Indian journal of research in pharmacy and biotechnology issue 5

Indian Journal of Research in Pharmacy and Biotechnology
ISSN: 2320-3471 (Online)
ISSN: 2321-5674 (Print)

Editor
B.Pragati Kumar, M.Pharm, Assistant Professor,
Nimra College of Pharmacy

Consulting editor
Dr. S Duraivel, M.Pharm, Ph.D., Principal,
Nimra College of Pharmacy

Associate Editors
Mr. Debjit Bowmick, M.Pharm., (Ph.D)
Assistant Professor, Nimra College of Pharmacy
Mr. Harish Gopinath, M.Pharm., (Ph.D)
Assistant Professor, Nimra College of Pharmacy
Dr. M. Janardhan, M.Pharm., Ph.D.
Professor, Nimra College of Pharmacy
Dr. A. Ravi Kumar, M.Pharm., Ph D.
Professor, Bapatla College of Pharmacy

Editorial Advisory Board
Dr.Y.Narasimaha Reddy, M. Pharm., Ph D.
Principal, University college of Pharmaceutical
Sciences, Kakatiya University, Warangal.
Dr.V.Gopal, M. Pharm., Ph D.

Dr. Biresh Kumar Sarkar,
Asstt.Director (Pharmacy),
Kerala
Dr. M.Umadevi, M.Sc. (Agri), Phd

Principal, Mother Theresa Post Graduate & Research
Institute of Health Sciences,Pondicherry-6

Research Associate, Tamil Nadu Agricultural
University, Coimbatore

Dr. J.Balasubramanium, M. Pharm., Ph D.

Dr. V.Prabhakar Reddy, M. Pharm., Ph D.

General Manager, FR&D
R A Chem Pharma Ltd., Hyderabad

Principal, Chaitanya College of Pharmacy Education &
Research, Warangal

Dr.P.Ram Reddy, M. Pharm., Ph D.

Dr. S.D.Rajendran, M. Pharm., Ph D.
Director, Pharmacovigilance, Medical Affairs,
Sristek Consultancy Pvt. Ltd, Hyderabad

General Manager, Formulation,
Dr.Reddy’s Laboratory, Hyderabad

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ISSN: 2321-5674 (Print)
S.No.
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Contents
Evaluation of the association of rs8052394 of metalothionein-1a gene with type 2 diabetes mellitus in
Nepalese population
Surya Prasad Sharma, Bishal Khatiwada, Binita Dhakal, Uddhav Timilsina
a novel RP- HPLC method development and validation of atorvastatin and fenofibrate in bulk and
pharmaceutical dosage forms
Vinjam Swathi, Nanda Kishore Agarwal, Kumari Jyothsna
Analytical method development and validation for the simultaneous estimation of Lamivudine,
Zidovudine and Efavirenz by RP-HPLC in bulk and pharmaceutical dosage forms
Sindhura D, Nanda Kishore Agarwal
Novel RP-HPLC method development and validation of Losartan potassium and Amlodipine drugs in
pure and pharmaceutical dosage forms
Kumari Jyothsna, Chandana N, Vinjam Swathi
Formulation and In-Vitro evaluation of Ornidazole gastroretentive tablets by using low density swellable
polymers
Abeda Aqther, B. Pragati kumar, Peer Basha
Formulation and evaluation of Fenofibrate tablets prepared by employing bioavailability enhancement
technique
Siva kothapally, Pragati Kumar Bada, Harish G
Formulation and e valuation of famotidine fast dissolving tablets by direct compression method
B.Venkateswarlu, B.Pragati Kumar, Debjit Bowmik
Formulation and evaluation of matrix floating tablets of Ofloxacin and Tinidazole combination
Syed Peer Basha, Pragati Kumar B, Duraivel S, Abeda Aqther
Formulation and In-Vitro evaluation of Terbutaline sulphate sustained release tablets
Rajeswari Kola, Deepa Ramani N, Pragati Kumar B
Evaluation of antidiabetic activity of Methanolic extract of flowers of Cassia siamea in Alloxan induced
diabetic rats of basal diet and maida mixed diet
Pushpavathi P, Janarthan M, Firasat Ali
Formulation and evaluation of transdermal patches of anti-hypertensive drug metoprolol succinate
Koteswararao P, Duraivel S, Sampath Kumar KP, Debjit Bhowmik
Neuropharmacological screening of ethanolic extract of Nelumbo nucifera gaertner seeds
Sirisha Chowdary G
A review on use of genetically engineered microorganisms for bioremediation of environmental
pollutants and heavy metals
Mariz Sintaha
Hepatoprotective effect of hydroalcoholic extract of Ocimum gratissimum leaves on Rifampicin-Isoniazid
induced rats
Sreenu Thalla,Venkata Ramana K, Delhiraj N
Analytical method development and validation of Amitriptyline hydrochloride and Chlordiazepoxide in
tablet by RP-HPLC
Neeli Sujatha, K Haritha Pavani
Evaluation of hepatoprotective activity of Sapindus emarginatus vahl pericarp extract against anti
tubercular drugs induced liver damage in rats
Shoba Rani J, Janarthan M, Firasat Ali
Evaluation of anti-diabetic and hepato protective activity of 95% methanolic extract of Terminalia
tomentosa bark by using albino rats
Srilakshmi P, Janarthan M, Zuber Ali M
Formulation of mouth dissolving tablets of Naproxen
Rajesh Reddy K, Nagamahesh Nandru, Desam Asha Latha, Srinivasa Rao Chekuri
Preparation of immediate release Atorvastatin and sustained release matrix tablets of Gliclazide using
retardant hydroxypropyl methyl cellulose
Vinod Raghuvanshi, Jayakar B, Debjit Bhowmik, Harish G, Dureivel S
Phytochemical sreening and antidiabetic antioxidant effect of Ecbolium ligustrinum flowers extracts
Ranjitsingh B Rathor, Rama Rao D, Prasad Rao
Development and validation of assay method for meloxicam tablets by RP-HPLC
K. Ranjith, M.V.Basaveswara Rao, T.E.G.K.Murthy
RP-HPLC development and validation of assay and uniformity of dosage units by content uniformity
for in house lamivudine and abacavir combined tablet
K. Ranjith, M.V.Basaveswara Rao, T.E.G.K.Murthy
Validation of a simple and rapid HPLC method for the determination of Metronidazole and Norfloxacin
in combined dosage form
SK Asma Parveen, Chandana Nalla

Volume 1 Issue 5

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Page No.
570-575

576-582

583-588

589-596

597-601

602-608

609-613
614-620
621-624
625-628

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635-642
643-648

649-654

655-659

660-663

664-667

668-671
672-675

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679-681
682-685

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ISSN: 2321-5674 (Print)
24

Analytical method development and validation for the estimation of olmesartan medoxomil by RP-UPLC
in bulk and pharmaceutical dosage forms
Farhana pattan, K.Haritha Pavani, A.Kiran Kumar, N.Sunny Babu, K.V.Kalyan Kumar, Chandana N
Formulation development and evaluation of Gliclazide gel using water soluble Sodium carboxy methyl
cellulose polymer
Vaibhav Kumar Mishra, Shashi Shekhar Tripathi
Analytical method development and validation of Gemifloxacin and Ambroxol in solid dosage form by
reverse phase high performance liquid chromatography
Md Haseena Begum, Nanda Kishore Agarwal and Duraivel S
Evaluation of the anti hyperglycemic activity of methanolic extract of root of heliotropium indicum in
streptozotocin and alloxan induced diebetic rats
Aqheel MA , Janardhan M, Durrai vel S
Novel RP-HPLC method development and validation of Metformin and Pioglitazone drugs in pure and
pharmaceutical dosage forms
Alekhya Pallapolu, Aneesha A
Carbon nano tube: a review
K.Shailaja, Tahseen Sameena, S.P.Sethy, Prathima Patil, Md. Owais Ashraf
Stability indicating RP-HPLC method for the simultaneous determination of Candesartan cilexetil and
hydrochlorothiazide in bulk and dosage forms
Veeranjaneyulu D, Aneesha A, Nandakishore Agarwal
A review on enhancement of solubility and disolution rate of bcs class-ii drug by solid dispersion and
nonaqeous granulation technique
Chakravarthi V, Duraivel S
Evaluation of anti-infl ammatory activity of Canthium parviflorum by In-Vitro method
Kandikattu Karthik, Bharath Rathna Kumar P, Venu Priya R, Sunil Kumar K , Ranjith Singh.B.Rathore
Evaluation of nephro protectiveactivity of methanolic extract of seeds of Vitis vinifera against Rifampicin
and Carbontetra chloride induced nephro toxicity in wistar rats
Kalluru Bhargavi, N Deepa Ramani, Janarthan M, Durraivel S
Analytical method development and validation of estimation method for Sotalol hydrochloride tablets
by using RP-HPLC
G Abirami, K Anand Kumar, T.Veterichelvan, Arunateja Muvva
A study on role of demographic factors in small investors’ savings in stock market
Durga Rao P V, Chalam G V and Murty T N
Morbidity pattern among the elderly population in a south Indian tertiary care institution: analysis of a
retrospective study
Narayan V, Chandrashekar R
Testicular gene expression profiling of Phenytoin treated albino rats using cdna microarray
Rajkumar R, Vathsala Venkatesan, Sriram Thanigai
Formulation and in-vitro evaluation of Lornoxicam immediate release and Diclofenac sodium sustained
release bilayered tablets
B. Manikanta Anil, K. Narendra Kumar reddy

692-696

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Formulation and evaluation of cefpodoxime proxetil sustained release matrix tablets
Divya Palparthi, K. Narendra Kumar Reddy

758-760

40

Formulation and development of sustained release matrix tablet using natural polymers
L. P. Hingmire, D. M. Sakarkar

761-764

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Volume 1 Issue 5

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701-706

707-710

711-716

717-719
720-724

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732-735

736-740

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744-747

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Sharma et.al


ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online)

