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METHOD DEVELOPMENT AND VALIDATION FOR THE
ESTIMATION OF ANTI-CANCER DRUG ERLOTINIB IN
MARKETED PHARMACEUTICAL DOSAGE FORMS BY RP-
HPLC
Kalepu Swathi*, Pathange Revathi Bai, Saleha Nuzhath, Sofia Jabeen, Yusra Fatima, Amgooth
Sharadha
ABSTRACT
The study set out to perfect an RP-HPLC technique for the measurement of Erlotinib in both bulk and
pharmaceutical dose forms that was quick, easy, and reliable. This particular chromatographic
separation was run on a Symmetry ODS (C18) RP Column that measured 250 mm in diameter by 4.6 mm
in internal diameter and had a 5 m particle size. In order to determine the eluents, a Phosphate Buffer
and Methanol mixture (46:54 v/v) was used, and the UV detector was set to a wavelength of 206 nm
(pH-3.2). The temperature in the column was kept at room temperature. According to the ICH criteria,
we investigated the following validation parameters: system appropriateness; linearity; precision;
accuracy; specificity; limit of detection (LOD); limit of quantitation (LOQ); and robustness. Erlotinib had a
retention time of 3.52 minutes and 25 seconds. Additional testing of the assay showed that the limit of
detection for Erlotinib was 0.08 g/ml and the limit of quantitation was 0.24 g/ml. Erlotinib was shown to
have an overall success rate of between 98% and 102%. Erlotinib's %RSD was determined to be suitable
for precision and moderate precision. Erlotinib was tested for linearity from 60% to 140%, and the
results were R2 = 0.9993, an intercept of 48313x, and a slope of 71969. The procedure was quick, exact,
sensitive, and precise; as a result, it ma use in quality control labs and the pharmaceutical industry for
regular testing of Erlotinib-containing drugs.
key words: RP-HPLC, erlotinib, accuracy, robustness, linearity, ICH guidelines, are some of the here.
INTRODUCTION
Erlotinib is a tyrosine kinase receptor inhibitor
used to treat pancreatic and non-small cell lung
cancer that has progressed or spread. Serum
aminotransferase increases following erlotinib
treatment are common, and clinically evident
acute liver damage occurs seldom. Erlotinib1 is
an antineoplastic quinazoline derivative.
Erlotinib binds reversibly to the intracellular
catalytic domain of epidermal growth factor
receptor (EGFR) tyrosine kinase, preventing
EGFR phosphorylation and the signal
transduction events and tumorigenic
consequences associated with EGFR activation
by competing with adenosine triphosphate.
Erlotinib is a quinazoline molecule that has an
amino group (3-ethynylphenyl) at the 4-position
and two 2-methoxyethoxy groups at the 6- and
7-positions. It acts as a protein kinase
Bojjam Narasimhulu , Pharmacy College for Women, Road Number 2, Vinayak Nagar, Vani Nagar, Saroor
Nagar West, Sayeedabad, Hyderabad, Telangana 500059
Department: Pharmaceutical Chemistry ,Mail id: swathi.kalepu@gmail.com

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inhibitor and an epidermal growth factor
receptor antagonist in addition to being an
antineoplastic drug. It is an aromatic ether, an
aromatic acetylenic molecule, and an amino
compound with a secondary amine group,
making it a member of the quinazoline family.
Erlotinib2 is prescribed for patients whose
epidermal growth factor receptor (EGFR)
cancers have either exon 19 deletions or exon
21 (L858R) replacement mutations and have
progressed or spread to other parts of the body.
For patients with locally advanced,
unresectable, or metastatic pancreatic cancer,
in addition to initial therapy. For patients with
NSCLC whose tumors have additional EGFR
mutations, the safety and effectiveness of
Erlotinib3 have not been demonstrated. Also, it
shouldn't be used at the same time as platinum-
based chemotherapy. N-(3-ethynyl phenyl)-6, 7-
bis(2-methoxy ethoxy)quinazolin-4-amine is the
IUPAC name for Erlotinib. Erlotinib's molecular
structure is as shown below.
Fig-1: Chemical Structure of Erlotinib
According to the literature review31-35, only a
handful of spectrophotometric and RP-HPLC
techniques have been published for estimating
Erlotinib alone or in combination with other
medications. Here, we make an effort to create
a straightforward, cost-effective RP-HPLC
technique for estimating Erlotinib in bulk form
and commercially available pharmaceutical
dosage forms in accordance with ICH
Guidelines30.
EXPERIMENTAL
Table-1: List of Instrument used
S. No. Instruments/Equipments/Apparatus
1. HPLC with Empower2 Software with Isocratic with UV-Visible Detector
(Waters).
2. T60-LAB INDIA UV – Vis spectrophotometer
3. Electronic Balance (SHIMADZU ATY224)
4. Ultra Sonicator (Wensar wuc-2L)
5. Thermal Oven
6. Symmetry ODS RP C18,5m, 15mm x 4.6mm i.d.
7. PH
Analyzer (ELICO)
8. Vacuum filtration kit (BOROSIL)

