METHOD VALIDATION AND STABILITY METHOD OF TELMISARTAN AND EFONIDIPINE IN RPHPLC METHOD

q
“DEVELOPMENT AND VALIDATION OF STABILITY INDICATING
RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF
EFONIDIPIN HYDROCHLORIDE ETHANOLATE AND TELMISARTAN
IN SYNTHETIC MIXTURE”
GUIDED BY
ANJANA PADHIYAR
PREPARED BY:
ANKUR S. GODHAT
Department of Pharmaceutical Quality Assurance
GUJARAT TECHNOLOGICAL UNIVERSITY
Chandkheda, Ahmadabad – 382424 - Gujarat

CONTENT
• Introduction
• Drug Profile
• Review of Literature
• Rationale
• Aim and Objective
• Materials and Equipment
• Experimental Work
• Stability indication RP-
HPLC Method
• FTIR
• Validation of Method
• References
• Paper Publication
2

INTRODUCTION OF DISEASE [1]
4
 Stage 2 hypertension is defined as a systolic BP of ≥140 mmHg
diastolic BP of ≥90 mmHg.
 A combination of healthy lifestyle changes and BP-lowering
medication (two first-line agents of different classes) is the
recommended care plan for patients with stage 2 hypertension. If
patient’s BP goal is met after one month of treatment, they can be
reassessed in 3-6 months.
 If BP goal has not been achieved after one month of treatment, a
different medication or titration should be considered along with
monthly follow-ups with their physician until BP is controlled.

INTRODUCTION OF DRUG
(EFONIDIPINE HYDROCHLORIDE ETHANOLATE) [2]
5
 Efonidipine hydrochloride ethanolate, sold under the trade name
Landel. It is a CCB with long-lasting vasodilator actions and little reflex
tachycardia.
 Efonidipine hydrochloride ethanolate improves endothelial function in
patients with hypertension when compared with doses of nifedipine that
result in comparable decreases in mean blood pressure Therefore, we
hypothesized that Efonidipine hydrochloride ethanolate therapy may
simultaneously improve endothelial dysfunction, adipocytokine profiles,
and other metabolic parameters in nondiabetic patients with
hypertension.
 MoA : This drug inhibits the L-type and T-type calcium channels,
thereby leading to vasodilation and decreased automaticity of the heart.
Efonidipine hydrochloride ethanolate exerts negative chronotropic
effects, decreasing heart rate. Acting on SA node cells by inhibiting T-
type calcium channel activity, Efonidipine prolongs the late phase-4
depolarization of the sinoatrial node action potential, decreasing heart
rate.

INTRODUCTION OF DRUG (TELMISARTAN) [3]
 Telmisartan, sold under the trade name Micardis among others, is a
medication used to treat high blood pressure, heart failure, and diabetic
kidney disease. It is a reasonable initial treatment for high blood pressure.
It is taken by mouth. Versions are available as the combination
telmisartan/hydrochlorothiazide and telmisartan/amlodipine.
 Common side effects include upper respiratory tract infections, diarrhea,
and back pain. Serious side effects may include kidney problems, low blood
pressure. It is an angiotensin II receptor antagonist and works by blocking
the effects of angiotensin II.
 MoA : Telmisartan blocks the vasoconstrictor and aldosterone-secreting
effects of angiotensin II by selectively blocking the binding of angiotensin II
to the AT1 receptor in many tissues, such as vascular smooth muscle and
the adrenal gland.
6

TECHNIQUES OF METHOD DEVLOPMENT
7

INTRODUCTION OF ANALYTICAL METHOD [4-12]
 Analytical chemistry is divided into two branches:
 Qualitative: A qualitative analysis provides information about the identity of
atomic or molecular species or functional groups in sample.
 Quantitative: A quantitative analysis provides numerical information as to the
relative amount of one or more of these components.
 Analytical methods development and validation play important roles in the
discovery, development and manufacture of pharmaceuticals with the objectives. To
qualify and quantify the active pharmaceutical ingredients in bulk as well as
dosage form To establish impurity profile and limit of allowable impurities in
dosage form.
8

9
INTRODUCTION OF HPLC METHOD
 HPLC is an analytical technique widely used for identification, separation, detection
and quantification of various drugs and its related degradants.
 High performance liquid chromatography (HPLC), is a separation technique based
on a solid stationary phase and a liquid mobile phase. Most of the drugs in multi-
component dosage forms can be analysed by HPLC method because of the several
advantages like rapidity, specificity, accuracy, precision and ease of automation in
this method. HPLC method eliminates tedious extraction and isolation procedures.
 Principle of separation:
 The principle of separation in normal phase mode and reverse phase mode is
adsorption. When mixtures of components are introduced in to a HPLC column,
they travel according to their relative affinities towards the stationary phase. The
component which has more affinity towards the adsorbent travels slower. The
component which has less affinity towards the stationary phase travels faster. Since
no two components have the same affinity towards the stationary phase, the
components are separated.

CONT…..
 Different modes of separation in HPLC:
1) Normal phase mode.
2) Reverse phase ion pair chromatography.
3) Reversed phase mode.
4) Size exclusion chromatography.
10

Method development by RP-HPLC
11
 Reversed-phase chromatography is the mainly used in chromatographic
mode, it is used to separate neutral molecules in solution based on their
hydrophobicity.
 As the name suggested that, reversed-phase chromatography is the
reverse of normal-phase chromatography in the intelligence that it involves
the employ of a polar mobile phase and a non-polar stationary phase.
 It ensures that a decrease in the polarity of the mobile phase results in a
decreases in solute retention.