EVALUATION OF THE ASSOCIATION OF rs8052394 OF METALOTHIONEIN-1A
GENE WITH TYPE 2 DIABETES MELLITUS IN NEPALESE POPULATION
Surya Prasad Sharma1,2* ,Bishal Khatiwada1,2, Binita Dhakal1,2, Uddhav Timilsina1
1. Department of Biotechnology, College for Professional Studies, Kathmandu, Nepal
2. Department of Biotechnology, Sikkim Manip al University, Gangtok, Sikkim
*Corresponding author: suryapdsharma@gmail.com
ABSTRACT
Polymorphisms in metallothionein-1A gene associated with the risk of type 2 diabetes mellitus and its
complications. Metallothioneine (MT) as a potent antioxidant can affect energy metabolism. The present study
was undertaken to investigate the association between MT gene polymorphism and type 2 diabetes mellitus.
Polymorphism in rs8052394 of Metallothioneine 1A gene at lys51Arg is the most prevalent mutation in T2DM.
Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) has been found to be a
reliable and effective tool to identify the specific gene alteration that is responsible for the development of T2DM
and its complication. The aim of study was to identify the mutation in specific part of rs8052394 of MT1A gene
(lys51Arg substitution) in diabetic population by PCR-RFLP technique. All together 62 diabetic samples were
collected and DNA extraction was performed according to protocol of D.K. Lahari et al. Amplification of
fragment with MT1A gene at 51th position of amino acid performed in a XP Thermocycler using primers
Forward: 5’-ACTAAGTGTCCTCTGGGGCTG 3’ and Reverse: 5’-AATGGGTCACGGTTGTATGG 3’ of
MT1A gene cleaved by pstI enzyme. The restriction fragments obtained were electrophoreses in a 2% agarose gel
and were visualized using transilluminator. Mutation of MT1A gene was present in 40.32% of 62 patients, out of
which 19.35%was of ≤50 yrs of age group. On comparing the mean age of two category of genotype (AA and
AG/GG, correlation is statistically significant with p=0.044 (CI=95%).This is the first time that the mutation
positions in MT1A gene Lys51Arg substitution have been studied in Nepalese population with Type II diabetic.
Since Nepal is geographically located between two countries (India and China) with around 30% of world’s total
diabetic cases, researches in this subject seems to be of a rationale work.
Keywords: Diabetes Mellitus type II, SNP, rs8052394, Metallothioneine 1A, PCR-RFLP
2009). In general the MT is known to modulate three
INTRODUCTION
fundamental processes:
Diabetes mellitus is a metabolic disorder in which
person is characterized by the high blood sugar either
1) The release of gaseous mediators such as hydroxyl
because the body does not produce enough insulin, or
radical or nitric oxide;
because cells do not use the insulin that is produced
2) Apoptosis, and
(World Health Organisatio, 1999). The classical
3) The binding and exchange of heavy metals such as
symptoms of diabetes are polyuria (frequent urination),
zinc, cadmium or copper.
polydipsia (increased thirst) and polyphagia (increased
hunger) (Cooke, 2008). Almost one in 10 of the world
Metallothionein and Its Relationship with Diabetes:
population already has this condition, or can be expected
Metallothioneins (MTs) are a group of intracellular metalto develop it during their lifetime, with prevalence rates
binding and cysteine-enriched proteins and are highly
forecast to double within the next 15 years (Florence,
inducible in many tissues in response to various types of
2003). According to WHO (2000A.D.), at least 171
stress. Although it mainly acts as a regulator of metal
million people worldwide suffer from diabetes, or 2.8% of
homeostasis such as zinc and copper in tissues, MT also
the population (Wild, 2004).
acts as a potent antioxidant and adaptive (or stress) protein
Metallothionein 1A (MT1A), mRNA: Metallothionein
(MT) is a sulfhydryl- and cysteine-rich protein found in
microorganisms, plants and all invertebrate and vertebrate
animals. Metallothioneins are a group of ubiquitous lowmolecular-weight proteins that have functional roles in
cell growth, repair and differentiation. These are those
family of proteins with low molecular mass and high
affinity to certain metal ions (Cai, 2007). They are
implicated primarily in metal ion detoxification, in that
they are essential for the protection of cells against the
toxicity of cadmium, mercury and copper (Higashimoto,
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to protect cells and tissues from oxidative stress. Diabetes
affects many Americans and other populations, and its
development and toxic effect on various organs have been
attributed to increased oxidative stress. Studies showed
that zinc-induced or genetically enhanced pancreatic MT
synthesis prevented diabetes induced by chemicals such as
streptozotocin and alloxan, and zinc pretreatment also
prevented spontaneously developed diabetes. Since
diabetic complications are the consequences of organ
damage caused by diabetic hyperglycemia and
hyperlipidemia through oxidative stress, whether MT in
nonpancreatic organs also provides a preventive effect on

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diabetic toxicity has been recently investigated. It has
been demonstrated that overexpression of cardiac MT
significantly prevented diabetes-induced cardiomyopathy.
Likewise, over expression of renal MT also prevented
diabetes-induced renal toxicity. In addition, it was found
that MT as an adaptive protein is over expressed in several
organs in response to diabetes. Therefore, the biological
importance of diabetes-induced MT in diabetic
complications and subsequent other pathogenesis was
further explored. This polymorphism is the result of a
nucleotide change A to G at position 55231329 of
chromosome 16. Similarly 225 number position of mRNA
sequence, and it results in the substitution of Lysine (k) by
Arginine (r) at 51 position of the MET1A protein. In the
wild type, codon AAA codes for Lysine but in mutated
type, codon AAA changes to AGA hence it codes for
different amino acid Arginine.
After analysis by MUpro we found that protein
structure stability was decrease due to this polymorphism.
Among the seven identified SNPs: rs8052394,
rs11076161, rs8052334, rs964372, rs7191779, rs708274
and rs10636. Significant associations of MT1A rs8052394
(G alteration) with T2DM and decreased serum SOD
activity were established. The other six SNPs were not
significantly associated with T2DM. However, SNPs
rs964372 and rs10636 were found to be significantly
associated with increased serum triglyceride and
neuropathy among T2DM individuals. It is a restriction
enzyme isolated from an E. coli strain that carries the PstI
gene from Providencia stuartii 164 (ATCC 49762).
According to Nepal Diabetic Association, the number of
people suffering from diabetes above 40 years in urban
areas has climbed up to 19% of special note is that there
will be a 67% increase in prevalence of diabetes in
developing countries from 2010-2030 (Shaw, 2010).
According to UN, 246 million people in the world
are suffering from diabetes and approximately half of that
fall in Nepal, India, China and other Asian countries.
Increase in the incidence of diabetes mellitus is the 4th
leading cause of death in world. Each year 3.8 million
people die from diabetes and its related complication like
cardiomyopathy, stroke, nephropathy, neuropathy, eye
disease etc and the gene metallothionein 1A may be
responsible for such complications. It has also been found
that polymorphism in metallothionein 1A gene may be
responsible for even inducing diabetes not only its
complications. Polymorphism in single nucleotide in
metallothionein 1A leads for to decrease in level of SOD
which may be due to the death of pancreatic β cells
(Grarup, 2007). Metallothionein (MT) isoforms I and II
are polypeptides with potent antioxidative and antiinflammatory properties (Lina, 2008) and once occur
polymorphism; it causes in the alteration in the normal
function of the protein and causes different complications
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like aging, neuropathy, retinopathy, stroke etc. Since
diabetes and its complications are the 4th leading cause of
death, it needs to be diagnosed soon. The most rapid
results could be achieved by using molecular methods
including real-time PCR, single-strand-conformation
polymorphism analysis (SSCP) (Lina, 2008) multiplexallele-specific
PCR
(MASPCR),
mass
spectrophotometry, Allele-Specific Hybridization etc. But
the PCR-RFLP approach has several advantages of being
cheap, robust and simple to both perform and interpret,
basically requiring PCR and electrophoresis set up. Genes
significantly associated with developing type 2 diabetes,
include TCF7L2, PPARG, FTO, KCNJ11, NOTCH2,
WFS1, CDKAL1, IGF2BP2, SLC30A8, JAZF1, and
HHEX (Lina, 2008; Shoelson, 2006). Within all these
family, Metallothionine and its different isoforms have a
relationship with T2DM and other disease like
neuropathy, hyperlipidemia etc. Among different MT, the
isoform MT 1A with SNP rs8052394 is found to have a
significant relationship with T2DM (Lina, 2008). The
magnitude and trends in diabetes and the polymorphism in
MT1A gene at rs8052394 are epidemiologically important
to monitor, the estimation of the burden of disease is
programmatically relevant in shaping policies for
screening and treatment.
METHODS
Study Population: 63 diabetic subjects (Male: 30 Female:
32) were randomly selected within Kathmandu Valley
Population. Samples collected were reported as T2DM
from Kathmandu Model Hospital, Bhrikutimandap
Samjhana Laboratory, Mangalbazzar.
Study Design: A cross sectional study was designed to
explore the polymorphism in rs8052394 of MT 1A gene in
T2DM patients.
Study Site: Department of Biotechnology, College for
Professional Studies, Kathmandu, Nepal.
Selection of Sample: Samples reported as T2DM
according to WHO criteria 2006
Criteria to Confirm T2DM
1) Age above 40 years
2) Fasting Blood Glucose: >110mg%
3) Post-prandial Glucose: >140mg%
Data Processing and Analysis: Data will be analyzed
manually as well as using SPSS and interpreted according
to frequency distribution and percentage. The statistical
tool chi-square and t test will be applied to analyze the
data. Data will be presented in tables and figures.
Clinically and epidemiologically relevant information
from each patient including, age, sex, dietry habits was
obtained.
DNA extraction from T2DM samples:
D.K Lahiri et.al method: 1% Agarose Gel
Electrophoresis of Extracted DNA: 1% agarose gel was

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prepared with 0.5 µg/ml if Ethidium Bromide (EtBr) in it.
6 µl if each extracted DNA sample and the loading dye
mixed in the ratio 5:1 was loaded on the wells and run for
an hour at 80-90 Volt on Tris Acetate EDTA (TAE)
buffer. The DNA bands were observed under UV transilluminator.
PCR Amplification of rs8052394 Specific Exon
Fragment of MT1A Gene: The primary task for
performing the PCR is to standardize the reaction mixture
and to optimize the PCR conditions for the reaction to
occur accurately so that the DNA is amplified efficiently.
The reaction and the PCR programme were standardize to
precisely amplify the rs8052394 specific Exon fragment
of MT1A gene in the DNA sample by PCR using the
primers to get a 283 bp amplified product.
Forward: 5’ACTAAGTGTCCTCTGGGGCTG 3’
Reverse: 5’AATGGGTCACGGTTGTATG3’
2% Agarose gel electrophoresis of PCR amplified
products: To check if the specific DNA segments have
been amplified or not, the PCR products were
electrophoresed on 2% agarose gel(0.8gm of
agarose+40ml of 1X TAE buffer+0.5µg/ml of EtBr) in 1X
TAE buffer along with 100bp DNA ladder(Fermentas) for
1 hour at 90 volt.
Since the specific PCR primers amplified a 283bp PCR
product, single DNA band was observed lying in between
200 and 300bp as indicated by DNA marker when viewed
on UV transilluminator. The PCR amplified products were
stored at 4°C.
Restriction Digestion of PCR amplified


Products
Steps:

Incubation at 37°C for 3 hours

Heat Inactivation at 65°C for 20 minutes

Holding at 4°C forever.
RESULTS
Genomic DNA was extracted from blood cells.
The extracted DNA was run through 1% agarose gel
electrophoresis. PCR product was run through 2% agarose
gel electrophoresis. Finally digested product was run
through 3% agarose gel electrophoresis. Further the
incubation of the reaction mixture was carried out in the
thermocycler and the following program was used; Then
the digested product was analyzed by agarose gel
electrophoresis.
3%Agarose gel Electrophoresis of digested products:
After digestion of the 283bp fragment obtained by PCR, to
check for three possible genotypes, 15µl of digested
products were mixed with 3µl of loading dye and loaded
on the wells of 3%agarose gel in 1XTAE buffer along
with 100bp DNA ladder for 1 hour at 90 volts. The result
was viewed under UV transilluminator for number of
DNA fragments obtained. The amplified product was
subjected to digestion by PstI restriction enzyme. After
digestion of the 283 bp fragment by PCR, 165bp, 283bp
the restriction enzyme digested fragment was run on 3%
agarose gel electrophoresis. The gel picture below depicts
the band pattern for genotypes. Genotypic distribution was
in accordance with Hardy-Weinberg Equilibrium when
analysed by PopGene.S2 software with χ2=1.7494, df=1
and P>0.05.

