SR.

Page
CONTENT
NO No.
1. INTRODUCTION
1.1. Introduction to UV Methods
1.2. Introduction to Hypertension
1.3. Introduction to Antihypertensive Drugs
1.4. Drug Profile of Amlodipine Besylate
1.5. Introduction to Analytical Method Validation
1.6. References
2. LITERATURE REVIEW
2.1. Official Analytical Methods for Amlodipine Besylate
2.2. Reported Analytical Methods for Amlodipine Besylate
2.3. Reported Analytical Methods for Amlodipine Besylate with other Drug
2.4. References
3. AIM OF THE PRSENT WORK
DEVELOPMENT AND VALIDATION OF UV METHOD FOR
4.
AMLODIPINE BESYLATE TABLET
4.1. Experimental Work
4.2. Results and Discussion
4.3. Conclusion
 LIST OF ABBREVIATIONS

Abbreviations Full Form

°C Degree Centigrade
CV Coefficient of Variance
µ Micron
µL Microliter
µg Microgram
IP Indian Pharmacopoeia
BP British Pharmacopoeia
USP United States Pharmacopoeia
JP Japanese Pharmacopoeia
EP European Pharmacopoeia
ACN Acetonitrile
CC Calibration Curve
GR Guaranteed Reagent
Con. Concentration
CS Calibration Standard
LQC Lower Quality Control
MQC Middle Quality Control
LLOQ Lower Limit of Quantification
C max Maximum concentration
DE Dry Extract
ULOQ Upper Limit of Quantification
gm. Gram
HPLC High Performance Liquid chromatography
IS Internal Standard
LC Liquid Chromatography
Log P Partition coefficient
UPH Ultra Performance Heater
LT Long term
m/z Mass to Charge Ratio
Min Minute
mg Milligram
 ml Millilitre
mM Millimolar
ng Nanogram
mg/ml Milligram/milliliter
e.g., Example
Sr. No. Serial Number
Ref. No. Reference Number
RSD Relative Standard Deviation
SD Standard Deviation
Temp. Temperature
v/v Volume/Volume
Vol. Volume
R2 Correlation Co efficient
LOD Limit of Detection
LOQ Limit of Quantification
VIL Vildagliptin
MET Metformin Hydrochloride
ICH International conference of Harmonization
RF Retention Factor
RT Retention Time
v/v/v Volume/Volume/Volume
% Percentage
MeOH Methanol
HCl Hydrochloric acid
LC-MS Liquid Chromatography-Mass Spectrometry
CHAPTER - 1
INTRODUCTION
 PAGE

INTRODUCTION TO UV METHODS
1. INTRODUCTION:
The spectrophotometer has well been called the workhorse of the modern laboratory. In particular, ultraviolet
and visible spectrophotometer is the method of choice in most laboratories concerned with the identification
and measurement of organic and inorganic compounds in a wide range of products and processes - in nucleic
acids and proteins, foodstuffs, pharmaceuticals and fertilizers, in mineral oils and in paint. In every branch of
molecular biology, medicine and the life sciences, the spectrophotometer is an essential aid to both research
and routine control. Modern spectrophotometers are quick, accurate and reliable and make only small demands
on the time and skills of the operator. However, the user who wants to optimize the functions of his instrument
and to be able to monitor its performance in critical areas will need to understand the elementary physics of the
absorption process as well as the basic elements of spectrophotometer design.

2. SPECTROSCOPY
Definition:
Spectroscopy is defined as the study of interaction of EMR with matter. It is used for analysis of wide range of
samples.
Spectrum:
A plot of the response as a function of wavelength or more commonly frequency is referred to as a spectrum.
Spectrometry:
It is the measurement of these responses and an instrument which performs such measurements is
a spectrometer or spectrograph,
Common types:
• Fluorescence spectroscopy
• X-ray spectroscopy and crystallography
• Flame spectroscopy
1- Atomic emission spectroscopy
2- Atomic absorption spectroscopy
3- Atomic fluorescence spectroscopy
• Plasma emission spectroscopy
• Spark or arc emission spectroscopy
• UV/VIS spectroscopy
• IR spectroscopy
• Raman spectroscopy
• NMR spectroscopy
• Photo thermal spectroscopy
• Thermal infra-red spectroscopy
• Mass Spectroscopy

3. UV- VISIBLE SPECTROSCOPY:

Spectroscopically, visible light behaves in a similar way as UV light. Hence, the techniques of UV
spectroscopy and Visible spectroscopy are studied together.
The UV-Visible spectroscopy is concerned with the UV & Visible regions of the EMR which ranges between
200-800nm.
UV wavelength range is 200-400 nm.
Visible region wavelength range is 400-800 nm.
 PAGE

4. PRINCIPLE:
The principle involved in UV-Visible spectroscopy is absorption spectroscopy. The principle of UV-Visible
spectroscopy is based on the fundamental law of absorption called Beer-Lambert’s law. This law governs the
absorption of radiation by an absorbing medium (dilute solution).
Beer’s law:
According to Beer’s law, when a beam of monochromatic radiation passes through an absorbing medium, the
intensity radiation decreases exponentially with an increase in the concentration of the absorbing medium.
In other words, absorbance is directly proportional to the concentration of the absorbing
substance.
Lambert’s law:
According to Lambert’s law, the rate of decrease in the intensity of the radiation (I) with the thickness of the
medium (t) is directly proportional to the intensity of the incident light.
5. Instrumentation:
The different components present in the spectrophotometer are as follows:
5.1. Source of light:
In all sources excitation is done by passing electrons through a gas and these collisions between electrons and
gas molecules may result in electronic, vibrational and rotational excitation in the gas molecules. When the
pressure of the gas is low, only line spectra are emitted. But, if the pressure of gas is high, band spectra and
continuous spectra will be obtained. Examples - Tungsten lamp, Hydrogen discharge lamp, Xenon discharge
lamp, Deuterium lamps, Mercury arc etc.
Tungsten lamp:It can offer sufficient intensity.
Carbon arc lamp: It can provide high intensity light.
5.2. Filters and Monochromators:
These can convert the polychromatic light into monochromatic light.
Filters:
Filters are used to permit a certain band of wavelength. The simplest type of filter is the
absorption filter. Most commonly colored glass filters are used. They absorb a broad
portion of the spectrum (complementary colors) and transmit other portions (its own
color).
These are two types:
1. Absorption filters
2. Interference filters
Advantages

 The filters are inexpensive.

 They possess technical simplicity.

Disadvantages

 The filters are restricted to the visible region only.

 They are not very good wavelength selectors.
 PAGE

 Not useful for research purposes as they allow broad bandwidth. So there are
more chances of deviation from Lambert beer’s law.

Monochromators: A monochromator is an optical device that is used to select a narrow

band of a wavelength of light. It may be a quartz prism or grating.

Uses of monochromators

 Monochromators are used for spectral scanning i.e. varying wavelength of

radiation over a range.
 They can be used for the UV-visible region

1. Prism type
2. Grating type
Prism types:
These two types:
a. Dispersive type of prisms
b. Littrow type of prisma
Grating types:
a. Diffraction grating
b. Transmission grating
5.3. Sample cells:
These are also called as ‘cuvettes.’
Sample containers or cuvettes may be made up of

1. Quartz
2. Borosilicate
3. Plastic

 Only quartz is transparent in both UV & visible regions (200-700nm range).

