ties namely impurity-1 and impurity-2 at a concentration
of 1.5 µg·mL–1 and degradants. The stress conditions
employed for degradation study includes photolytic (car-
ried out as per ICH Q1B), thermal (100˚C), acid hy-
drolysis (1M HCl), base hydrolysis (2 M NaoH) and
oxidation (6% H2O2). All stressed samples of gefitinib
the late eluting degradants. Assays are carried out for
stress samples against qualified reference standard and
the mass balance (% assay + % of impurities + % of
degradation products) is calculated for all the samples.
The precision of
by injecting six individual preparations of (0.5 mg·mL–1)
gefitinib spiked with 0.03% each impurity. The % RSD
for percentage of each impurity is calculated.
The intermediate precision (ruggedness) of th
evaluated by different analyst using different column,
different day and different analyst in the same laboratory.
Precision is determined through repeatability (intra-day)
and intermediate (inter-day) precision and calculated the
% RSD for the area of each component.
Sensitivity is deter
tection (LOD) and limit of quantification (LOQ) for ge-
fitinib, impurity-1 and impurity-2 estimated based on
signal-to-noise ratio method, by injecting a series of di-
lute solutions with known concentration. The precision
study is also carried out at the LOQ level by injecting six
individual preparations of impurity-1 and impurity-2 and
calculated the % RSD for the areas of each component.
Linearity test solutions from
to test concentration are prepared by diluting the impu-
rity stock solution to the required concentrations. For
assay method test solutions from 50% to 150% with re-
spect to test concentration are prepared by diluting the
stock solution to the required concentrations. The corre-
lation coefficient, slope and Y-intercept of the calibration
curve are calculated for the both related substances and
assay methods.
A known amount
spiked to the previously analysed samples at LOQ (100%
sample + 0.03% impurities), 100 (100% sample + 0.15%
impurities) and 150% (100% sample + 0.225% impuri-
ties) of the analyte concentration (0.5 mg·mL–1). The
percentage of recoveries for impurity-1, impurity-2 are
calculated. A known amount of gefitinib stock solution
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 re-
coveries is calculated.
To determine the r
he solution stability of gefitinib and its related impuri-
ent stress condi-
ons suggested the following degradation behavior. Ta-
egradation in Acid Stress Condition
efitinib is exposed for degradation with time in 1 M
efitinib is exposed for degradation with time in 2 M
on is ob-
resolution between each component is evaluated. The
flow rate of the mobile phase is 0.5 mL·min–1. To study
the effect of flow rate on the resolution, 0.03 units
changed i.e. 0.3 and 0.7 mL·min–1. The effect of column
temperature on resolution is studied at 35˚C and 45˚C
instead of 40˚C. In the all above varied conditions, the
components of the mobile phase are held constant.
2.4.7. Solution Stability and Mobile Phase Stabili
ties are carried out by leaving spiked sample solution
tightly capped volumetric flask at room temperature for
48 h. Impurity content is determined for every 6 h inter-
val up to the study period. Mobile phase stability is also
carried out for 48 h by injecting the freshly prepared
sample solutions for every 6 h interval. Impurity content
is checked in the test solutions. Mobile phase prepared is
kept constant during the study period.
3. Results and Discussion
3.1. Specificity and Stress Stu
Stress studies on gefitinib under differ
ble 2.
3.1.1. D
HCl upon heating for 6 h and no degradation is
3.1.2. Degradation in Base Stress Condition
NaOH upon heating for 6 h and no degradati
3.1.3. Degradation in Peroxide Stress Condition
Gefitinib is gradually undergone degradation with time
in 6% H2O2 upon heating for 2 h and prominent degrada-
tion is observed as gefitinib N-Oxide. Gefitinib is sensi-
tive to oxidative condition and is degraded into unknown
impurities by oxidation using 6% H2O2. Gefitinib has
shown significant sensitivity towards oxidative treatment.
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 in-
dicate the N-Oxide of gefitinib. The chemical name of
the degradant at RT ~ 20.0 min could be N-(3-chloro-4-
quinazolin-4-amine N oxide (Figure 4).
3.1.4. Degradation in Neutral Water Stress Condition
Gefitinib is exposed water heating for 8 hours at 80˚C,
no degradation is observed.
3.1.5. Photolytic Stress Condition
Gefitinib is exposed to light for an overall illumination of
1.2 million Klux hours and an integrated near ultraviolet
energy of 200-watt hours/square meter (w/mhr) (in photo
stability chamber), no degradation is observed.
Table 1. System suitability report.
Compound USP resolution
(RS )
USP tailing
No. of theoretical
plates (USP
tangent method)
Impurity-1--- 1.2 9998
Impurity-210.842 1.4 12115
Gefitinib 6.440 1.2 9099
Table 2. Summary of forced degradation results.