Table.1. PCR reaction mixture with component and volume
Reagent Stock
Final Concentration
Volume/ Reaction
10XPCR buffer
2.5µl
25mM Mgcl2
1.5mM
2.0µl
10 µM (Each) dNTPs
200 µM
0.5µl
100 µM Reserve primer
0.4 µM
1.0µl
5 units/ µl.Taq polymerase
0.4 µM
0.2µl
Nuclease free D/W
1 Units/ µl
12.8µl
Total Master Mix
20 µl
DNA extract
5 µl
Total
25 µl

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Table.2.The following program was standardized for the amplification reaction
Step
Temperaure (oC)
Time
Initial Denaturation
95
5 Minutes
Denaturation
95
1 minutes
Primer Annealing
55.7
45 seconds
Extension
72
45 seconds
Cycle repeat from step 2 (35 cycles)
Final extension
72
7 minutes
Holding
4
Forever
End
Table.3.The reaction mixture for restriction digestion
Master Mix for Restriction Digestion
Pst I
0.5 µl
10X Buffer ‘O’
1.5 µl
Nuclease free distilled water
3.0 µl
PCR product
10 µl
Table.4.Genotype Distribution in Nepalese Population
Allotypes
Band Patterns

Frequency
Total sample (a)

GG
118
165
1
62

GA
118
165
283
24

AA
283
37

Table.5. Distribution of genotypic frequency of SNP rs8052394
SNP
Group
CASE no
Genotype
X2
P
62
Rs8052394
Case
GG GA AA 1.7494 0.79035
1
24
37
Table6: Agewise distribution in accordance to mutation
Age
< 50
51-60

61
33.87
22.58
43.55
Percentage
21
14
27
Total Number

Figure.1.WHO distributions of diabetic people

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Figure.2.Outline of mechanisms by which MT
coordinate with Zn prevents diabetes development and
diabetic complications rs8052394 of MET1A gene


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Figure.3.Gender wise distribution of patients

Figure.6. 3% Agarose Gel Electrophoresis of digested product by PSTL restriction enzymes
DISCUSSION
The present study revealed an association of MT
genes with T2DM. SNP rs8052394 detected in this study
was highly polymorphic. The χ2 goodness-of-fit test
showed that the genotypic distribution of rs8052394 SNP
was not deviated from the Hardy- Weinberg equilibrium
(P >0.05), suggesting the suitability of this sample pool
for genetic analysis. The allelic frequency distribution
analysed by PopGene software showed that ‘G’ allele
(p=0.79035) is high in Nepalese Population whereas ‘A’
allele (q=0.20965) is less in frequency. The gender wise
distribution of rs8052394 of MT1A gene mutated and wild
type or differently mutated strains. Among male subjects
30 (48.39%) weresuffering from diabetes and 32(51.61%)
were from female subjects. This shows no any significant
relationship between the prevalence of diabetes mellitus
and gender.
Agewise distribution of the T2DM patient: Agewise
distribution of the patient shows that 33.87% people were
less than 50 years, 22.58 were between 51-60 and 43.55
%were >61. In a given diabetic population, the chance of
alteration on different genes are possible which are
responsible for the induction of T2DM (Shoelson, 2006;
Lyssenko, 2008). But the role of MT1A is most widely
studied due to the importance of this gene. The mutation
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in MT1A gene
is a major mechanism for the
development of diabetes and its complications. The most
common mutation is the Lys51Arg substitution in MT1A
gene, which is present in approximately 36.13% of the
diabetic population and is associated with relatively high
level induction of the diabetes and its complication (Lina,
2008). But however it should be remembered that at
normal condition MTIA gene acts as antioxidant therefore
prevents diabetes (Papouli, 2000). The most frequent
mutation patterns of diabetic patient of MT1A gene occurs
at rs8052394 fragment at aminoacid51 (36.13%) of
MT1Agene. But mutation in MT1A gene occurs in a
higher frequency than any other gene and is regarded as to
be most important in diagnosing diabetes (Lina Yang,
2008).
CONCLUSION
The study of genotype frequency distribution for
the MT1A polymorphism in rs8052394 in Nepalese
population from Nepal for the first time will definitely
serve as a major achievement in understanding the
molecular level of mechanism and effects of the gene
mutation which varies in different geographical region of
world. We found that rs8052394 of MT1A gene mutation
at amino acid 51 accounted 40.3% among which the
mutation is more commonly found on the age group less

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than 50years. To the best of our knowledge frequency of
the MT1Agene lys51arg mutation has not been previously
determined in Nepalese diabetic population. The higher
percentage (40.3%) of common mutation in MT1A
definitely highlights the importance of the 51st amino acid
for development of diabetes and its complication.
BIBLIOGRAPHY
American diabetes association, Report: Type 2 diabetes in
children and adolescents. Diabetes care 2000, 23:381-9
Cai L, Diabetic cardiomyopathy and its prevention by
metallothionein:
experimental
evidence,
possible
mechanisms and clinical implications, Curr Med Chem,
14, 2007, 2193–2203.
Cooke DW, Plotnick L, Type 1 diabetes mellitus in
pediatrics, Pediatr Rev Nov, 29 (11), 2008, 374–384.
Florence Demenais, Timo Kanninen, Cecilia M. Lindgren,
A meta-analysis of four European genome screens (GIFT
Consortium) shows evidence for a novel region on
chromosome 17p11.2–q22 linked to type 2 diabetes,
Human Molecular Genetics, 12(15), 2003, 1865–1873.


Mikkel Faurschou1, Milena Penkowa, Claus Bøgelund
Andersen, Henrik Starklint4 and Søren Jacobsen: The
renal metallothionein expression profile is altered in
human lupus nephritis. Arthritis Research & Therapy, 10,
2008, 761.
Papouli E, Defais M, Larminat F, Over expression of
metallothionein-II sensitizes rodent cells to apoptosis
induced by DNA cross-linking agent through inhibition of
NF-kappaβ activation, J Biol Chem, 277, 2000, 47644769.
Shoelson SE, Lee J, Goldfine AB, Inflammation and
insulin resistance, J Clin Invest, 116 (7), 2006, 1793–801
Wild S, Roglic G, Green A, Sicree R, King H, Global
prevalence of diabetes: estimates for 2000 and projections
for 2030, Diabetes Care, 2004, 27(5), 1047–1053.
World Health Organisation & Department of Non
communicable Disease Surveillance (1999). Definition,
Diagnosis and Classification of Diabetes Mellitus and its
Complications.

Grarup N, Andersen G, Gene-environment interactions in
the pathogenesis of type 2 diabetes and metabolism.
Current Opinion in Clinical Nutrition & Metabolic Care,
10, 2007, 420–426.
Higashimoto Minoru, Isoyama Naohiro, Ishibashi Satoshi,
Inoue Masahisa, Takiguchi Masufumi, Suzuki Shinya,
Ohnishi Yoshinari, Sato Masao:Tissue dependent
preventive effect of metallothionein against DNA damage
in dyslipidemic mice under repeated stresses of fasting or
restraint, Life Sciences, 84, 2009, 569-575
J. E. Shaw, R. A. Sicree, and P. Z. Zimmet, Global
estimates of the prevalence of diabetes for 2010 and 2030.
Diabetes Research and Clinical Practice, 87(1), 2010, 4–
14.
Lahari D. K., Steve Bye, Nurenberger Jr J. J., Mario E.,
Hondes D. and Crisp M, A non organic and non enzymatic
extraction methods gives high yields of genomic DNA
from whole blood samples than do nine other methods
tested, J. Biochem. Biophys Methods, 25, 1992, 193–205
Li X, Cai L, Feng W, Diabetes and Metallothionein, Mini
Rev Med Chem, 7, 2007, 761–768.
Lina yang, Polymorphisms in metallothionein-1 and -2
genes associated with the risk of type 2 diabetes mellitus
and its complications, Am J Physiol Endocrinol Metab,
294, 2008, 987-992.
Lyssenko V, Jonsson A, Almgren P, Clinical risk factors,
DNA variants, and the development of type 2 diabetes,
The New England Journal of Medicine, 359 (21), 2008,
2220–2232.
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A NOVEL RP- HPLC METHOD DEVELOPMENT AND VALIDATION OF ATORVASTATIN AND
FENOFIBRATE IN BULK AND PHARMACEUTICAL DOSAGE FORMS
Vinjam Swathi, Nanda Kishore Agarwal, Kumari Jyothsna
Nimra College of Pharmacy, Jupudi, Vijayawada, A.P, India
*Corresponding author: Email: swathi.vinjam90@gmail.com, Phone +91-9490776192
ABSTRACT
The present investigation describes about a simple, economic, selective, accurate, precise reverse phase high
performance liquid chromatographic method for the simultaneous estimation of Atorvastatin and Fenofibrate in
pure and pharmaceutical dosage forms. Atorvastatin and Fenofibrate were well separated using a Thermohypersil
BDS C18 column of dimension 100 × 4.6, 5µm and Mobile phase consisting of Methanol: Water (Adjusted with
orthophosphoric acid to pH-2) in the ratio of 40:60v/v at the flow rate 1 ml/min and the detection was carried out
at 274nm with PDA detector. The Retention time for Atorvastatin and Fenofibrate were found to be 1.438, 2.949
respectively. The developed method was validated for recovery, specificity, precision, accuracy, linearity
according to ICH guidelines. The method was successfully applied to Atorvastatin and Fenofibrate combination
pharmaceutical dosage form.
KEY WORDS: RP-HPLC, Atorvastatin, Fenofibrate, Accuracy, Precision.
Atorvastatin and Fenofibrate in bulk and Pharmaceutical
1. INTRODUCTION
dosage forms as per ICH guidelines.
Atorvastatin
((3R,
5R)-7-[2-(4fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2The goal of this study is to develop rapid, economical
ylpyrrol-1-yl]-3, 5-dihydroxyheptanoic acid) is a member
HPLC method for the analysis of Atorvastatin and
of the drug class known as statins. It is used for lowering
Fenofibrate in combined dosage form using most
cholesterol. Atorvastatin is a competitive inhibitor of
commonly employed column (C18) and simple mobile
hydroxymethylglutaryl-coenzyme-A
(HMG-CoA)
phase preparation.
reductase, the rate-determining enzyme in cholesterol
In the present proposed work a successful attempt
biosynthesis via the mevalonate pathway. HMG-CoA
had been made to develop a method for the simultaneous
reductase catalyzes the conversion of HMG-CoA to
estimation of Atorvastatin and Fenofibrate pharmaceutical
mevalonate. It acts primarily in the liver. Decreased
dosage form and validate it. From the economical point of
hepatic cholesterol levels increases hepatic uptake of
view and for the purpose of routine analysis, it was
cholesterol and reduces plasma cholesterol levels.
decided to develop a more economical RP-HPLC method
Fenofibrate
(propan-2-yl
2-{4-[(4with simple mobile phase preparation for the estimation of
chlorophenyl) carbonyl] phenoxy}-2 methyl propanoate)
Atorvastatin and Fenofibrate combinational dosage form.
is a drug of the fibrate class. It is mainly used to reduce
The method would help in estimate of drugs in single run
cholesterol levels in patients at risk of cardiovascular
which reduces the time of analysis and does not require
disease. Like other fibrates, it reduces low-density
separate method for each drug. Thus, the paper reports an
lipoprotein (LDL) and very low density lipoprotein
economical, simple and accurate RP-HPLC method for the
(VLDL) levels, as well as increasing high-density
above said pharmaceutical dosage forms.
lipoprotein (HDL) levels and reducing triglycerides level.
2. MATERIALS AND METHODS
It is used alone or in conjunction with statins in the
Quantitative HPLC was performed on a high
treatment
of
hypercholesterolemia
and
performance liquid chromatograph -Waters e2695Alliance
hypertriglyceridemia. It lowers lipid levels by activating
HPLC system connected with PDA Detector 2998 and
peroxisome proliferator-activated receptor alpha (PPARα).
Empower2 Software. The drug analysis data were
PPARα activates lipoprotein lipase and reduces apoprotein
acquired and processed using Empower2 software running
CIII, which increases lipolysis and elimination of
under Windows XP on a Pentium PC and Thermohypersil
triglyceride-rich particles from plasma.
BDS C18 column of dimension 100 × 4.6, 5µm particle
Literature survey revealed that very few methods
size. In addition an analytical balance (DENVER 0.1mg
have been reported for the analysis of Atorvastatin and
sensitivity), digital pH meter (Eutech pH 510), a sonicator
Fenofibrate combinational dosage forms which include
(Unichrome associates UCA 701) were used in this study.
UV spectroscopy, Reverse Phase High performance
Standards and chemicals used: Pharmaceutical grade
Liquid Chromatography, Densitometric method, HPTLC
Atorvastatin and Fenofibrate were kindly supplied as a gift
methods. The present study illustrate development and
sample by Dr.Reddy’s Laboratory, Hyderabad, and
validation of simple, economical, selective, accurate,
Andhra Pradesh, India. Methanol was of HPLC grade and
precise RP-HPLC method for the determination of
Purchased from E. Merck, Darmstadt, Germany. Ortho
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Phosphoric Acid was analytical reagent grade supplied by
Fischer Scientific Chemicals. Water HPLC grade was
obtained from a Milli-QRO water purification system.
Atorvastatin and Fenofibrate Tablets available in the
market as Lipicure-TG (intas pharmaceuticals, Sikkim,
India.)
in composition of Atorvastatin (10mg),
Fenofibrate (150mg).
Preparation of mobile phase: Transfer water into
1000ml of beaker dissolve and diluted volume with water.
Then adjust its pH to 2 with Ortho Phosphoric Acid
(OPA). The Water adjusted pH to 2 with OPA: Methanol
(60:40 v/v) and filtered through 0.45µ membrane filter
and degassed by sonication.
Preparation
of
calibration
standards:
10mg
Atorvastatin and 16mg Fenofibrate was taken into a 10, 50
ml of volumetric flask and add 10ml of Diluent and
sonicated for 10 minutes and made up with Diluent. It was
further diluted to get stock solution of Atorvastatin and
Fenofibrate (To get 0.02 ppm and 0.32 ppm solution
respectively). This is taken as a 100% concentration.
Working standard solutions of Atorvastatin and
Fenofibrate was prepared with mobile phase. To a series
of 10 ml volumetric flasks, standard solutions of
Atorvastatin and Fenofibrate in the concentration range of
0.01-0.03µg/ml and 0.16-0.48µg/ml were transferred
respectively.
System suitability: System suitability are an integral part
of chromatographic system. To ascertain its effectiveness,
certain system suitability test parameters were checked by
repetitively injecting the drug solutions at 100%
concentration level for Atorvastatin and Fenofibrate to
check the reproducibility of the system. At first the HPLC
system was stabilized for 40 min. One blank followed by
six replicate analysis of solution containing 100% target
concentration of Atorvastatin and Fenofibrate were
injected to check the system suitability. To ascertain the
system suitability for the proposed method, a number of
parameters such as theoretical plates, peak asymmetry,
and retention time were taken and results were presented
in Table 1.
Recommended procedure:
Calibration curves for Atorvastatin and Fenofibrate:
Replicate analysis of solution containing 0.01-0.03µg/mL,
0.16-0.48µg/mL of Atorvastatin and Fenofibrate sample
solutions respectively were injected into HPLC according
to the procedure in a sequence and chromatograms were
recorded. Calibration curves were constructed by plotting
by taking concentrations on X-axis and ratio of peak areas
of standards on Y-axis and regression equation were
computed for both drugs and represented in Table .6
Analysis of marketed formulation: The content of ten
tablets was weighed accurately. Their average weights
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were determined. Powder of tablets equivalent to one
tablet weight (521.1mg) were weighed and taken in a 50
ml volumetric flask, dissolved in diluents, shaken and
sonicated for about 20 minutes then filtered through 0.45µ
membrane filter. The filtered solution was further diluted
(5 to 50ml) in the diluents to make the final concentration
of working sample equivalent to 100% of target
concentration. The prepared sample and standard solutions
were injected into HPLC system according to the
procedure. from the peak areas of Atorvastatin and
Fenofibrate the amount of the drugs in the sample were
computed. The contents were calculated as an average of
six determinations and experimental results were
presented in Table 4. The representive standard and
sample chromatograms were shown in fig.2 and fig.3.
Validation study of Atorvastatin and Fenofibrate: An
integral part of analytical method development is
validation. Method validation is the process to confirm
that the analytical procedure employed for a specific test
is suitable for its intended use. The newly developed RPHPLC method was validated as per International
Conference on Harmonization (ICH) guidelines for
parameters like specificity, system suitability, accuracy,
linearity, precision (repeatability), limit of detection
(LOD), limit of Quantification (LOQ) and robustness.
Specificity: The effect of wide range of excipients and
other additives usually present in the formulation of
Atorvastatin and Fenofibrate in the determination under
optimum conditions were investigated. The specificity of
the RP-HPLC method was established by injecting the
mobile phase and placebo solution in triplicate and
recording the chromatograms. The common excipients
such as lactose anhydrous, microcrystalline cellulose and
magnesium state have been added to the sample solution
injected and tested.
Precision: precision study of sample (Losartan potassium
and Amlodipine) was carried out by estimating
corresponding responses 6 times on the same day for the
100% target concentration. The percent relative standard
deviation (%RSD) is calculated which is within the
acceptable criteria of not more than 2.0.
Linearity: The linearity graphs for the proposed assay
methods were obtained over the concentration range of
0.01-0.03µg/ml
and
0.16-0.48µg/ml
(50-150%)
Atorvastatin and Fenofibrate respectively. Method of least
square analysis is carried out for getting the slope,
intercept and correlation coefficient, regression data
values and the results were presented in Table 2. The
representative chromatograms indicating the sample were
shown in fig.2&3. A calibration curve was plotted
between concentration and area response and statistical
analysis of the calibration curves were shown in fig. 6&7.

