“STABILITY INDICATING RP-HPLC AND UV METHODS FOR THE
SIMULTANEOUS ESTIMATION OF LOSARTAN POTASSIUM AND
AMLODIPINE IN BULK DRUGS AND DOSAGE FORMULATION”
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA,
BANGALORE.
BY
Ms. RAMYA GAVINI
Under The Guidance of
Dr. SANGAMESH B. PURANIK
P.G. DEPARTMENT OF PHARMACEUTICAL ANALYSIS
EAST WEST COLLEGE OF PHARMACY, BANGALORE – 560091
2010 – 2012
1
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE –ІІ
REGISTRATION OF SUBJECT FOR DISSERTATION
1.0
NAME AND ADDRESS OF
RAMYA GAVINI
THE CANDIDATE
DEPARTMENT OF PHARMACEUTICAL ANALYSIS,
EAST WEST COLLEGE OF PHARMACY,
#63,1 PHASE,BEL LAYOUT,BHARATHNAGAR,
VISHWANEEDAM EAST PO.OFF. MAGADI ROAD,
BANGALORE-560 091
KARNATAKA.
2.0
NAME OF THE INSTITUTION
EAST WEST COLLEGE OF PHARMACY,
#63,1 PHASE,BEL LAYOUT, BHARATHNAGAR,
VISHWANEEDAM EAST PO.OFF. MAGADI ROAD,
BANGALORE-560 091
KARNATAKA.
3.0
COURSE OF STUDY & SUBJECT
MASTER OF PHARMACY
(PHARMACEUTICAL ANALYSIS)
4.0
5.0
DATE OF ADMISSION
27th AUGUST 2010
TITLE OF THE TOPIC:
“STABILITY INDICATING RP-HPLC AND UV METHODS FOR THE SIMULTANEOUS
ESTIMATION OF LOSARTAN POTASSIUM AND AMLODIPINE IN BULK DRUGS AND
DOSAGE FORMULATION”
2
6.0
BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED FOR STUDY:
Stability is defined as the capacity of a drug substance or drug product to remain within
established specifications to maintain its identity, strength, quality, and purity throughout the retest or expiration dating periods. The purpose behind stability testing of a drug is that, to monitor
quality of a drug product which may vary with time because of the influence of many of the
environmental conditions such as temperature, light, humidity etc. The results are applied in
developing manufacturing processes and selecting proper packaging, storage conditions, product's
shelf life and expiry dates.
Stress testing of the drug can help to identify the degradation products which can help to
establish degradation pathways and the intrinsic stability of the molecule and validate stability
indicating power for analytical procedures used.
Validation of a method indicates to establish documented evidence that the system is doing
what is purpose to do. Validation is necessary when a method or a procedure is going to be used by a
manufacturing company or to be published in any Pharmacopoeias. The validated assay methods will
be more accurate, precise and reproducible.
Basic criteria for new method development of stability indicating assay method:

The drug or drug combination may not be official in any pharmacopoeias.

A proper stability indicating analytical procedure for the drug may not be available in
the literature due to patent regulations.