12
HPLC Techniques
Normal phase
Mode
Stationary phase : Polar
Mobile phase : Non polar
Reverse phase Mode
Stationary phase : Non
polar
Mobile phase : Polar

13
Fig. A schematic diagram of HPLC equipment.

VALIDATION OF ANALYTICAL METHOD [6-16]
14
 Validation is a process of establishing documented evidence, which
provides a high degree of assurance that a specific activity will
consistently produce a desired result or product meeting its
predetermined specifications and quality characteristics.
 It is an act of proving that any Procedure, process, equipment, material,
activity or System Perform as expected under given set conditions and
also give required accurate result.

15
 The Discussion of the Validation of analytical procedures is directed to the
four most common types of analytical procedures.
 Identification tests
 Quantitative tests for impurities content
 Limit tests for the control of impurities
 Quantitative tests of the active moiety in samples of drug substance on drug
product on other selected components in the drug product
 Various validation parameters are available to validate the method :
 Precision
 Accuracy
 Linearity
 Range
 Limit of Detection
 Limit of Quantification
 Selectivity
 Specificity
 Robustness

Introduction of stability indicating method [21]
16
 Stability indicating method is Quantitative analytical methods that are based
on the characteristic structural, chemical or biological properties of each active
ingredient of a drug product and that will distinguish each active ingredient
from its degradation products so that the active ingredient content can be
accurately measured.
 The purpose of Stability testing is to provide evidence on how the quality of a
drug substance or Product varies with time under the influence of a variety of
environment factor such as temperature, humidity , and light.
 Importance of stability indicating analytical method :
 To determine the self life of the drug and drug product.
 To determine the storage condition of product.
 To identify reaction which are involved in the degradation of a product.
 To establish the retest period for the drug product.

Cont........
17
 Force Degradation Studies:
 It is technique where different stress condition are applied over drug
substances and which in turn different degradation product are produced.
 These studies are called as stress degradation studies.
 These method are mainly used for determination of stability of molecule under
accelerated conditions.
1. Hydrolytic degradation
 Acid hydrolysis.
 Basic hydrolysis .
2. Oxidative degradation.
3. Thermal degradation.
4. Photolytic degradation.

Drug profile of Efonidipine hydrochloride ethanolate [22]
INTRODUCTION
CAS Number 111011-53-1
Description
Efonidipine hydrochloride ethanolate is a calcium channel
blocker of the dihydropyridine class, commercialized by
Shionogi & Co. (Japan). Initially, it was marketed in 1995 under
the trade name, Landel. The drug has been shown to block T-
type in addition to L-type calcium channels 1,2. It has also
been studied in atherosclerosis and acute renal failure 2. This
drug is also known as NZ-105, and several studies have been
done on its pharmacokinetics in animals
Structure
Chemical Formula C34H38N3O7P
19

Mol. Weight 631.666 gm/mol
IUPAC Name
2-(Phenyl-(phenyl methyl)amino)ethyl 5-(5,5-dimethyl-2-oxo-1,3-
dioxa-2λ5-phosphacyclohex-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-
1,4-dihydropyridine-3-carboxylate.
Categories
 Calcium Channel Blockers
 Antihypertensive Agents
Solubility
It is poorly soluble in water and freely soluble in methanol, very
slightly soluble in octanol and isopropylacetate
Mechanism
Of Action
This drug inhibits the L-type and T-type calcium channels, thereby
leading to vasodilation and decreased automaticity of the heart.
Efonidipine hydrochloride ethanolate exerts negative chronotropic
effects, decreasing heart rate. Acting on SA node cells by inhibiting
T-type calcium channel activity, Efonidipine prolongs the late
phase-4 depolarization of the sinoatrial node action potential,
decreasing heart rate.
logP 5.35
pKa 19.49
Melting point 151℃
20

Drug profile of Telmisartan [23]
INTRODUCTION
CAS Number 144701-48-4
Description
Telmisartan is an angiotensin II receptor antagonist (ARB)
used in the management of hypertension. Generally,
angiotensin II receptor blockers (ARBs) such as telmisartan
bind to the angiotensin II type 1 (AT1) receptors with high
affinity, causing inhibition of the action of angiotensin II on
vascular smooth muscle, ultimately leading to a reduction in
arterial blood pressure. Recent studies suggest that
telmisartan may also have PPAR-gamma agonistic properties
that could potentially confer beneficial metabolic effects.
Structure
Chemical Formula C33H30N4O2
21

Mol. Weight 514.629 gm/mol
IUPAC Name
2-(4-{[4-Methyl-6-(1-methyl-1H-1,3-benzodiazol-2-yl)-2-propyl-
1H-1,3-benzodiazol-1-yl]methyl}phenyl)benzoic acid
Categories
 Angiotensin II receptor blockers
 calcium channel blockers
Solubility
Practically insoluble in water and is soluble in acetone, ether, and
chloroform.
Mechanism
Of Action
Telmisartan blocks the vasoconstrictor and aldosterone-secreting
effects of angiotensin II by selectively blocking the binding of
angiotensin II to the AT1 receptor in many tissues, such as
vascular smooth muscle and the adrenal gland.
Characteristics White to slightly yellowish solid
logP 6.66
pKa 4.1
Melting point 261 °C to 263 °C
22