 Glass & plastic are suitable for the visible region only.
 Glass is not suitable for the UV region because it absorbs UV radiation i.e. it is not
transparent in the UV region.
 Plastic cells are not used for organic solvents.

Sample volume:

Small volume à<0.5 ml

Large volume à5-10 ml
Shape of cell: Cylindrical or rectangular cells
 PAGE

Material:
For visible region colour corrected fused glass
For UV region cells which are made up with quartz.
5.4. Detectors:
In UV/Visible spectrophotometers these can be called as photometric detectors.
Types:
1. Photo voltaic cell or barrier layer cell
2. Photo emissive cells or photo tubes
3. Photo multiplier tubes
6. Conclusion:
By using this equipment can determine the lambda max, which is unique for each and every compound.

Description of a UV Spectrophotometer
A) Single-beam system

UV radiation is given off by the source. A convex lens gathers the beam of radiation and focuses it on the inlet
slit. The inlet slit permits light from the source to pass, but blocks out stray radiation. The light then reaches
the monochromator, which splits it up according to wavelength. The selected radiation from the
monochromator passes through the sample cell to the detector, which measures the intensity of the radiation
reaching it By comparing the intensity of radiation before end after it passes through the sample, it is possible
to measure how much radiation is absorbed by the sample at the particular wavelength used. The output of the
detector is usually recorded on graph paper.

B) Double-beam system

The radiation from the source is allowed to pass via a mirror system to the monochromator unit. The function
of the monochromator is to allow a narrow range of wavelengths to pass through an exit slit. The radiation
 PAGE

coming out of the monochromator through the exit slit is received by the rotating sector which divides the
beam into two beams, one passing through the reference and the other through the sample cell.

After passing through the sample and reference cells, the light beams are focused on the detector. The output of
the detector is connected to a phase sensitive amplifier which responds to any change in transmission through
sample and reference. The phase sensitive amplifier transmits the signals to the recorder which is followed by
the movement of the pen on chart. The chart drive is coupled to the rotation of the prism and thus the
absorbance or transmittance of the sample is recorded as a function of wavelength.

INTRODUCTION TO HYPERTENSION

Key facts

 An estimated 1.28 billion adults aged 30–79 years worldwide have hypertension, most (two-thirds)
living in low- and middle-income countries
 An estimated 46% of adults with hypertension are unaware that they have the condition.
 Less than half of adults (42%) with hypertension are diagnosed and treated.
 Approximately 1 in 5 adults (21%) with hypertension have it under control.
 Hypertension is a major cause of premature death worldwide.
 One of the global targets for noncommunicable diseases is to reduce the prevalence of hypertension by
33% between 2010 and 2030.

Overview

Hypertension (high blood pressure) is when the pressure in your blood vessels is too high (140/90 mmHg or
higher). It is common but can be serious if not treated.

People with high blood pressure may not feel symptoms. The only way to know is to get your blood pressure
checked.

Things that increase the risk of having high blood pressure include:

 older age
 genetics
 being overweight or obese
 not being physically active
 high-salt diet
 drinking too much alcohol

Lifestyle changes like eating a healthier diet, quitting tobacco and being more active can help lower blood
pressure. Some people may still need to take medicines.
 PAGE

Blood pressure is written as two numbers. The first (systolic) number represents the pressure in blood vessels
when the heart contracts or beats. The second (diastolic) number represents the pressure in the vessels when
the heart rests between beats.

Hypertension is diagnosed if, when it is measured on two different days, the systolic blood pressure readings
on both days is ≥140 mmHg and/or the diastolic blood pressure readings on both days is ≥90 mmHg.

Risk factors

Modifiable risk factors include unhealthy diets (excessive salt consumption, a diet high in saturated fat and
trans fats, low intake of fruits and vegetables), physical inactivity, consumption of tobacco and alcohol, and
being overweight or obese.

Non-modifiable risk factors include a family history of hypertension, age over 65 years and co-existing
diseases such as diabetes or kidney disease.

Symptoms

Most people with hypertension don’t feel any symptoms. Very high blood pressures can cause headaches,
blurred vision, chest pain and other symptoms.

Checking your blood pressure is the best way to know if you have high blood pressure. If hypertension isn’t
treated, it can cause other health conditions like kidney disease, heart disease and stroke.

People with very high blood pressure (usually 180/120 or higher) can experience symptoms including:

 severe headaches
 chest pain
 dizziness
 difficulty breathing
 nausea
 vomiting
 blurred vision or other vision changes
 anxiety
 confusion
 buzzing in the ears
 nosebleeds
 abnormal heart rhythm

If you are experiencing any of these symptoms and a high blood pressure, seek care immediately.

The only way to detect hypertension is to have a health professional measure blood pressure. Having blood
pressure measured is quick and painless. Although individuals can measure their own blood pressure using
automated devices, an evaluation by a health professional is important for assessment of risk and associated
conditions.

Treatment

Lifestyle changes can help lower high blood pressure. These include:

 eating a healthy, low-salt diet

 losing weight
 PAGE

 being physically active

 quitting tobacco.

If you have high blood pressure, your doctor may recommend one or more medicines. Your recommended
blood pressure goal may depend on what other health conditions you have.

Blood pressure goal is less than 130/80 if you have:

 cardiovascular disease (heart disease or stroke)

 diabetes (high blood sugar)
 chronic kidney disease
 high risk for cardiovascular disease.

For most people, the goal is to have a blood pressure less than 140/90.

There are several common blood pressure medicines:

 ACE inhibitors including enalapril and lisinopril relax blood vessels and prevent kidney damage.
 Angiotensin-2 receptor blockers (ARBs) including losartan and telmisartan relax blood vessels and
prevent kidney damage.
 Calcium channel blockers including amlodipine and felodipine relax blood vessels.
 Diuretics including hydrochlorothiazide and chlorthalidone eliminate extra water from the body,
lowering blood pressure.

Prevention

Lifestyle changes can help lower high blood pressure and can help anyone with hypertension. Many who make
these changes will still need to take medicine.

These lifestyle changes can help prevent and lower high blood pressure.

Do:

 Eat more vegetables and fruits.

 Sit less.
 Be more physically active, which can include walking, running, swimming, dancing or activities that
build strength, like lifting weights.

o Get at least 150 minutes per week of moderate-intensity aerobic activity or 75 minutes per week
of vigorous aerobic activity.
o Do strength building exercises 2 or more days each week.
 Lose weight if you’re overweight or obese.
 Take medicines as prescribed by your health care professional.
 Keep appointments with your health care professional.

Don’t:

 eat too much salty food (try to stay under 2 grams per day)
 eat foods high in saturated or trans fats
 smoke or use tobacco
 drink too much alcohol (1 drink daily max for women, 2 for men)
 miss or share medication.
 PAGE

Reducing hypertension prevents heart attack, stroke and kidney damage, as well as other health problems.

Reduce the risks of hypertension by:

 reducing and managing stress

 regularly checking blood pressure
 treating high blood pressure
 managing other medical conditions.
 PAGE

INTRODUCTION TO ANTIHYPERTENSIVE DRUGS

Antihypertensives are medications prescribed to treat hypertension, or high blood pressure. The
goal of antihypertensive therapy is to prevent the complications associated with high blood
pressure, such as stroke and heart attack. Studies have shown that reducing blood pressure by 5
mmHg can significantly lower the risk of stroke by 34% and the risk of ischemic heart disease
by 21%. Moreover, lowering blood pressure can also decrease the likelihood of developing
dementia, heart failure, and mortality from cardiovascular disease.

