INDIAN JOURNAL OF RESEARCH IN PHARMACY AND BIOTECHNOLOGY

by LAMP marker Showkat Hussain Ganie, Deepak Yadav, Altaf Ahmad, Anis Chadhry, Mohd Asif 765-771 2 Comparative in-vitro dissolution study of five brands of Diclofenac sodium delayed

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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

Professor, Nimra College of Pharmacy

Dr A Ravi Kumar, M.Pharm., Ph D

Professor, Bapatla College of Pharmacy

Editorial Advisory Board

Principal, University college of Pharmaceutical

Sciences, Kakatiya University, Warangal

Dr Biresh Kumar Sarkar, Asstt.Director (Pharmacy),

Kerala

Dr.V.Gopal, M Pharm., Ph D

Principal, Mother Theresa Post Graduate & Research

Institute of Health Sciences,Pondicherry-6

Dr M.Umadevi, M.Sc (Agri), Phd Research Associate, Tamil Nadu Agricultural

University, Coimbatore

Dr J.Balasubramanium, M Pharm., Ph D

General Manager, FR&D

R A Chem Pharma Ltd., Hyderabad

Principal, Chaitanya College of Pharmacy Education &

Research, Warangal

General Manager, Formulation,

Dr.Reddy’s Laboratory, Hyderabad

Dr S.D.Rajendran, M Pharm., Ph D

Director, Pharmacovigilance, Medical Affairs, Sristek Consultancy Pvt Ltd, Hyderabad

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INDIAN JOURNAL OF RESEARCH IN PHARMACY AND BIOTECHNOLOGY

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Indian Journal of Research in Pharmacy and Biotechnology is a bimonthly journal, developed and published in collaboration with Nimra College of Pharmacy, Ibrahimpatnam, Vijayawada, Krishna

District, Andhra Pradesh, India-521456

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1 Authentication of traditional crop Kalongi (Nigella sativa L.) by LAMP marker

Showkat Hussain Ganie, Deepak Yadav, Altaf Ahmad, Anis Chadhry, Mohd Asif

765-771

2 Comparative in-vitro dissolution study of five brands of Diclofenac sodium delayed release

tablets in QbD environment

V Malleswari Bai, M Prasada Rao, M Chandana, K Naga Harini, B Naga Deepthi, K Thirumala

Devi1, P Lakashmana Rao, Vinay U rao and J Naga raja

772-777

3 Development and validation of a stability indicating HPLC method for analysis of Altretamine

in bulk drug and pharmaceutical formulations

M Karimulla Santhosh, A Sreedevi, L Kalyani, A Lakshmana Rao

6 Method development and validation for the simultaneous estimation of Ofloxacin and

Tinidazole in bulk and pharmaceutical dosage form by reverse phase HPLC method

Y.Bhargav, K Haritha Pavani, S Amareswari

797-802

7 Evaluation of nephro protective activity of methanolic extract of seeds of Vitis vinifera against

Rifampicin and carbon tetra chloride induced nephro toxicity in wistar rats

Kalluru Bhargavi, N Deepa Ramani, Janarthan M, Duraivel S

803-807

8 Method development and validation for the simultaneous estimation of Atazanavir and

Ritonavir in tablet dosage form by RP-HPLC

Nuli Vasavi, Afroz Patan

808-814

9 Evaluation of anti arthritic activity of aqueous extract of Hibiscus Platinifolius in albino rats

Marri Praveen, M.Janarthan

815-818

10 Some H.R methodology/ techniques for costs reduction in companies to improve profit

M Sarkar, B K Sarkar, M D Gora, S C Verma

819-821

11 Analytical method development and validation of Artesunate and Amodiaquine hydrochloride

in tablet dosage form by RP-HPLC

P RajaRao, Nanda Kishore Agarwal

822-827

12 Analytical method development and validation for the simultaneous estimation of

Rabeprazole sodium and Itopride hydrochloride in bulk and pharmaceutical dosage forms by

RP-HPLC

Syed Shaheda, Nanda Kishore Agarwal

828-834

13 Formulation and evaluation of herbal anti-dandruff shampoo

Anusha Potluri*, Harish G, B Pragathi Kumar, Dr Durraivel

835-839

Paracetamol and Tapentadol by RP-HPLC in bulk and pharmaceutical dosage forms

V.Praveen Kumar Reddy, Aneesha, D.Sindhura, M.Sravani, Thandava Krishna Reddy

840-845

15 Protective role of methanolic extract of Polygonum glabrum willd against Cisplatin and

Gentamycin induced nephrotoxicity in Albino rats

Radha.B, Janarthan M, Durraivel S

846-849

Rosuvastatin and Finofibate in tablet dosage form by reverse phase high performance liquid

chromatography

M Sumalatha, K.Haritha Pavani

850-856

17 A new development and validated RP-HPLC method for the assay and related substances of

Itraconazole in capsule dosage form

Sarvani Paruchuri, Haritha Pavani K

857-865

18 Evalution of anti urolithiatic activity of aqueous extract of stem core of Musa paradisiaca

againest ethylene glycol and ammonium chloride induced urolithiasis on wistar rats

Thirumala K, Janarthan M, Firasat Ali M

866-868

19 Preparation and characterization of bioadhesive vaginal gel of Propranolol hydrochloride

Hardeep Singh Dhaliwal, Dhruba Sankar Goswami

869-874

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Indian Journal of Research in Pharmacy and Biotechnology

20 Topical herbal analgesic and anti arthritic (max-relief) versus Diclofenac in symptomatic

treatment of osteoarthritis of the knee: a randomized controlled trial

Md Q Azam, Abdallah A Al-Othman, Mir Sadat-Ali, Ahmed A Tantawy

875-880

21 Analytical method development and validation for the estimation of Olmesartan medoxomil

by RP-UPLC in bulk and pharmaceutical dosage forms

Farhana Pattan, Haritha Pavani, Chandana N, Karimulla M

881-885

22 Modern hygienic industrial canteen amenity: A change factor for healthy physical work

environment of the work force in Indian industrial units

TN Murty, GV Chalam, Md Aasif Siddique Ahmed Khan, T Abhinov and T Abhilash

886-892

23 Design and development of Metformin hydrochloride Trilayered sustained release tablets

Venkateswara Rao T, Bhadramma N, Raghukiran CVS and Madubabu K

893-897

24 Amlodipine: the upcoming threat to Periodontist

Sivaranjani, Vineet Kashyap, S.P.K.Kennedy Babu, Ajish Paul K,

26 Development and optimization of Diltiazem hydrochloride loaded microspheres by using

different Eudragit polymers

V Kamalakkannan, K.S.G.Arul Kumaran

908-914

27 Formulation, characterization and optimization of Methotrexate loaded sodium alginate

chitosan Nanoparticles using 3 2 factorial design

S.Daisy Chella Kumari, C.B.Tharani , N.Narayanan , C.Senthil Kumar

915-921

28 Strategies in Dendritic architecture for drug delivery – An over review

Pandurangan Dinesh Kumar, Palanirajan Vijayaraj Kumar, Govindaraj Saravanan

922-934

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Authentication of traditional crop Kalongi (Nigella sativa L.) by LAMP marker

Showkat Hussain Ganie1, Deepak Yadav1, Altaf Ahmad2, Anis Chadhry1, Mohd Asif1*

1 Department of Ilmul-Advia, Faculty of Medicine, Jamia Hamdard, New Delhi-110062 India

2 Department of Botany, Faculty of Science, Jamia Hamdard, New Delhi-110062

*Corresponding author: Email: profasif2012@gmail.com, Phone: 8860142069

ABSTRACT

Nigella sativa, commonly known as kalongi is an important drug of traditional system of

medicine, commonly used against cough, fever, abdominal disorders, skin infections, paralysis and jaundice Because of increased demand and high price, there are chances that the herb could be adulterated in the trade by other related species Therefore, a reliable authentication method is needed

to facilitate identification of this genuine material from its adulterants To carry out the work, the market samples were procured from the crude-drug markets of different geographical regions of India The RAPD derived LAMP technique was employed for the characterization of different accessions of Kalongi 19 primers were used of which one unique band, common in all accessions were eluted, cloned and sequenced LAMP primers were designed and LAMP product formation was detected at 60°C.Out of 25 primers, 19 primers amplified a total of 524 reproducible, clear and scorable bands One monomorphic RAPD fragment present in all the accessions, amplified by OPAA-09 primer, was

developed into LAMP marker for identification of N Sativa The primers successfully amplified the genome of kalongi while as in negative control (Catharanthus roseus) there was no amplification The

LAMP markers developed in this study may provide guidance for the authentication of plant materials traded as Kalongi

Key words: Adulteration; Authentication; loop mediated isothermal amplification (LAMP);