Review of Literature
 Official
Drugs Method Abstract Summary Ref. No
Efonidipine
hydrochloride
ethanolate
RP-HPLC IP
2018
Column: silica gel column 25 cm x 4 mm
Mobile phase :Acetonitrile and a buffer solution
prepared by dissolving 1.32 g of ammonium
phosphate in 900 ml of water,
Flow rate: 1.0 ml/min
Wavelength: 250 nm
24
Telmisartan RP-HPLC IP
2018
Column: stainless steel column 12.5 cm x 4.0
mm, packed with octadecylsilane bonded to
porous silica (5 μm),
Mobile phase : dissolve 2.0 g of potassium
dihydrogen phosphate and 3.8 g of sodium
pentane sulphonate monohydrate in water,
adjusted to pH 3.0 with ortho phosphoric acid
and dilute to 1000 ml with water
Wavelength: 230 nm
25
24

 Non - official
Efonidipine
hydrochloride
ethanolate
RP-HPTLC Column:silica gel 60 F254 aluminum plates
Mobile Phase: ethyl acetateto dichloromethane to
triethylamine (3:2:0.5 v/v)
Wavelength:251 nm.
26
Efonidipine
hydrochloride
ethanolate
RP-HPLC Column: C18 column (250 × 4.6 mm, 5 μ),
Mobile Phase: ammonium acetate buffer and acetonitrile
Wavelength:254 nm.
27
Efonidipine
Hydrochloride
Ethanolate
(EFD
RP-HPLC Column: C18, 5.0 mm column
Mobile Phase: acetonitrile and water ratio was selected
85: 15
Wavelength: 254 nm.
LOQ: 2.06 μg/Ml
Linearity range: 20-140 μg/ml
28
25

Efonidipine
hydrochloride
ethanolate
HPLC Column: C18 column (4.6 × 250 mm), packed with 5 μm particles
Mobile Phase: acetonitrile and 0.020 mol/l KH2PO4 (pH 2.5)
buffer in the ratio of 85:15 v/v
Wavelength:235 nm.
29
Telmisartan UHPLC Column: C18 column (4.6 x 50mm, 2.7μm)
Mobile Phase: acetonitrile: 50mM ammonium acetate buffer in
the ratio of (45: 55 v/v), pH adjusted to 4.5 with acetic acid
Wavelength:290 nm.
Flow rate:0.5 ml/min
Linearity range: 100-300 μg/ml
30
26

Telmisartan RP-HPLC Column: C18 column (150 X 4.6 mm , particle size 3.5μm).
Mobile Phase: Pentane sulphonic acid sodium salt mono
hydrate, add 1ml of Perchloric acid and adjust the pH-2.7±0.05
with Triethyl amine: Methanol in the ratio 40: 60 v/v
Wavelength:230 nm.
31
Telmisartan RP-HPLC Column: C18, column (250×4.6mm)
Mobile Phase: 0.025M potassium dihydrogen phosphate :
acetonitrile : methanol (45:50:5)
Wavelength:216 nm , Flow rate:1 ml/min
Linearity range: 100 to 500 ng/ml
LOQ: 83 𝜇g/ml , LOD: 27 𝜇g/ml
32
Telmisartan RP-HPLC Column: C18, column (250×4.6mm, particle size 5μm)
Wavelength:216 nm , Flow rate: 0.8ml/min
Injection volume : 10 μl
Run time : 10 min.
33
27

Telmisartan RP-HPLC Column: C18(150x4.6 mm, 5μm)
Mobile Phase: Acetonitrile and (0.1ml Phosphoric acid and
0.2ml Try Ethyl Amine in100mlof Triple distilled Milli-Q-
water) Buffer (35:65 v/v)
Wavelength:234 nm.
Linearity range: 0.1 – 0.6 mg/ml
retention time : 5.332 minutes
34
Telmisartan
and
Chlorthalidone
RP-HPLC Column:C -18 column (250 × 4.6mm ID,5 μm)
Mobile Phase: Acetonitrile : Methanol (85:15v/v)
Wavelength:242 nm.
Flow rate: 1 ml/min
Retention times: Telmisartan – 3.96 min
Chlorthalidone – 2.63 min
Linearity range: Telmisartan – 16-56 μg/ml
Chlorthalidone – 5-17.5 μg/ml
35
28

Telmisartan
and
Cilnidipine
HPTLC Mobile Phase: Toluene: Methanol: Glacial acetic acid (8: 2: 1,
v/v/v)
Wavelength:260 nm.
retention factor : Telmisartan : 0.38 ± 0.004
Cilnidipine : 100.79 ± 1.38
36
Chlorthalidone
,Telmisartan
and Benidipine
HCl
RP-HPLC Column: C18 (25 cm × 0.46 cm) Hypersil BDS column
Mobile Phase: .Buffer (pH 3.0): Methanol (50:50 v/v)
Wavelength:230 nm.
Flow rate: 1 ml/min
retention time : Chlorthalidone : 4.887 min
Telmisartan : 8.813 min
Benidipine : 6.690 min
Linearity range: Chlorthalidone : 6.25-18.75 μg/ml
Telmisartan : 20-60 μg/ml
Benidipine : 2-6 μg/ml
37
29

Telmisartan
and
amlodipine
RP-HPLC Column: C18 Column (100 x 4.6 mm, 5μ.)
Mobile Phase: Buffer (pH was adjusted to 3.6) and Acetonitrile
taken in the ratio 60:40
Wavelength:234 nm.
Flow rate: 1 ml/min
38
Bisoprolol
fumarate and
Telmisartan
RP-HPLC column : C18 (4.6 x 250 mm) column
Mobile Phase: methanol and water (75:25 v/v)
Flow rate: 1 ml/min
retention time : Bisoprolol : 5.7 min
Telmisartan : 7.6 min
Linearity range: Telmisartan – 5-25 μg/ml
Bisoprolol – 40-200 μg/ml
39
30