CLASSIFICATION OF ANTIHYPERTENSIVE DRUGS

A. Diuretic agents
a. Thiazide diuretics, hydrochlorothiazide, chlorothiazide.
b. Loop diuretics, furosemide, ethacrynic, bumetanide
c. Potassium sparing diuretics, spironolactone, amiloride.

B. Calcium channel blockers

Calcium channel blockers are medications that prevent calcium from entering muscle cells in the
walls of arteries.
a. Dihydropyridines:
 Amlodipine
 Cilnidipine
b. Non-Dihydropyridines:
 Verapamil
 Diltiazem

C. ACE inhibitors
Angiotensin-converting enzyme (ACE) is an enzyme that plays a key role in the regulation of
blood pressure by converting angiotensin I to angiotensin II, a potent vasoconstrictor.
 Captopril
 Perindopril

D. Angiotensin II receptor antagonist

Angiotensin II receptor antagonists are drugs that inhibit the activation of angiotensin receptors
by blocking the binding of angiotensin II, a potent vasoconstrictor, to its receptors.
 Azilsartan
 Candesartan

E. Adrenergic receptor antagonists

 Beta blockers – acebutolol, atenolol, bisoprolol.
 Alpha blockers- doxazosin, indoramin, prazosin.
 Mixed Alpha + Bita blockers- bucindolol, labetalol.

F. Vasodilators
Vasodilators are medications that work by directly relaxing the smooth muscle of arteries, thereby making
it easier for blood to flow through them. They are typically reserved for use in hypertensive emergencies
or as a last resort when other medications have failed, and are often used in combination with other drugs
rather than as a standalone treatment.

G.Renin inhibitors
Renin inhibitors are a class of antihypertensive drugs that target the renin-angiotensin system by
inhibiting the enzyme renin, which catalyzes the conversion of angiotensinogen to angiotensin I.
Aliskiren is a renin inhibitor that has been approved for use in treating hypertension, and it is
 PAGE

usually used as a first-line therapy for this condition. By inhibiting renin, aliskiren reduces the
production of angiotensin II, a potent vasoconstrictor, which leads to relaxation of blood vessels
and decreased blood pressure.

H.Aldosterone receptor antagonists:

 Eplerenone
 Spironolactone
 Clinical rational

 Amlodipine is a medication used to treat high blood pressure in adults and children aged 6
years and older. It can also be used to treat certain types of chest pain (angina) and coronary
artery disease, which is the narrowing of blood vessels that supply blood to the heart.
 Clinical studies suggest that amlodipine besylate is a well-tolerated drug for the treatment of
mild to moderate hypertension with a lower incidence of side effects.
 Amlodipine belongs to a class of medications known as calcium channel blockers, which
work by relaxing the blood vessels and reducing the workload on the heart, leading to a
decrease in blood pressure.
 By increasing the supply of blood to the heart, it also helps to control chest pain. When taken
regularly, amlodipine can effectively control chest pain.

DRUG PROFILE OF AMLODIPINE BESYLATE

Table 1.1. Drug Profile of Amlodipine Besylate

PHYSICOCHEMICALPROPERTY: -
Drug Amlodipine besylate
Category Antihypertensive

Structural Formula

Chemical Formula C26H31ClN2O8S

3-Ethyl 5-methyl 2- [(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-6-
IUPAC Name methyl-1,4-dihydro-3,5-pyridinedicarboxylate benzenesulfonate

Molecular Weight 567.05 g/mol

Amlodipine besylate is a white crystalline powder that is slightly
State & Solubility soluble in water and sparingly soluble in ethanol.
 PAGE

Indian Pharmacopeia
United State Pharmacopeia
Pharmacopeia Status British Pharmacopeia
European Pharmacopeia
Density 1.227 g/cm2
Melting Point 199-201o C

PKa 8.6
Log P 1.64
PHARMACOLOGICAL PROPERTY: -
Amlodipine besylate belongs to a category of drugs known as calcium
channel blockers. It reduces blood pressure by dilating the blood
Mechanism of Action vessels, which reduces the workload on the heart. Additionally, it
manages chest pain by improving blood flow to the heart.
Amlodipine besylate can be utilized either on its own or alongside
other medications that lower blood pressure and treat angina to
Indication and Use address hypertension, chronic stable angina, and coronary artery
disease.
 If you experience headaches while taking Amlodipine besylate,
ensure you get sufficient rest and hydration.
 Feeling dizzy is a possible side effect of Amlodipine besylate. If
Adverse Reaction you feel dizzy, cease your current activity and sit or lie down until
you feel better.
 Flushing may occur while taking Amlodipine besylate. Consider
reducing your consumption of coffee, tea, and alcohol to alleviate this
symptom.
 A rapid, pounding heartbeat may be a side effect of Amlodipine
besylate. Consult your healthcare provider if this persists or is
concerning.
 Swollen ankles can be a potential side effect of Amlodipine
besylate. If you experience this symptom, contact your healthcare
provider for further evaluation.
 Diltiazem and Amlodipine besylate should not be taken together
without medical supervision as diltiazem may increase the
concentration of Amlodipine besylate in the body.
 Certain antifungal medications may interact with Amlodipine
besylate.
 Some antibiotics may interact with Amlodipine besylate. It is
recommended to consult your healthcare provider before starting
any new antibiotic treatment.
 Medications used to treat erection problems may interact with
Drug Interaction Amlodipine besylate. Consult your healthcare provider before
taking any medication for erectile dysfunction.
 Cholesterol-lowering medications may interact with Amlodipine
besylate. It is advised to discuss any new cholesterol medication
with your healthcare provider.
 Drugs that modulate the immune system may interact with
Amlodipine besylate. It is recommended to consult your healthcare
provider before taking any new medication that affects the immune
system.
 PAGE

INTRODUCTION TO ANALYTICAL METHOD VALIDATION

Validation is a documented evidence which gives high degree of assurance that a given procedure will
produce a product which comprises of pre-determined specifications and quality characteristics.
Validation is a basic requirement to ensure quality and reliability of the results for all analytical
applications.

The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended
purpose, determine by means of well-documented experimental studies.

Typical validation parameters are listed below.

 Linearity

 Range

 Accuracy

 Precision

 Specificity

 Limit of detection (LOD)

 Limit of Quantification(LOQ)

Linearity and Range

Linearity of an analytical method is its ability to produce results that are directly, proportional to
the concentration of analyte in samples. The range of the procedure is an expression of the
lowest and highest levels of analyte that have been demonstrated to be determinable with
acceptable precision, accuracy and linearity. These characteristics are determined by application
of the procedure to a series of samples having analyte concentration spanning the claimed range
of the procedure. When the relationship between response and concentration is not linear,
standardization may be provided by means of a calibration curve. ICH recommends that for the
establishment of linearity a minimum of 5 concentrations normally used.

Accuracy

Accuracy is the closeness of test results obtained by that method to the true value. In case of
assay of a drug substance accuracy may be determined by application of the analytical method to
an analyte of known purity (e.g. reference standard) or by comparison of the results of the
method with those of a second well characterized method, the accuracy of which has been stated
or defined. Accuracy is calculated as the percentage of recovery by the assay of the known
added amount of analyte in the sample, or as the difference between the mean and the accepted
true value, together with confidence intervals.
 PAGE

The ICH documents recommended that accuracy should be assessed using a minimum of nine
determinations over a minimum of three concentrations levels, covering the specified range (i.e.,
three concentrations and three replicates of each concentration).