Molecular markers; kalongi; RAPD

1 INTRODUCTION

The traditional knowledge of herbal

medicine is widespread- ranging from tribal folklore

use to age-old practices and closely guarded recipes

handed down from generation to generation, to

highly evolved systems of medicine like Ayurveda,

Unani and Siddha These systems have served the

humanity through the centuries and it is certain that

they will continue to be in use for times to come

However, in the process of urbanization the contact

with nature was cut off and, consequently, the

knowledge about the identification of medicinal

plants deteriorated to a great extent Additionally,

the crude drugs sold in the market are adulterated,

sophisticated or substituted by quite unrelated plant

materials The adulteration of market samples is one

of the greatest drawbacks in promotion of herbal

products (Dubey, 2004) Plant samples in the market

are stored under undesirable conditions over the

years and often contain a mixture of other plant

species (Khatoon, 1993), thus, adversely affecting

their bio efficacy The efficacy of many of the drugs

has become suspect because of the adulterated, dried

raw materials profusely available in the indigenous

market (Anonymous, 1996) Very often the identity

of market drugs is taken for granted without

subjecting the plant material to stringent methods of

botanical identification This result in the loss of

therapeutic potential of the preparations if the plant

used is adulterated or substituted It results in the

production of misleading or overlapping data on

phytochemical, pharmacological, pharmacognostical and clinical aspects

Nigella sativa (Family: Ranunculaceae) is

considered one of the most important medicinal herb used in various Indigenous System of Medicine The plant cultivated almost all over India, is an annual herb with linear- lance late leaves Pale blue flowers are solitary, fruit is capsule; seeds are black, flattened, angular and funnel shaped Seeds of the herb are mostly used in medicine Many formulations containing Kalongi as a single drug or

in combination with other drugs are available in Indian market The important Unani formulations using the drug are “Anquriya Kabir, Habe-e-Halteet, Roghan Kalan, Qairuti Arad Karsana, Mujun Nankhaw” Traditionally the drug is used against cough, fever, abdominal disorders, skin infections, paralysis and jaundice (Paarakh, 2010) Seed oil is used as a local anaesthetic (Paarakh, 2010) As far

as its pharmacological activities are concerned, the drug is hypoglycaemic, hypocholestermic (Bamosaet, 2002) and antioxidant (Kanter, 2003) Thymoquinone (constituent of seed oil extract) is antitumor, found to kill the pancreatic cancer cells and its derivatives are used in blood, skin and breast cancers (Paarakh, 2010)

The aqueous decoction of kalonji revealed significant antibacterial potential against

Streptococcus mutans, Streptococcus morbillorium, Streptococcus sanguis, Streptococcus intermedius,

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Mohd Asif et.al Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online)

Klebsiella ozaenae, Aeromonas hydrophila, and

Streptococcus salivarius (Chaudhry and Tariq,

2008) Considering the medicinal importance of

Kalongi, the authentication of this herb is highly

mandatory DNA fingerprinting techniques are very

useful for correct identification of taxa Among

various DNA markers, loop-mediated isothermal

amplification (LAMP) is convenient because the

reaction could be conducted under isothermal

conditions, thereby facilitating amplification and the

results could be achieved in less than 1 hr

To the best of our knowledge, no attempt

has been made to characterize this herb using

molecular biology To fill this gap, we employed

LAMP markers to authenticate the said drug For

this, samples were collected from the crude drug

dealers of North and West India

2 MATERIALS ANDMETHODS

2.1 Plant material: The samples of N Sativa were

collected from crude drug dealers of Delhi,

Kolkatta, Uttarakhand and Uttar Pradesh Voucher

specimens of these samples were prepared and kept

in the Herbarium, Department of Botany, Hamdard

University, New Delhi, 110062 The seeds are

stored in seed bank, Department of Botany,

Hamdard University, New Delhi, 110062 The

identified specimens were compared with

authenticated voucher specimens preserved in the

herbarium of National Institute of Science and

Information Resources (NISCAIR) The seeds were

used for DNA isolation

2.2 DNA Isolation: The modified CTAB protocol

of Doyle and Doyle (1990) and purification kit

(HiPurA, India) were used to extract DNA from the

overnight soaked seeds

2.2.1 Reagents and Solutions: CTAB extraction

buffer (2M Sodium Chloride, 100mM TrisHCl (pH

8), 20 mM EDTA) 0.2% β-mercaptoethanol,

Chloroform:Isoamylalcohol (24:1), absolute alcohol,

3M Potassium acetate, Isoamyl alcohol All the

chemicals chemical were of analytic grade

Enzymes (Taq polymerase, Bst polymerase and

RNAase A), Taq buffer, MgCl2 and dNTPs were

purchased from Bangalore Genei (Bangalore, India)

2.2.2 Protocol: In order to avoid surface

contamination, the seeds were washed with 0.2%

Cetrimide for 3 min, followed by treatment with

0.5% streptomycin sulphate and 0.5% bavistine for

5 min each After these treatments the seeds were

rinsed with 70% alcohol for 1 min andfinally

washed with sterile double distilled water and kept

overnight 1g seeds were pulverized to fine powder

by liquid nitrogen in a chilled mortar and pestle

followed by the addition of 100 mg of poly vinyl

pyrollidone (PVP, insoluble) and 10 ml pre-heated

CTAB buffer (containing 0.2 % β-mercaptoethanol) The slurry was transferred into autoclaved 50 ml centrifuge tube and incubated at 60oC for 1 hr 10 ml

of Chloroform, Isoamyl alcohol (CHCl3: IAA, 24:1) was added to the centrifuge tubes and mixed carefully for 15 min

The content was centrifuged at 8000 rpm for

15 min at 15oC The upper phase was transferred into new autoclaved centrifuge tubes 10µl of RNAase was added and the tubes were incubated at

37oC for 30 min 10 ml of CHCl3: IAA (24:1) was added carefully and the tubes were centrifuged at

8000 rpm for 15min at 15oC The upper phase was transferred again into autoclaved centrifuge tube and 0.5 vol of 3M Potassium acetate (pH 5.2) was added For DNA precipitation equal volume of chilled isopropanol (chilled absolute ethanol was

also used) was used and the tubes were kept at -20oC for 2 hrs It was recentrifuged at 8000 rpm for 15 min at 4oC The supernatant was discarded and the pellet was washed with 70% ethanol, air dried and dissolved in 250 µl of sterile water The DNA thus obtained was purified by DNA purification (HiPurA, India) kit according to manufacturer’s instructions

Amplification: The PCR was carried out in 20 µl

reaction volume containing 50ng DNA, 0.5 u/µl Taq DNA polymerase, 1.66 mM MgCl2, 30 pmol 10-mer primers, 200 µM of each dNTPs, 2x Taq polymerase buffer with minor changes as described by Shaik et

al (2006) The final volume was made-up with sterile MilliQ water The amplifications were carried out in DNA thermal cycler (Eppendorf, Germany) The PCR amplification conditions for RAPD consisted of initial step of denaturation at 94°C for 4 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 35°C for 1 min, extension at 72°C for 2 min, followed by final extension at 72°C for 10 min The amplified DNA was loaded on 1.2% agarose gel

in 0.5x TBE buffer containing 10 µl of EtBr (10mg/ml) and photographed using gel documentation system (UVP, Germany) Twenty 10-mer RAPD primer series OPAA, purchased from Qiagen, USA and five (BG series) from Bangalore Genei (India) were screened

2.4 Gel purification, Cloning and sequencing of RAPD amplified product: RAPD fragment was

excised from the gel with a sharp and sterile scalpel

to avoid any contamination Elution of DNA from agarose gel was carried out using MiniElute® kit from Qiagen (USA) following manufacturer’s instructions and the product was run on 1.2% gel along with the Gene Rular DNA ladder, to check the presence of the desired product The eluted DNA was ligated into pGEM®-T easy vector (Promega,

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USA) Ligation reaction was carried out in 10μl

reaction containing 10x ligase buffer (5.0 μl),

pGEM®-T easy vector (0.5 μl), T4 DNA Ligase

(1.0 μl) and PCR product 3.0 μl) and the final

volume was set up with autoclaved Milli Q water

Competent cells of E coli DH5α were prepared by

CaCl2 method (Sambrook et al 2001) and 5μl of the

ligation mixture was mixed with an aliquot of 100 μl

competent cells The transformed cells were plated

on LB-X-gal/Amp plates and the recombinant

colonies were selected through blue-white

screening The plasmid was isolated from the

positive bacterial colonies using plasmid isolation

kit (Qiagen, Germany) The cloned fragments were

sequenced using T7 primers through the centre for

Genomic Application, New Delhi, India

2.5 Design of LAMP Oligonucleotides: A total set

of four LAMP primers were designed using Primer

(http://primerexplorer.jp/elamp3.0.0/index.html) for

the specific detection of PCR product The primer

set consisted of two outer (F3 and B3), and two

inner (FIP and BIP); the inner primers cover two

distinct sequences of the target (F1c/B1c and

F2c/B2c) Sequences of the LAMP primers are

given in table 1

2.6 LAMP reaction: The LAMP reaction was

carried out in a 25 µl reaction volume containing

60pmol each of the primers FIP and BIP, 10 pmol

each of the outer primers F3 and B3, 8mM MgSO4,

1.4mM dNTPs, 0.8M betaine, 10 units of the Bst

DNA polymerase and 2 µl of DNA template The

optimum temperature for the LAMP reaction was

60°C 1μlSYBR Green-Ι dye was added at the end

of the reaction Visual inspection for amplifications

was performed through observation of colour

change following addition of 1μl of SYBR Green I

(fluorescent dsDNA intercalating dye) to the tube

3 RESULTS

RAPD reaction was performed in order to find out unique specie specific monomorphic bands present in all the samples, meant for LAMP analysis Twenty five 10-mer RAPD primers were used of which six did not amplify the DNA Each RAPD reaction was repeated thrice and only reproducible bands were taken in to account A total