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Accuracy (Recovery studies): The accuracy of the
method is determined by calculating recovery of
Atorvastatin and Fenofibrate by the method of addition.
Known amount of Atorvastatin and Fenofibrate at 50%,
100%, 150% is added to a pre quantified sample solution.
The recovery studies were carried out in the tablet in
triplicate each in the presence of placebo. The mean
percentage recovery of Atorvastatin and Fenofibrate at
each level is not less than 99% and not more than 101%.


(±5), mobile phase composition (±5%), and pH of the
buffer solution.
LOD and LOQ: Limit of detection is the lowest
concentration in a sample that can be detected but not
necessarily quantified. Under the stated experimental
conditions. The limit of quantification is the lowest
concentration of analyte in a sample that can be
determined with acceptable precision and accuracy. Limit
of detection and limit of quantification were calculated
using
following
formula
LOD=3.3(SD)/S
and
LOQ=10(SD)/S, where SD= standard deviation of
response (peak area) and S= average of the slope of the
calibration curve.

Robustness: The robustness is evaluated by the analysis
of Atorvastatin and Fenofibrate under different
experimental conditions such as making small changes in
flow rate (±0.2 ml/min), λmax (±5), column temperature

Table 1: optimized chromatographic conditions and system suitability parameters for proposed method
S.NO
Parameter
Chromatographic conditions
1.

Instrument

Waters e2695 Alliance HPLC with Empower2 software

2.

Column

thermohypersil C18, (5μ, 150 x 4.6mm)

3.

Detector

PDA Detector 2998

4.
5.

Diluents
Mobile phase

Methanol
Water(adjusted pH 2.0 with OPA): methanolo (60:40 v/v)

6.

Flow rate

1ml/min

7.

Detection wavelength

274nm

8.

Temperature

35°c

9.

Injection volume

5µl

10.

Retention time
Atorvastatin
Fenofibrate
Theoretical plate count

12.

13.

2.949

Atorvastatin
Fenofibrate
Tailing factor
Atorvastatin
Fenofibrate
Resolution factor

11.

1.438

2552
3000
1.17
1.59
9.38
Table 2: Specificity study

S.NO.

Name of the solution

Retention time in min

1.

Blank

No peaks

2.

Atorvastatin

1.438

3.

Fenofibrate

2.949

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S.NO

1.

2.

S.NO

1.

2.


Table 3: results of precision study
Injection number

Sample

Atorvastatin

Fenofibrate

Precission
RT
1.443
1.438
1.442
1.382
1.441
1.444

1
2
3
4
5
6
Mean
%RSD(NMT 2.0)
1
2
3
4
5
6
Mean
%RSD(NMT 2.0)

Peak area
2054782
2054809
2065165
2060368
2041300
2043795
2053370
0.50
3515822
3503597
3509064
3507668
3511717
3513906
3510296
0.1

2.977
2.961
2.973
2.970
2.973
2.877

Table 4: Recovery data of the proposed Atorvastatin and Fenofibrate
Sample
Spiked Amount
Recovered Amount
%Recovered
(µg/ml)
(µg/ml)
10.04
10.02
100.16
Atorvastatin
19.983
19.94
100
29.937
29.85
99.3
158.47
158.82
100.33
Fenofibrate
316.538
317.59
100.33
474.20
476.32
100.33

%Average
recovery
100%

100.33%

Table 5: Robustness results of Atorvastatin and Fenofibrate
S.NO

sample

1.

parameters
Flow rate
(±0.2)

Optimized

Atorvastatin

Flow rate
(±0.2)
2.

35°C

1ml/min

Fenofibrate
Temperature
(±5°C)

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35°C

Peak area
2187263
6100287

Plate count
3021
2723

1.2
30
35
40
0.8
1

1.209
1.357
1.438
1.211
2.508
2.940

1639328
1565593
2043083
1639328
3198139
3507208

2856
2943
2552
2734
3037
3234

2.495
2.488

2378287
2519528

2810
2853

35

2.949

3527161

3222

40

Temperature
(±5°C)

RT
1.799
1.436

1.2
30

1ml/min

Used
0.8
1

3.684

2643655

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Table 6: linearity data of the Atorvastatin and Fenofibrate
S.NO

sample

1.

Atorvastatin

2.

Fenofibrate

Linearity level
(µg/ml)
0.1
0.15
0.2
0.25
0.3
0.01
0.015
0.02
0.025
0.03

Peak area
1024859
1530720
2028343
2552721
3061292
1756257
2634474
3501613
4352056
5252030

Slope

Y-intercept

r²

20379

1640.2

0.999

34837

15635

0.999

Table.7: Limit of Detection and Limit of Quantification
Parameter
Atorvastatin
Fenofibrate
Limit of detection(LOD)
0.00013µg/mL
0.00210µg/mL
Limit of Quantification(LOQ)
0.00046µg/mL
0.0070µg/mL

Fig.1: Structure of Atorvastatin

Fig.3: Chromatogram of Blank solution

Fig.2 Structure of Fenofibrate

Fig. 4: Typical Chromatogram of standard

Fig. 5: Typical chromatogram of Atorvastatin and Fenofibrate in marketed formulation

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ATORVASTATINy = 20379x +
1640.2
r² = 0.999

3000000

Area

2000000
1000000

Linear
(Area)

0
0

100

200

CONC.

Fig. 6: Linearity of Atorvastatin

15635
r² = 0.999

6000000
AREA

4000000
AREA

FENOFIBRATE34837x +
y=
4000000
Area

2000000

Linear (Area)
0
0

100

200

CONC.

3. RESULTS AND DISCUSSION

Fig. 7: Linearity of Fenofibrate
0.03µg/ml for Atorvastatin and 0.16-0.48µg/ml for
Fenofibrate.

Reverse phase HPLC method was preferred for
the determination of Atorvastatin and Fenofibrate.
Preliminary experiments were carried out to achieve the
best chromatographic conditions for the simultaneous
determination of the drug substances. Several column
types and lengths were tried considering other
chromatographic parameters. C18 column with a 4.6 mm
inner diameter and 5µm particle size was chosen. The
detection wave length was selected as 274nm with PDA
detector. Chromatographic conditions were optimized by
changing the mobile phase composition and buffers used
in mobile phase.