Precision

Precision is the degree of agreement among individual test results when the method is applied
repeatedly to multiple samplings of a homogenous sample. Precision of an analytical method is
usually expressed as the standard deviation (SD) or relative standard deviation (RSD)
(coefficient of variation) of a series of measurements.

Precision may be measure of either the degree of reproducibility or of repeatability of the

analytical method under normal operating conditions. Precision of ananalytical method is
determined by assaying a sufficient number of aliquots of a homogenous sample to be able to
calculate statistically valid estimates of SD or RSD. The ICH documents recommend that
repeatability should be assessed using a minimum of nine determinations covering the specified
range for the procedure.

Repeatability

The precision of the analytical method when repeated by the same analyst under set of
laboratory conditions, the only difference being the sample. The repeatability of a test procedure
is assessed by carrying out complete separate determinations on the separate samples of the same
homogeneous sample and this will provide a measure of the precision of the procedure under
normal laboratory operating conditions.

Reproducibility

When the procedure is carried out by different analysts in different laboratories using different
equipments, reagents and laboratory settings. The reproducibility of a test procedure is
determined by evaluating the samples from the same homogeneous sample, the analytical data
will provide information about the reproducibility of the test procedure under validation.

Specificity

The instruments ability to measure or identify or measure the analyte with out any interference
from sample matrix, impurities, precursors or degradation products.

Limit of detection (LOD)

It is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitied
as an exact value, under the stated experimental conditions. The detection limit is usually
expressed as the concentration of analyte (e.g.percentage parts per million) in the sample.
Determined by the analysis of samples with known concentration of analyte and by establishing
the minimum level at which the analyte can be reliably detected.
 PAGE

LOD 3.3 SD/slope of calibration curve

Where, SD-Standard deviation of intercepts

Limit of Quantification (LOQ)

It is the lowest amount of analyte in a sample that can be determined with acceptable precision
and accuracy under the stated experimental conditions. Quantification limitis expressed as the
concentration of analyte (e.g .percentage, parts per billion) in the sample. For instrumental and
non-instrumental methods, the quantification limit is generally determined by the analysis of
samples with known concentration of analyte and by establishing the minimum level at which
the analyte can be determined with acceptable accuracy and precision.

LOQ=10* SD/ slope of calibration curve

Where, SD =Standard deviation of intercepts

 PAGE

REFERENCES:

1. INTRODUCTION TO UV METHODS
1. Souney PF, Matthews SJ. Comprehensive Pharmacy Review.2nd edition.Harwal; 1994. pp. 765–777.
2. Espinosa Bosch M, Ruiz Sánchez AJ, Sánchez Rojas F, Bosch Ojeda C. Analytical methodologies for
the determination of omeprazole: an overview. J Pharma and Biomed Anal. 2007;44(4):831–844.
3. Dhumal SN, Dikshit PM, Ubharay II, Mascarenhas BM, Gaitonde CD. Individual UV-
spectrophotometric assays of trazodone hydrochloride and omeprazole from separate pharmacetical
dosages. Indian Drugs. 1991;28(12):565–567.
4. Sastry CSP, Naidu PY, Murty SSN. Spectrophotometric methods for the determination of omeprazole
in bulk form and pharmaceutical formulations.Talanta. 1997;44(7):1211–1217.
5. Özaltin N, Koçer A. Determination of omeprazole in pharmaceuticals by derivative spectroscopy. J
Pharma and Biomed Anal. 1997;16(2):337–342.

2.INTRODUCTION TO HYPERTENSION

1.Tripathi KD. Essential Medical of Pharmacology, 7th ed, Jaypee Brothers Publishers, New Delhi,
2013; 275 – 277.

2.Gupta S. Review of Pharmacology, 14th ed, Jaypee Brothers Publishers, New Delhi, 2015; 189- 191.

3.Drug profile, Amlodipine Besylate, October2020.

4.ICH Harmonization Tripartite Guideline. Validation of Analytical procedure: Text and Methodology Q2
(R1). International conference on Harmonization (ICH) Geneva, Switzerland, 2005.

5.O’Neil MJ, The Merck Index, An encyclopedia of Chemical, Drugs and Biological, New jersey,
Published by Merck Research Laboratories, Division of Mercks and Co., Inc. Whitehouse Station, 14th ed,
2006, 83-84,1715-16.

7.WHO

3. INTRODUCTION TO ANALYTICAL METHOD VALIDATION

1.ICH Harmonization Tripartite Guideline. Validation of Analytical procedure: Text and Methodology Q2
(R1). International conference on Harmonization (ICH) Geneva, Switzerland, 2005.

2. European Medicines Agency

ICH Topic Q 2 (R1) Validation of Analytical Procedures: Text and Methodology

 PAGE

CHAPTER - 2
LITERATURE
REVIEW
Sr. Ref.
No. Drug Pharmacopoeia Description
No.
HPLC PAGE
Stationary Phase: A Stainless-steel column
15cm×3.9mm 5µ, packed with
octadecylsilane bond to porous silica(5μm).
Amlodipine
1 IP 2018 Mobile Phase: Acetonitrile: Methanol:7.0 ml 1
Besylate (API)
Triethylamine in 1000 ml Water, (15:35:50
v/v/v).
Flow Rate: - 1 ml/min
Detection: - UV at 237 nm.
HPLC
Stationary Phase: A Stainless-steel column
15cm×3.9mm 5µ, packed with
Amlodipine
octadecylsilane bond to porous silica(5μm).
Besylate
2 IP 2018 Mobile Phase: - Acetonitrile: Methanol:7.0 2
(Tablet)
ml Triethylamine in 1000 ml Water,
(15:35:50 v/v/v).
Flow Rate: - 1 ml/min
Detection: - UV at 237 nm
HPLC
Stationary Phase: 3.9-mm ´ 15-cm; 5-mm
Amlodipine packing L1
3 Besylate (API) USP 2020 Mobile Phase: Methanol, acetonitrile, and 3
Buffer (35:15:50)
Flow Rate: - 1.0 ml/min
Detection: - UV at 237 nm
HPLC
Stationary Phase: 3.9-mm ´ 15-cm; 5-mm
Amlodipine
packing L1
Besylate
4 USP 2020 Mobile Phase: Methanol, acetonitrile, and 4
(Tablet)
Buffer (35:15:50)
Flow Rate: 1.0 ml/min
Detection: UV at 237 nm
HPLC
Stationary Phase: Phenomenex, Kinetex
C18 (100 mm × 4.6 mm, 2.6 μm).
Mobile Phase: Mobile Phase A :0.03M
KH2PO4, adjusted to pH 3.0 with H3PO4:
Amlodipine
5 Besylate (API) BP 2020 methanol (45: 55 %v/v), Mobile Phase B 5
:0.03M KH2PO4, adjusted to pH 3.0with
H3PO4: methanol (30:70 %v/v)
(Gradient mode for 0-8 min mobile phase A
is 100 %v/v and mobile phase B is 0 %v/v,
for 8- 13 min mobile phase A is 100-0 %v/v
and mobile phase B is 0-100 %v/v, for 13-34
min
mobile phase A is 0 %v/v and mobile phase
B is 100 %v/v, for 34-35 min mobile phase
A is 0-100 %v/v and mobile phase B is 100-
0
%v/v, for 35-45 min mobile phase A is 100
%v/v and mobile phase B is 0 %v/v.)
Flow Rate: 0.6 ml/min
Detection: UV at 238 nm