32
 Various analytical methods are reported for the analysis of individual
drug of Efonidipine hydrochloride ethanolate and Telmisartan and
also available in combination with other drug as per the literature
review.
 RP-HPLC method is not available for the simultaneous estimation of
Efonidipine hydrochloride ethanolate and Telmisartan in synthetic
mixture.
 There is no method available on stability indicating RP-HPLC method
for the simultaneous estimation of Efonidipine hydrochloride
ethanolate and Telmisartan in synthetic mixture.
 As stability indicating RP - HPLC method is mainly use for the
estimation of the API along with the impurities generated by force -
degradation study.
 So, it is worthwhile to develop & validate stability indicating method
RP - HPLC method for simultaneous estimation of Efonidipine
hydrochloride ethanolate & Telmisartan in synthetic mixture.

AIM AND OBJECTIVES
34
 Aim
 The aim of the present work to develop and validate a new simple, precise,
accurate, and reliable stability indicating RP-HPLC method for the
simultaneous estimation of Efonidipine hydrochloride ethanolate and
Telmisartan in Synthetic mixture.
 Objectives
 To develop stability indicating RP-HPLC method for simultaneous
estimation of Efonidipine hydrochloride ethanolate and Telmisartan in
Synthetic mixture.
 Approach to build up a suitable stability indicating method for Efonidipine
hydrochloride ethanolate and Telmisartan and to get a thought of how drug
substance or product degrades, degenerates & behaves under different
condition (force degradation).
 Applying the newly developed validated analytical method for the
estimation of Efonidipine hydrochloride ethanolate and Telmisartan in
Synthetic mixture.

MATERIAL AND EQUIPMENT
36
 Equipment used in method
Name Make/Model
UV Visible
Spectrophotometer
Shimadzu 1800
HPLC CYBERLAB LC100
FTIR Shimadzu
Ultrasonicator HINTRON CD4820
Analytical Weight
balance
Skyway
PH meter LABTRONICS LT-
14
ISO : 2008
Volumetric flask
(graduated)
10,25,50,100 ml
NAME MAKER
Methanol Merck
Acetonitrile Merck
Water Milli – Q -
Water
Di – Sodium Hydrogen
orth Phosphate
S.D fine
chemical
Pvt Ltd.,
Ortho
phosphoric acid
S.D fine
chemical
Pvt Ltd.,
 Chemicals used in method

Melting point determination
37
 Melting point of Drugs
Sr.No. APIs
Melting point (˚C)
Reported Measured
1 Efonidipine Hydrochloride
ethanolate
150 to 151 °C 149 - 150˚C
2 Telmisartan 261 to 263°C 261 - 262˚C
 Melting Point Determination of Efonidipine Hydrochloride and Telmisartan:
Melting point of Efonidipine Hydrochloride and Telmisartan have been determined by
using melting point apparatus.

FTIR
38
 Identification by FTIR Spectroscopy:
1. IR Spectra of Efonidipine Hydrochloride ethanolate :

39
Sr. No. Functional Group Frequency
1 C-H 2920
2 C-N 1257
3 C=C 1603
4 C=O 1757
5 N-H 2869
6 N-O 1522
7 P-O 1906
8 P=O 1014
9 C-O 1266
• IR Spectral interpretation of Efonidipine Hydrochloride ethanolate
Structure of Efonidipine Hydrochloride ethanoate

CON….
40
 Identification by FTIR Spectroscopy:
2. IR Spectra of Telmisartan :

41
• IR Spectral interpretation of Telmisartan
Sr. No. Functional Group Frequency
1 C-H 2850
2 C-N 1267
3 N-H 2915
4 C=O 1577
5 C=C 1696
6 C-O 1316
Structure of Telmisartan

CON….
42
 Solubility Study
The solubility of Efonidipine Hydrochloride and Telmisartan practically
determined separately by taking 100 mg of both the drugs in 100 ml volumetric
flasks, adding required quantity of solvent at room temperature and shaken for
few minutes. Solubility data for each study was observed and recorded.
Description Terms Relative Quantities of solvent for parts of solute
Very Soluble
Less than1 parts
Freely Soluble
From 1 to 10 parts
Soluble
From 10 to 30 parts
Sparingly Soluble
From 30 to 100 parts
Slightly Soluble
Very Slightly Soluble
Practically Insoluble
More than 10000 parts
 Solubility table as per IP 2010specification

CON….
43
 Solubility data of Efonidipine Hydrochloride
ethanolate and Telmisartan
Solvent Efonidipine Hydrochloride
ethanolate
Telmisartan
Water poorly soluble Practically Insoluble
0.1 N HCl Slightly soluble Slightly soluble
0.1 N NaOH soluble Slightly Soluble
Methanol soluble soluble
Ethanol soluble soluble

CON….
44
 Method development
 Selection of wavelength (λmax)
• The sensitivity of the HPLC method that uses UV detection depends
upon the proper selection of the wavelength.
• An ideal wavelength is one that gives good response for the drugs to
be detected.
• Efonidipine Hydrochloride and Telmisartan in Methanol, were
scanned in the UV-Visible range of 200 to 400 nm against blank
separately Then suitable wavelength for the detection of Efonidipine
Hydrochloride and Telmisartan was selected at 230 nm by
overlapping the spectrum of both the drugs.