The experimental results were shown in table 6
and fig.6&7. The % recovery of Atorvastatin and
Fenofibrate was found to be in the range of 99.5 to 100 %
& 99 to 100.33% respectively. As there was no
interference due to excipients and mobile phase, the
method was found to be specific. As both compounds
pass the peak purity, the method was found to be specific.
The method was robust and rugged as observed from
insignificant variation in the results of analysis by
changes in Flow rate, column oven temperature, mobile
phase composition and wave length separately and
analysis being performed by different analysts.

Different experiments were performed to optimize
the mobile phase but adequate separation of the drugs
could not be achieved. By altering the pH of buffer results
a good separation. Different proportions of solvents were
tested. Eventually the best separation was obtained by the
isocratic elution system using a mixture of water (adjusted
the pH to 2 with OPA): methanol (60:40, v/v) at a flow
rate of 1 ml/min. a typical chromatogram for simultaneous
estimation of the two drugs obtained by using a above
mentioned mobile phase. Under these conditions
Atorvastatin and Fenofibrate were eluted at 1.438min and
2.949minutes respectively with a run time of 5 minutes.
The representative chromatogram of this simultaneous
estimation shown in fig. 3 & 4 and results were
summarized in Table 1.

The results were shown in Table 5. The LOD and
LOQ values were calculated based on the standard
deviation of the response and the slope of the calibration
curve at levels approximately the LOD and LOQ. The
limit of detection was obtained as 0.00013µg/mL for
Atorvastatin and 0.00210µg/mL for Fenofibrate. The limit
of quantitation was obtained as 0.00046µg/mL for
Atorvastatin and 0.0070µg/mL for Fenofibrate which
shows that the method is very sensitive. The results were
shown in Table. 7.

The Methanol and water (pH 2 with OPA) (40:60,
v/v) was chosen as the mobile phase. The run time of the
HPLC procedure was 5 minutes at flow rate of 1ml/min
was optimized which gave sharp peak, minimum tailing
factor. The system suitability parameters were shown in
Table 1 were in within limit, hence it was concluded that
the system was suitable to perform the assay. The method
shows linearity between the concentration range of 0.01-

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4. CONCLUSION
A new validated RP-HPLC method has been developed
for the quantitative and Qualitative determination of
Atorvastatin and Fenofibrate in tablet dosage forms in
bulk and pharmaceutical dosage forms was established.
The method was completely validated shows satisfactory
results for all the method validation parameters tested and
method was free from interferences of the other active
ingredients and additives used in the formulation. Infact
results of the study indicate that the developed method
was found to be simple, reliable, accurate, linear,
sensitive, economical and reproducible and have short run
time which makes the method rapid. Hence it can be
concluded that the proposed method was a good approach

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for obtaining reliable results and found to be suitable for
the routine analysis of Atorvastatin and Fenofibrate in
Bulk drug and Pharmaceutical formulations.

Linda
LN:
Reviewer
guidance-Validation
of
chromatographic methods, Center for drug evaluation and
research, 1994, 1-30.

5. ACKNOWLEDGEMENT
The authors would like to thank beloved parents
and all my well wishers, one and all who have helped me
directly and indirectly in completing this project work.

Lloyd R.Synder, Joseph J.Kirkland, Joseph L.Glajeh,
Practical HPLC method development, 2nd Edition. 1997,114.

REFERENCES
D A Shah, KK Bhatt, RS Mehtha, MB Shankar, TR
Gandhi, Development and validation of a RP-HPLC
method for determination of Atorvastatin calcium and
Aspirin in a capsule dosage form, Indian Journal of
Pharmaceutical Sciences, 69(4), 2007, 546-549.
Deepti Jain, N Jain, R Raghuwanshi,Development and
validation of RP-HPLC method for simultaneous
estimation of Atorvastatin calcium and Fenofibrate in
tablet dosage forms, Indian Journal of Pharmaceutical
Sciences,70(2), 2008, 263-265.
Gadewar CK, Mundik MB, Chandekar NA, Mahajan NM,
Telgoti PD,RP-HPLC Method for Simultaneous
Estimation of Atorvastatin Calcium Ezetimibe in
Pharmaceutical Formulation, Asian Journal of Research in
Chemistry, 3(2), 2010, 485-490.
Hirave Rupali. V, Bendgude Ravindra.D, Maniyar
Mithun.G, Kondavar Manish.S, Patil Sandeep.B,
Spectrophotometric method for Simultaneous estimation
of Atorvastatin Calcium & Fenofibrate in tablet Dosage
Form, International Journal of Drug Development &
Research, 5(1), 2013, 38-42.
ICH-Q2A, Text on Validation of Analytical Procedures,
ICH Harmonized Tripartite Guideline, Geneva, 1995, 2-3.
ICH-Q2B, Validation of Analytical Procedures:
Methodology, ICH Harmonized Tripartite Guideline,
Geneva, 1996, 1-8.
Krishna R. Gupta, Sonali S.Askarkar, Prashanth R.Rathod,
Sudhir G.wadodkar, Validated spectrophotometric
determination of Fenofibrate in Formulation, Der
Pharmacia Sinica, pelagia research library, UK, 1(1),
2010, 173-178.

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Nagavalli D, Balipaka Srinivas, Kalyan Chakravarthy C,
A validation analytical method development for the
simultaneous estimation of Metformin hydrochloride and
Fenofibrate in pure and in tablet dosage form,
International research journal of pharmacy, 2(12), 2011,
146-149.
Onkar S. Havele, Shweta S.Havele, Simultaneous
Determination of Atorvastatin calcium and Pioglitazone
hydrochloride in Its Multicomponent Dosage Forms by
UV Spectrophotometry, International Journal of Pharmacy
and Pharmaceutical Science Research, 1(2), 2011, 75-79.
P.
N.
Dhabale,
D.S.Gharge,
Simultaneous
spectrophotometric estimation of Atorvastatin and
Fenofibrate in bulk drug and dosage form by using
simultaneous equation method. International Journal of
ChemTech Research, 2 (1), 2010, 325-328.
R.J.Hamilton and Swell, Introduction to HPLC, 2nd
Edition, 2-94.
Rajasekaran A, Sasikumar R, Dharuman J, Simultaneous
RP-HPLC method for the stress degradation studies of
Atorvastatin calcium and Ezetimibe in multicomponent
dosage form, Ars Pharmaceutica, Spain,US.52(3), 2011,
12-18.
Rupali Hirave, S.D.Bhinge, S.M.Malipatil, A.S.Savali,
RP-HPLC method for simultaneous estimation of
Atorvastatin Calcium and Fenofibrate in tablet dosage
forms, Journal of PharmacyResearch, 3 (10), 2010, 2400.
T.Depan, K Paul Ambethkar, G.Vijaya Lakshmi,
M.D.Dhanaraju, Analytical Method Development And
Validation of RP-HPLC For Estimation of Atorvastatin
Calcium And Fenofibrate in Bulk Drug and Tablet Dosage
forms, European Journal of Applied sciences, 3 (2), 2011,
35-39.


Page 582

Sindhura and Agarwal et.al



ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE
SIMULTANEOUS ESTIMATION OF LAMIVUDINE, ZIDOVUDINE AND
EFAVIRENZ BY RP-HPLC IN BULK AND PHARMACEUTICAL DOSAGE FORMS
Sindhura D*, Nanda Kishore Agarwal
Department of pharmaceutical analysis, Nimra College of Pharmacy
*Corresponding Author: dasarisindhura.pharma@gmail.com, Phone no: 9951089007
ABSTRACT
A simple rapid, accurate, precise and reproducible validated reverse phase HPLC method was developed for
the determination of Lamivudine, Zidovudine and Efavirenz in bulk and pharmaceutical dosage forms. The
quantification was carried out using Symmetry C18 (250 X 4.6 mm, 5 µm) column run in isocratic way using
mobile phase comprising of methanol and water in the ratio of 65:35 v/v and a detection wavelength of 250nm,
and injection volume of 20µL, with a flow rate of 1.0mL/min. The retention times of Lamivudine, Zidovudine and
Efavirenz was found to be 2.519, 3.015 and 24.103. The method was validated in terms of linearity, precision,
accuracy, LOD, LOQ and robustness in accordance with ICH guidelines. The linearity ranges of the proposed
method lies between 0.080 mg/mL to 0.120 mg/mL, which is equivalent to 80% to 120% and with correlation
coefficient of r2=0.9995,0.9994 and 0.9993 for Lamivudine, Zidovudine and Efavirenz. The assay of the proposed
method was found to be 99.98%, 99.96% and 100.14%. The recovery studies were also carried out and mean %
Recovery was found to be 100.7%, 100.28%, 100.45%. The % RSD from reproducibility was found to be <2%.
The proposed method was statistically evaluated and can be applied for routine quality control analysis of
Lamivudine, Zidovudine and Efavirenz in bulk and in Pharmaceutical dosage form.
Key Words: Lamivudine, Zidovudine, Efavirenz, RP-HPLC, Symmetry C18, Tablets, Validation.
1. INTRODUCTION
Lamivudine is 4-amino-1-[(2R, 5S)-2(hydroxymethy1)1, 3- oxathiolan-5-yl]-1, 2- dihydro
pyramidine-2-one.The molecular weight is 229.26,
molecular formula is C8H11N3O3S. It is an enantiomer of
dideoxy analogue of cytidine. Zidovudine is 3' azido-3'deoxythymidine. The molecular weight is 267.24,
molecular formula is C10H13N5O4.It is a thymidine
analogue. Both Lamivudine and Zidovudine inhibits the
HIV reverse transcriptase enzyme competitively and acts
as a chain terminator of DNA synthesis and is used in the
treatment of both types of HIV I and HIV II virus and
chronic hepatitis B.Efavirenz is chemically (S)-6-chloro4-(cyclopropylethynyl)-1,4-dihydro4-(trifluoromethyl)2H-3,1- benzoxazin-2-one. The molecular weight is
315.67, molecular formula is C14H9ClF3NO2.It diffuses

Figure.1.Structure of
Lamivudine

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into the cell where it binds adjacent to the active site of
reverse transcriptase. This produces a conformational
change in the enzyme that inhibits function.
Literature review reveals very few methods are
reported for the assay of Lamivudine, Zidovudine and
Efavirenz in Tablet dosage forms using RP-HPLC
method. The reported HPLC methods were having
disadvantages like high flow rate, more organic phase and
use of costly solvents. The proposed RP-HPLC method
utilizes economical solvent system and having advantages
like better retention time, less flow rate, very sharp and
symmetrical peak shapes. The aim of the study was to
develop a simple, precise, economic and accurate RPHPLC method for the estimation of Lamivudine,
Zidovudine and Efavirenz in Tablet dosage forms.