The existing analytical procedures may require expensive reagents and solvents. It may also
involve cumbersome extraction and separation procedures and these may not be reliable.
Stability indicating assay method provides the support to track the quality of the product from
time to time.
3
Methods for analyzing drugs in dosage forms can be developed, provided one has knowledge
about the nature of the sample, its molecular weight, polarity, ionic character and the solubility
parameter. Method development involves
considerable trial
and
error procedures. The most
difficult problem usually is where to start, what type of column is worth trying with what kind of
mobile phase.
The following is a suggested method development scheme for a typical HPLC-UV
related substance method.
1. To define the goals for method development (e.g., what is the intended use of the method), and to
understand the chemistry of the analytes and the drug product.
2. To develop preliminary HPLC conditions to achieve minimally acceptable separations. These
HPLC conditions will be used for all subsequent method development experiments.
3.To develop a suitable sample preparation scheme for the drug product
4.To determine the appropriate standardization method and the use of relative response factors
in calculations.
5.To identify the “weaknesses” of the method and optimize the method through experimental
design. Understand the method performance with different conditions, different instrument set ups
and different samples.
6. To complete method validation according to ICH guidelines as mentioned in Q2 (R1).
Losartan potassium and Amlodipine combination is available as marketed formulation and prescribed
by physician for the treatment of hypertension. A few of the brands available in the market are
AMLOZAAR (MICRO CARSYON), LOZADIP (ALKEM), NUSAR-AM LOW (EMCURE).
4
DRUG PROFILE:
Losartan Potassium¹
Amlodipine²
PARAMETERS
LOSARTAN POTASSIUM¹
AMLODIPINE²
IUPAC name
(2-butyl-4-chloro-1-{[2’-(1Htetrazole-5-yl)biphenyl]methyl}
-1H-imidazole-5-yl) methanol
monopotassium salt.
(RS)-3-ethyl 5-methyl 2-[(2-amino
ethoxy) methyl]-4-(2-chlorophenyl)
-6-methyl-1, 4-dihydropyridine-3,
5-dicarboxylate.
Molecular formula
C22H23ClKN6O
C20H25ClN2O5
Molecular weight
461.91g/ml
408.87g/ml
Solubility
Freely soluble in water
Freely soluble in water
Category
Angiotensin II receptor antagonist
Calcium channel blocker
Bioavailability
25-35%
64-90%
Half life
1.5 to 2hours
30 to 50hours
Excretion
Renal 13-25%
Renal
PKa
---
8.6
Routes
oral
Oral
5
PHARMACOLOGY
LOSARTAN :
MECHANISM OF ACTION
Losartan is a selective, competitive angiotensin II receptor type 1 (AT1) receptor
antagonist, reducing the end organ responses to angiotensin II. Losartan administration results in a
decrease in total peripheral resistance (after load) and cardiac venous return (preload) All of the
physiological effects of angiotensin II, including stimulation of release of
aldosterone, are
antagonized in the presence of losartan..
DRUG INTERACTIONS
Concurrent use with NSAIDs may further worsen renal function. Cimetidine. Phenobarbital
and other enzyme inducers may decrease levels of losartan and its active metabolite. Ketoconazole
inhibits the conversion of losartan to its active metabolite.
AMLODIPINE :
MECHANISM OF ACTION
Amlodipine is a dihydropyridine calcium antagonist (calcium ion antagonist or slowchannel blocker) that inhibits the transmembrane influx of calcium ions into vascular smooth muscle
and cardiac muscle. Experimental data suggest that amlodipine binds to both dihydropyridine and
nondihydropyridine binding sites. The contractile processes of cardiac muscle and vascular smooth
muscle are dependent upon the movement of extracellular calcium ions into these cells through
specific ion channels.
DRUG INTERACTIONS
In patients with severe coronary artery disease, amlodipine can increase the frequency
and severity of angina or actually cause a heart attack on rare occasions. This phenomenon usually
occurs when first starting amlodipine, or at the time of dosage increase.
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6.2 REVIEW OF LITERATURE :
A literature survey was carried out for the stability indicating assay methods for the
combination of losartan potassium and amlodipine. It was found that no methods have been
reported for this combination. The collection of references have been reported below:

Permender Rathee, Sushila Rathee, Dharmender Rathee and Hema Chaudhar3, have developed
a new stability indicating UV-spectroscopic methods for the simultaneous assay of losartan
potassium (LP) and hydrochlorothiazide (HCZ) in bulk drugs and tablet dosage forms by using
ELICO SL 160 double beam UV-VIS spectrophotometer. Diluent used was 0.01N HCl and
the methods used were simultaneous equation and Q-analysis, the λmax for LP and HCZ in the
solvent were found to be 227.4nm, 270.4nm and 256.4nm, 270.4nm for method A and method
B respectively. Beer’s law obeyed in the range of 2.02-27.27µg/ml, 3.03-27.27µg/ml and 5.055.50µg/ml, 3.03-27.27µg/ml for LP and HCZ for method A and method B respectively,
linearity range was found to be 4.04-22.22µg/ml for LP and 6.06-27.27µg/ml for HCZ. LOD
and LOQ were found to be 0.233µg/ml and 0.186µg/ml for LP and 0.708µg/ml, 0.563µg/ml
for HCZ respectively.

