ANALYTICAL SCIENCES MARCH 2012, VOL. 28 305
Erlotinib hydrochloride, 6,7-bis(2-methoxyethoxy)-N-(3-
ethynylphenyl)quinazolin-4-amine (Tarceva, R 1415, CP 358774,
OSI 774, NSC 718781), is a drug used to treat non-small cell
lung cancer, pancreatic cancer and several other types of cancer.
It is a tyrosine kinase inhibitor, which acts on the epidermal
growth factor receptor (EGFR). Erlotinib hydrochloride is an
orally active small molecule epidermal growth factor receptor
(HER1/EGFR) inhibitor approved in the United States and
Switzerland for the treatment of refractory locally advanced or
metastatic non-small cell lung cancer.1 Erlotinib was synthesized
from 6,7-bis-(2-methoxyethoxy)quinazolin-4-(3H)-one, which
in turn was prepared in an efficient way from commercially
available 3,4-dihydroxy benzaldehyde. Further, the compound
was reacted with thionyl chloride at reflux temperature to obtain
4-chloro-6,7-bis-(2-methoxyethoxy)quinazoline; this, upon
treatment with 3-ethynylaniline in DMF yielded erlotinib·HCl.2
The raw material and intermediates formed during the synthesis
may be present in bulk drug as an impurity; according to the
USFDA such impurities present at the levels of 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, unreacted raw materials and
intermediates, since they may possess unwanted toxicological
effects. Hence, thorough monitoring of related substances is of
higher importance for controlling the quality of erlotinib in the
final product. HPLC is the technique of choice for the separation
and estimation of related impurities in pharmaceutical
formulations. A thorough literature survey has indicated several
HPLC methods for the determination of erlotinib·HCl in
pharmaceutical formulations as well as in biological fluids using
ultra-violet and mass spectra detection.3
8 The HPLC method
was developed for the assay of erlotinib in the presence of
degradation products.9 The analysis of erlotinib and its
metabolites is conducted in rat tissue sections by MALDI
quadrupole time-of-flight mass.10 A specific method for
determination of erlotinib (OSI-774) and its metabolite
desmethyl erlotinib (OSI-420) in human plasma involves using
tandem mass spectrometry.7 All of these
methods were used for estimating erlotinib in pharmaceutical
formulations, bulk drugs and their metabolites in biological
fluids. A literature survey revealed that there is no HPLC
method for a simultaneous estimation of erlotinib and its
process-related impurities. The present work describes a simple
and rapid isocratic HPLC method for the simultaneous
determination of erlotinib and its process-related impurities.
This method is suitable for the quality assessment of erlotinib in
bulk drugs in the presence of process impurities. The developed
method was validated with respect to accuracy, precision,
linearity and ruggedness.
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 Corp. (USA), HPLC-grade acetonitrile and
ammonium acetate procured from Merck India Ltd. (India).
The HPLC system included two LC-10AT VP pumps, an
SPD-M10A VP photo-diode array detector, a CTO-10AS VP
oven and SCL-10A VP controller (all from Shimadzu, Japan).
A reverse-phase Inertsil ODS-3V (GL Sciences Inc., Japan)
column (25 cm × 4.6 mm i.d.; particle size, 5 μm) was used for
separation, and chromatograms were integrated using Class vp
The mobile phase was 1% ammonium acetate and acetonitrile
(55:45 v/v); before delivering into the column it was filtered
through a 0.45-μm nylon filter and degassed. The analysis was
carried out under isocratic condition using a flow rate of
2012 © The Japan Society for Analytical Chemistry
† To whom correspondence should be addressed.
