METHOD DEVELOPMENT

VALIDATION, AND
SIMULTANEOUS
ESTIMATION OF
AMLODIPINE BESYLATE
AND TELMISARTAN BY
RP-HPLC.
LIST OF ABBREVIATIONS
Avg Average
C.F Conversion Factor
Diln Dilution
FDA Food and Drug Administration
HPLC High Performance Liquid Chromatography
ICH international Conference Of Harmonization
LC Liquid Chromatography
LC-MS Liquid Chromatography-Mass Spectroscopy
LOD Limit of detection
LOQ Limit of quantitation
NMT Not more than
NLT Not less than
ODS Octyl deactivated silane
ppm Parts Per Million
RSD Relative Standard Deviation
RT Retention time
RP-HPLC Reverse Phase High Performance Liquid Chromatography
SPL Sample STD Standard
SD Standard Deviation
CONTENTS:
1.INTRODUCTION
2. LITERATURE REVIEW
3. OBJECTIVE AND PLAN OF WORK
4. DRUG PROFILE
5. MATERIALS AND INSTRUMENTS USED
6. METHOD DEVELOPMENT AND OPTIMIZATION
7. METHOD VALIDATION
8. CHROMATOGRAMS
9. RESULTS AND DISCUSSION
10. SUMMARY AND CONCLUSION
11. BIBLIOGRAPHY
INTRODUCTION:
Analytical chemistry is the science to analyze morphologies, compositions, and
quantities of analytical targets. These analytical results have played critical
roles from the understanding of basic science to a variety of practical
applications, such as biomedical applications, environmental monitoring,
quality control of industrial manufacturing, and forensic science, to name a
few (P.D.Sethi,2001). Modern analytical chemistry is dominated by instrumental
analysis. There are so many different types of instruments today that it can
seem like a confusing array of acronyms rather than a unified field of study.
Many analytical chemists focus on a single type of instrument. Academics tend
to either focus on new applications and discoveries or on new methods of
analysis. An effort to develop a new method might involve the use of a tunable
laser to increase the specificity and sensitivity of a spectrometric method.
Many methods, once developed, are kept purposely static so that data can
be compared over long periods of time.
SPECTROSCOPY

Atomic Absorption And Emission Spectroscopy(AAS/AES):

To analyse alkali and alkaline earth metals in dilute solution, natural liquids, and
extracts at trace levels.
Ultraviolet-Visible Spectroscopy (UV/Vis):
To analyse molecular (organic) and ionic species capable of absorbing at UV
or Visible wavelengths in dilute solutions.
Fourier Transform Infrared Spectroscopy (FT-IR): To analyse only molecular
compounds (organic compounds, natural products, polymers, etc.)
Nuclear Magnetic Resonance Spectroscopy (NMR)
To identify and characterize the organic and inorganic compounds.
Microwave Spectroscopy:
To analyzse simple gaseous molecules in Far IR region, to study their stereo
chemistry.
Electron Spin Resonance Spectroscopy (ESR):
To study the formation and life time of the free radicals formed in organic
reactions and also finds applications in biological works.
CHROMOTOGRAPHY:

Introduction:
Chromatography (from Greek: chroma, colour and "grafein" to
write) is the collective term for a family of laboratory techniques for the
separation of mixtures. It involves passing a mixture dissolved in a
"mobile phase" through a stationary phase, which separates the
analyte to be measured from other molecules in the mixture and allows
it to be isolated(P.D.Sethi, 2001). Chromatography may be preparative
or analytical. Preparative chromatography seeks to separate the
components of a mixture for further use (and is thus a form of
purification). Analytical chromatography normally operates with
smaller amounts of material and seeks to measure the relative
proportions of analytes in a mixture. The two are not mutually exclusive
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY METHOD :