CON….
45
 UV spectrum of Efonidipine Hydrochloride and Telmisartan

CON….
46
 Preparation of solutions
 Preparation of phosphate buffer solution
4.2568 gm of di-sodium hydrogen orthophosphate was weighed and sufficient
water (HPLC grade) was added to dissolve it. Then sonicate for 10 min. Then 1ml
of tri ethanol amine was added, the final volume was made up to 1000ml with
water and adjusted the pH to 3.5 with ortho phosphoric acid.
 Preparation of mobile phase:
Methanol, Buffer and Acetonitrile were mixed in the ratio of 65:30:5 and
sonicated for 20minutes, Filtered with 0.45 µ membrane filter.
 Preparation of 0.1N NaOH:
Accurately weighed 4gm of NaOH was transferred in100 ml volumetric flask and
diluted up to mark with methanol. From above solution take10 ml and
transferred in 100 ml volumetric flask and diluted up to the mark with
methanol.

CON….
47
 Preparation of 0.1N HCL:
Concentrated HCl (0.86ml)was transferred in 100 ml volumetric flask and dilute
up to the mark with methanol.
 Preparation of Hydrogen peroxidesolution (3%)
10ml Hydrogen peroxide was transferred in100ml volumetric flask and dilute up
to the mark with methanol.
 Preparations of working standard solution of Efonidipine Hydrochloride
and of Telmisartan
40mg of Efonidipine Hydrochloride and 40 mg of Telmisartan were accurately
weighed and transferred in to a separate 50 ml volumetric flask and sufficient
mobile phase was added to dissolve the drug. The final volume was made up to
50ml with mobile phase(primary stock solution). Pipette out 2ml from the above
stock solution into a 50ml volumetric flask and the final volume was made up to
the mark with the mobile phase. The solution contains 40 µg/ ml and 40 µg/ ml
concentration of Efonidipine Hydrochloride and of Telmisartan respectively

CON….
48
 Preparation of Sample solution Efonidipine Hydrochloride and of
Telmisartan:
20 tablets were weighed and powdered, tablets powder equivalent to 40mg of
Efonidipine Hydrochloride and 40mg of Telmisartan was transferred in to a 50 ml
volumetric flask, sufficient amount of mobile phase was added and dissolved by
20 minutes ultra sonication. Then made the volume up to the mark with the
mobile phase and filtered with 0.45 µ filter paper. Pipette out 2 ml from the above
solution and diluted to 50ml with the mobile phase The solution contains 40 µg/
ml and 40 µg/ml concentration of Efonidipine Hydrochloride and of Telmisartan
respectively.

49
Sr no Mobile phase Ratio Remarks
1 methanol: water 50:50 More noise peaks are observed
2 methanol: water 60:40 More noise peaks are observed
and eluted peaks are broad in
shape.
3 Phosphate buffer : methanol
(pH-4.5)
50:50 peak shape is not good
(pH-4.5)
20:80 Peak shape are not clear and
Tailing of peaks are observed
(pH-4)
40:60 peaks are elated, but failed in
tailing factor
(pH-4)
45:55 peaks are eluted, but failed in
tailing factor
(pH-3.5)
35:65 sharp peaks will not be
appearing
8 Phosphate buffer : ACN: methanol
(pH-3.5)
35:15:50 Peaks are eluted properly, but
tailing are observed
9 Phosphate buffer : ACN: methanol
(pH-3.5)
30:5:65 Clear peak observed, indicate
the good separation

CON….
50
 Trial - 1
 Chromatographic conditions and Selection of Mobile Phase Selection:
HPLC : LC 100 MODEL
Column : C 18 column (150x4.6mm, 5µm).
Flow Rate : 1.0 ml/min
UV Detection : 230nm
Injection Volume : 20 µl
Temperature : 25C
Run Time : 20 min.
Mobile phase : Methanol: Water (50:50)
Diluents : Mobile phase.

CON….
51
 Result:
 More noise peaks are observed and drug is not eluted.

CON….
52
 Trial - 2
HPLC : LC 100 MODEL
Temperature : 25oC
Run Time : 20 min.
Mobile phase : Methanol: Water (60:40)

CON….
53
 Result:
 peaks are observed but eluted peaks are not properly separated and more
tailing are observed.

CON….
54
 Trial - 3
HPLC : LC 100 MODEL
Temperature : 25oC
Run Time : 20 min.
Mobile phase : Buffer: Methanol (50:50); pH4.5.

CON….
55
 Result:
 Telmisartan and Efonidipine Hydrochloride peaks are eluted but of poor
resolution observed and Tailing of peaks are observed.

CON….
56
 Trial - 4
HPLC : LC 100 MODEL
Temperature : 25oC
Run Time : 20 min.
Mobile phase : Buffer: Methanol (20:80); pH4.5.

CON….
57
 Result:.
 Peak shape are not clear and resolution observed and Tailing of peaks are
observed.

CON….
58
 Trial - 5
HPLC : LC 100 MODEL
Temperature : 25oC
Run Time : 20 min.
Mobile phase : Buffer : methanol (40:60); pH-4

CON….
59
 Result:
 Telmisartan and Efonidipine Hydrochloride peaks are eluted, but failed in
tailing factor and also retention time both peaks are being more.

CON….
60
 Trial - 6
HPLC : LC 100 MODEL
Temperature : 25oC
Run Time : 20 min.
Mobile phase : Buffer : methanol (44:55); pH-4

CON….
61
 Result:
 Telmisartan and Efonidipine Hydrochloride peaks are eluted, but failed in tailing
factor and also retention time both peaks are being more.

CON….
62
 Trial - 7
HPLC : LC 100 MODEL
Temperature : 25oC
Run Time : 10 min.
Mobile phase : Buffer: Methanol and (35:65); pH3.5.