of 524RAPD bands (table 2) were obtained and to develop the LAMP marker, we analyzed the nucleotide sequences of 5 species-specific RAPD

amplicons, consisting of 5 DNA fragments for N

saitva From the resulting nucleotide sequences, one

unique RAPD amplicon from primer OPAA-9 has been registered in the NCBI Gene Bank dbGSS, and used to develop RAPD derived LAMP marker (Fig 1) The specific amplicon of 600bp (fig.2) RAPD

fragment, specific for all the accessions of N sativa,

was used for designing primers of LAMP reaction

The reaction was carried out using genomic DNA as a template to determine the optimal temperature and reaction time and to evaluate the use of primers LAMP product formation was detected at a temperature range of 60−64°C and consequently, 60°C was considered to be the optimal reaction temperature for the LAMP assay The tube containing the amplified products were visualised in the presence of fluorescent intercalating dye SYBR Green I under UV transilluminator In case of positive amplification, the original colour of the dye was changed into green that was judged under natural light as well as under UV light (302 nm) with the help of UV transilluminator In case of negative control

(Catharanthus roseus) there was no amplification,

the original orange colour of the dye was retained (Figure.3)

Figure.1 RAPD profile of Kalonji (N sativa) representing specific band present in all accessions amplified with

OPAA-09 primer.Lane M, molecular marker 200-1700 bp, Lanes K1–K5 corresponds to the 5 accessions (K1- Kolkatta, k2- Govindpuri- New Delhi, K3- KhariBaowli- Delhi, K4- Aligarh- Uttar Pradesh, K5- Dehradun-

Uttarakhand)

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Figure.2.Nucleotide sequence of RAPD amplicon (600 bp) of N sativa used for development LAMP marker

Figure.3.Analysis of LAMP under UV light (A) and natural light (B) 1-5 accessions of Nigella sativa.(1- Kolkatta,

2- Govindpuri- New Delhi, 3- KhariBaowli- Delhi, 4- Aligarh- Uttar Pradesh, 5- Dehradun- Uttarakhand, C-

Negative control (Catharanthus roseus)

Table 1.Primer Sequences used in this study

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4 DISCUSSION

Correct identification of plants forming

the drug is a prerequisite and fundamental to

whole realm of medicine and science Most of

the regularity guidelines and pharmacopoeia

recommend macro- and microscopic evaluation

and chemical analysis of botanical material for

quality and quantity control and standardization

macroscopic identification of plant materials is

based on parameters like shape, size, texture,

colour, surface feature, odour, taste and other

organoleptic characters that are compared to

standard reference materials Though this

method is simple and direct, its accuracy and

authenticity which are sometimes subjective,

depends on examiners

examinations are done to study comparative

microscopic inspection of broken as well as

powdered, crude, botanical materials to reveal

the characteristics of tissue structure and

arrangement of cells in different organs and

tissues Chemical authentication establishes a

chemical composition of plant, which is used

for differentiation The variation of chemical

composition may hinder the authentication, and

in some cases, may be misleading if the samples

are adulterated Moreover, it is difficult to

distinguish closely related species due to similar

chemical compounds Molecular or DNA-based

markers are now becoming a popular means for

the identification of medicinal plants (Yip,

2007)

Molecular markers have the advantage

composition is unique for each individual and is

least affected by age (Kumble, 2003),

conditions (Macbeath and Schreiber, 2000),

harvest, storage and processing of the samples

(Schweitzer, 2003) Molecular markers are not

stage and tissue specific and thus can be

detected at any stage of development Ours is

the first attempt to characterize Kalongi using

LAMP markers Although in earlier reports

RAPDs were used for the authentication

purpose (Rivera-Arce, 2007; Shinde, 2007;

Hammad and Qari 2010; Ganie, 2012),

however, these markers are very sensitive and

reproducibility problem Therefore, in the present study RAPDs were converted into LAMP markers These markers are very specific and highly reproducible because these markers could amplify a specific gene from the

nucleotide difference (Parida, 2008) As the reaction is carried out under optimal and isothermal conditions, therefore, there are negligible chances of inhibition reaction at the later stage of amplification compared with the PCR

The results can be easily monitored by checking the turbidity obtained from the precipitate and most importantly the detection

of the desired gene could be completed in a single step by incubating mixture of gene sample, primers, DNA polymerase with strand displacement activity and substrates at constant temperature (Parida, 2008) In our study, the amplification was not detected when the concentration of the template was 0.3 ng, however when the template concentration was

in the range of 0.8-1.5 ng, amplifications occurred; therefore, it was thought that DNA concentration of 0.8 ng is the detection limit in

N sativa Such type of results was also

observed in the studies of P Ginseng (Sasaki,

2008) in which 0.5 ng of template was the detection limit

The optimized reaction parameters that showed positive results were 10 ng template DNA, 10pmol of outer primers (F3 and B3), 60pmol of each of forward internal and backward outer primers (FIP and BIP), 20 mM reaction buffer, 10 mmMgSO4, 0.8 M betaine

and 10 units of Bst DNA polymerase The

optimum temperature for the reaction was set at 60°C, which is considered optimum for the

activity of Bst DNA polymerase The use of

LAMP markers for the authentication of medicinal plants, although is rare; however, there are some recent reports in which the technique has been successfully applied for the identification purposes and some of which

include Curcuma longa (Sasaki and Nagumo;

Catharanthus roseus (Choudhry, 2011)

We have developed LAMP, a rapid, highly sensitive, and specific method for the

authentication of N sativa The present study

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could be applicable to identify N Sativa to

differentiate the herb from the spurious and

adulterated drugs sold in the market in the name

of geniuine drugs Efforts are in progress to

develop LAMP markers to medicinal plants in

authenticating the medicinal plants

5 CONCLUSION

LAMP technology could be efficiently used to

characterize the medicinal plants like of N

sativa By analysing the genetic profiling it is

possible to identify the said drug; this

identification of authenticN sativa devising

from the spurious and adulterated drugs

ACKNOWLEDGMENTS

This work was financed by the CCRUM,

AYUSH, Ministry of Health and Family

Welfare, Government of India

REFERENCES

Anonymous, The Useful Plants of India PID,

CSIR, 1996, New Delhi

Bamosa A.O, Ali B.A, Al-Hawsawi Z.A, The

effects of thymoquinone on blood lipids on rats,

Indian J Physio Pharmacol, 46, 2002,

195-201

Chaudhary A.A, Hemant, Mohsin M, Ahmad A,

amplification (LAMP)-based technology for

authentication of Catharanthus roseus (L.) G

Don Protoplasma, 249, 2012, 417-422

Chaudhry N.M.A, Tariq P, In-vitro anti

bacterial activities of Kalonji, Cumin and Poppy

Seed Pak J Bot, 40, 2008, 461-467

Doyle J.J, Doyle J.J, Isolation of plant DNA

from fresh tissue, Focus, 12, 1990, 13-15

Dubey N.K, Kumar R, Tripathi P, Global

promotion of herbal medicine: India’s

opportunity Curr Sci 86, 2004, 37-41

Ganie S.H, Srivastava P.S, Narula A, Ali Z,

Sharma M.P., Authentication of shankhpushpi

by RAPD markers Eurasia J Biosci, 6, 2012,

39-46

Hammad I, Qari S.H, Genetic diversity among

Zygophyllum (Zygophyllaceae) populations

based on RAPD analysis Genet Mol Res 9,

2010, 2412-2420

Kanter, M., Meral, I.,Dede, S., Effects of

Nigella sativa L and Urticadioica L on lipid

peroxidation, antioxidant enzyme systems and some liver enzymes in CCl4-treated rats J Vet Med Physiol Pathol Clin Med 50, 2003, 264-

268

Khatoon, S., Mehrotra, S., Shome, U., Mehrotra, B.N., Analysis of commercial Ratanjot; by TLC fluorescence fingerprinting Int J Pharmacol 31, 1993, 269-277

Kumble, K.D., Protien microarrays, new tools for pharmaceutical development Analyt Bio Chem 377, 2003, 812-819

MacBeath, G., Schreiber, S.L., Printing proteins

as microarrays for high-throughput function determination Sci., 289, 2000, 1760-1763

Paarakh, P.M., Nigella sativa Linn.- A

Comprehensive Review Ind J Nat Prod Resour 1, 2010, 409-429

Parida, M., Sannarangaiah, S., Dash, P.K., Rao, P.V.L., Morita, K., Loop mediated isothermal amplification (LAMP): a new generation of

perspectives in clinical diagnosis of infectious diseases Rev Med Virol 18, 2008, 407-421 Rivera-Arce, E., Gattuso, M., Alvarado, R., Zarate, E., Aguero, J., Feria, I., Lozoya, X., Pharmacognostical studies of the plant drug