HPLC was introduced commercially in 1969 and since then it has undergone extensive
modifications and innovation which lead to its emergence as the foremost analytical tool
for quantitative analysis. HPLC is a type of liquid chromatography that employs a liquid
mobile phase and a very finely divided stationary phase. In order to obtain a satisfactory
flow rate liquidmust be pressurized to a few thousands of pounds per square inch. The
rate of distribution of drugs between stationary and mobile phase is controlled by
diffusion process. If diffusion is minimized, a faster and effective separation can be
achieved. The technique of high performance liquid chromatography is so called
because of its improved performance when compared to classical column
chromatography. Advances in column technology, high pressure pumping system and
sensitive detectors have transformed liquid column chromatography into high speed,
efficient, accurate and highly resolved method of separation.
 Principle:

Principle of separation in HPLC The principle of separation in normal phase mode and
reverse phase mode is adsorption. When a mixture of components is introduced in to a
column, they travel according to their relative affinities towards stationary phase. The
component which has more affinity towards the adsorbent travels slower. The
component which has less affinity towards stationary phase travels faster. Since no two
components have the same affinity towards the stationary phase, the components are
separated
Solvent delivery system:

Solvent delivery system: The mobile phase is pumped under pressure from
one or several reservoirs and flows through the column at a constant rate.
Eluting power of the mobile phase is determined by its overall polarity, the
polarity of the stationary phase and the nature of the sample components.
For normal phase separations eluting power increases with increasing
polarity of the solvent but for reversed phase separations, eluting power
decreases with increasing solvent polarity. Optimum separating conditions
can be achieved by making use of mixture of two solvents. Someother
properties of the solvents, which need to be considered for a successful
separation, are boiling point, viscosity, detector compatibility, flammability
and toxicity. The most important component of HPLC in solvent delivery
system is the pump, because its performance directly effects the retention
time, reproducibility and detector sensitivity. Among the several solvent
delivery systems (direct gas pressure, pneumatic intensifier, reciprocating
etc.) reciprocating pump with twin or triple pistons is widely used, as this
system gives less baseline noise, good flow rate reproducibility etc
SOLVENT DEGASSING
SYSTEM:
Solvent degassing system The constituents of the mobile
phase should be degassed and filtered before use. Several
methods are employed to remove the dissolved gases in
the mobile phase. They include heating and stirring,
vacuum degassing with an aspirator, filtration through 0.45
filters, vacuum degassing with an air-soluble membrane,
helium purging ultra sonication or purging or combination
of these methods. HPLC systems are also provided an
online degassing system, which continuously removes the
dissolved gases from the mobile phase
 SAMPLE INTRODUCTION
SYSTEM:

SAMPLE INTRODUTION SYSTEM:

Two means for analyte introduction on the column are injection in to a
flowing stream and a stop flow injection. These techniques can used
with a syringe or an injection valve. Automatic injector is a
microprocessor-controlled be version of the manual universal injector.
Usually, up to 100 samples can be loaded in to the auto injector tray.
The system parameters such as flow rates, gradient, run time, volume to
be injected, etc. are chosen, stored in memory and sequentially
executed on consecutive injections. Column and Column-packing
material.
TYPES OF HPLC
TECHNIQUES:
Types of HPLC techniques
i) Based on Modes of Chromatography
(a) Normal Phase chromatography
(b) Reverse Phase chromatography
ii) Based on principle of separation
(a) Adsorption chromatography
(b) Ion exchange chromatography
(c) Ion pair chromatography
(d) Size exclusion chromatography
(e) Affinity chromatography
(f) Chiral phase chromatography.
iii) Based on elution technique
(a) Isocratic separation
(b) Gradient separation
iv) Based on the scale of operation
(a) Analytical HPLC,
(b) Preparative HPLC.
METHOD VALIDATION:

The developed methods were validated by

following steps(R.Synder,1997).