Chitlange SS, Kiran Bagri and Sakarkar DM4, have developed and validated a RP-HPLC
method for the simultaneous estimation of amlodipine besylate (AMLB) and valsartan (VAT)
by using a gradient HPLC (Merck Hitachi) with L-7400 UV-detector. A C18 (Kromasil,
250×4.6mm) column was used, mobile phase used was a combination of acetonitrile:
phosphate buffer (0.02M, pH-3.0) in the ratio of 56:44v/v at a flow rate of 1ml/min
respectively and the eluent was monitored at 234nm, retention time for AMLB and VAT was
found to be 3.07 and 6.20min, respectively, linearity range for AMLB and VAT were found to
be 1-40µg/ml. The LOQ and LOD of AMLB and VAT were found to be 0.089µg/ml,
0.03µg/ml and 0.054µg/ml, 0.018µg/ml, respectively.

Kavitha J, Muralidharan S5, have developed and validated a RP-HPLC method for the analysis
of atenolol, hydrochlorothiazide and losartan potassium in tablet formulation by using
Shimadzu HPLC with SPD M-10AVP Photodiode Array Detector. A phenomenex C18
column (250×4.6mm) was used, mobile phase consisted of acetonitrile: 50mM potassium
dihydrogen ortho phosphate (pH-3.5) ratio 50:50v/v with a flow rate of 1.0ml/min.
Sulphadoxine
was
selected
as
internal
standard,
retention
time
of
atenolol,
hydrochlorothiazide, losartan potassium and internal standard were found to be 5.550, 3.280,
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7.370 and 12.397sec, respectively and detection wavelength was 270nm, linearity range for
atenolol, hydrochlorothiazide and losartan potassium were found to be 10-20, 2.5-15, 560µg/ml, LOD and LOQ of atenolol, losartan potassium, hydrochlorothiazide were found to be
10, 5, 7ng/ml and 30, 15, 20ng/ml, respectively.

Mustafa Celebier, Mustafa Sinan Kaynak, Sacide Altinoz, Selma Sahin 6, have developed and
validated a HPLC method for the simultaneous estimation of amlodipine(AMD) and valsartan
(VAL) in their combined dosage forms and for drug dissolution studies. The HPLC system
consisted of Waters 2996 Photodiode Array Detector, C18 column was used with mobile
phase of phosphate buffer (pH-3.6): acetonitrile: methanol (46:44:10v/v/v), flow rate was
1min/ml and the detection wavelength was 240nm, linearity range for amlodipine was 0.150µg/ml and for valsartan was 0.05-50µg/ml, retention time for amlodipine and valsartan were
found to be 7.1min and 3.4min, respectively, LOD and LOQ for VAL and AMD were found
to be 0.02µg/ml, 0.05µg/ml and 0.05µg/ml, 0.1µg/ml, respectively.

Patel AI, Oza CK, Prajapati JP, Vyas AJ and Mehta P7, have developed and validated a RPHPLC method for the simultaneous estimation of losartan potassium and perindopril erbumine
in its tablet form using HPLC JASCO PU-2080 plus with JASCO UV-2075 plus UV-Visible
detector and C18 column (250mm×4.5mm) with mobile phase of acetonitrile: water
(50:50v/v), pH was adjusted to 3.2 with 1% o-phosphoric acid, flow rate was 1.0ml/min and
the detection wavelength was 210nm, retention time of losartan potassium and perindopril
erbumine was found to be 6.7min and 4.5min, respectively with linearity range of 2-18µg/ml
for both the drugs, LOQ of losartan potassium and perindopril erbumine were found to be
0.109µg/ml and 0.041µg/ml and LOD of losartan potassium was found to be 0.035µg/ml,
respectively.