Mimosa tenuifloraecortex J Ethnopharmaco

113, 2007, 400-408

Sasaki, Y., Komatsu, K., Nagumo, S., Rapid

detection of Panax ginseng by loop-mediated

isothermal amplification and its application to authentication of Ginseng Biol Pharm Bull,

31, 2008, 1806-1808

Sasaki, Y., Nagumo, S., Rapid identification of

Curcuma longa and C aromatic by LAMP

Biol Pharm Bull, 30, 2007, 2229-2230

Schweitzer, B., Predki, P., Synder, M., Microarrays to characterize protein interactions

on a whole-protoeme scale Proteomics 3,

2003, 190-199

Shaik Y.B, Castellani M.L, Perrella A, Conti

F, Salini V, Tete S, Madhappan B, Vecchiet

J, De Lutiis M.A, Caraffa A, Cerulli G, Role of quercetin (a natural herbal compound) in allergy

Trang 11

and inflammation, J Biol Regul Homeost

Agents, 20(3-4), 2006, 47-52

Shinde M, Dhalwal K, Mahadik K.R, Joshi,

K.S, Patwardhan B.K, RAPD Analysis for

Determination of Components in Herbal

Medicine, Evi Based Complemen Alterna Med, 4, 2007, 21-23

Yip P.Y, Chau C.F, Mak C.Y, Kwan H.S, DNA methods for identification of Chinese medicinal materials, J Chin Med, 2, 2007, 1-19

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Malleswari et.al Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online)

Comparative in-vitro dissolution study of five brands of Diclofenac sodium delayed

release tablets in QbD environment

V Malleswari Bai* 1 , M Prasada Rao 1 , M Chandana 1 , K Naga Harini 1 , B Naga Deepthi 1 , K Thirumala Devi 1 ,

P Lakashmana Rao 1 , Vinay U rao2 and J Naga raja 1

1 Department o f Pharmaceuitical Analysis, Medarametla Anjamma Masthanrao College of Pharmacy, Narasarao

Pet, Guntur district, Andhra Pradesh, India

2 Institutes of Pharmaceutical Sciences, Hyderabad-500049

*Corresponding author: E.Mail:malleswari.v4@gmail.com

ABSTRACT

Diclofenac sodium tablets are available as delayed release tablets in the market Delayed release tablets are typically produced by coating the tablet with enteric coating polymers These polymers provide the resistance of drug release in acidic environment of stomach and allow the drug to be released in alkaline environment of the intestine A large number of enteric polymers are available which provide excellent protection to drug release in acidic environment However, each polymer dissolves at different alkaline pH For e.g Eudragit L-100 dissolves at pH 6 and above while Eudragit S-100 dissolves at pH 6.5 and above HPMC Phthalate P5.5 dissolves at pH 5.5 and above while HPMC Phthalate P dissolves at

pH 6 and above Hence, for the same drug the bioavailability can subtly but significantly change based on which enteric polymer is used to provide the delayed release The aim of the current work was to comparatively evaluate five brands of Diclofenac sodium enteric coated tablets and determine which brands may be equivalent to each other based on in vitro testing Comparative dissolution profile testing was carried out in pH 5.5, pH 6 and pH 6.8 buffers It was determined that brand 1 and 5 are equivalent to each other while brands 2, 3 and 4 are equivalent to each other Similarity factor f2 was used for comparing the dissolution profiles Alcohol dumping studies indicated that only brand 1 was able to withstand the enteric effect at 40% level of alcohol All other marketed brands fail the alcohol dumping test This indicates that patients may have to counsel not to concomitantly consume alcohol while on Diclofenac sodium delayed release tablets

Key words: Diclofenac sodium, Quality by design (QbD), Delayed release, Dissolution test

INTRODUCTION

Quality by Design (QbD) is a concept first

outlined by well-known quality expert Joseph M

Juran in various publications, most notably Juran on

Quality by Design Juran believed that quality could

be planned, and that most quality crises and problems

relate to the way in which quality was planned in the

first place While Quality by Design principles has

been used to advance product and process quality in

every industry, and particularly the automation

industries, they have most recently been adopted by

the U.S Food and Drug administration (FDA) as a

vehicle for the transformation of how drugs are

discovered, developed, and commercially

manufactured (Juran, 1992)

MATERIALS AND METHODS

Development of a predictive dissolution method:

The effects of dissolution medium pH, stirring speed,

volume of the dissolution medium, type of apparatus

used were systemically evaluated to develop the

predictive dissolution method

Effect of dissolution medium: An initial attempt at

developing the discriminating dissolution method that

would be predictive of in vivo performance was made

using USP apparatus 2 Effect of dissolution medium pHof both the innovator product and the four brands were subjected to dissolution testing using USP apparatus 2 at 50 rpm in 900 mL of various media including water, 0.1 N HCl, pH 5.5 phosphate buffer, and pH 6.8 phosphate buffer The drug release of the marketed samples in comparison with the innovator at different time intervals was obtained in all the mediums The similarity factor of the brands using innovator product as the reference is calculated

Effect of dissolution medium volume: The drug

release of innovator products and all the marked brands was evaluated using pH 1.2(0.1N HCl) dissolution medium volumes of1000ml and 500ml The stirring speed was 50 rpm in each case The drug release profile of the marketed samples in comparison with the innovator product at various time intervals were obtained in all two volumes The similarity factor of the brands using innovator product as the reference is calculated

Effect of stirring speed: Dissolution testing of all the

marketed samples and the innovator product was conducted at25, 50, 75, and 100 rpm in 900 mL of pH 1.2(0.1NHCl) dissolution medium The similarity

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factor of the brands using innovator product as the

reference is calculated

Effect of type of dissolution apparatus: Dissolution

testing of all the marketed samples and the innovator

product was conducted using both USP1 (Basket) and

USP2 (Paddle) apparatus in 900ml of pH

1.2(0.1NHCl) dissolution medium The drug release

profiles of all the marketed samples in comparison

with the innovator product at various time intervals

were obtained The similarity factor of the brands

using innovator product as the reference is calculated

QbD development process includes:

 Begin with the target product profile that

describes the use, safety and efficacy of the

product

 Defining a target quality profile that will be

used by formulators and process engineers as

a quantitative surrogate for aspects of clinical

safety and efficacy during product

development

 Gather relevant prior knowledge about the

substance, potential excipients and process

operation

 Design a formulation and identify the quality

attributes to the final product that must be

controlled to meet the target product quality

profile

 Design a manufacturing process to produce a

final product having this critical material

attributes

 Identify the critical process parameters and

raw material attributes that must be controlled

to achieve these critical material attributes of

the final product

 Establish a control strategy for the entire

process that may include input material

controls, process controls and monitors design

space around individual or multiple unit

operation and/ or final product tests

 Continually monitor and update the process to

assure consistent quality

Quality by Design for drug release

Two primary aspects:

1 Clinical relevance of release and stability

specifications

2 Correlation between process parameters

and ability to achieve specifications (and

therefore remain clinically relevant)

Dissolution testing and drug release:

 Dissolution testing has been widely used as

the primary tool to evaluate drug release

 Dissolution is the process by which a solid solute enters a solution, and is characterized

by rate (amount dissolved by time)

 In the pharmaceutical industry, it may be defined as the amount of drug substance that goes into solution per unit time under standardized conditions of liquid/solid interface, temperature and solvent composition

 Dissolution is the quality control measure and potential to provide in sight into the in vivo performance of the drug product

 In vitro release test that predicts the drug in vivo would be optimal and highly desirable

 A variety of designs of apparatus for dissolution testing have been proposed and tested, varying from simple beaker with stirrer

to complex systems

techniques are required for different dosage forms because of significant differences in formulation design and the physicochemical properties of the drugs

 Dissolution tests have been developed for various drug delivery systems including immediate release solid dosage forms, several controlled release solid dosage forms and many novel and special dosage forms

Most of the tests with recommended apparatus and other specifications are now available as compendial standards in Pharmacopoeias and are used in

pharmaceutical analysis and drug development for the various drug delivery systems

RESULTS AND DISCUSSION

Five brands of Diclofenac sodium delayed release tablets 50 mg were procured from the market and subjected to assay and comparative dissolution profile testing as per USP guidelines for determining

in vitro equivalence of modified release products

Assay of Diclofenac sodium delayed release tablets:

Twenty tablets were weighed and crushed using mortar and pestle Quantity of powder equivalent to

100 mg of Diclofenac sodium was weighed accurately and transferred to 100 ml volumetric flask Approximately 70 ml of methanol AR grade was added and syndicated for 15 minutes The volume was made up to 100 ml with methanol and filtered From the clear filtrate and aliquot equivalent to 100 ppm was pipette out and transferred to 10 ml volumetric flask The volume was made up to 10 ml with Methanol (10 µg/ml solution) The absorbance of this

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solution was measured on UV spectrophotometer at

276 nm wavelength The drug content was calculated

by simultaneously measuring the absorbance of a

standard 10 µg/ml solution of Diclofenac sodium The

assay values for all five brands are given in Table 1

Disintegration test for Enteric coated tablets (IP):