HPLC
 PAGE

REPORTED ANALYTICAL METHODS FOR AMLODIPINE

Table 2.2. Reported Methods for Amlodipine Besylate

Sr. Ref.
No. Drug Methods Description
No.
Electrode: CNT
Amlodipine modified edge plane pyrolytic graphite
Besylate in working electrode. Platinum wire
1 Voltametric 7
human urine auxiliary electrode and an Ag/AgCl
reference electrode
Linearity: 5.0-10 to 1.0-10 M
Stationary phase: column Zodiac C18
column (250mm x 4.6 mm, 5µ)
Mobile phase: Methanol: Water:
2 Amlodipine RP-HPLC 8
Acetonitrile (60:20:20, v/v/v)
Detection: UV 245 nm
Flow rate: 1 ml/min
Stationary phase: 4.6 x 150 mm
column packed with octadecyl bonded
silica, particle size 5 um
Amlodipine in
RP-HPLC Mobile phase: 0.05 M phosphate
3 serum 10
buffer solution (pH3.1): acetonitrile
(65:35, v/v)
Flow rate: 1 ml/min
Detection: UV 237 nm
Stationary phase: Accurasil ODS
5um C18 column (250 x 4.6 mm)
Amlodipine Mobile phase: 0.1%orthophosphoric
4 HPLC 11
Besylate acid (pH 3): acetonitrile (20:80, v/v)
Flow rate: 1 ml/min
Detection: UV 238 nm
Stationary phase: Hypersil BDS C18
column (50 x 4.6 mm)5.0 µm,
Amlodipine in Mobile phase: Methanol: ammonium
LC-MS/MS
5 human plasma format (pH 4.5), (80:20, v/v) 12
Flow rate: 1 ml/min
Detection: Mass spectrometric
detection that enables detection
Stationary phase: The DBFFAP, 30
Gas m x 0.53 mm ID, 1.0 µm column.
Amlodipine
6 Chromatography Carrier gas: Nitrogen 13
Besylate
Flow rate:40 ml/min
Detector temp.: 260 C
 PAGE

REPORTED ANALYTICAL METHODS FOR AMLODIPINE BESYLATE WITH

OTHER DRUGS

Table 2.3. Reported Methods for Amlodipine Besylate with Other Drugs

Sr. Ref.
Drug Method Description
No. No.
Solvent: Methanol
Amlodipine Wavelength: UV at
UV
Besylate, Telmisartan: 292.8 nm
Spectroscopy
Telmisartan Amlodipine Besylate: 238.5 nm
(simultaneous
1 and Hydrochlorothiazide: 271.2 nm 14
equation
Hydrochloro- Linearity:
method)
thiazide. Telmisartan: 4-24 µg/ml
Amlodipine Besylate :4-24 µg/ml
hydrochlorothiazide: 4-24 µg/ml
Solvent: Methanol
UV Wavelength: UV at 290 nm
Amlodipine Linearity:
Spectroscopy
2 and Valsartan Amlodipine Besylate :10-80 µg/ml 15
(first derivative
Method) Valsartan: 20-180 µg/ml

Solvent: Methanol
Amlodipine
Uv Spectroscopy Wavelength: UV at
Besylate and
(simultaneous Amlodipine: 237 nm
3 Olmesartan 16
equation Olmesartan Medoxomil: 259 nm
Medoxomil
method). Linearity: Amlodipine: 5-35 µg/ml
tablet
Olmesartan Medoxomil: 5-40 µg/ml
Solvent: Acetonitrile and water
Olmesartan
Wavelength: UV at
Medoxomil Uv Spectroscopy
Olmesartan Medoxomil: 265
and (Absorption
4 Amlodipine Besylate: 360 nm 17
Amlodipine correction
Linearity:
Besylate method)
Olmesartan Medoxomil: 2-32μg/ml
tablet
Amlodipine Besylate :2-20 μg/ml
Solvent: Methanol
Amlodipine Ultraviolet Wavelength: UV at
Besylate and Spectroscopy Amlodipine Besylate: 261 nm
5 atorvastatin (simultaneous atorvastatin: 246 nm 18
calcium in equation Linearity:
tablet method) Amlodipine Besylate: 0.5-30µg/ml
atorvastatin: 0.5-30 µg/ml
Solvent: water
Atorvastatin Wavelength: UV at
Ultraviolet
calcium and Atorvastatin Calcium: 243 nm
Spectroscopy
6 Amlodipine Amlodipine Besylate: 247 nm 19
(absorption ratio
Besylate Linearity:
method)
Atorvastatin Calcium:10-60 µg/ml
Amlodipine Besylate :10-60 µg/ml
 PAGE

Solvent: 0.1 M HCl

Amlodipine Wavelength: UV at Amlodipine
Uv Spectroscopy
7 Besylate and Besylate: 252 nm Nevirapine: 295 nm 20
(absorption ratio
nevirapine in bulk Linearity: Amlodipine Besylate: 3-7
method)
and µg/ml
tablet Nevirapine: 3-7 µg/ml

Solvent: Methanol
Losartan Uv Spectroscopy Wavelength: UV at
8 potassium and (simultaneous Losartan potassium: 208 nm 21
Amlodipine equation Amlodipine Besylate: 237.5 nm
Besylate in method) Linearity:
tablet Losartan potassium: 2-20 µg/ml
Amlodipine Besylate: 2-20 µg/ ml
Solvent: Methanol
Amlodipine Wavelength: UV at Amlodipine
UVSpectroscopy
9 Besylate and Besylate: 237 nm Celecoxib: 252 nm 22
(simultaneous
celecoxib Linearity:
estimation Method)
tablet Amlodipine Besylate: 2-12 µg/ml
Celecoxib: 2-12 µg/ml

Stationary phase: A Li Chrosorb

C18 column (250 mm x 4.6 mm, 10
10 Amlodipine μm) 23
Besylate and RP-HPLC Mobile phase: Acetonitrile: 0.5M
Lisinopril sodium acetate buffer (25:75, v/v)
Flow rate: 1.5 ml/min
Detection: UV 215 nm
Stationary phase: C18 column (250
× 4.6 mm, i.e.) packed with 5 um
Amlodipine particle size (Milford Massachusetts
Besylate and USA)
atorvastatin Mobile phase: Acetonitrile:
HPLC
calcium in binary phosphate buffer, adjust pH 3, (45:55,
11 24
mixture v/v)
Flow rate: 1 ml/min
Detection: Fluorescence at excitation
wavelengths 361 and 274 nm and
emission wavelengths 442 and 378
nm for AM and AT
Stationary phase: C18 column
Amlodipine (FORTIS TM 250 mm x 4.6 mm, 5
12 Besylate and RP-HPLC μm) 25
Furosemide tablet Mobile phase: Water: ACN (50:50,
v/v)
Flow rate: 1.0 ml/min
Detection: UV 238 nm
Amlodipine Stationary phase: C18 bonded phase
13 Besylate and HPLC i.e., CAPCELL PACK Col No. 26
Olmesartan
Medoxomil tablet AKAD 05395 (4.6 mm X250mm)
 PAGE