• Accuracy
• Precision
• Specificity
• Limit of detection
• Limit of quantitation
• Linearity of range
• Ruggedness
• Robustness
PREPARATION OF MOBILE
PHASE:

Preparation of Buffer 3.8954g of disodium

hydrogen phosphate and 3.4023g of potassium
dihydrogen phosphate in 1000 ml volume of
distilled water and pH is adjusted to 4 with
orthophosphoric acid. Mobile- phase 580 ml of
acetonitrile and 420 ml of buffer were added in a
beaker to give 1000ml. Finally the pH was adjusted
to 4.0 by adding orthophosphoric acid. Mobile-
Phase Ratio Buffer: Acetonitrile (42: 58 % V/V
METHOD
DEVELOPMENT AND
OPTIMIZATION
The method development by RP-HPLC method for the
estimation of Amlodipine Besylate and Telmisartan is
• Development of suitable mobile phase.
• Optimization of the chromatographic conditions.
• Selection of suitable detection of wavelength.
• Preparation of standard calibration curve of Amlodipine and
Telmisartan Assay of pure mixed standards and formulation.
• Validation of the developed method
SOLUBILITY:
Solubility of drugs in different solvents at different pH conditions which
is useful while selecting diluents for standard solution and extraction
solvents for test solutions. According to Indian pharmacopoeia and
literature review collected Amlodipine Besylate and Telmisartan are
very Slightly soluble in water, Acetonitrile. Sparingly soluble in alcohol.
Then it was checked for different dilutions of Acetonitirile and
Disodium hydrogen Phosphate buffer and finally at PH - 4 the mixture
was chosen for present work.

STABILITY:
STABILITY Stability of drug with storage conditions .This
helps to adopt suitable and adequate precaution
while handling drug substances and its solutions.
SELECTION OF
CHROMATOGRAPHIC METHOD
FOR SEPARATION
Depending on nature of sample (ionic or neutral molecule), its solubility and
molecular weight, chromatographic method is to be selected. The drugs are
polar in nature and so reverse phase chromatographic technique were
selected for the present study.

DETECTION OF WAVELENGTH (λmax

Selection of detection of wavelength is a critical step
in the analytical method. The conditions for the
development of the assay method for the choice of
detection wavelength was based on the scanned
absorption for Amlodipine Besylate and Telmisartan
METHOD DEVELOPMENT TRIALS

Five trials on composition of buffer and organic phase were done to decide
the ultimate composition of mobile phase.

TRIAL – 1 The Trial 1 was performed by operating HPLC with acetonitrile and
phosphate buffer 80:20 % v/v with flow rate of 1.5 ml/min.

TRIAL -- 2 was performed by operating HPLC with Acetonitrile and

phosphate buffer ratio 75:25% v/v with flow rate of 1ml/min.

TRIAL – 3 was performed by operating HPLC with acetonitrile and

phosphate buffer 70:30 %v/v with flow rate of 1 ml/min.
TRIAL – 4 was performed by operating HPLC with Acetonitirile and phosphate
buffer65:35 %v/v with flow rate of 1.0ml/min.

TRIAL – 5 The trial was performed by operating HPLC with Acetonitirile and
phosphate buffer (pH –4) 58:42 %v/v with flow rate of 1.0ml/min.
The peaks of Amlodipine Besylate and Telmisartan were good and retention time of
both peaks was 4.320 and 5.320, there is a good resolution of 3.771. Among the 5
trials, the 5 th trial was selected as the peaks are good with less retention time.
OPTIMIZED CHROMATOGRAPHIC
CONDITION:

Stationary phase : Phenomenex Luna C18 (250 × 4.6 mm, 5µm)

Mobile phase : Acetonitrile : Phosphate buffer(58:42% v/v) P H :4
Flow rate : 1 ml/min.
Column Temperature : Room temperature.
Volume of Injection : 20µl.
Detection of Wavelength : 236 nm.
Run time : 8 min.
Mode of Operation : Isocratic
PREPARATION OF SOLUTIONS

Preparation of buffer solution:

3.8954g of disodium hydrogen phosphate and 3.4023g of potassium
dihydrogen phosphate in 1000 ml volume of distilled water and pH is adjusted
to 4 with orthophosphoric acid.
Preparation of Mobile Phase:
Mobile phase is prepared by mixing 580 ml of acetonitrile and 420 ml of buffer
(58:42).
PREPARATION OF STANDARD STOCK SOLUTION :
An accurately weighed quantity of 5 mg of Amlodipine besylate and 40 mg of
Telmisartan were transferred into a 100 ml volumetric flask. Dissolved with 25 ml
of mobile phase and diluted to required volume with mobile phase, having the
concentration of 0.4 mg/ml of Telmisartan and 0.05 mg/ml of Amlodipine
besylate
Preparation of Standard Solution

Preparation of Standard Solution From the standard stock solution 5 ml is pipetted out into
100 ml volumetric flask and made up the volume with mobile phase, having the
concentration of 0.02 mg/ml of Telmisartan and 0.0025 mg/ml of Amlodipine besylate.

PREPARATION OF SAMPLE SOLUTION

REPARATION OF SAMPLE SOLUTION Twenty tablets were weighed and ground to a fine powder. An
amount of powder equivalent to 40 mg of Telmisartan and 5 mg of Amlodipine besylate were
weighed accurately and transferred into a 100 ml volumetric flask containing 25 ml of mobile
phase and sonicated for 30 min. and diluted to 100 ml with mobile phase, then the solution was
filtered through 0.45 µm membrane filter and 5 ml of filtrate taken into 100 ml volumetric flask and
made up to the volume with mobile phase. The standard stock solution is diluted to the working
concentration equivalent to that of sample. 20 µl of the standard and sample are injected
separately and chromatograms are generated, with peak area obtained for standard and
sample.
METHOD VALIDATION

After the method development the method was validated in terms of parameters
like Accuracy, Precision, Specificity, Linearity, Ruggedness, and Robustness, stability
etc,

SYSTEM SUITABILITY PARAMETERS

For system suitability, five replicates of standard solutions of Amlodipine and
Telmisartan were injected and studied the suitability parameters like Plate number
(N), Resolution (R) and Relative retention time (α), and Peak symmetry of samples
(As) were studied with the help of standard chromatograms.
RESULTS AND DISCUSSION

After several trails with various solvents, mobile phase system composed of acetonitrile and
phosphate buffer of PH 4.0 in the proportion of 58: 42 v/v. respectively was chosen for the
simultaneous estimation of Amlodipine Besylate and Telmisartan and in combined dosage
form by RP-HPLC. This mobile phase composition offered maximum resolution for the drug at
the detection wavelength of 236nm.The column used was C18 pheno-menex luna (250 ×
4.6mm) with flow rate of 1.0 ml/min and UV Detection was carried out. The chromatogram
of Amlodipine Besylate and Telmisartan reference standard were presented in
chromatogram 7 and chromatogram - 8 respectively. The individual peaks of Amlodipine
Besylate and Telmisartan were identified by knowing the retention time 4.320 and 5.320
minutes respectively.
 SUMMARY AND CONCLUSION:
RP-HPLC method was developed and validated as per ICH guidelines for the estimation of
Amlodipine and Telmisartan. Simultaneous Estimation of Amlodipine and Telmisartan were
carried out by RP- HPLC using Phosphate buffer (PH 4.0): Acetonitrile (42:58) and column
Phenomenex Luna C-18(250*4.6 mm, 5um) as a stationary phase and peak was observed at
236 nm which was selected as a wavelength for quantitative estimation. After the
development of the method, it was validated for specificity, linearity, precision, accuracy,
robustness and ruggedness studies. The system suitability parameter also reveals that the
values within the specified limit for the proposed method. Theoretical plate for Amlodipine was
found to be 3594, for Telmisartan it was found to be 3334. The precision of the System and
Method were checked and found to be within limits. This indicates that the method is precise.
The robustness of the method was checked by varying PH
change in buffer solution, variation in temperature and
found that the system suitability parameters were within
limit at all variable conditions, hence the method is
robust. Based on the results observed, it was concluded
that proposed method can be used for routine analysis of
Amlodipine and Telmisartan.