The DT test for enteric coated tablets as described in

IP was performed for 6 tablets of each brand and it

was observed that all brands pass this test

Comparative dissolution testing in 0.1N HCl: It is

mandatory for all delayed release products to show <

10% drug release in 0.1N HCl when in vitro

dissolution testing is performed for 2 hours in this

medium The results for the dissolution testing of the

5 brands are given in Table 2 and shown in Figure.1

All five brands passed the criteria of <10% in two

hours in pH 1.2 medium

Comparative dissolution testing in pH 5.5: pH 5.5

simulates the duodenum and upper intestinal portion

Comparative dissolution testing was conducted in pH

5.5 acetate buffer for 1 hour (Figure.2)

The dissolution profiles were statistically

compared by calculating the similarity factor (f2) The

f2 factor for brand 2, 3, 4 and 5 was calculated by

comparing with brand 1 Only brand 5 showed f2 >

50 Hence this may be considered as equivalent to

brand 1 for dissolution profile testing in pH 5.5 The

comparing with brand 2 Brand 3 and 4 showed f2 >

50 Hence these may be considered as equivalent to

brand 2 for dissolution profile testing in pH 5.5 The

comparing with brand 3 Brand 5 showed f2 > 50

Hence this may be considered as equivalent to brand 3

for dissolution profile testing in pH 5.5 The f2 factor

for brand 1, 2, 3 and 5 was calculated by comparing

with brand 4 Brand 2 showed f2 > 50 Hence this may

be considered as equivalent to brand 4 for dissolution

profile testing in pH 5.5 The f2 factor for brand 1, 2,

3 and 4 was calculated by comparing with brand 5 as

standard Only brand 1 showed f2 > 50 Hence this

may be considered as equivalent to brand 5 for

dissolution profile testing in pH 5.5

simulates the duodenum and upper intestinal portion

Comparative dissolution testing was conducted in pH

6.0 Phosphate buffer for 1 hour (Figure.3)

The dissolution profiles were statistically

compared by calculating the similarity factor (f2) The

comparing with brand 1 Only brand 5 showed f2 >

50 Hence this may be considered as equivalent to brand 1 for dissolution profile testing in pH 6.0 The f2 factor for brand 1, 3, 4 and 5 was calculated by comparing with brand 2 Brand 3 and 4 showed f2 >

50 Hence these may be considered as equivalent to brand 2 for dissolution profile testing in pH 6.0 The f2 factor for brand 1, 2, 4 and 5 was calculated by comparing with brand 3 Brand 2 and 5 showed f2 >

50 Hence these may be considered as equivalent to brand 5 for dissolution profile testing in pH 6.0

simulates the middle and lower portion of the gut Comparative dissolution testing was conducted in pH 6.0 Phosphate buffer for 1 hour (Figure.4)

The dissolution profiles were statistically compared by calculating the similarity factor (f2) The f2 factor for brand 2, 3, 4 and 5 was calculated by comparing with brand 1 Brand 2 and 4 showed f2 >

50 Hence these may be considered as equivalent to brand 4 for dissolution profile testing in pH 6.8 The f2 factor for brand 1, 2, 3 and 4 was calculated by comparing with brand 5 Brand 1, 2 and 3 showed f2 >

50 Hence these may be considered as equivalent to brand 5 for dissolution profile testing in pH 6.8

The multimedia dissolution study indicates that the differences in the rate and extent of dissolution between different brands are significantly more at pH 5.5 and pH 6 than at pH 6.8 This may most likely be due to the fact that each brand may have been coated with enteric materials of different chemistries having different solubility profiles in

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alkaline pH For e.g the reported solubility of

Eudragit L100 is pH 6 and above while that of

Eudragit L100-55 is pH 5.5 and above Consequently,

tablets coated with L-100-55 will show significantly

faster and more complete dissolution at pH 5.5 as

compared to Eudragit L-100 All pH dependent

enteric polymers completely dissolve at pH 6.8 and

above Hence the difference between the brands is

significantly reduced in case of dissolution profile

testing in pH 6.8 buffer

In order to simulate the way the dosage form is

exposed to pH change in vivo, dissolution profile

testing for all brands was conducted by using the in

situ pH change method The in situ pH change method

of dissolution testing indicates that all five brands

achieve > 80% release within 30 minutes of reaching

the pH 6.8 However, dissolution profiles for Brand 1

and brand 5 are significantly faster at pH 6 than those

of brands 2, 3 and 4 Hence, from this study it may be

predicted that Brands 1 and 5 may show therapeutic

equivalence to each other while brands 2, 3 and 4 may

be equivalent to each other But brand 1 and 5 may

not show therapeutic equivalence to brands 2, 3 and

4.(Figure.7)

Alcohol dumping study: For modified release

products, different types of polymers are used to get

the same kind of effect Each of these polymers has

different solubility profiles in commonly used solvents The probability that this may affect the intended release profile in vivo is very genuine if we consider that patients may consume alcohol when under treatment with modified release products The solubility profile of the polymer in alcohol may adversely affect the release rate of the drug from the dosage form and the actual drug release may be entirely different from the intended release

Hence, the US FDA in its latest guidelines has mandated that the alcohol dumping studies should be carried out for modified release products in order to demonstrate that the dosage form is able to perform within its specified standards even in presence e of significant levels of alcohol For the five marketed brands of Diclofenac sodium delayed release tablets, a comparative alcohol dumping study was performed in 0.1N HCl without alcohol, and 0.1N HCl with 5%, 10% 20% and 40% v/v of alcohol respectively The dissolution profile testing was carried out for two hours The study indicates that only Brand 1 is able to maintain the enteric effect of < 10% release in acidic

pH even in presence of 40% alcohol All other brands fail the alcohol dumping test at 40% level of alcohol Brand 2 fails the test even at 10% and 20% level of alcohol (Figure.8)

Table.1 Assay values for all five Brands of Diclofenac sodium 50 mg delayed release tablets

Figure.1 Dissolution profiles for 5 brands of Diclofenac

sodium delayed release tablets in 0.1N HCl

Figure.2.Comparative dissolution profile of five brands in

ph 5.5

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Figure.3.Comparative Dissolution Profile In Ph 6.0

Figure.7 Dissolution profile by in situ ph change method Figure.8.Alcohol Dumping study for Diclofenac sodium

delayed release tablets market brands SUMMARY AND CONCLUSION

Diclofenac sodium tablets are available as

delayed release tablets in the market Delayed release

tablets are typically produced by coating the tablet

with enteric coating polymers These polymers

provide the resistance of drug release in acidic

environment of stomach and allow the drug to be

released in alkaline environment of the intestine A

large number of enteric polymers are available which

provide excellent protection to drug release in acidic

environment However, each polymer dissolves at

different alkaline pH For e.g Eudragit L-100

dissolves at pH 6 and above while Eudragit S-100

dissolves at pH 6.5 and above HPMC Phthalate P5.5

dissolves at pH 5.5 and above while HPMC Phthalate

P dissolves at pH 6 and above Hence, for the same drug the bioavailability can subtly but significantly change based on which enteric polymer is used to provide the delayed release

The aim of the current work was to comparatively evaluate five brands of Diclofenac sodium enteric coated tablets and determine which brands may be equivalent to each other based on in vitro testing Comparative dissolution profile testing was carried out in pH 5.5, pH 6 and pH 6.8 buffers It was determined that Brand 1 and 5 are equivalent to each other while brands 2, 3 and 4 are equivalent to

Trang 17

each other Similarity factor f2 was used for

comparing the dissolution profiles

Alcohol dumping studies indicated that only

brand 1 was able to withstand the enteric effect at

40% level of alcohol All other marketed brands fail

the alcohol dumping test This indicates that patients

may have to counsel not to concomitantly consume

alcohol while on Diclofenac sodium delayed release tablets

ACKNOWLEDGEMENT

The authors are grateful thanks to Indian Pharmaceutical Sciences, Arabindo pharma lim, EMCO industries-hyd, FMC-US, Ashaland specialty chemicals-US for providing gift samples of Diclofenac Sodium

REFFERECES

1 Juran JM, Juran on Quality by Design, The Free

Press, A Division of Macmillan, Inc., New York,

1992, 407-425

2 Kearney PM, Baigent C, Godwin J, Halls H,

Emberson JR, Patrono C: Do selective cyclo-

oxygenase-2 inhibitors and traditional non-steroidal

anti-inflammatory drugs increase the risk of

atherothrombosis? Meta-analysis of randomised trials,

BMJ, 3, 2006, 1302

3 Solomon DH, Avorn J, Sturmer T, Glynn RJ,

Mogun H, Schneeweiss S: Cardiovascular outcomes

in new users of coxibs and Nonsteroidal

anti-inflammatory drugs: high-risk subgroups and time

course of risk, Arthritis Rheum, 54(5), 2006,

1378-89

4 FitzGerald GA, Patrono C, The coxibs, selective

inhibitors of cyclooxygenase-2, N Engl J Med, 345(6),

2001, 433-42

5 Graham DJ: COX-2 inhibitors, other NSAIDs, and

cardiovascular risk: the seduction of common sense,

JAMA, 296(13), 2006, 1653-6

6 Brater DC, Renal effects of

cyclooxygyenase-2-selective inhibitors, J Pain Symptom Manage, 23(4

Suppl), 2002, 15-20

7 Sigma Aldrich Gan TJ: Diclofenac: an update on its

mechanism of action and safety profile, Curr Med

Res Opin, 26(7), 2010, 1715-31

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Lakshmana Rao et.al Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online)