CON….
63
 Result:
 Peaks are eluted properly, but tailing are observed.

CON….
64
 Trial - 8
HPLC : LC 100 MODEL
Temperature : 25oC
Run Time : 4 min.
Mobile phase : Buffer: ACN: Methanol (35:15:50), pH-3.5.

CON….
65
 Result:
 Peaks are eluted properly, but tailing are observed

CON….
66
 Trial - 9
HPLC : LC 100 MODEL
Temperature : 25oC
Run Time : 4 min.
Mobile phase : Buffer: ACN: Methanol (30:5:65), pH-3.5.

CON….
67
 Conclusion :
 Efonidipine Hydrochloride and Telmisartan are eluted at 2.078 and 3.238
respectively, efficiency parameters are indicate the good separation,
asymmetric. So this method was selected for further analysis

Validation of developed Method

69
• The specificity of the method was established by injecting the blank and standard
solution. It is observed that there is no interference of the blank with principal peaks,
hence the method was specifically for these two drugs.
1. Specificity
Chromatogram of blank solution

70
Chromatogram of Efonidipine Hydrochloride and Telmisartan Standard
Chromatogram of Efonidipine Hydrochloride and Telmisartan synthetic mixture

71
• The working standard solution was injected 5 times into the HPLC,
chromatograms were recorded and measured the responses for the major
peaks. System suitability parameters such as retention time, theoretical
plates and asymmetric factor. Then the percentage of RSD of all the
parameters were calculated and presented in the table 6 the percentage RSD.
2. System suitability

72
EFH Area TEL Area
EFH
Theoretical
plates
TEL
Theoretical
plates
EFH
Tailing
factor
TEL
Tailing
factor
4208.745 400.587 3568.305 4836.127 1.276 1.105
4208.746 3965.74 3586.231 4836.241 1.285 1.118
4198.754 3985.67 3528.97 4863.727 1.256 1.126
4184.764 3978.54 3594.212 4758.963 1.274 1.113
4207.841 3998.52 3567.422 4698.521 1.293 1.133
AVG 4201.770 3986.86 3569.028 4798.716 1.2768 1.119
SD 10.39799 15.9219 25.1889 68.2888 0.01388 0.0109
% RSD
0.247467 0.3993 0.7057 1.4230 1.0872 0.9769

73
• Appropriate volume from the stock solution was diluted to get the final concentration
of 20, 30, 40, 50, 60 µg/ml for Efonidipine Hydrochloride and 20, 30, 40, 50, 60
µg/ml for Telmisartan. Then the chromatogram was recorded for each concentration,
plot the graph concentration versus area. The calibration graph was shown in figure.
3. Linearity
Overlay chromatogram of different concentration

74 Calibration curve of Efonidipine Hydrochloride
Sr. No.
Efonidipine Telmisartan
Concentration
(µg/ml)
Area
(n=3)
Concentration
(µg/ml)
Area
(n=3)
1 20 2208.745 20 2207.745
2 30 3208.746 30 3134.746
3 40 4208.745 40 4194.554
4 50 5308.746 50 5284.563
5 60 6498.754 60 6403.641
1 2 3 4 5
X 20 30 40 50 60
Y 2208.745 3208.746 4208.745 5308.746 6498.754
y = 106.8x + 14.74
R² = 0.9985
0
1000
2000
3000
4000
5000
6000
7000
AREA
EFO

75
Calibration curve of Telmisartan
1 2 3 4 5
(µg/ml) 20 30 40 50 60
area 2207.745 3134.746 4194.554 5284.563 6403.641
y = 1054.2x + 28.40
R² = 0.9988
0
1000
2000
3000
4000
5000
6000
7000
AREA
TEL
 From the linearity study, Efonidipine Hydrochloride and Telmisartan were
found to be linear in the concentration range from 20 - 60 µg/ml and 20 -
60 µg/ml. Correlation coefficient values (R2) were found to be 0.9993 and
0.9996 for Efonidipine Hydrochloride and Telmisartan respectively.
Results revealed that good correlation exists between the concentration
of the sample and their area.

76
 Precision study was carried out by injecting a sample solution in to HPLC without
changing the assay procedure and the results are shown the % RSD is less than 2 %
for Efonidipine Hydrochloride and Telmisartan. The low RSD value indicated that the
method was precise.
1. Intermediate precision:
• Intermediate precision was performed by injecting the sample solution in to HPLC at
three different days, different analysts and in different instruments.
• This study was performed with a minimum of three replicate measurements of
sample solution at 0 hrs, 8 hrs and 16 hrs in a same day.
2. Reproducibility:
• Reproducibility studies were done in two laboratories and the results were
compared.
• All the precision study results presented in the table 7.
4. Precision

77
Parameters Sampling
time
EFH TEL
Amount
present (mg)
Amount
present (%)
RSD
(%)
Amount
present (mg)
Amount
present (%)
RSD
%
Intra day
Precision
0 hrs 40.67 99.02 0.0920 40.62 100.97 1.4542
2nd hrs 40.45 99.93 0.9449 40.37 100.62 0.5498
4th hrs 40.78 100.79 0.3633 40.60 100.83 0.7566
Inter day
Precision
I st Day 40.28 100.92 0.4993 40.55 100.42 0.7712
2nd day 40.95 100.71 0.3197 40.63 101.06 0.6141
3rd day 40.56 99.50 0.1257 40.70 101.64 0.1250
Reproducibility Analyst -1 40.95 100.45 0.1907 40.63 101.07 0.8081
Analyst -2 40.78 100.87 0.1197 40.61 100.94 0.6498
Table - 7: Precision results for Efonidipine Hydrochloride and Telmisartan