with particle size 5µm

Mobile phase: 0.05 M Pot.
Dihydrogen phosphate: ACN (50:50
v/v), pH (6.8)
Flow rate: 1 ml/min
Detection: UV 238 nm
Stationary phase: C18 (250×4.6mm,
Amlodipine 5 µ) column
14 Besylate and HPLC Mobile phase: Acetonitrile: water in 27
Nebivolol the ratio (40:60, v/v) adjusted pH5.0
Hydrochloride in adjusted
Tablets Flow rate: 1 ml/min
Detection: UV 268 nm
Stationary phase: C18 (250 mm ×
Amlodipine 4.6 mm i.d., 5μm) column
15 Besylate and HPLC Mobile phase: phosphate 28
perindopril Buffer (pH 5.0): Acetonitrile (70:30
arginine tablet v/v)
Flow rate: 1 ml/min
Detection: UV 240 nm
Stationary phase:
A Brownlee C-18, 5 μm column
Amlodipine Mobile phase:
16 Besylate and RP-HPLC 0.02 M potassium dihydrogen 29
Indapamide phosphate–methanol (30+70, v/v)
Tablets total pH-adjusted to 3 using o-
phosphoric acid was used.
Flow rate: 1 ml/min
Detection: UV 242 nm
Amlodipine Stationary phase: The Phenomenex
Besylate and C-18 column (250 mm × 4.6 mm, 5
17 perindopril, RP-HPLC μm). 30
erbumine, Mobile phase: acetonitrile: methanol:
indapamide tablet water (30:20:50, v/v/v
Flow rate: 1 ml/min
Detection: UV 215 nm
Stationary phase: C18 column
Amlodipine (4.6x250 mm id., particle size of 5
18 Besylate and RP-HPLC μm). 31
Indapamide Mobile phase:
Tablets acetonitrile: methanol (50: 40 % v/v)
Flow rate: 1, 1.2, 1.4 ml/min
Detection: UV 215 nm
Stationary phase: C18 column (250
Amlodipine mm × 4.6 mm i.e., particle size 5 μm)
19 Besylate & RP-HPLC Mobile phase: Water: Acetonitrile 32
Irbesartan (60:40, v/v) adjust pH 3.5
Flow rate: 1 ml/min
Detection: UV 245 nm

Stationary phase: aluminium plates

 PAGE

precoated with silica gel 60Fâ??254

Amlodipine (10×10)
Besylate and Mobile phase: toluene: ethyl acetate:
20 metoprolol HPTLC methanol: triethylamine (4:1:1:0.4 33
succinate Tablets v/v/v).
Rf value:
Amlodipine Besylate: 0.39±0.02
metoprolol succinate: 0.59±0.02
Detection: UV 254 nm
Stationary phase: Phenomenex C18
column (Luna 5 μ, 100A, 250x4.6
mm; California, USA) protected with
Amlodipine a Phenomenex C18 guard column
Besylate & (4.0x3.0 mm; California, USA).
Olmesartan HPLC Mobile phase: phosphate buffer (pH
21 34
Medoxomil 4.0, 0.04
mol/L): methanol: acetonitrile
(40:45:15, v/v/v)
Flow rate: 0.8 ml/min
Detection: 234 nm for AMLO and
205 nm for OLM
Wavelength:
Amlodipine Amlodipine Besylate: 458 nm
22 Besylate and Spectrofluorimetry Telmisartan: 675 nm 35
Telmisartan Log P Value:
Amlodipine Besylate: 3.0
Telmisartan: 6.66
 PAGE

REFERENCES:
1.Indian Pharmacopoeia Vol. II. New Delhi: Controller of Publication, Govt. of India, Ministry of Health
and Family Welfare Ghaziabad, 2018,1045-1046.

2. USP 43 NF 38 United State Pharmacopoeia, Vol. III, U.S. Pharmacopeia convention. Twin brook
Parkway, Rockville, MD,2020,1150-1151.

3. USP 43 NF 38 United State Pharmacopoeia, Vol. III, U.S. Pharmacopeia convention. Twin brook
Parkway, Rockville, MD,2020, 1150-1151.

4. British Pharmacopoeia, Vol.I. The Department of Health, Social Services and Public Safety, London
Her Majesty's. Stationary office, 2020, 138-139.

5. British Pharmacopoeia, Vol.I. The Department of Health, Social Services and Public Safety, London
Her Majesty's. Stationary office, 2020, 138-139.

6. Rajendra N. Goyal. Sunita Bishnoi.: Voltammetric determination of amlodipine besylate in human

urine and pharmaceuticals. Bioelectrochemistry, 2010, 79, 234- 240.

7. B. Lakshmi, K.Rama Krishna and K.N. Jayaveera. Development and validation of RP- HPLC method
for the determination of amlodipine. Int. J. Appl. Biol. Pharm., 2015, 6(1), 225-231.

8. Mohamed Alaama. ABM Helal Uddin, Huda Jamilah Mohamad, Noor SyafawatiAmiruddin and SA
Abbas.: Development and validation of reversed phase high performance liquid chromatographic method
for determination of amlodipine. Trop.J. Pharm. Res., 2015.14 (4), 663-669.

9. Mohamed Alaama, ABM Helal Uddin, Huda Jamilah Mohamad, Noor Syafawati Amiruddin and SA
Abbas. Development and validation of reversed phase high performance liquid chromatographic method
for determination of amlodipine. Trop. J. Pharm. Res., 2015.14 (4), 663-669.

10. Kanakapura Basavaiah, Umakanthappa Chandrashekar and Paregowda Nagegowda.:

Spectrophotometric and high performance liquid chromatographic determination of amlodipine besylate in
pharmaceuticals. Science Asia., 2005, 31, 13-21.

11. Jignesh Bhatt, Sadhana Singh, Gunta Subbaiah, Bhavin Shah, Sandeep Kambli and Suresh Ameta.: A
rapid and sensitive liquid chromatography-tandem mass spectrometry (LC- MS/MS) method for the
estimation of amlodipine in human plasma. Bio. Chrom., 2007, 21, 169-175.

12. V. Anil Kumar, G. Aravind, I. Srikanth, A. Srinivasara, Ch. Dharma Raju: Novel analytical method
development and validation for the determination of residual solvents in amlodipine besylate by gas
chromatography. Der. Pharma. Chemica., 2012, 4(6), 2228-2238.

13. Delhiraj N, CH Narisimaraju BH and Anbazhagan S.: Simultaneous spectrophotometric estimation of

telmisartan, amlodipine besylate and hydrochlorthiazide in pharmaceutical dosage form. Int. Jou. Chem.
Phys. Sci., 2012, 3(3), 34-36.

14. Nashwah Gadallah Mohamed.: Simultaneous determination of amlodipine and valsartan. Ana. Chem.
Ins., 2011, 6, 53-59.
 PAGE

15. Kardile D.P., Kalyane N.V., Thakkar T.H., Patel M.R., Moradiya R.K.:Simultaneous estimation of
amlodipine besylate and Olmesartan medoxomil drug formulations by HPLC and UV-spectrophotometric
methods. J. Pharm. Sci. & Res..2010, 2(9), 599-514.

16. Pournima Patil, Vaishali Barge, Harinath More, Sachin Piswikar.: Spectrophotometric method for
simultaneous determination of Olmesartan medoxomil and amlodipine besylate from tablet dosage form,
Int. J. Curr. Pharm. Res., 2011,3(2), 74-79.