Development and validation of a stability indicating HPLC method for analysis of

Altretamine in bulk drug and pharmaceutical formulations

M Karimulla Santhosh, A Sreedevi, L Kalyani, A Lakshmana Rao *

V.V Institute of Pharmaceutical Sciences, Gudlavalleru, Andhra Pradesh, India

Key Words: HPLC, Altretamine, Stability, Formulation

INTRODUCTION

Altretamine (Figure 1) is a synthetic cytotoxic

antineoplastic agent (Neil, 2006) Chemically it is

N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-2,4,6-triamine Altretamine is indicated for use as a single

agent in the palliative treatment of patients with

persistent or recurrent ovarian cancer following

first-line therapy with a cisplatin and/or alkylating

agent-based combination (Wiernik, 1992) Altretamine is

structurally related to the alkylating agents Its precise

mechanism of action is unknown but hydroxy methyl

intermediates in the metabolism process are possibly

the reactive species, and may act as alkylating agents

(Rhoda, 1995) Altretamine interferes with the growth

of cancer cells and slows their growth and spread in

the body

Literature survey revealed that few HPLC

methods (Ghiorghis, 1991; Barker, 1994) were

reported for the determination of Altretamine But no

stability indicating HPLC method was reported

Hence the objective of this method is to develop and

validate a simple, rapid and accurate stability

indicating HPLC method (Snyder, 1997) in

accordance with ICH guidelines (ICH Q2(R1), 2005;

ICH Q1A(R2), 2003) for the determination of

Altretamine in bulk sample and its pharmaceutical

formulations

Chemicals and solvents: The working standard

of Altretamine was provided as gift sample from

Spectrum Labs, Hyderabad, India The market

formulation CANTRET capsules (Altretamine 50

grade acetonitrile and water were purchased from E.Merck (India) Ltd, Mumbai, India Potassium dihydrogen phosphate, orthophosphoric acid and triethylamine of AR grade were obtained from S.D Fine Chemicals Ltd, Mumbai, India

performance liquid chromatographic method for quantitative determination of Altretamine using Waters HPLC system on Hypersil BDS C18 column (100 mm x 4.6 mm I.D., particle size 5 µm) was used The instrument is equipped with

an auto sampler and UV detector A 10 μL rheodyne injector port was used for injecting the samples Data was analyzed by using Empower 2 software

Chromatographic conditions: A mixture of

phosphate buffer pH 3.1 and acetonitrile (90:10, v/v) was found to be the most suitable mobile phase for ideal chromatographic separation of Altretamine The solvent mixture was filtered through 0.45 μ membrane filter and sonicated before use It was pumped through the column at

a flow rate of 1.0 mL/min Injection volume was

10 µL and the column was maintained at a

equilibrated by pumping the mobile phase through the column for at least 30 minutes prior

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to the injection of the drug solution The detection

of the drug was monitored at 227 nm The run

time was set at 6 minutes

Preparation of phosphate buffer pH 3.1: 2.72

grams of potassium dihydrogen phosphate was

weighed and transferred into a 1000 mL beaker

and dissolved 1 mL of triethylamine solution was

added to the above solution and diluted to 1000

mL with HPLC water pH was adjusted to 3.1

with orthophosphoric acid solution

Preparation of mobile phase and diluent: 900

mL of phosphate buffer was mixed with 100 mL

of acetonitrile and was used as mobile phase The

solution was degassed in an ultrasonic water bath

for 5 minutes and filtered through 0.45 µ filter

under vacuum The mixture of 800 mL of water

and 200 mL of acetonitrile was used as diluent

Preparation of standard solution: 10 mg of

Altretamine was accurately weighed, transferred

to 10 mL volumetric flask and is dissolved in 7

mL of the diluent Sonicated the solution for few

minutes to dissolve the drug completely Then it

is filtered through 0.45 μ filter and the volume is

made up to 10 mL with diluent to get a

concentration of 1 mg/mL stock solution Further

pipetted 1.0 mL of the above stock solution into a

10 mL volumetric flask and diluted up to the

concentrations

Preparation of sample solution: Twenty

powdered A quantity of the powder equivalent to

10 mg of Altretamine was accurately weighed,

transferred to 10 mL volumetric flask and is

dissolved in 7 mL of the diluent Sonicated the

solution for few minutes to dissolve the drug

completely Then it is filtered through 0.45 μ

filter and the volume is made up to 10 mL with

diluent to get a concentration of 1 mg/mL stock

solution Further pipetted 1.0 mL of the above

stock solution into a 10 mL volumetric flask and

diluted up to the mark with diluent to obtain

required concentrations of Altretamine in

pharmaceutical dosage forms Inject 10 µL of the

above solution into the HPLC system All

experiments were conducted in triplicate

Linearity: Several aliquots of standard solution

of Altretamine was taken in different 10 mL volumetric flasks and diluted up to the mark with diluent such that the final concentrations of Altretamine were in the linearity range of 25-150 µg/mL Evaluation of the drug was performed with UV detector at 227 nm, peak area was recorded for all the peaks The response for the drug was linear and the regression equation was found to be y=19094x-10685 and correlation coefficient value of Altretamine was found to be 0.999 The results show that an excellent correlation exists between peak area and concentration of drug within the concentration range indicated

Limit of detection and limit of quantification:

The limit of detection (LOD) and limit of quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solution using the developed HPLC method The LOD and LOQ for Altretamine were found to be 0.46 μg/mL and 1.39 μg/mL respectively

System suitability: System suitability parameters

like retention time, theoretical plates and tailing factor were calculated and compared with standard values

Accuracy: The accuracy of the method was

assessed by recovery study of Altretamine in the dosage form at three concentration levels A fixed amount of preanalyzed sample was taken and standard drug was added at 50%, 100% and 150% levels The standard concentration was fixed as

100 μg/mL and three concentration levels of 50 μg/mL, 100 μg/mL and 150 μg/mL were added to the standard concentration Each level was repeated three times The content of Altretamine per capsule was calculated The percentage recovery ranges from 99.62-100.27% and the mean recovery of Altretamine was 99.92% and the recovery values of Altretamine indicate the method is accurate

Precision: The precision was determined for

Altretamine in terms of system and method

precision For system precision evaluation,

%RSD for Altretamine was 0.32% (limit %RSD

< 2.0%) In addition, the method precision was

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studied and the %RSD for Altretamine was

0.77% (limit %RSD < 2.0%)

Ruggedness and robustness: The ruggedness of

the method was determined by carrying out the

experiment on different instruments by different

operators using different columns of similar

types Robustness of the method was determined

by making slight changes in the chromatographic

conditions like changes in flow rate and mobile

phase composition It was observed that there

were no marked changes in the chromatograms,

which demonstrated that the HPLC method so

developed is rugged and robust

Solution stability: The stability of solution under

study was established by keeping the solution at

room temperature for 24 hrs The result showed

no significant change in concentration and thus

confirms the stability of the drug in the mobile

phase used for the analysis

Analysis of the marketed formulations: The

determination of Altretamine in pharmaceutical

formulatons of Altretamine capsules 10 µL of

each standard and sample solution were injected

and from the peak area of Altretamine, amount of

drug present in samples were computed The

result of assay undertaken yielded 99.98% of

label claim of Altretamine The assay obtained is

more than 99% and no interference of impurity

peak observed in Altretamine peak

Acidic d egradation s tudies: To 1 mL of stock

solution of Altretamine, 1 mL of 2N hydrochloric

acid was added and refluxed for 30 mins at 600C

The resultant solution was diluted to obtain 100

µg/mL solution and 10 µL solution were injected

into the system and the chromatograms were

recorded to assess the stability of sample

Alkaline d egradation s tudies: To 1 mL of

stock solution of Altretamine, 1 mL of 2N

sodium hydroxide was added and refluxed for

30 mins at 600C The resultant solution was

diluted to obtain 100 µg/mL solution and 10 µL

solution were injected into the system and the

chromatograms were recorded to assess the

stability of sample

Oxidative degradation studies: To 1 mL of

stock solution of Altretamine, 1 mL of 20% hydrogen peroxide (H2O2) was added separately The solutions were kept for 30 mins at 600C The resultant solution was diluted to obtain

100 µg/mL solution and 10 µL solution were injected into the system and the chromatograms were recorded to assess the stability of sample