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Table-2: List of Chemicals Used
S.No. Name
Specifications
Manufacturer/Supplier
Purity Grade
1. Doubled distilled water 99.9% HPLC Sd fine-Chem ltd; Mumbai
2. HPLC Grade Water 99.9% HPLC Sd fine-Chem ltd; Mumbai
3. Methanol 99.9% HPLC Loba Chem; Mumbai.
4. Hydrochloric Acid 99.9 A.R. Sd fine-Chem ltd; Mumbai
5. Acetonitrile 99.9% HPLC Loba Chem; Mumbai.
6. Sodium Hydroxide 99.9 A.R. Sd fine-Chem ltd; Mumbai
7. Ethanol 99.9 A.R. Sd fine-Chem ltd; Mumbai
8. Octanol 99.9 A.R. Sd fine-Chem ltd; Mumbai
Wavelength Selection
How to Prepare a Sample and a Standard for
Analysis by UV-Spectrophotometry
After transferring 25 mg of Erlotinib standard
into a 25 ml volumetric flask, we dissolved it in
mobile phase and brought the volume up to 25
ml. The aforementioned solution was further
diluted by placing 0.5 ml in a 10 ml volumetric
flask and filling it up with mobile phase4.
Chromosome Separation Process
Improvement:
Several methods were used to fine-tune the
chromatographic conditions5. (Varying
conditions such as column type, mobile phase,
flow rate, detection wavelength, and sample
diluents.
Table-3: Trials for Method Development
Column Used Mobile Phase Flow
Rate
Wave
length
Observation Result
Symmetry ODS (C18)
RP Column, 250 mm x
4.6 mm, 5µm
Methanol :
Acetonitrile = 20 :
80
1.0ml/min 206nm Very Low
response
Method
rejected

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Symmetry ODS (C18)
RP Column, 250 mm x
4.6 mm, 5µm
Methanol : Water
= 70 : 30
1.0ml/min 206nm Low response Method
rejected
Symmetry ODS (C18)
RP Column, 250 mm x
4.6 mm, 5µm
Acetonitrile:
Water = 50 : 50
1.0ml/min 206nm Tailing peaks Method
rejected
Symmetry ODS (C18)
RP Column, 250 mm x
4.6 mm, 5µm
Phosphate
Buffer :
Acetonitrile =
85:15 (pH-4.8)
1.0ml/min 206nm Resolution
was not good
Method
rejected
Symmetry ODS (C18)
RP Column, 250 mm x
4.6 mm, 5µm
Phosphate
Buffer : Methanol
= 65:35 (pH-4.0)
1.0ml/min 206nm Tailing peak Method
rejected
Symmetry ODS (C18)
RP Column, 250 mm x
4.6 mm, 5µm
Phosphate
Buffer : Methanol
= 46:54 (pH-3.2)
1.0ml/min 206nm Nice peak Method
accepted
Preparation of 0.01M Potassium dihydrogen
orthophosphate Solution:
Potassium dihydrogen orthophosphate,
weighing around 1.36086 grams, was weighed
and transported to a 1000 ml beaker, where it
was dissolved in HPLC Grade water and diluted
to 1000 ml. Diluted orthophosphoric acid was
used to bring the pH level down to 3.20.
Phase I: Mobile Device Preparation
In an ultrasonic water bath for 15 minutes, 460
ml of phosphate buffer (0.05M) pH 3.20 was
thoroughly combined with 540 ml of HPLC
Grade Methanol to remove all traces of air. In
order to filter the fluid, a 0.45 m filter was used
in a vacuum filtration6 system.
Discussion and Results
Process Planning Frequency Choice