Figure.2. Structure of Zidovudine

Figure.3.Structure of
Efavirenz

Page 583



UV-3000 LABINDIA double beam with UV win
5software UV-VISIBLE spectrophotometer with 1cm
matched quartz cells. Schimadzu HPLC equipped with
SPD 20A UV-VIS detector and the column used was
SYMMETRY C18 (250*4.6mm, 5µ). The data acquisition
was performed by using LC solutions software.
2.1. Chemicals and reagents: Lamivudine, Zidovudine
and Efavirenz pure samples were obtained from Mylan
Laboatories, Hyderabad, India and dosage form “DuovirE” marketed by CIPLA was purchased from local
pharmacy. Other chemicals all are of HPLC grade.
2.2. Preparation of mobile phase: A suitable quantity of
degassed mixture of methanol and water in the ratio of
65:35 v/v was prepared and filtered through 0.45µ filter
under vacuum filtration.
2.3. Preparation of standard solution: Standard
solution of Lamivudine, Zidovudine and Efavirenz
(600μg/ml, 150 μg/ml,300μg/ml) was prepared by
dissolving 60mg of Efavirenz, 15mg of Lamivudine and
30mg of Zidovudine working standard in 50ml of diluent
with sonication and made up to 100ml with the same
diluent.
2.4. Preparation of sample solution: Five tablets were
weighed and finely powdered and a powder quantity
equivalent to 150mg Lamivudine, 300mg of Zidovudine
and 600mg of Efavirenz were accurately weighed and
transferred to a 100ml volumetric flask and 50ml of
diluent was added to the same. The flask was sonicated for
30 min and volume was made up to the mark with diluent.
Transferred 5ml of solution into a 50ml volumetric flask
and dilute up to the mark with diluent so as to obtain a
concentration of 150,300,600 μg/mL mixed well and
injected. The amount present in each tablet was calculated
by comparing the area of standard Efavirenz, Lamivudine,
Zidovudine and tablet sample.

2.5. Method optimization: The chromatographic
separation was performed using Symmetry C18
(250×4.6mm, 5µm) column. For selection of mobile
phase, various mobile phase compositions were
observed for efficient elution and good resolution.
The mobile phase consisting of methanol and water
in the ratio of 65:35v/v was found to be the optimum
composition for efficient elution of analyte. The
mobile phase was injected to the column at a flow
rate of 1.0 ml/min for 35min. The column
temperature was maintained at 30oC. The analyte was
monitored at 250 nm using UV-detector. The
retention time of the drugs was found to be 2.519,
3.015 and 24.103min. Mobile phase was used as
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diluent during the standard and test samples
preparation.
3. RESULTS
3.1. Method Validation
3.1.1. System suitability: System suitability tests are an
integral part of method validation and are used to ensure
adequate performance of the chromatographic system.
Retention time (RT), number of theoretical plates (N) or
column efficiency and tailing factor (T) were evaluated for
five injections of standard solution at a solution of
100µg/ml of Lamivudine, Zidovudine and Efavirenz. The
results are tabulated in the table no-2 and the
chromatogram was shown in the figure no- 4.
3.1.2. Specificity: Specificity is the ability of analytical
method to measure accurately and specifically the analyte
in the presence of components that may be expected to be
present in the sample. The specificity of method was
determined by spiking possible impurities at specific level
to standard drug solution (100ppm). The diluent and
placebo solutions were also injected to observe any
interference with the drug peak. The results are tabulated
in the table no-3 and the chromatogram was shown in the
figure no- 5, 6.
3.1.3. Linearity: Linearity is the ability of the method to
produce results that is directly proportional to the
concentration of the analyte in samples with given range.
The linearity of Lamivudine, Zidovudine and Efavirenz
was in the concentration range of 80-120%.From the
linearity studies calibration curve was plotted and
concentrations were subjected to least square regression
analysis to calculate regression equation. The regression
coefficient was found to be 0.9995 for Lamivudine,
0.9994 for Zidovudine and 0.9993 for Efavirenz and
shows good linearity for three drugs. The results are
tabulated in the table no-4 and the chromatogram was
shown in the figure no- 7, 8, 9.
3.1.4. Accuracy: Accuracy is the closeness of results
obtained by a method to the true value. It is the measure of
exactness of the method. Accuracy of the method was
evaluated by standard addition method. Recovery of the
method was determined by spiking an amount of the pure
drug (80%,100% ,120%) at three different concentration
levels in its solution has been added to the pre analyzed
working standard solution of the drug. The results are
tabulated in the table no-5, 6, 7.
3.1.5. Precision: The precision of the analytical method
was studied by analysis of multiple sampling of
homogeneous sample. The Precision expressed as
standard deviation or relative standard deviation.


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3.1.6. System precision: System precision was performed
by injecting a standard solution of Lamivudine,
Zidovudine and Efavirenz at working concentrations five
times. The results are tabulated in the table no-8.
3.1.7. Method precision: Method precision was
performed by analyzing a sample solution of Lamivudine,
Zidovudine and Efavirenz by injecting six replicates of the
same sample preparations at a concentration of
100ppm/mL. The results are tabulated in the table no-9.


3.1.8. Robustness: Robustness shows the reliability
of an analysis with respect to deliberate variations in
method parameters. If measurements are susceptible
to variations in analytical conditions, the analytical
conditions should be suitably controlled or a
precautionary statement should be included in the
procedure.

Table.1.Optimized chromatogram conditions for Lamivudine, Zidovudine and Efavirenz
Column
Symmetry C18 (250*4.6mm,5µ)
Mobile phase
Methanol: Water(65:35)
Flow rate
1.0 ml/ min
Wavelength
250 nm
Injection volume
20 l
Column temperature
30o C
Run time
35 min
Table.2. System suitability Data for Lamivudine, Zidovudine and Efavirenz
Lamivudine
Zidovudine
Peak area
Peak area
1
2027423
1501204
2
2025853
1500977
3
2026576
1501113
4
2025030
1501308
5
2027292
1502391
Average
2026435
1501399
SD
1005
568
%RSD
0.05
0.04
Theoretical plates
6615
7512
Tailing factor
1.25
1.32
Retention time
2.519
3.015
S.No

Peak Name
Lamivudine
Zidovudine
Efavirenz

Table.3.Specificity Data for Lamivudine, Zidovudine and Efavirenz
Retention Time (Minutes)
2.541
3.001
22.403

Efavirenz
Peak area
3138895
3142519
3150529
3154259
3158911
3149023
8251
0.26
4500
1.12
24.103

RT ratio
1.00
1.18
8.82

Table.4. Linearity Data for Lamivudine, Zidovudine and Efavirenz
Lamivudine
Zidovudine
Efavirenz
Level
Con. (mg/ml)
Peak area
Con.(mg/ml) Peak area
Con.(mg/ml) Peak area
80%
0.07959
1576950
0.08005
1169205
0.08095
2521629
90%
0.08855
1761010
0.09010
1299358
0.08846
2753981
100%
0.10350
2040551
0.10360
1504208
0.10290
3198155
110%
0.10810
2121575
0.10850
1565537
0.10853
3342493
120%
0.11950
2357552
0.12015
1735081
0.11925
3701006
Slope
19338642.0219
14201079.1883
30526500.1606
Intercept
40602.8714
27753.3632
50253.3069
Correlation coefficient
0.9997
0.9997
0.9997
R square
0.9995
0.9994
0.9993

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Page 585



S.No
1
2
3
4
5
Average
SD
% RSD

S.No
1
2
3
4
5
6
Average
SD
% RSD


Table.5. System precision of Lamivudine, Zidovudine and Efavirenz (8)
Lamivudine
Zidovudine
Efavirenz
Area
RT
Area
RT
Area
RT
2027423
2.540
1501204
3.044
3138895
25.192
2025853
2.540
1500977
3.043
3142519
25.100
2026576
2.544
1501113
3.045
3150529
25.061
2025030
2.541
1501308
3.043
3154259
24.954
2027292
2.542
1502391
3.042
3158911
24.559
2026435
1501399
3149023
1005
568
8251
0.05
0.04
0.26
Table.6. Method precision of Lamivudine, Zidovudine and Efavirenz(9)
Lamivudine Assay
RT
Zidovudine
RT
Efavirenz
(%)
Assay (%)
Assay (%)
99.5
2.539
99.3
3.019
99.3
100.5
2.539
99.6
3.019
100.4
99.5
2.541
99.4
3.024
99.3
99.6
2.539
99.8
3.020
99.8
99.8
2.537
99.5
3.020
99.9
100.0
2.540
99.9
3.021
99.9
99.8
99.6
99.8
0.4
0.2
0.4
0.4
0.2
0.4

RT
22.841
22.869
22.940
22.941
22.889
22.941
-

Table.7. Variation in flow rate, column temperature and buffer for Lamivudine (10)
Lamivudine
Flow (mL/min)
Temperature(oC)
Buffer
Low
High
Low
High
Low
High
%RSD
0.05
0.12
0.30
0.03
0.05
0.25
Retention time
3.094
2.556
2.814
2.808
2.809
2.821
Plate count
5.54
6.10
5124
5029
5954
4054
Tailing factor
1.20
1.21
1.20
1.26
1.32
1.22
Parameter

Table.8.Variation in flow rate, column temperature and buffer for Zidovudine (11)
Zidovudine
Flow (mL/min)
Temperature(oC)
Buffer
Low
High
Low
High
Low
High
%RSD
0.14
0.17
0.19
0.06
0.04
0.18
Retention time
3.598
3.000
3.316
3.310
3.291
3.351
Plate count
4818
6010
4523
6842
4898
5818
Tailing factor
1.14
1.21
1.18
1.18
1.27
1.25
Parameter

Table.9.Variation in flow rate, column temperature and buffer for Efavirenz(12)
Efavirenz
Flow (mL/min)
Temperature(oC)
Buffer
Low
High
Low
High
Low
High
%RSD
0.43
0.17
0.23
0.15
0.31
0.23
Retention time
25.251
20.889
23.399
23.677
21.621
26.840
Plate count
4558
5518
5326
5692
5558
4558
Tailing factor
1.32
1.18
1.23
1.32
1.31
1.31
Parameter

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Page 586



Figure.4.Standard chromatogram

Figure.6.Chromatogram for Specificity

Figure.8.Linearity plot for Zidovudine
4. DISSCUSSION
4.1. System suitability: From the system suitability
studies it was observed that retention time of Lamivudine,
Zidovudine and Efavirenz was found to be 2.519, 3.015
and 24.103 min. % RSD of peak area was found to be
0.05.0.04 and 0.26. Theoretical plates were found to be
more than 4000. USP tailing factor was found to be 1.25,
1.32 and 1.12 for Lamivudine, Zidovudine and Efavirenz.
All the parameters were within the limit.
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Figure.5.Sample chromatogram

Figure.7. Linearity plot for Lamivudine

Figure.9.Linearity plot for Efavirenz
4.2. Specificity: The Chromatograms of Standard and
Sample are identical with nearly same Retention time.
There is no interference with blank and placebo to the
drugs. Hence the proposed method was found to be
specific. 4.3. Linearity: From the Linearity data it was
observed that the method was showing linearity in the
concentration range of 80-120μg/ml for Lamivudine,
Zidovudine and Efavirenz. Correlation coefficient was
found to be 0.9995, 0.9994 and 0.9993 for three
compounds.

Page 587



4.4. Accuracy: The recoveries of pure drug from the
analyzed solution of formulation were in the range of
99.3%-101.6%, which shows that the method was
accurate.
4.5. Precision
4.5.1. System precision: The percentage relative standard
deviation (RSD) for the peak area of Lamivudine,
Zidovudine and Efavirenz were 0.05, 0.04 and 0.26.
4.5.2. Method precision: The percentage relative
standard deviation for the assay values of Lamivudine,
Zidovudine and Efavirenz were 0.4, 0.2 and 0.4.
4.6. Ruggedness: Comparison of both the results obtained
for two different Analysts shows that the method was
rugged for Analyst-Analyst variability. The %RSD for
intermediate precision for Lamivudine, Zidovudine and
Efavirenz was found to be 0.4, 0.2 and 0.4.
4.7. Robustness



forms, Journal of Pharmacy Research, 4(10), 2011, 37663768.
Pishawikar SA, Jadhav SD, Bhatia M.s and Thamake SL,
UV spectrophotometric method for the estimation of
Lamivudine, Zidovudine and Nevirapine in pure and tablet
formulation, Asian Journal of Research in Chemistry
Issue, 4(3), 2010, 1-3.
M. Kumar, B. Jayakar, C. Saravanan and M. V.
Kumudhavalli, RP-HPLC method for simultaneous
estimation of Lamivudine and Zidovudine in pure and
tablet formulation, J. Chem. Pharm. Res, 1(2), 2010, 478481.
Palani Venkatesh, M.Ruthu and Y.Padmanabha Reddy,
Normal phase HPTLC method for the simultaneous
analysis of Lamivudine and Zidovudine in fixed dose
combination tablets, Journal of Pharmaceutical Analysis,
2(2), 2012, 152-155.