Thermal degradation s tudies: The standard

A l t r e t a m i n e solution w a s placed in oven at

1050C for 6 hrs to study thermal degradation The resultant solution was diluted to obtain 100 µg/mL solution and 10 µL solution were injected into the system and the chromatograms were recorded to assess the stability of the

sample

Hydrolytic d egradation s tudies: Stress testing

under hydrolytic conditions was studied by refluxing the s t a n d a r d A l t r e t a m i n e

s o l u t i o n in water for 6 h r s at a temperature

of 60ºC The resultant solution was diluted to obtain 100 µg/mL solution and 10 µL solution

chromatograms were recorded to assess the

stability of the sample

Photolytic degradation studies: The photolytic

stability of the drug Altretamine was studied by exposing the standard Altretamine solution to UV light by keeping the beaker in UV chamber for 7 days or 200 Watt hours/m2 in photo stability chamber The resultant solution was diluted to obtain 100 µg/mL solution and 10 µL solution

chromatograms were recorded to assess the stability of sample

In the present work, a simple, accurate and precise stability indicating HPLC method has been optimized, developed and validated for the determination of Altretamine in pharmaceutical formulations with UV detector by using Hypersil BDS C18 column (100 mm x 4.6 mm I.D., particle size 5 µm) in isocratic mode with mobile phase composition of phosphate buffer pH 3.1: acetonitrile (90:10, v/v) and pH adjusted to 3.1 with orthophosphoric acid The use of phosphate buffer and acetonitrile in the ratio of 90:10, v/v resulted in peak with good shape and resolution

Trang 21

The flow rate was 1.0 mL/min and the drug

component was measured with UV detector at

227 nm The results of optimized HPLC

conditions were shown in Table 1

The method was linear in the range of

25-150 µg/mL for Altretamine with correlation

coefficient of 0.999 The linearity results were

shown in Table 2 and the linearity curve of

Altretamine was shown in Figure 2 The %

recoveries of Altretamine were found in the range

of 99.62-100.27% and the % mean recovery was

found to be 99.92% for Altretamine, which

indicate the method is accurate The results of

recovery studies were shown in Table 3 The

%RSD for system precision and method precision

for Altretamine were found to be 0.32 and 0.77,

which indicate the method is precise The results

of precision studies were shown in Table 4 and

Table 5 The retention time of Altretamine was

2.533 min, cuts down on overall time of sample

analysis and the method was more cost effective

as it utilizes very less quantity of mobile phase

The number of theoretical plates was 4253 and

tailing factor was 1.49 for Altretamine, which

indicates efficient performance of the column

Typical chromatogram of drug Altretamine was

shown in Figure 3

demonstrated by the absence of any interfering

peaks at the retention time of the drug The

limit of detection and limit of quantification for

Altretamine were found to be 0.46 μg/mL and

1.39 μg/mL, which indicate the sensitivity of

the method A system suitability test was

performed to evaluate the chromatographic parameters and the summary of system suitability parameters were shown in Table 6 Validated method was applied for the determination of Altretamine in commercial formulations The % assay was found to be 99.98% for Altretamine and the assay results were shown in Table 7

HPLC studies of Altretamine under different stress conditions indicated the following degradation behavior In acidic degradation, the degradation product of Altretamine was appeared

at retention time of 2.522 min and the % degradation is 9.08% In alkaline degradation, the degradation product of Altretamine was appeared

at retention time of 2.522 min and the % degradation is 6.96% In oxidative degradation, the degradation product of Altretamine was appeared at retention time of 2.504 min and the % degradation is 7.40% In thermal degradation, the degradation product of Altretamine was appeared

at retention time of 2.498 min and the % degradation is 5.27% In hydrolytic degradation, the degradation product of Altretamine was appeared at retention time of 2.503 min and the % degradation is 0.21% In photolytic degradation, the degradation product of Altretamine was appeared at retention time of 2.498 min and the % degradation is 1.05% The results of analysis are given in Table 8 The typical chromatograms of degradation behavior of Altretamine in different stress conditions are shown in Figure 4 to Figure 9.

Figure.1 Molecular structure of Altretamine

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Figure.2 Calibration curve of Altretamine

Figure.3 Typical chromatogram of Altretamine

Figure.4 Acidic degradation chromatogram of Altretamine

Figure.5 Alkaline degradation chromatogram of Altretamine

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Figure.6 Oxidative degradation chromatogram of Altretamine

Figure.7 Thermal degradation chromatogram of Altretamine

Figure.8 Hydrolytic degradation chromatogram of Altretamine

Figure.9 Photolytic degradation chromatogram of Altretamine

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Table.1 Optimized chromatographic conditions of Altretamine

Table.2 Linearity results of Altretamine

Table.4 System precision data of Altretamine Table.5 Method precision data of Altretamine

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Table.7 Assay results of Altretamine

% Degradation

% of active drug present after degradation

The present study represents the first report

that deals with the development of a stability

indicating HPLC method for determination of

Altretamine This study is a typical example for

development of a stability indicating assay established

by following the recommendations of ICH guidelines

The proposed method showed acceptable accuracy,

precision, selectivity and wide linear concentration

range The results of analysis proved that the method

is suitable for the determination of Altretamine in bulk

and capsule dosage forms without any interference

from the degradation products and it is recommended

for routine quality control analysis of the Altretamine

in pharmaceutical formulations

REFERENCES

Barker IK, Crawford SM and Fell AF, Determination

of Altretamine in human plasma with

high-performance liquid chromatography, Journal of Liquid

Chromatography B, 660(1), 1994, 121-126

Ghiorghis A and Talebian AH, High-pressure liquid

chromatography separation of potential impurities of

Altretamine, Journal of Liquid Chromatography,

14(12), 1991, 2331-2349

ICH Harmonised Tripartite Guideline, Stability Testing of New Drug Substances and Products, Q1A(R2), International Conference on

Harmonization, 2003, 1-18

ICH Harmonised Tripartite Guideline, Validation of analytical procedures: Text and methodology, Q2(R1), International Conference on Harmonization, 2005, 1-

13

Neil OJM, The Merck Index, An Encyclopedia of Chemicals Drug and Biologicals, 14th Ed., Merck Research Laboratories, Division of Merck and Co Inc., White House Station, NJ, 2006, 57

Rhoda LC and Diana F, Altretamine: A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in cancer chemotherapy, Drugs, 49(6), 1995, 932-953

Snyder LR, Kirkland JJ and Glajch JL, Practical HPLC Method Development, 2nd Ed., New York, John Wiley and Sons, 1997, 184-185

Wiernik PH, Hexamethylmelamine and low or moderate dose cisplatin with or without pyridoxine for treatment of advanced ovarian carcinoma: a study of the eastern cooperative oncology group, Cancer Investigation, 10(1), 1992, 1-9

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Delhi Raj et.al Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online)

A review of various analytical methods on Atrovastatin

N.Delhiraj*, P.Ashok,U.Ravikiran,P.Abhinandhana

Department of pharmaceutical analysis, A.S.N Pharmacy College, Tenali, Andhra Pradesh

*Corresponding author E.Mail:pharmaraj1981@gmail.com

ABSTRACT

There is a review of analytical methods for atorvastatin, such as spectrophotometry, derivative

spectrophotometry and various chromatographic procedures such as, high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC), and liquid

chromatography tandem mass spectrometry (LC-MS) This review is based on representative publications that were published between 2000 and 2013

Key Words: hypolipideamic drugs, HMG-COA analysis, review, spectrophotometry, chromatography

INTRODUCTION

Atorvastatin is the most efficacious and best

tolerated hypolipideamic drugs introduced in

1980.They competitively inhibit conversion of

3-hydroxy 3-methyl glutaryl coenzyme and

(HMG-COA) to mevalonate Atorvastatin is the most

efficacious and best tolerated hypolipideamic drugs

They competitively inhibit conversion of hydroxy

3-methyl glutaryl coenzyme and (HMG-COA) to

mevalonate (rate limiting step in cholesterol (CH)

synthesis) by the HMG-COA reductase Therapeutic

doses reduce cholesterol synthesis by 20-50% This

results in compensatory increases in Low density

lipoprotein (LDL) receptor expression on liver cell

leads to increased receptor mediated uptake and

catabolism of Intermediate density lipoprotein (IDL)

and LDL Over long term feedback induction of

HMG-COA reductase tends to increase CH synthesis

but a steady is finally attained with a dose dependent

lowering of LDL-CH levels The daily dose of

Atorvastatin for lowering LDL-CH levels by 30-35%

is 10 mg Morever at their maximum recommended

doses atorvastatin can reduce CH up to 45-55%.5 The

more efficacious atrovastatin given at their higher

doses effectively reduce Triglycerides (TGs) (by

25-35%) when they are moderately raised but not when

they are markedly raised Because HMG-COA

reductase activity is maximum at midnight, statins are

administered at bed time to obtain maximum

effectiveness All statins except rosuvastatin are

metabolized primarily by CYP3A4 Inhibitors and

inducers of this isoenzyme respectively increase and

decrease statin blood levels (Tripathi,KD 2008)

methods: A novel, precise, accurate and rapid

isocratic reversed-phase high performance liquid

method was developed, optimized and validated

for simultaneous determination of rosuvastatin

and atorvastatin in human serum using naproxen sodium as an internal standard Effect of different experimental parameters and various particulate columns on the analysis of these analytes was

separation for rosuvastatin and atorvastatin and best resolution was achieved with Brownlee analytical C18 column (150×4.6 mm, 5 μm) using methanol-water (68:32, v/v; pH adjusted to 3.0 with trifluoroacetic acid) as a mobile phase at a flow rate of 1.5 ml/min and wavelength of 241

nm The calibration curves were linear over the concentration ranges of 2.0-256 ng/ml for rosuvastatin and 3.0-384 ng/ml for atorvastatin The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for rosuvastatin were 0.6 and 2.0 ng/ml while for atorvastatin were 1.0 and 3.0ng/ml, respectively All the analytes were separated in less than 7.0 min The proposed method could be applied for routine

interaction studies and pharmacokinetics studies (Shah Y, 2011)