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Both the standard and the sample were
dissolved in the mobile phase7 solvent before
being diluted with the same solvent to create
their respective stock solutions.
(Once all variables have been fine-tuned) UV
analysis. It scanned the ultraviolet spectrum
from two hundred to four hundred nanometers.
In order to accurately estimate the Erlotinib
concentration in an HPLC UV detector, this has
been done to determine its maximum
concentration. The ultraviolet (UV) spectrum
scan is included on the next page.
Fig-2: UV spectrum for Erlotinib
In scanning the Erlotinib solution, we found that
its maximum intensity was visible at 206 nm.
The Best Conditions for Chromatography,
Summarized
Below is a summary of the experimentally
determined Optimal Chromatographic
Conditions8:
Table-4: Summary of Optimised Chromatographic Conditions
Mobile phase Phosphate Buffer : Methanol = 46:54 (pH-3.2)
Column Symmetry ODS (C18) RP Column, 250 mm x 4.6
mm, 5µm
Column Temperature Ambient
Detection Wavelength 206 nm
Flow rate 1.0 ml/ min.
Run time 08 min.
Temperature of Auto sampler Ambient
Diluent Mobile Phase
Injection Volume 10µl
Type of Elution Isocratic
Retention time 3.622 minutes

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SUMMARY AND CONCLUSION
Different chromatographic settings were
utilized, and the findings were published in
earlier chapters, in order to establish a precise,
linear, specific, and suitably stable indicating
RP-HPLC technique for analysis of Erlotinib. It's
easy to get consistent results with isocratic
elution since it simply calls for a single pump
and a flat baseline separation. In light of that, it
was chosen as the method of choice for this
investigation rather than gradient elution.
Several column options exist for RP-HPLC,
however in this instance It was decided that the
Symmetry ODS (C18) RP Column, 250 mm x 4.6
mm, 5m was the best option.
The form of the peaks, the resolution, and the
absorbance of the column were all satisfactory.
After analyzing the API's solubility in the
available solvents, we settled on a mobile phase
and diluent for sample preparation (methanol,
Acetonitrile, dichloromethane, water, 0.1N
NaOH, 0.1NHCl). The typical medicine
arrangement was analyzed, and a recognition
wavelength between 200 and 400 nm was
selected. Erlotinib's U.V. spectrum reveals that
several HPLC tasks benefit from being improved
within a wavelength range of 206 nm. In
addition, it was found that the optimal testing
conditions were a stream rate of 1 ml/min and
an infusion volume of 10 l. The results show
that the developed method is remarkable,
another ideal technique for test and steadiness-
related contamination considerations that may
aid the investigation of Erlotinib in diverse
definitions.
Erlotinib, both in its active pharmaceutical
ingredient (API) form and its commercially
available pharmaceutical dosage form, may
now be tested using a sensitive and specific RP-
HPLC method. The suggested RP-HPLC method
has excellent influence, accuracy, and
reproducibility, thus it should be promoted.
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Development and validation of RP-HPLC
technique for the quantification of Erlotinib in
pharmaceutical formulation, Arabian Journal of
Chemistry, Volume 10, Supplement 1, February
2017, Pages: S1138-S1144, S. T. Latha, S.
Ananda Thangadurai, M. Jambulingam, K.
Sereya, D. Kamala kannan, M. Anilkumar.
To cite this article: V. Kalyana Chakravarthy*
and D. Gowri Sankar, "Development and
Validation of RP-HPLC Method for Estimation of
Erlotinib in Bulk and its Pharmaceutical
Formulation," Rasayan Journal of Chemistry,
Volume 4, Issue 2 (2011), Pages 393-399.
For example: "Development and validation of
RP-HPLC and UV technique for Erlotinib
hydrochloride tablets," by R. Vijay Amirtharaj1,*
and S. Lavanya1, in the IP International Journal
of Comprehensive and Advanced
Pharmacology, 2021;6(3):144-151.
Journal of Drug Delivery and Therapeutics;
2013; 3(1):50-54 VS Saravanan*, Bojja
Mallikarjuna Rao, Analytical Method
Development and Validation for the
Determination of Erlotinib Hydrochloride Bulk
and in Pharmaceutical Dosage Form.
Indian Journal of Pharmaceutical Education and
Research 2021; 55(2s):s589-s594. Purvini K1,
Chethan IA2, Jaishree Vaijanathappa2,*, Novel
Analytical Method Development, Validation,
and Stability Study of Anticancer Drug Erlotinib
in Tablet Dosage Form by RP-UFLC.