78
• This study was performed using a minimum of 3 concentration levels, each in
triplicate determinations.
• 50%, 100% and 150% from the label claim of Efonidipine Hydrochloride and
Telmisartan was taken in to a 50 ml volumetric flask and sufficient mobile
phase was added, sonicated 20 min for dissolving the drugs, final volume was
adjusted up to the mark with the mobile phase.
•
• Pipette out 2 ml from the above solution into a 100 ml volumetric flask and
final volume was adjusted up to the mark with the mobile phase.
• Then chromatogram was recoded in triplicate and the results are shown in
table – 8.
5. Accuracy

79
Concentration
(%)
Added AMT
amount
(mg)
Amt recovered
(mg)
Amt recovered
(%)
EFH / TEL EFH TEL EFH TEL EFH TEL
50 20 20 20.65 20.94 99.90 100.20
100 40 40 40.17 40.49 99.03 101.19
150 60 60 60.83 60.87 99.49 99.11
 The percentage recoveries of the three concentrations (50%, 100% and 150%)
were found to be close to 100%, indicative of high accuracy of this method.
Table - 8: Recovery results for Efonidipine Hydrochloride and Telmisartan

80
• The LOD and LOQ of the developed method were determined by analyzing
progressively low concentration of the standard solution using the developed
methods.
• The LOD is the concentration of the analyte that gives a measurable response
(signal to noise ratio 3.3).
• The LOQ is the lowest concentration of the analyte, which gives a response
that can be accurately quantified (signal to noise ratio of 10).
• LOD and LOQ of Efonidipine Hydrochloride and Telmisartan was found to be
0.359561 ng/ml, 0.08403 ng/ml and 1.08958 ng/ml, 0.254658 ng/m (Table
12).
6. LOD and LOQ

81
• Calibration curve was repeated for five times and the standard deviation of the
intercept was calculated. Then LOD and LOQ were calculated as follows,
Where, SD = Standard deviation of intercepts of 6 calibration curves
Slope = Mean slope of the 5 calibration curves
Parameter Efonidipine
Hydrochloride
Telmisartan
SD 1113.106 515.5502
Slope 10215.91 20244.82
LOD (µg/mL) 0.359561 0.084037
LOQ (µg/mL) 1.08958 0.254658
Table : LOD and LOQ Data of Efonidipine Hydrochloride and Telmisartan

82
• Robustness of the method were determined by changing the method
parameters (wavelength ± 1nm from 1nm, Flow rate ± 0.1 (Chromatogram
no. 26,27), pH ± 0.05 and the mobile phase ratio ± 2 %), the results were
presented in Table 11. Based on the results of these studies show, the small
changes made to the method procedure, but it will not affect the method
results so this method is robust.
7. Robustness

83
EFP TEL
Parameters Amount
present
(mg)
Amount
present
(%)
RSD
%
Amount
present
(mg)
Amount
Present
(%)
RSD
%
Wavelength
(nm)
210 40.04 98.60 0.1139 12.64 101.16 0.0549
240 40.57 101.11 0.1237 12.63 101.11 0.0504
Flow Rate
(ml/min)
1.3 40.87 100.57 0.3725 40.61 100.94 0.4278
1.1 40.90 100.58 0.7906 40.65 101.23 0.0153
Mobile phase
(% of
(Methanol)
67 40.99 100.59 0.3907 40.65 101.27 0.1750
63 40.86 100.97 0.09942 40.58 100.66 0.3853
pH 3.55 40.76 99.75 1.1828 40.64 101.18 0.0634
3.45 40.30 100.06 1.3808 40.63 101.08 0.0801
Table - 11: Results observed by changing the wavelength ± 1nm

84
• Stability of drug solution was determined by keeping the drug solution for 3
days at room temperature. The chromatogram was recorded by injecting
the sample solution at once per day and calculated the amount of drug
present. There is no significant degradation was observed. The stability test
results (Table 13) are indicating the drug solutions are stable up to 3 days at
room temperature.
8. Stability Studies
EFP TEL
Parameters
Amount
present
(mg)
Amount
present
(%)
Amount
present (mg)
Amount
Present (%)
Day 1 40.12 98.60 40.65 101.25
Day 2 40.38 100.87 40.64 101.17
Day 3 40.88 100.77 40.64 101.16
Table – 13: Stability studies of Efonidipine Hydrochloride and Telmisartan

85
• Sample solution was prepared according to the above described procedure. Then the
solution was injected in to the HPLC and calculated the amount of Efonidipine
Hydrochloride, Telmisartan present in the each tablet by using the above mentioned
formula, results are present in the table.
Assay
Amt present %Amt present
Efonidipine
Hydrochloride
AVG 40.4721 100.0944108
SD 2.977577 1.195515343
% RSD 1.194388 1.194387712
Telmisartan
AVG 40.54347 100.3477463
SD 0.091207 0.729659142
% RSD 0.727131 0.727130573
Table – 14: Assay results for Efonidipine Hydrochloride and Telmisartan

1. Acid Degradation
87
• Acid decomposition studies were performed by Refluxing 1 ml of stock solution was
transferred in to 10 ml of volumetric flask. 2 ml of 0.1 N Hydrochloride solution and 2
ml of 0.1 N NaOH was added and mixed well and put for 4 hrs. Then neutralize with
2ml 0.1N NaOH to stop the degradation further. Now make up volume with mobile
phase Then the volume was adjusted with diluents to get 40μg/ml for Efonidipine
Hydrochloride and 40μg/ml for Telmisartan.
Acid Degradation Blank