Srinivasa Rao K and Srinivas K8, have developed a RP-HPLC method for the simultaneous
determination of losartan potassium [LP] and ramipril in tablet dosage forms by using
Shimadzu HPLC with UV/VIS detector. A C18 column was used with mobile phase of
acetonitrile: methanol: 10mM tetra butyl ammonium hydrogen sulphate [30:30:40%v/v/v],
flow rate was 1.0ml/min and detection wavelength was 210nm, retention time of LP and
ramipril were 4.7 and 3.3min with linearity range of 0.04-100µg/ml and 0.2-300µg/ml for LP
and ramipril, LOD and LOQ for LP and ramipril were found to be 0.071µg/ml, 0.275µg/ml
and o.11µg/ml, 0.322µg/ml, respectively.
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6.3 OBJECTIVES OF THE STUDY :
The objective of the present study is to establish and generate inheriting stability
data for losartan and amlodipine through stress degradation studies under ICH recommended stress
conditions (like temperature, light, solvent system, exposure to UV radiations) and develop stability
indicating assay method. ICH recommend a generation of stability data through stress degradation
studies for life saving drugs as they are key to understand the fate of drugs exposed under various
stringent conditions, right from the production up to their consumption by the patients as medication.
7.0
MATERIALS AND METHODS :
 All experiments shall be carried out in the Department Of Pharmaceutical Analysis, East West
College of pharmacy, Bangalore.
 Pure samples of losartan potassium and amlodipine shall be procured from industries involved
in bulk manufacturing of these drugs.
 Dosage formulation shall be procured from local market.
 The methods shall be developed and validated in pharmaceutical analysis lab of East West
College of Pharmacy.
 The methods shall be first developed, and then validated as per ICH guidelines, and then the
method shall be applied to the formulations.
 HPLC instrument Shimadzu Prominence LC-20A, Binary Gradient HPLC System with Luna
C18 column shall be used.
 UV-VIS spectrophotometer Shimadzu 1800 with matched quartz shall be used for measuring
absorbance for losartan potassium and amlodipine solutions.
7.1 SOURCES OF DATA :
 References from library-East West College of Pharmacy
 www.google.com
 www.sciencedirect.com
 www.drugupdate.com
 www.rxlist.com
 www.pharmainfo.net
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SOURCES OF EXPERIMENTAL DATA :
Experimental investigation in the laboratory.
UV-spectrophotometer, HPLC.
7.2 METHODS :
Degradation studies
a) Hydrolytic studies : Acid decomposition studies will be performed by heating the solution of drug
in 0.1N Hcl at 80ºc for 12hrs.The studies in alkaline conditions will be done using 0.1M NaOH
followed by heating at 80ºc for 12hrs. For study in neutral conditions, drug will be dissolved in
aqueous medium.
b) Oxidative studies :The studies will be performed using initially 3% and than 10% H2O2 and
heating at 80ºc for 12hrs.
c) Photolytic studies: The studies will be performed by keeping the sample in photostability chamber
or direct sunlight for 48hrs.
d) Thermal (dry heat) studies: susceptibility of the drug to the dry heat will be studied by exposing
the solid drug to 100ºc for 12 to 14hrs.
7.3 Does the study require any investigation to be conducted on patients and animals?
No
7.4 Has the ethical clearance been obtained from your institution in case of 7.3?
Not applicable
8.0
REFERENCES:
1. www.drugbank.com (http://www.chemicalland21.com/lifescience/uh/losartan potassium.htm)
2011 May22;10:30.
2. www.drugbank.com (http://www.drugbank.ca/drugs/DB00381) 2011 May22;11:00.
3. Permender Rathee, Sushila Rathee, Dharmender Rathee, Hema Chaudhary. Stability indicating
UV-spectroscopic methods for simultaneous determination of losartan potassium and
hydrochlorothiazide in pharmaceuticals. Eurasian J Anal Chem 2009;4(1):98-109.
4. Chitlange SS, Kiran Bagri, Sakarkar DM. Stability indicating RP-HPLC method for
simultaneous estimation of valsartan and amlodipine in capsule formulation. Asian J Res
10
Chem 2008;1(1):15-18.
5. Kavitha J, Muralidharan S. Development and validation of new method for atenolol,
hydrochlorothiazide and losartan potassium by RP-HPLC. Int J ChemTech Res 2010;2(2):
880-4.
6. Mustafa Celebier, Mustafa Sinan Kaynak, Sacide Altinoz, Selma Sahin. HPLC method
development for the simultaneous analysis of amlodipine and valsartan in combined dosage
forms and invitro dissolution studies. Braz J Pharm Sci 2010;46(4):761-8.
7. Patel AI, Oza CK, Prajapati JP, Vyas AJ, Mehta P. RP-HPLC method for the determination of
losartan potassium and perindopril erbumine in combined tablet dosage form. Int J Pharm and
BioSci 2011;2(1):709-15.
8. Srinivasa Rao K, Srinivas K. RP-HPLC method for the determination of losartan potassium
and ramipril in combined dosage form. Indian J Pharm Sci 2010;72(1):1-4.
9. Permender Rathee, Sushila Rathee, Dharmender Rathee, Hema Chaudhary. Simultaneous
estimation of amlodipine besylate and lisinopril dihydrate as API and tablet dosage forms. Int J
PharmTech Res 2010;2(1):556-62.
10. Bharat Chaudhary G, Ashok Patel B. Simultaneous spectroscopic estimation of atorvastatin
calcium and amlodine besylate in tablet dosage forms. Int J ChemTech Res 2010;2(1):633-9.
11. Santaji Nalwade, Vangala Ranga Reddy, Dantu Durga Rao, Inabathina Koteswara Rao. Rapid
simultaneous determination of telmisartan, amlodipine besylate and hydrochlorothiazide in a
combined poly pill dosage forms by stability indicating ultra performance liquid
chromatography. Sci Pharm 2011;79:69-84.
12. Deanne Hetzog L, Jennifer Finnegan McCafferty, Xueguang fang, Jeffrey Tyrrell R, Robert
Reed A. Development and validation of a stability-indicating HPLC method for
the
simultaneous determination of losartan potassium, hydrochlorothiazide and their degradation
products. J Pharm Biomed Anal 2002;30:747-60.
13. Priyanka Patil R, Sachin Rakesh U, Dhabale PN, Burade KD.RP-HPLC method for the
simultaneous estimation of losartan potassium and amlodipine besylate in tablet formulation.
Int J ChemTech Res 2009;1(3):464-9.
14. ICH of technical requirements for registration of pharmaceuticals for human use document.
Validation of analytical procedure: Text and methodology ICH Q2 (R1). www.bioforum.org
(http://www.bioforum.org.il/uploads/editor/karen/q2_r1-step4.pdf) 2011 May 19; 3:59.
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9.0
SIGNATURE OF THE CANDIDATE
(RAMYA GAVINI)
10.0
REMARKS OF THE GUIDE
11.0
NAME AND DESIGNATION OF GUIDE
11.1
SIGNATURE
11.2
HEAD OF THE DEPARTMENT
11.3
SIGNATURE
12.0
REMARKS OF THE PRINCIPLE
12.1
SIGNATURE
Dr. SANGAMESH B. PURANIK
PROFESSOR AND HOD,
DEPT. OF PHARMACEUTICAL ANALYSIS,
EAST WEST COLLEGE OF PHARMACY,
BANGALORE-560 091.
Dr. SANGAMESH B. PURANIK
PROFESSOR AND HOD
DEPT. OF PHARMACEUTICAL ANALYSIS,
EAST WEST COLLEGE OF PHARMACY,
BANGALORE-560 091.
Prof. K.A. SRIDHAR
PRINCIPAL
EAST WEST COLLEGE OF PHARMACY,
BANGALORE.
12