As the % RSD of retention time and asymmetry
were within limits for variation in flow rate (± 0.1
ml). Hence the allowable flow rate should be
within 0.9 ml to 1.1 ml.
were within limits for variation (+ 20C) in column
oven temperature. Hence the allowable variation
in column oven temperature is + 20C.
were within limits for variation (+ 2 %) in
composition of mobile phase. Hence the allowable
variation in mobile phase composition is ± 2 %.
All the system suitability parameters are within
limits for variation (±2nm) in wavelength. Hence
the allowable variation in wavelength is ± 2nm.
The results obtained were satisfactory and are in
good agreement as per the ICH guidelines.

Sigonda, Yogendra Singh and Rajiv Sharma, Normal
phase HPTLC method for the simultaneous analysis of
Lamivudine, Stavudine and Nevirapine in fixed dose
combination tablets, Journal of Pharmaceutical and
Biomedical Analysis, 54(3), 2010, 445-450.

4.8. Acknowledgement: The authors thankful to Mr. K.
Srinivasa Rao (AGM), Mr. Jyothibasu Director, Mylan
Laboratories for providing necessary facilities to carry out
the research work.
5. CONCLUSION

Jacqueline De Souza, Eunice Kazue Kano, Eunice Emiko
Mori Koono, Simone Grigoleto Schramm, Valentina Porta
and Sílvia Storpirtis, LC–UV Methodology for
Simultaneous Determination of Lamivudine and
Zidovudine in Plasma by Liquid–Liquid Extraction,
Chromatographia, 2(69), 2009, 231-235.








Finally it concludes that all the parameters are
within the limits and meet the acceptance criteria of ICH
guidelines for method validation. The proposed method
was simple, accurate, specific, precise, robust, rugged and
economical. Hence this method is validated and can be
used for routine sample analysis
REFERENCES
Balamuralikrishna K, Mahendra K. And B. Syama
Sundar, RP-HPLC method for the simultaneous estimation
of Efavirenz, Lamivudine and Zidovudine in tablet dosage
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Hemanth Kumar and Anil Bhandari, Reverse-phase high
performance liquid chromatographic method for
simultaneous determination of plasma zidovudine and
nevirapine with UV detection at 260 nm, Journal of
Pharmaceutical and Biomedical Analysis, 29(6), 2002,
1081-1088.
Sindhuri and Versha Parcha, High performance liquid
chromatographic method was developed for the
simultaneous estimation of lamivudine, zidovudine and
nevirapine in pharmaceutical dosage forms, Journal of
Chromatography, 7(2) 2013, 353-362.

Ashok Peepliwal, Sagar D. Vyawahare and Chandrakant
G. Bonde, Quantitative analysis of Zidovudine containing
formulation by FT-IR and UV spectroscopy, Anal.
Methods, 11, 2010, 1756-1763.
Beckett AH, Stenlake JB, Practical pharmaceutical
chemistry. Vol. II. New Delhi: CBS Publisher and
Distributors, 1986, 13-17.
Skoog, DA Holler, FJ Nieman, T.A. Principles of
instrumental analysis, Thomson Brooks/Cole, Fifth
edition. 1998, 329-335.

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Jyothsna et.al



NOVEL RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF LOSARTAN
POTASSIUM AND AMLODIPINE DRUGS IN PURE AND PHARMACEUTICAL
DOSAGE FORMS
Kumari Jyothsna, Chandana N, Vinjam Swathi
Nimra College of Pharmacy, Vijayawada, A.P, India

*Corresponding author: Email: jyothsna.s.k24@gmail.com, Phone +91-9492940598
ABSTRACT
A fast, robust and accurate RP-HPLC method was developed and validated for simultaneous determination of
Losartan potassium and Amlodipine in tablets. The mobile phase was mixture of aqueous Tri ethyl amine with pH
2.0 and Acetonitrile(70:30), effluent flow rate monitored at 1.0 ml/min. the stationary phase was C18 column,
3µm(4.6×250mm). The solutions of standard and the sample were prepared in methanol. The retention times was
found to be 2.916min and 5.214min for Losartan potassium and Amlodipine respectively at 246nm. Calibration
graphs constructed at their wavelengths of determination were linear in the concentration range of 50-150µg/ml.
The percentage assay for Losartan potassium and Amlodipine were found to be 101% and 100%respectively. The
method was validated and it was found to be accurate, precise, linear and reproducible as per ICH guidelines.
Keywords: RP-HPLC, Losartan potassium, Amlodipine, Accuracy, Precision, Linearity.
1. INTRODUCTION
Losartan potassium is a [2-butyl-4-chloro-1-({4[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-1Himidazol-5-yl]methanol
monopotassium
salt.
It
competitively inhibits the binding of angiotensin II to AT1
in many tissues including vascular smooth muscle and the
adrenal glands. Inhibition of angiotensin II binding to AT1
inhibits its AT1-mediated vasoconstrictive and
aldosterone-secreting effects and results in decreased
vascular resistance and blood pressure. Losartan
potassium is 1,000 times more selective for AT1 than
AT2. Inhibition of aldosterone secretion may increase
sodium and water excretion while decreasing potassium
excretion. It is effective for reducing blood pressure and
may be used to treat left ventricular hypertrophy and
diabetic nephropathy.
Amlodipine
(3-ethyl
5-methyl
2-[(2aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4dihydropyridine-3,5-dicarboxylate) decreases arterial
smooth
muscle
contractility
and
subsequent
vasoconstriction by inhibiting the influx of calcium ions
through L-type calcium channels. Calcium ions entering
the cell through these channels bind to calmodulin.
Calcium-bound calmodulin then binds to and activates
myosin light chain kinase (MLCK). Activated MLCK
catalyzes the phosphorylation of the regulatory light chain
subunit of myosin, Inhibition of the initial influx of
calcium decreases the contractile activity of arterial
smooth muscle cells and results in vasodilation. The
vasodilatory effects of amlodipine result in an overall
decrease in blood pressure. Another possible mechanism
is that amlodipine inhibits vascular smooth muscle
carbonic anhydrase I activity causing cellular pH increases
which may be involved in regulating intracelluar calcium
influx through calcium channels.
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Literature survey revealed that very few methods
have been reported for the analysis of Losartan potassium
and Amlodipine combinational dosage forms which
include UV spectroscopy, High performance Liquid
Chromatography, HPTLC methods. The present study
illustrate development and validation of simple, selective,
accurate, economical precise RP-HPLC method for the
determination of Losartan potassium and Amlodipine in
bulk and Pharmaceutical dosage forms as per ICH
guidelines.
The aim of this study is to develop rapid,
economical HPLC method for the analysis of Losartan
potassium and Amlodipine in combined dosage form
using most commonly employed column (C18) and simple
mobile phase preparation. In the present work a successful
attempt had been made to develop a method for the
simultaneous estimation of Losartan potassium and
Amlodipine pharmaceutical dosage form and validate it.
The method would help in estimation of drugs in single
run which reduces the time of analysis and does not
require separate method for each drug. Thus, the paper
reports an economical, simple and accurate RP-HPLC
method for the above said pharmaceutical dosage forms.
Equipments used: Quantitative HPLC was performed on
a high performance liquid chromatography -Waters
e2695Alliance HPLC system connected with PDA
Detector 2998 and Empower2 Software. The drug analysis
data were acquired and processed using Empower2
software running under Windows XP on a Pentium PC
and Agilent Zorbax C18, (3μ, 250 x 4.6mm). In addition
an analytical balance (DENVER 0.1mg sensitivity), digital
pH meter (Eutech pH 510), a sonicator (Unichrome
associates UCA 701) were used.

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Standards and chemicals used: Pharmaceutical grade
Losartan potassium and Amlodipine were kindly supplied
as a gift sample by Dr.Reddy’s Laboratory, Hyderabad,
India. Acetonitrile and methanol was of HPLC grade,
Purchased from E. Merck, Darmstadt, Germany.
Triethylamine was analytical reagent grade supplied by
Fischer Scientific Chemicals. Water HPLC grade was
obtained from a Milli-QRO water purification system.
Losartan potassium and Amlodipine Tablets available in
the market as Losar-A (Unichem pharmaceuticals,
Himachal pradesh, India.) in composition of Losartan
potassium (50mg) Amlodipine (5mg).
Preparation of mobile phase: Transfer water into
1000ml of beaker dissolve and diluted volume with water.
Then adjust its pH to 2.0 with Trithylamine. The Water
adjusted pH to 2 with Triethylamine: acetonitrile (70:30
v/v) and filtered through 0.45µ membrane filter and
degassed by sonication.
Preparation of calibration standards: 10mg Losartan
potassium and 10mg Amlodipine was taken into a 50, 10
ml of Volumetric flask and add 10ml of Diluent and
sonicated for 10 minutes and made up with Diluent.It was
further diluted to get stock solution of Losartan potassium
and Amlodipine (To get 0.2 ppm and 0.02 ppm solution
Respectively). This is taken as a 100% concentration.
Working standard solutions of Losartan potassium and
Amlodipine was prepared with mobile phase. To a series
of 10 ml volumetric flasks, standard solutions of Losartan
potassium and Amlodipine in the concentration range of
0.1-0.3µg/ml and 0.01-0.03µg/ml were transferred
respectively.
System suitability: System suitability is an integral part
of chromatographic system. At first the HPLC system was
stabilized for 40 min. One blank followed by six replicate
analysis of solution containing 100% target concentration
of Losartan potassium and Amlodipine were injected to
check the system suitability. To ascertain the system
suitability for the proposed method, a number of
parameters such as theoretical plates, retention time were
taken and results along with optimized chromatographic
conditions were presented in Table 1.
Recommended procedure:
Calibration curves for Losartan potassium and
Amlodipine: Replicate analysis of solution containing
0.1-0.3µg/ml and 0.01-0.03µg/ml of Losartan potassium
and Amlodipine sample solutions respectively were
injected into HPLC according to the procedure in a
sequence and chromatograms were recorded. Calibration
curves were constructed by plotting by taking
concentrations on X-axis and ratio of peak areas of
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standards on Y-axis and regression equation were
computed for both drugs and represented in Table 2.
Analysis of marketed formulation: The content of Ten
tablets were weighed accurately. Their average weights
were determined. Powder of tablets equivalent to two
tablets weight(600.04mg) were weighed and taken in a 50
ml volumetric flask, dissolved in diluents, shaken and
sonicated for about 20 minutes then filtered through
0.45µ membrane filter. The filtered solution was further
diluted (5 to 50ml) in the diluent to make the final
concentration of working sample equivalent to 100% of
target concentration. The prepared sample and standard
solutions were injected into HPLC system according to the
procedure. From the peak areas of Losartan potassium and
Amlodipine the amount of the drugs in the sample were
computed. The contents were calculated as an average of
six determinations and experimental results were
presented in Table 3. The representive standarad and
sample chromatograms were shown in fig. 4and fig.5.
Validation study of Losartan potassium and
Amlodipine: An integral part of analytical method
development is validation. Method validation is the
process to confirm that the analytical procedure employed
for a specific test is suitable for its intended use. The
newly developed RP-HPLC method was validated as per
International Conference on Harmonization (ICH)
guidelines for parameters like specificity, system
suitability, accuracy, linearity, precision (repeatability),
limit of detection(LOD), limit of Quantification(LOQ)
and robustness.
Specificity: The effect of wide range of excipients and
other additives usually present in the formulation of
Losartan potassium and Amlodipine in the determination
under optimum conditions were investigated. The
specificity of the RP-HPLC method was established by
injecting the mobile phase and placebo solution in
triplicate and recording the chromatograms. The common
excipients such as lactose anhydrous, microcrystalline
cellulose and magnesium state have been added to the
sample solution injected and tested.
Precision: precision study of sample (Losartan potassium
and Amlodipine) was carried out by estimating
corresponding responses 6 times on the same day for the
100% target concentration. The percent relative standard
deviation (%RSD) is calculated which is within the
acceptable criteria of not more than 2.0.
Linearity: The linearity graphs for the proposed assay
methods were obtained over the concentration range of
0.1-0.3µg/ml and 0.01-0.03µg/ml (50-150%) Losartan
potassium and Amlodipine respectively. Method of least
square analysis is carried out for getting the slope,