17. Devi Ramesh, S Ramakrishna.. New spectrophotometric methods for simultaneous determination of
amlodipine besylate and atorvastatin calcium in tablet dosage forms. Int. J. Pharm. Sci., 2010, 2(4), 215-
219.

18. Shyni Bernard, Molly Mathew, K.L.Senthilkumar, K.N.Girija.: Simultaneous estimation of

atorvastatin calcium and amlodipine besylate by uv spectrophotometric method using hydrotropic
solubilization. Hyg. J. D.Med., 2013, 5(1), 105-112,

19. Sathis Kumar Dinakaran, Narayudu Yandamuri, L P S Nainesha Allada.: Spectrophotometric method
development and validation for amlodipine besylate and nevirapine in bulk and tablet dosage form using
absorption ratio method, Worl. Jou. Pharm. Pharma. Sci., 2014, 3(7), 1720-1730.

20. Priyanka R Patil, Sachin U Rakesh, PN Dhabale, and KB Burade.: Simultaneous UV

spectrophotometric method for estimation of losartan potassium and amlodipine besylate in tablet dosage
form. Asian J. Research Chem., 2009, 2(1), 183-187.

21. Manish. S. Kondawar., Development and Validation of Analytical Method for Simultaneous
Estimation of Amlodipine Besylate and Celecoxib in Pure and Combined Dosage Form. Research J.
Pharm. and Tech 2020; 13(9):4280-4284.

22. Lily P. Peikova, Boyka G. Tsvetkova.: Investigations and HPLC assay of model formulations
containing amlodipine besylate and lisinopril. Int. J. Pharm. Sci. Rev. Res., 2013, 20(1), 11- 15.

23. Bahia A. Moussa, Asmaa A. el-Zaher, Marianne A. Mahrouse and Maha S. Ahmed. Simultaneous
determination of amlodipine besylate and atorvastatin calcium in binary mixture by spectrofluorimetric
and HPLC coupled with fluorescence detection. Ana. Chem. Ins., 2013, 8, 107-115.

24. Fevziye O. Simsek, and Mustafa Sinan Kaynak: Determination of amlodipine and furosemide with
newly developed and validated RP-HPLC method in commercially available tablet dosage forms. Trop. J.
Pharm. Res., 2012, 32 (2), 145-158.

25. Kardile D.P., Kalyane N.V., Thakkar T.H., Patel M.R., Moradiya R.K.; Simultaneous estimation of
amlodipine besylate and Olmesartan medoxomil drug formulations by HPLC and UV-spectrophotometric
methods. J. Pharm. Sci. & Res., 2010, 2 (9), 599-514.

26. Madhuri Ajay Hinge, Jigar Arjunbhai Patel, Rajvi J. Mahida.: Spectrophotometric and high
performance liquid chromatographic determination of amlodipine besylate and nebivolol hydrochloride in
tablets dosage form. Pharm. Methods., 2016, 7(1), 1-7.

27. Bhagirath k. Patel., Analytical method development and validation for simultaneous estimation of
amlodipine besylate and perindopril arginine in combined pharmaceutical dosage form. Pharmaceutical
and biological evaluations.,2016, 3 (3),343-350.
 PAGE

28. Deval B. Patel.,Simultaneous Estimation of Amlodipine Besylate and Indapamide in a Pharmaceutical

Formulation by a High Performance Liquid Chromatographic (RP-HPLC) Method. Sci Pharm., 2012; 80:
581–590.

29. Patel et al., A new RP–HPLC method for simultaneous quantification of perindopril erbumine,
indapamide, and amlodipine besylate in bulk and pharmaceutical dosage form. Future Journal of
Pharmaceutical Sciences, (2020) 6:80.

30. Diren S.Y., Development and Validation of an HPLC Method Using an Experimental Design for
Analysis of Amlodipine Besylate and Enalapril Maleate in a Fixed-dose Combination. Turk J Pharm Sci
2021;18(3):306-318.

31. Mazharuddin M. Shaikh, Stavan Master & Abrar M. Chaudhary: Development and validation of RP-
HPLC method for simultaneous estimation of amlodipine besylate and irbesartan. Int. bull. drug res.,
2014, 4(7), 1-15.

32. P. S. Jain*, S. B. Bari and S. M. K. Patel, J. Surana., development and validation of HPTLC method for
simultaneous determination of amlodipine besylate and metoprolol succinate in bulk and tablet. Indian J
Pharm Sci, 2012, 74 (2): 152-156.

33. Sahin Selma., Determination of in vitro Dissolution Profiles of Amlodipine Besylate and Olmesartan
Medoxomil Using a New "HPLC Method“. Revista de chimie-Bucharest- original edition- 64(1):27-30.

34.S. Neeli., Development of dissolution test method for a telmisartan/amlodipine besylate combination
using synchronous derivative spectrofluorimetry. Brazillian Journal of Pharmaceutical Science.2014, 50
(2): 329-336.
CHAPTER - 3
AIM OF THE
PRESENT
WORK
3. AIM OF THE PRESENT WORK
 Amlodipine Besylate is from Anti-Hypertensive (calcium channel blocker) class that is used
for treatment of high blood pressure.
 Extensive literature survey reveals that number of analytical methods are available for
estimation of Amlodipine Besylate in single & combination dosage form.
 So, aim of my present work is to develop sensitive, robust, accurate and precise UV method
determining the % Drug Release (In vitro) of Amlodipine Besylate drugs in tablet.
 To Validate method of UV for estimation of method for estimation of Amlodipine Besylate
as per ICH guideline, Q2 (R1).
Chapter – 4
UV –SPECTRO
PHOTOMETRIC
METHOD
 3. DEVELOPMENT AND VALIDATION OF UV METHOD FOR AMLODIPINE
BESYLATE TABLET.

EXPERIMENTAL WORK

MATERIAL AND REAGENTS

1. Amlodipine besylate – Active Pharmaceutical Ingredient Supplied by BRILLIANT

LIFESCINCES PVT. LTD. (Dangarva)

2. Amlodipine besylate tablets ( 5 mg) and Placebo Supplied by BRILLIANT

LIFESCINCES PVT. LTD. (Dangarva)

 Working standard Used:

No. Name % Potency Valid up to
Amlodipin besylate
1. 99.47 Sep 2024

 API Used:
Name Batch No.
Amlodipine besylate ALK5AYP16B

 Sample used:

No. Name Batch no. Manufacturing date

Amlodipine besylate tablet
1. EP20-098-476 Dec – 2021
5mg

 Chemicals and reagents used

Table 4.1. List of Chemicals and Reagent

Sr.
No. Name Make Grade

1 Methanol Actylis HPLC

2 Water Milli-Q HPLC
INSTRUMENTS LIST

Table 4.2. List of Instruments

Name of the
Make Model
instrument

Analytical
Electronic Scaletec Analytical Balance SAB-224 CL
Balance

UV-Visible
Spectrophotom ELICO SL-159
eter

UV Spectrophotometric Specifications:

Detector : UV -visible

Measurement Mode : Photometric

Wavelength Maxima : 361nm

 ASSAY

Standard solution : Weigh accurately about 50 mg of Amlodipine WS and shake with 70 ml of

methanol for 30 minutes. Add sufficient methanol to produce 100.0 ml, mix, then filtrate the
solution, take 5.0 ml of filtrate solution then add sufficient methanol to produce up to 50 ml.

