ANALYTICAL TECHNIQUE:

1) RP-HPLC (REVERSED PHASEJ HIGH PRESSURE LIQUID CHROMATOGRAPHY):

Reversed phase chromatography uses polar solvents as mobile phase and non-polar
solvent as stationary phase. This method is developed according to the ICH
Guidelines. This method is used to measure the concentration of the afatinib in the
short period of time. This high speed method is used as it eliminates or reduces the
cost or waste that is used in the preparation of the raw material.
USE OF THIS METHOD: It is used for the qualitative analysis of afatinib in bulk and
pharmaceutical products. The main aim of this method is to develop a precise,
simple, accurate, rapid method for analysis of afatinib. Afatinib dimaleate has UV
absorption maxima at 258 nm. Hence conventional reverse phase HPLC has been
selected for its estimation in pharmaceutical dosage forms.
REQUIREMENTS:
 REAGENTS: Acetonitrile, methanol HPLC grade (Qualigens) and ortho-
Phosphoric acid (Rankem)
 CHROMATOGRAPHIC CONDITIONS:
a) Column: X-Terra RP-8, (250mmx4.6mm, 5µ)
b) Mobile Phase: Solvent-A is potassium dihydrogen
orthophosphate buffer system adjusted pH-3.0 with o-
phosphoric acid and Solvent-B is Acetonitrile: methanol
(70:30v/v)
c) Diluent: Water: Acetonitrile (30:70 v/v)
d) Flow rate: 1.0 ml/min.
e) Run time: 20 min
f) Temperature: Ambient. Injection
g) volume: 10 μl
h) Detection wavelength 258 nm
i) Retention time: 10.558 min

 EXPERIMENTAL: Quantitative HPLC was performed on Agilent liquid

Chromatography, with PDA detector equipped with automatic injector with
injection volume 20 µl, and 2693 pump. X-Terra RP-8, 250mmx4.6mm, 5µ was
used. The HPLC system was equipped with LC solution Software.

PREPARATION OF STANDARD AND SAMPLE SOLUTION:

 STANDARD DRUG SOLUTION: Take 150.2mg of afatinib into 50ml volumetric
flask and dissolve into 30ml of diluent and by the process of sonication,
sonicated it for 15min and then made up the final volume up to 50ml. Then
take 5ml of the solution in the 50ml of volumetric flask and dilute to get
0.3004mg/ml solution.
 SAMPLE SOLUTION: Take 20 tablets and crushed in a pestle and mortar.
Weigh accurately and transfer 300.4mg of Gilotrif tablet (Afatinib marketed
preparation) powder into 50ml volumetric flask. Dissolve into 30 ml of
 diluent. For 1hr allow the mixture to stand with intermittent sonication for
complete solubility. Now filter the solution through a 0.45 μm membrane
filter. Made the final up to 50ml with the help of diluent. Then take 5ml of the
solution into 50ml volumetric flask to get a solution of 0.6008mg/ml which is
used as working solution.
 WORKING STANDARD SOLUTION: Take 1ml of the Standard stock solution in
10 ml volumetric flask. Made volume up to 10 ml with mobile phase to get a
concentration of 300.4µg/ml.
ANALYSIS OF AFATINIB TABLET: To perform this, marketed preparation of afatinib
i.e .Gilotrif tablets which contains afatanib salt of strength 20mg form is taken.
STEPS:
a) Weight accurately and take 29.56mg of the afatanib dimaleate in 50ml
volumetric flask and mixed it with 30ml of the diluent.
b) Allow the mixture to stand for 30minutes with intermittent sonication for the
drug to be completely soluble.
c) Filter the above prepared solution through a 0.45 μm membrane filter.
d) Dilute the above filtered solution with 50ml diluent to obtain a stock solution
of 0.6mg/ml and this solution work as working solution.
e) The results obtained from this is similar to the statistically accepted criterion.
VALIDATION OR SUBSTANTIATION METHODS:

 SYSTEM SUITABILITY: It is used to ensure that our system is working perfectly before
starting the analysis on HPLC or any other analyzing technique. It is required to done
before every sample analysis. Also it is used to check the specifications of a liquid
chromatographic system. The testing limits for the system stability should comply to
the guidelines issued by CDER (Centre For Drug Evaluation and Research).
 SPECIFICTY: It is ability of the analytical technique to distinguish between the
analyte(s) and other components in the matrix of sample. In case of an HPLC
method, specificity is done by complete separation of the different peaks originated
from the sample matrix.
 PRECISION: The precision of an analytical procedure expresses the closeness of
agreement between a series of measurements obtained from multiple sampling of
same sample under the prescribed conditions.
 LINEARITY: Validation for linearity requires the preparation and analysis of a set of
several independently prepared solutions. As an example, according to ICH
guidelines, HPLC method linearity is normally based on five concentration levels
between 70% and 130% of the nominal concentration, each to be injected three
times.
 ROBUSTNESS: The robustness of a method refers to its stability in the face of minor
alterations in operating conditions. To assess the method's robustness, deliberate
modifications were made to the experimental conditions at three different levels,
focusing on variations in retention time and chromatographic response.
CONCLUSION: From the above studies, RP-HPLC method is considered as simple, accurate
and rapid method for quantitative analysis of afatinib in bulk and pharmaceutical dosage
form. The results which is obtained after analysis is accurate and the proposed method was
found to be Linear, precise and accurate for the quantitative estimation of Afatinib in tablet
formulations.