Separation and Determination of Process-Related Impurities of Erlotinib
Using Reverse-Phase HPLC with a Photo-Diode Array Detector
Chandrashekara KARUNAKARA, Udupi APARNA, Venkateshappa CHANDREGOWDA, and
Chandrasekara G. REDDY†
Vittal Mallya Scientific Research Foundation (VMSRF), #94/3 & 94/5, 23 Cross, 29 Main, BTM II Stage,
A simple and rapid reverse-phase high-performance liquid chromatographic (HPLC) method for the simultaneous
separation and determination of erlotinib and its process-related impurities in bulk drugs has been developed. Five
process-related impurities of erlotinib have been separated on an Inerstsil ODS-3V (C18) column and detected at 254 nm
using a photo diode array (PDA). This HPLC method was successfully applied to the analysis of erlotinib bulk drug. The
recoveries of erlotinib and process-related impurities were in the range of 92.86
106.23%, and found to be specific,
precise and reliable for the determination of unreacted raw materials, intermediates in the reaction mixtures and bulk drugs.
(Received June 22, 2011; Accepted December 3, 2011; Published March 10, 2012)
306 ANALYTICAL SCIENCES MARCH 2012, VOL. 28
1.0 ml/min at 30°C. Chromatograms were recorded at 254 nm
using a PDA detector.
Solutions of (1000 μg/ml) erlotinib and its process-related
impurities were prepared by dissolving known amounts of
components in a mobile phase. These solutions were further
diluted to determine the accuracy, precision, linearity and limit
of detection and limit of quantification.
Results and Discussion
Optimization of chromatographic conditions
Figure 1 shows the synthetic process for the synthesis of
erlotinib. It can be seen from Fig. 1 that there are five
compounds that include the starting material and intermediates
that could be present as a potential impurity in erlotinib. The
present study was aimed at developing a chromatographic
system capable of eluting and separating erlotinib and its
All of the impurities and erlotinib were subjected to separation
by reverse-phase HPLC using different columns and mobile
phases. The separation and peak shapes were good on Inertsil
ODS-3V (4.6 × 250 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 is shown in
Fig. 2. It is evident from Fig. 2 that all of the compounds were
eluted and well separated with good peak shapes and resolutions.
The UV wavelength 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 the retention times of individual
compounds and studying the absorption spectra using a PDA
detector (Fig. 3). The developed method was validated with
respect to accuracy, precision, linearity and robustness.
To demonstrate the specificity of the method, erlotinib bulk
drug was spiked with known amounts of impurities, and
chromatographed. All of the impurities were well separated
from erlotinib; the chromatographic peak purity and the
homogeneity were evaluated with a PDA detector. The peaks
with a flat-top indicated that erlotinib has a homogeneous peak
with no impurities embedded in it. Also, the specificity was
checked by stressing pure erlotinib under UV light at 254 nm,
and under extreme conditions, such as 0.1 N NaOH, 0.3 N HCl
and 3% H2O2 at 60°C for 24 h. Under UV and acidic conditions
there was no change in the purity, but in alkaline and oxidative
conditions the degradation products were formed. However,
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. It can be seen
from the HPLC chromatogram of erlotinib bulk drug (Fig. 4)
that the process-related impurities were well separated from
The system suitability was evaluated by using 0.1% of all
impurities spiked to erlotinib (100 μg/ml), and evaluated by
making five replicate injections. The system suitability
parameters (retention time (tR), relative retention time (RRT)
and tailing factors) were evaluated, and are recorded in Table 1.
The linearity of the detector response to different concentrations
of impurities was studied by analyzing erlotinib spiked with
each impurity at seven levels ranging over 0.1
similarly, the linearity of erlotinib was studied by preparing
standard solutions at seven different levels, ranging over
300 μg/ml. The data were subjected to statistical analysis
using linear-regression model. The correlation coefficients (R2)
for erlotinib and its impurities were in the range of 0.995
3 - ethynylaniline, DMF
90 - 130 °C
E (Erlotinib HCl)
Fig. 1 Process for the synthesis of erlotinib.
Fig. 2 Typical chromatogram of erlotinib (E) spiked with 25 ppm of
each impurity (E0, E1, E2, E3 and E4).