Sample solution :Take 20 tablet of amlodipine besylate and powdered take E.q 220 mg of
amlodipine powder and transfer in to 100 ml volumetric flask add 70 ml of methanol then
sonicate for 30 minutes and add sufficient methanol to produce 100 ml. & filter the solution then
take 10 ml of filtrate solution and transfer into 20 ml volumetric flask add sufficient methanol to
produce up to 20 ml.

Blank solution : 5 ml methanol

Measure the absorbance at about 361 nm for Amlodipine using Blank solution as blank

INSTRUMENTS LIST

Table 4.2. List of Instruments

Name of the
Make Model
instrument

Analytical
Electronic Scaletec Analytical Balance SAB-224 CL
Balance

UV-Visible
Spectrophotom ELICO SL-159
eter

UV Spectrophotometric Specifications:

Detector : UV -visible

Measurement Mode : Photometric

Wavelength Maxima : 361nm

2. VALIDATION PARAMETERS:

To Check the performance of analytical method developed for the determination of assay
content (Amlodipine) by various parameters of method validation.

2.1 Linearity & Range: To Check the linearity of different concentration of test for active drug
at 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml and 50 µg/ml in 10 µg/ml to 50 µg/ml specific
range. Perform assay of 5 different concentrations of active drug; taking assay concentration
of active drug as a middle concentration, and check the linear response by plotting graph.

2.3 Accuracy: To Check the recovery by 9 determinations over 3 different concentrations.

2.4 Precision: To Check the relative standard deviation of the repeatability of the method by 6
determinations of 100 % test concentration of the same batch by one analyst.

2.1 Linearity & Range:

Preparation of Test Stock Solution: Weigh accurately a quantity of the powder containing
about 10mg of Amlodipine and shake with 75 ml of methanol for 30 minutes.

1) Preparation of 10 µg/ml Test solution:

Take aliquot of 5 ml from TSS in a 50 mL volumetric flask and dilute upto 50 ml.
Measure the absorbance at about 361 nm using blank solution.

Preparation of 20µg/ml Test solution:

Take aliquot of 10 ml from TSS in a 50mL volumetric flask and dilute upto 50 ml
Measure the absorbance at about 361 nm using blank solution.

3) Preparation of 30 µg/ml Test solution:

Take aliquot of 15 ml from TSS in a 50 mL volumetric flask and dilute upto 50 ml.
 Measure the absorbance at about 361nm using blank solution.

4) Preparation of 40 µg/ml Test solution:

Take aliquot of 20 ml from TSS in a 50 ml volumetric flask and dilute upto 50 ml.
Measure the absorbance at about 361 nm using blank solution.

4) Preparation of 50 µg/ml Test solution:

Take aliquot of 25 ml from TSS in a 50 ml volumetric flask and dilute upto 50 ml.
Measure the absorbance at about 361 nm using blank solution.

2.2.2 Absorbance Measurement:

Absorbance measurement of three replicates of each solution starting from the lowest
concentration to highest concentration at 361 nm

Mode of Evaluation:
2.2.3
Note down the Absorbance data at 361 nm for each Solution and report the Regression
Coefficient (R2) value.

Acceptance Criteria:
2.2.4
There should be linear response in operating range and Regression Coefficient (R2)
should be more than 0.99.
Accuracy

It was determined by calculating the recovery of Amlodipine by UV method .To fixed amount
of test 80%, 100% and 120% amount of standard was added and the amount of standard added
was calculated using equation. Known amount standard solutions of Amlodipine (0.024, 0.03,
0.036 ) were added to a pre-quantified sample solution of Amlodipine (0.03 mg/ml).Each
solution was scanned in triplicate and the percentage recovery was calculated by measuring the
responses and fitting these value into

Precision

The repeatability of the proposed method was determined by measuring the corresponding
responses 3 times for 100% test concentration of Amlodipine. The intra-day and inter-day
precisions of the proposed method was determined by measuring the corresponding responses 3
times on the same day and on 3 different days over a period of 1 week for 3different
concentration of Amlodipine ( 20, 30 and 40 µg/ml ). The results were reported in terms of
relative standard deviation.
METHODVALIDATION

LINEARITY (n=3)
The calibration curves for Amlodipine was prepared as mentioned in section and responses
were measured at 361nm.Lambert-Beer ҆s law was followed in this concentration ranges.The data
for responses of Amlodipine at 361nm shown in table . The linearity curve for Amlodipine are
shown in Fig .The straight line equations and correlation coefficient for Amlodipine shown in
table.
Linearity For Amlodipine Besylate
Mean Response± SD % CV
Conc.(µg/ml)
10 0.0051±0.00010 1.9607

20 0.0067± 0.000057 0.8574

30 0.0086± 0.000153 1.7693

40 0.0108± 0.000102 0.9259

50 0.0136± 0.000200 0.8000

Absorbance
0.014

0.012 f(x) = 0.000189 x + 0.00307

R² = 0.997458952306489
0.01
Absorbance
0.008 Linear (Absorbance)
0.006

0.004

0.002

0
5 10 15 20 25 30 35 40 45 50 55

Calibration curve for Amlodipine (10-50µg/ml) at 361 nm

Drug Regression equation of Correlation Coefficient (R2)

Calibration curve
Amlodipine Y= 0.000x+0.003 0.999
 ACCURACY

Results of accuracy are shown in table___ for Amlodipine. The results show that the percentage
recoveries for Amlodipine was found to be in the range of ___.

Table. Results-Accuracy for S (-) Amlodipine Besylate

Levels of Sample (%) Recovery of Average

%CV
Addition No. Amlodipine Besylate (%)
1 99.19
First Levels 2 96.62 98.3 1.45
(80%)
3 98.96
1 98.45
Second Levels 2 99.71 99.3 0.76
(100%)
3 99.80
1 98.51
Third Levels 2 99.50 98.5 0.97
(120%)
3 97.58
Avg. 98.7 1.06

PRECISION
A) Repeatability ( n=6 )

The repeatability data for Amlodipine are shown in table___. The %CV was found to be
1.489% for Amlodipine.

Table Repeatability data for Amlodipine

Conc. of Absorbance
Amlodipine (n=6)
(µg/ml)
0.0111
0.0108
0.0110
20 0.0108
0.0112
0.0109
Mean 0.0109
SD 0.00016

%CV 1.489
B) INTRADAY PRECISION (n=3)

The data for intraday precision for Amlodipine is shown in table___. The %CV for
intraday precision was found to be ______.

Intraday
Concentration Mean response ± SD %CV
20 0.00516 ± 0.000516 1.117
30 0.00870 ± 0.00010 1.149
40 0.01366 ± 0.01132 1.114

C) INTERDAY PRECISION (n=3)

The data for interday precision for Amlodipine in table _____. The %CV for interday precision
was found to be 1.76525-1.9230 Amlodipine.

Conc. (µg/ml) Mean response ± SD %CV

20 0.0545 ± 0.00075 1.9230
30 0.1359 ± 0.00140 1.7625
40 0.1888 ± 0.001762 1.8280

SUMMARY OF VALIDATION PARAMETERS:

Table___ Summary of validation parameters

Parameter Amlodipine
Linearity(µg/ml) 10-50
Accuracy (% Recovery) 98.7±1.06
Precision(% CV )
Repeatability (n=6) 1.489
Intraday (n=3) 1.114-1.149

Interday (n=3) 1.7625-1.9230