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intercept and correlation coefficient, and the results were
presented in Table 2. A calibration curve was plotted
between concentration and area response and statistical
analysis of the calibration curves were shown in fig 6,7.
Accuracy (Recovery studies): The accuracy of the
method is determined by calculating recovery of Losartan
potassium and Amlodipine by the method of addition.
Known amount of Losartan potassium and Amlodipine at
50%, 100%, 150% is added to a pre quantified sample
solution. The recovery studies were carried out in the
tablet in triplicate each in the presence of placebo. The
mean percentage recovery of Losartan potassium and
Amlodipine at each level is not less than 99% and not
more than 101%.
Robustness: The robustness is evaluated by the analysis
of Losartan potassium and Amlodipine under different
experimental conditions such as making small changes in
flow rate (±0.2 ml/min), column temperature (±5).
LOD and LOQ: Limit of detection is the lowest
concentration in a sample that can be detected but not
necessarily quantified, under the stated experimental
conditions. The limit of quantification is the lowest
concentration of analyte in asample that can be determined
with acceptable precision and accuracy. Limit of detection
and limit of quantification were calculated using following
formula LOD=3.3(SD)/S and LOQ=10(SD)/S, where SD=
standard deviation of response (peak area) and S= average
of the slope of the calibration curve.
3. RESULTS AND DISCUSSION
Reverse phase HPLC method was preferred for
the determination of Losartan potassium and Amlodipine.
Preliminary experiments were carried out by changing the
mobile phase composition and buffers used in mobile
phase. Different experiments were performed to optimize
the mobile phase. By altering the pH of buffer results a
good separation. Different proportions of solvents were
tested. Eventually the best separation was obtained by the
isocratic elution system using a mixture of water (adjusted

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the pH to 2 with Triethyl amine): acetonitrile (70:30, v/v)
at a flow rate of 1 ml/min. Several column types and
lengths were tried considering other chromatographic
parameters. C18 column with 250 x 4.6mm length, inner
diameter and 3µm particle size was chosen. The detection
wave length was selected as 246nm with PDA detector. A
typical chromatogram for simultaneous estimation of the
two drugs obtained by using a above mentioned mobile
phase. Under these conditions Losartan potassium and
Amlodipine were eluted at 2.916min and 5.216min
respectively with a run time of 6 minutes. The
representative chromatogram of this simultaneous
estimation shown in fig. 3,4,5 and system suitability
results along with optimized chromatographic conditions
were summarized in Table 1.
The method shows linearity between the
concentration range of 0.1-0.3µg/ml for Losartan
potassium and 0.01-0.03µg/ml for Amlodipine. The
experimental results were shown in table 6 and fig 6, 7.
The % recovery of Losartan potassium and Amlodipine
was found to be in the range of 99.5 to 100 % & 99 to
100.33% respectively, the results wer shown in Table 4.
As there was no interference due to excipients and mobile
phase, the method was found to be specificresults were
shown inTable 2 and fig 3,4,5. The precssion obtained
was within the limits i.e., RSD<2 which would indicate
that the proposed method was quite precise and
reproducible and results were shown in Table 3. The
method was robust and rugged as observed from
insignificant variation in the results of analysis by
changes in Flow rate, column oven temperature. The
results were shown in Table 5. The LOD and LOQ values
were calculated based on the standard deviation of the
response and the slope of the calibration curve at levels
approximately the LOD and LOQ. The limit of detection
was obtained as 0.000633µg/mL for Losartan potassium
and 0.000069µg/mL for Amlodipine. The limit of
quantitation was obtained as 0.0021µg/mL for Losartan
potassium and 0.00023µg/mL for Amlodipine which
shows
the
method
is
very
sensitive.


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Jyothsna et.al



Table.1. Optimized chromatographic conditions and system suitability parameters for proposed method
Parameter
Chromatographic conditions
Instrument
Waters e2695 Alliance HPLC with Empower2 software
Column
Agilent Zorbax C18, (3μ, 250 x 4.6mm)
Detector
PDA Detector 2998
Diluents
Methanol
Mobile phase
Aqueous triethylamine(Ph 2.0): acetonitrile (70:30 v/v)
Flow rate
1ml/min
Detection wavelength
246nm
Temperature
Ambient(35°c)
Injection volume
5µl
Retention time
Losartan potassium
2.91
Amlodipine
5.21
Theoretical plate count
Losartan potassium
8216
Amlodipine
8081
Tailing factor
Losartan potassium
1.59
Amlodipine
1.43
Resolution factor
12.25
Table.2. Specificity study
Name of the solution
Retention time in min
Blank
No peaks
Losartan potassium
2.91
Amlodipine
5.21

Sample

Losartan potassium

Amlodipine

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Table.3. Results of precision study
Injection number
Precision
RT
Peak area
1
2.920
6126639
2
2.919
6167905
3
2.916
6160774
4
2.919
6188024
5
2.915
6138705
6
2.917
6127062
Mean
6156494
%RSD(NMT 2.0)
0.40
1
5.216
9971154
2
5.214
9966863
3
5.212
9970936
4
5.214
9990844
5
5.207
9954087
6
5.207
9963155
Mean
9969177
%RSD(NMT 2.0)
0.12


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Jyothsna et.al



Table.4. Recovery data of the proposed Losartan potassium and Amlodipine
Sample
SpikedAmount
RecoveredAmount
%Recovered
%Average
(µg/ml)
(µg/ml)
recovery
49.5
49.95
101
Losartan
101%
99
100.26
101
potassium
148.5
149.56
101
4.95
4.94
100
Amlodipine
100.33%
9.9
9.93
101
14.85
14.96
100
Table 5: Robustness results of Losartan potassium and Amlodipine
sample

parameters
Flow rate
(±0.2)

Losartan
potassium

Optimized
1ml/min

Temperature
(±5°C)

35°C

Flow rate
(±0.2)

1ml/min

Temperature
(±5°C)

35°C

Amlodipine

used
0.8
1
1.2
30
35
40
0.8
1
1.2
30
35
40

RT
3.408
2.916
2.910
3.408
2.916
2.908
5.933
5.214
5.166
5.953
5.214
5.167

Peak area
5159863
6100287
6134592
5159863
6100287
6141851
8217887
9833918
9942019
8217887
9833918
9954276

Plate count
7861
7868
7958
7794
7868
7862
7746
7660
7764
7751
7660
7765

Table.6.Linearity data of the Losartan potassium and Amlodipine
S.NO

1.

2.

S.NO
1.
2.

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sample

Losartan
potassium

Amlodipine

Linearity level
(µg/ml)
0.1
0.15
0.2
0.25
0.3
0.01
0.015
0.02
0.025
0.03

Peak area
3053242
4539421
6105323
7616656
9188085
4913960
7471465
9978009
12408307
19972978

Slope

Y-intercept

r²

19288

99780.88

0.9999

16616

61387.68

0.9999

Table.7.Limit of Detection and Limit of Quantification
Sample
LOD
Losartan potassium
0.000633µg/ml
Amlodipine
0.000069µg/ml

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LOQ
0.0021µg/ml
0.00023µg/ml

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Jyothsna et.al


Fig 1: Structure of Losartan potassium


Fig 2: Structure of Amlodipine

Fig.3: Chromatogram of Blank solution

Fig 4: Chromatogram of standard solution

Fig 5: Chromatogram of marketed formulation

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LOSARTAN POTASSIUM
10000000

r²=0.9999

8000000

Y=19288X+99780.8

AREA

6000000
4000000

LINEAR

2000000
0
0

50

100
CONC%

150

200

Fig. 6: Linearity of Losartan potassium

AMLODIPINE

16000000
14000000
12000000
10000000
8000000

r²=0.9999

AREA

Y=16616X+
61387.68

6000000
4000000
2000000
0

LINEAR

0

50

100
CONC%

150

200

Fig. 7: Linearity of Amlodipine
4. CONCLUSION
A new validated RP-HPLC method has been
developed for the quantitative and Qualitative
determination of Losartan potassium and Amlodipine in
tablet dosage forms in bulk and pharmaceutical dosage
forms was established. The developed HPLC technique is
precise, specific, robust and accurate. Results of analysis
of pharmaceutical formulations reveal that the proposed
methods are suitable for their analysis with virtually no
interference of the usual additives presented in
pharmaceutical formulations. This method is simple,
reliable, accurate, linear, sensitive, economical and
reproducible. Hence it can be concluded that the proposed
method was a good approach for obtaining reliable results
and found to be suitable for the routine analysis of
Losartan potassium and Amlodipine in Bulk drug and
Pharmaceutical formulations.
5. ACKNOWLEDGEMENT

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The authors would like to thank beloved parents
and all my well wishers, one and all who have helped me
directly and indirectly in completing this project work.
REFERENCES
AV Kasture and Madhuri Ramteke, Simultaneous UVspectrophotometric method for the estimation of atenolol
and amlodipine besylate in combined dosage form, Indian
J Pharm Sci, 68, 2006, 394-6
Chaudhari BG, Patel NM and Shah PB, Stability
indicating RP-HPLC
method for simultaneous
determination of atorvastatin and amlodipine from their
combination drug products, Chem Pharm Bull (Tokyo),
55(2), 2007, 241-6.
D. N. Vora and A. A. Kadav, Development and validation
of a simultaneous HPLC method for estimation of
Bisoprolol Fumarate and Amlodipine besylate from
tablets, Indian J Pharm Sci, 70(4), 2008, 542–546


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Jyothsna et.al


ICH, Q2A validation of analytical procedure:
Methodology International Conference on Harmonization,
Geneva, October 1994.
ICH, Q2B Validation of analytical procedure:
Methodology International Conference on Harmonization,
Geneva, March 1996.
Loyd R. Snyder, Practical HPLC Method Development,
4th ed, A-Wiley-inter Science publication, 1997, 234265.

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R. Sharma, S. Khanna and G. P. Mishra, RP-HPLC
method for simultaneous estimation of atenolol,
hydrochlorothiazide and losarton in tablet dosage form
Chem Sci Trans., 2013, 2(S1), S1-S6.
Ramya Gavini, S. B. Puranik, G. V. S. Kumar and K. A.
Sridhar, Simultaneous estimation of Amlodipine and
Losartan by UV-method in bulk drug and tablet dosage
formulation, Archives of Applied Science Research, 4 (5),
2012, 2206-2212.


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