Stress condition %Total impuriactive substance
purities +
% degradation p
Remarks ties Study time % Assay of Mass bal
(% assay + % im
Acid hydrolysis
(1 M HCI )
No degraducts were
0.15% 6 h 99.4 99.6 ation prod
Base hydrolysis
(2 M NaOH) 0.15% 6 h 99.4 99.6 No degradation product is
(15% H202) 14.0% 2 h 86.2 100.2 served fied as
Th ) 0.16% 168 h 99.5 99.7
1200 /HrNs
One major degradation ob-
and identi
N-oxide impurity of gefitnib
by LC-MS
No degradation products
ermal (100˚C
Light (photolytic
degradation) 0.15% KLUX99.7 99.7 o prominent degradation i
Copyright © 2011 SciRes. AJAC
3.1.6ss Condi
efitin to dry heat at 0˚C fono
tinib, impurity-1 and impu-
of area% of each impurity in precision
veloped analytical method
ediate pre study for gefiturity-1,
efitinib, impurity-1 and impu-
.01% (of analyte concentration, i.e.
. Thermal Stretion
10Gib exposedr 168 hours,
degradation is observed. The mass balance of stresse
samples is close to 99.5%.
The assay of gefitinib is unaffected in the presence of
two impurities and its degradants confirm the stability
indicating power of the developed method.
3.2. Method Validation
3.2.1. Precision
he %RSD of area of gefiT
rity-2 and %RSD
study are less than 5.0 confirming the good precision of
impurity-2 are less than 5.0, confirming the intermediate
precision of the method.
3.2.2. Sensitivity
he limit of detection of g
the de
. The %RSD obtained in
inib, impcision
rity-2 is 0.01 and 0
0.50 mg·mL) 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 vol-
ume. The % RSD for area of impurity-1 and impurity-2
are less than 5.0 for precision at LOQ level.
Figure 3. Linearity chart for gefitinib and its impurities.
Copyright © 2011 SciRes. AJAC
3.2.3. Linearity and Range
Calibration curve obtained by the least square regression
analysis between peak area and concentration exhibited
linear relationship with a correlation coefficient of 0.998
over the ranges tested. Linear calibration plot for related
substance method is obtained over the ranges tested, i.e.
LOQ to 0.225% for Impurity-1, Impurity-2 and LOQ to
0.15% for gefitinib. The correlation coefficient obtained
is greater than 0.998 for the two impurities and gefitinib.
% Y-intercept is below 5.0 as per acceptable validation
practices. The result shows an excellent correlation ex-
isted between the peak area and concentration of gefit-
inib and impurities. Linear calibration plot for assay de-
termination method is obtained over the calibration
ranges tested, i.e. 50% to 150% for gefitinib and the cor-
relation coefficient is found more than 0.999.The results
show an excellent correlation existed between the peak
area and concentration of gefitinib in assay determination
Figure 4. Mass spectrum of oxidation degradation impurity (Gefitinib N-oxide impurity) N-(3-chloro-4-fluoro-phenyl)-7-
methoxy-6-(3-morpholin-4-ylpropoxy) quinazolin-4-amine N oxide.
Copyright © 2011 SciRes. AJAC
Table 3. Linearity, regression results for impurities and gefitinib.
Impurity-1 I mpurit y-2
Parameter Gefitinib
Trend line equation Y = 221.31x 4758 Y = 147.38x 0.3698 Y = 307.72x 1.0334
Range 0.03% - 0.15% 0.03% - 0.15% 0.03% - 0.15%
Regression Coefficient 0.999 0.998 0.998
Slope 221.31 147.38 307.72
Intercept 0.4758 0.3698 1.0334
%Intercept with respect to 100%
conc. response 1.84 2.22 2.99
Residual sum of Squares 0.5688 0.5562 2.7990
method (Figure 4, Table 3).
3.2.4. Accuracy
The percentage recovery of impurity-1 and impurity-2 in
bulk drug samples ranged from 97.35-101.91. HPLC
chromatogram of Gefitinib bulk drug sample spiked with
the two impurities is shown in Figure 2 (Table 4).
3.2.5. Robustness
Analysis results for deliberately changed chromatogra-
phic conditions (flow rate and column temperature) re-
vealed that the resolution between closely eluting impu-
rities, namely gefitinib and impurity-2 is greater than 5.0,
illustrating the robustness of the method. Repeatability
and reproducibility results also within the % RSD of 5.0.
3.2.6. Solution Stability and Mobile Phase Stability
The %RSD of assay of gefitinib during solution stability
and mobile phase stability experiments is within 1.0. No
significant changes are observed in the content of impu-
rity-1 and impurity-2 during solution stability and mobile
phase stability experiments. 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.
Analysis is performed for different samples of gefit-
Table 4. Results study for related s.
inib (n = 3). The two impurities in these samples are less
than 0.1%.
of accuracycompound
Amount of impurity
added (μg) to the 100%
sample (n)
% Recovery
of impurity-1
% Recovery of
0.03 99.80 101.91
0.10 99.08 101.77
0.15 101.23 97.35
n = 3, number of determinations.
4. Conclusion
The degradation pathway of gefitinib is established as
per ICH recommendations. The proposed method is
validated as per ICH requirements. The isocratic RRLC
method developed can be used for stress studies and for
quantitative determination of related substance and assay
of gefitinib. The developed method is stability indicative
and can be employed in routine analysis of gefitinib
production samples and also to analyze stability samples.
5. Acknowledgements
The authors grateful to the management of Hetero labs
limited for the extensive support in achieving this work.
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