ANALYTICAL SCIENCES MARCH 2012, VOL. 28 307
Accuracy and precision
The accuracy of the method for impurities was checked by
spiking each impurity at three different concentration levels,
ranging over 0.1
1.0 μg/ml (0.1, 0.4 and 1.0 μg/ml) to the
erlotinib at a specified level (100 μg/ml). All estimations were
made in triplicate (n = 3); the recoveries for all five impurities
were found to be 92.86
106.23%. The precision of the method
for impurities was tested by injecting six individual preparations
of 100 μg/ml of erlotinib spiked with 0.1 μg/ml of each
impurity. The recoveries of each impurity were calculated, and
the RSD was found to be in the range of 1.07
The accuracy and precision for determining the assay of
erlotinib was checked at three different levels: i.e., 50, 100 and
200 μg/ml each, in triplicate. Also, the RSD values were found
to be below 1.0%. The intermediate precision is the inter-day
variation at the same concentration levels determined on
Fig. 3 Comparative UV absorption spectra of impurities (E0, E1, E2, E3 and E4) in standard and bulk drugs.
Fig. 4 Typical chromatogram of a bulk drug (Uk, unknown
308 ANALYTICAL SCIENCES MARCH 2012, VOL. 28
successive days. The inter-day variations calculated for three
concentration levels are expressed in terms of RSD, %. At each
concentration level, the RSD, % values were below 1.5%,
indicating a good intermediate precision.
In order to evaluate the robustness of the method, the influence
of a small and deliberate variation of the analytical parameters
on erlotinib and its impurities was studied. The robustness was
studied by varying ±0.2 ml of the flow rate, ±2 ml of the
acetonitrile composition in the mobile phase and ±2°C in the
column temperature to the actual method parameters. In all of
the above variations, test samples were injected in triplicate.
There was a slight change in the retention times of erlotinib and
its impurities upon changing the mobile-phase concentration,
but all peaks were well separated without affecting the accuracy
of the quantitative estimation of erlotinib and the impurities.
There was no significant change observed upon changing the
flow rate and the temperature. The result indicates that the
method is suitable for the separation and estimation of erlotinib
and its synthetic impurities.
Limit of detection and quantification
The limit of detection (LOD) and quantification (LOQ)
represent the concentrations of the analytes that would yield a
signal-to-noise ratio of 3 for LOD and 10 for LOQ, respectively.
LOD and LOQ were determined by measuring the magnitude of
the analytical background by injecting blank samples and
calculating the signal-to-noise ratio for each compound by
injecting a series of solutions until the S/N ratio is 3 for LOD
and 10 for LOQ. LOD and LOQ of all compounds lie in the
range of 0.016
0.029 and 0.045
0.095 μg/ml, respectively.
Analysis of samples
First, accurately weigh 200 mg of an erlotinib bulk drug
sample into a 100-ml volumetric flask, and dissolve in the
mobile phase and make up. This solution was used to estimate
impurities. The impurities in bulk drug samples were identified
by comparing the retention times and the UV spectral curves
with that of standard impurities. The results are recorded in
Table 2. Almost all impurities were 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 assay for
determining erlotinib when carried out by diluting the above
solution to 200 ppm with the mobile phase ranged from 99.67 to
A simple reverse phase-HPLC method was developed and
validated for the simultaneous estimation of erlotinib and its
process-related impurities in erlotinib bulk drug. This method is
selective, sensitive and precise for estimating erlotinib and its
process-related impurities, which may be present in trace levels
in bulk drugs.
We thank Dr. Anil Kush, CEO, Vittal Mallya Scientific Research
Foundation, for his encouragement and support.
Tables containing linearity data, recovery data, precision data,
intra-day and inter-day precision assay, robustness data and
assay results of bulk drug erlotinib and its impurities (Tables S1
S6) are reported in supporting information. This material is
available free of charge on the Web at http://www.jsac.or.jp/
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Table 1 System suitability data
Compound Abbrevia tion tR/min RRT Tailing factor λmax/nm (ε)
Table 2 Results of analysis of bulk drugsa
Impurities, % (w/w) ± %RSD
0.01 ± 1.29
0.02 ± 0.53
0.03 ± 3.53
0.03 ± 1.10
0.01 ± 1.03
0.04 ± 2.44
0.02 ± 1.21
0.01 ± 2.87
0.02 ± 0.23
0.03 ± 0.82
0.05 ± 2.87
0.03 ± 1.37
a. n = 3, average of three determinations.