88
Efonidipine Hydrochloride Acid Degradation Standard
Telmisartan Acid Degradation Standard

89
Efonidipine Hydrochloride and Telmisartan Acid Degradation Sample
Parameter Acid
Drug Efonidipine Hydrochloride Telmisartan
Area of Standard 4414.012 1356.255
Area of Degradation 3362.11 1163.484
% Degradation 22.57 17.07
Acid Degradation of Drug Efonidipine Hydrochloride and Telmisartan

90
• Basic decomposition studies were performed by refluxing 1 ml of stock solution was
transferred in to 10 ml of volumetric flask. 2 ml of 0.1 N NaOH solutions and 2 ml of
0.1 N HCl was added and mixed well and put for 3 hrs. After time period the content
was cooled to room temperature. Then the volume was adjusted with diluents to get
40μg/ml for Efonidipine Hydrochloride and 40μg/ml for Telmisartan.
2. Base Degradation
Base Degradation Blank

91
Efonidipine Hydrochloride Base Degradation
Telmisartan Base Degradation

92
Efonidipine Hydrochloride and Telmisartan Base Degradation Sample
Parameter Base
Base Degradation of drug Efonidipine Hydrochloride and Telmisartan

93
• Oxidative decomposition studies were performed by refluxing 1 ml of stock solution
was transferred in to 10 ml of volumetric flask. 2 ml of 3% Hydrogen peroxide
solutions was added and mixed well and put for 2 hrs at Room temperature. Then
the volume was adjusted with diluents to get 40μg/ml for Efonidipine Hydrochloride
and 40μg/ml Telmisartan.
3. Oxidative Degradation
Oxidation Degradation Blank

94
Efonidipine Hydrochloride Oxidation Degradation
Telmisartan Oxidation Degradation

95
Efonidipine Hydrochloride and Telmisartan Oxidation Degradation sample
Parameter Oxidation
Oxidative Degradation of Efonidipine Hydrochloride and Telmisartan

96
• Photo Degradation studies were performed 1 ml of stock solution was transferred in
to 10 ml of volumetric flask. The volumetric flask was keep in UV light for 24 hrs.
Then the volume was adjusted with diluents to get 40μg/ml for Efonidipine
Hydrochloride and 40μg/ml for Telmisartan.
4. Photo Degradation
Photo Degradation Blank

97
Efonidipine Hydrochloride Photo Degradation
Telmisartan Photo Degradation

98
Efonidipine Hydrochloride and Telmisartan Photo Degradation sample
Parameter Photo Degradation
Photo Degradation of Efonidipine Hydrochloride and Telmisartan

99
• Thermal Degradation studies were performed One ml of stock solution was
transferred in to 10 ml of volumetric flask. The volumetric flask was stored in oven at
105°C for 3 hrs. Then the volume was adjusted with diluents to get 40μg/ml for
Efonidipine Hydrochloride and 40μg/ml for Telmisartan.
5. Thermal Degradation
Thermal Degradation Blank

100
Efonidipine Hydrochloride Thermal Degradation
Telmisartan Thermal Degradation

101
Efonidipine Hydrochloride and Telmisartan Thermal Degradation sample
Parameter Thermal
Thermal Degradation of Efonidipine Hydrochloride and Telmisartan

102
S.No Parameters Observation Acceptance Criteria
1
Specificity Placebo
Interference
No peak was observed at the RT
of Efonidipine Hydrochloride
and telmisartan in the
chromatogram of placebo
On this chromatogram should not
show any peak at the RT of Efonidipine
Hydrochloride and telmisartan
2
System suitability
Efonidipine Hydrochloride
1.2768 Tailing factor: < 2.0
3569.028
Theoretical plates : >2000
0.7057 % RSD: <2.0
telmisartan
1.119 Tailing factor :< 2.0
4798.716
Theoretical plates: >2000
1.4230 % RSD: < 2.0
3
Linearity & Range
Efonidipine Hydrochloride 0.9993 Correlation coefficient: < 0.995
telmisartan 0.9996
4
Accuracy
99.90– 99.49% 98.0 – 102.0%
telmisartan 100.20 – 99.11%
5
Method Precision
1.45
% RSD : < 2.0
telmisartan 0.09

103
S.No Parameters Observation Acceptance Criteria
6 LOD LOQ
Efonidipine
Hydrochloride
0.359561 - 1.08958 Overall % RSD < 2.0
telmisartan 0.084037 - 0.254658
7
Robustness
Efonidipine
Hydrochloride
1.39 – 1.07 Tailing factor: < 2.0
4755 – 90856 Theoretical plates: >2000
0.1139– 1.3808 % RSD: < 2.0
telmisartan
1.00 – 1.05 Tailing factor: < 2.0
13715 - 16403 Theoretical plates: >2000
0.0549– 0.0801 % RSD: < 2.0
8
Solution stability
Efonidipine
Hydrochloride
98.60-100.77
% assay
telmisartan 101.25- 101.16

104
 The developed RP-HPLC method was specific, simple, sensitive, precise,
accurate and robust, for the detection of Efonidipine Hydrochloride
ethanolate and Telmisartan in synthetic mixture.
 The proposed method is quite simple and do not require any pre-
treatment of the drug and tedious extraction procedure.
 The method has a wider linear dynamic range with good accuracy and
precision.
 So the proposed method is used for the routine analysis of Efonidipine
Hydrochloride ethanolate and Telmisartan in pharmaceutical quality
control.
Conclusion

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