Two simple and accurate methods to determine atorvastatin calcium and ramipril in capsule dosage forms were developed and validated using HPLC and HPTLC The HPLC separation was achieved on a Phenomenex Luna C18 column (250 x 4.6 mm id, 5 microm) in the isocratic mode using 0.1% phosphoric acid- acetonitrile (38 + 62, v/v), pH 3.5 +/- 0.05, mobile phase at a flow rate of 1 ml/min The retention times were 6.42 and 2.86 min for atorvastatin calcium and ramipril, respectively Quantification was achieved with a photodiode

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array detector set at 210 nm over the

concentration range of 0.5-5 µg/ml for each, with

mean recoveries (at three concentration levels) of

100.06 +/- 0.49% and 99.95 +/- 0.63% RSD for

atorvastatin calcium and ramipril, respectively

The HPTLC separation was achieved on silica gel

60 F254 HPTLC plates using

methanol-benzene-glacial acetic acid (19.6 + 80.0 + 0.4, v/v/v) as the

mobile phase The Rf values were 0.40 and 0.20

respectively Quantification was achieved with

concentration range of 50-500 ng/spot for each,

with mean recoveries (at three concentration

levels) of 99.98 +/- 0.75% and 99.87 +/- 0.83%

RSD for atorvastatin calcium and ramipril,

respectively Both methods were validated

Harmonization guidelines and found to be simple,

specific, accurate, precise, and robust The mean

assay percentages for atorvastatin calcium and

ramipril were 99.90 and 99.55% for HPLC and

99.91 and 99.47% for HPTLC, respectively The

methods were successfully applied for the

determination of atorvastatin calcium and

ramipril in capsule dosage forms without any

interference from common excipients (Panchal

HJ, 2010)

Charged aerosol detector (CAD) detection

approach was applied in a new HPLC method for

the determination of three of the major statins

used in clinical treatment-simvastatin, lovastain

and atorvastatin The method was optimized and

the influence of individual parameters on CAD

response and sensitivity was carefully studied

Chromatography was performed on a Zorbax

Eclipse XDB C18 (4.6 mm x 75 mm, 3.5

microm), using acetonitrile and formic acid 0.1%

as mobile phase The detection was performed

using both charged aerosol detector (CAD) (20

pA range) and diode array detector-238

nm(DAD) simultaneously connected in series In

terms of linearity, precision and accuracy, the

method was validated using tablets containing

atorvastatin and simvastatin The CAD is

designated to be a non-linear detector in a wide

dynamic range, however, in this application and

in the tested concentration range its response was

found to be perfectly linear The limits of

quantitation (0.1 µg/ml) were found to be two times lower than those of UV detection (Patil

UP, 2010)

A simple, specific, accurate and precise high-performance thin-layer chromatographic method for analysis of Telmisartan and Atorvastatin calcium in fixed dose combination has been developed The method uses aluminium plates coated with silica gel 60 F254 as stationary phase and toluene: methanol (7: 3, v/v) as mobile phase Densitometric evaluation of the separated bands was performed at 280 nm The two drugs were satisfactorily resolved with RF values 0.50

± 0.01 and 0.29 ± 0.00 for Telmisartan and Atorvastatin calcium, respectively The respective calibration plots were found to be linear over the range 200–1000 and 200–700 ng/band for

respectively This method has been successfully validated and applied for the analysis of drugs in

pharmaceutical formulation (Novakova, 2009)

A number of analytical methods were reported for the estimation of atorvastatin and ramipril from their individual dosage forms or in combination with other drugs (Valiyare, 2004; Vachareau and Neirinck, 2000) Here successful

chromatographic method and spectroscopic methods were developed then validated for the analysis of combined dosage form of atorvastatin

atorvastatin is 247 nm and that of ramipril is 208

nm They intersect at 215 nm which is fixed as wavelength for reverse phase-high performance liquid chromatographic method (Joseph, 2008)

A simple, accurate and precise performance thin-layer chromatographic method has been developed for the estimation of Atorvastatin Calcium and Metoprolol Tartarate simultaneously from a capsule dosage form The method employed Silica gel 60F254 precoated plates as stationary phase and a mixture of Chloroform: Methanol: Glacial acetic acid (dil.)

Densitometric scanning was performed at 220 nm using Camag TLC scanner 3 The method was linear in the drug concentrations' range of 500 to

2500 ng/spot for Atorvastatin Calcium, also for

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Metoprolol Tartarate with correlation coefficient

of 0.984 for Atorvastatin Calcium and 0.995 for

Metoprolol Tartarate respectively The retention

factor for Atorvastatin Calcium was 0.45 ± 0.04

and for Metoprolol Tartarate was 0.25 ± 0.02

The method was validated as per ICH

(International Conference on Harmonization)

Guidelines, proving its utility in estimation of

Atorvastatin Calcium and Metoprolol Tartarate in

combined dosage form (Patole SM, 2011)

chromatographic method was developed and

validated for determination of atorvastatin in

pharmaceutical dosage forms, and for evaluation

of its stability in the solid phase Separation of

atorvastatin was successfully achieved on a C-18

column utilizing water acetonitrile at the

volumetric ratio of 48:52, adjusted to pH 2.0 with

80% ortho-phosphoric acid The detection

wavelength was 245 nm The method was

validated and the response was found to be linear

in the drug concentration range of 0.04 mg/mol -

0.4 mg/mol The mean values percentage relative

standard deviation+/- (RSD) of the slope and the

correlation coefficient were 8.192 +/- 0.260 and

0.999, respectively The RSD values for intra-

and interday precision were < 1.00% and 0.90%,

atorvastatin at 363 K in a relative humidity of

76.4% was observed to be autocatalytic first order

reaction The kinetic parameters were as follows:

k (where k represents the velocity constant; s (-1)

= (1.42 +/- 0.19) 10(-6); t (0.5) (where t (0.5)

represents the time needed for a 50% decay of

atorvastatin; days) = 32.82 +/- 0.9; t (0.1) (where

t (0.1) represents the time needed for a 10%

decay of atorvastatin; days) = 13.86 +/- 0.8

(Stains B, 2006)

A simple, precise and accurate

reversed-phase liquid chromatographic method has been

developed for the simultaneous estimation of

atorvastatin calcium and telmisartan in tablet

formulations The chromatographic separation

was achieved on (Waters symmetry C18, 250mm

x 4.6mm, 5μ) analytical column A mixture of

ammonium acetate (0.02M, pH 4.0 adjusted with

glacial acetic acid) and acetonitrile in ratio (40:60

v/v) at flow rate of 1.0ml/min and detector

wavelength 254 nm The retention time of atorvastatin calcium and telmisartan was found to

be 4.6 and 6.1 minutes respectively The validation of the proposed method was carried out for its specificity, linearity, accuracy, precision, limit of detection and quantification for both

developed method can be used for routine quality analysis of titled drugs in combination in tablet formulation (Suresh Kumar GV, 2010)

A simple, specific, accurate and stability indicating reversed phase high performance liquid chromatographic method was developed for the

calcium and amlodipine besylate in tablet dosage forms A Phenomenex Gemini C-18, 5 mm column having 250´4.6 mm i.d in isocratic mode, with mobile phase containing 0.02 M potassium

(30:10:60, v/v/v) adjusted to pH 4 using ortho phosphoric acid was used The flow rate was 1.0 ml/min and effluents were monitored at 240 nm The retention times of atorvastatin calcium and amlodipine besylate were 11.6 min and 4.5 min, respectively The calibration curves were linear in the concentration range of 0.08-20 µg/ml for atorvastatin calcium and 0.1-20 µg/ml for amlodipine besylate Atorvastatin calcium and

subjected to acid and alkali hydrolysis, chemical oxidation and dry heat degradation The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time values The proposed method was validated and successfully applied to the estimation of atorvastatin calcium and amlodipine besylate in combined tablet dosage forms (Shah D.A, 2008)

A reverse phase high performance liquid chromatographic method was developed for the simultaneous estimation of atorvastatin calcium and fenofibrate in tablet formulation The separation was achieved by Luna C18 column and methanol: acetate buffer pH 3.7 (82:18 v/v)

as mobile phase, at a flow rate of 1.5 ml/min Detection was carried out at 248 nm Retention time of atorvastatin calcium and fenofibrate was found to be 3.02+0.1 and 9.05+0.2 min,

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respectively The method has been validated for

linearity, accuracy and precision Linearity for

atorvastatin calcium and Fenofibrate were in the

range of 1-5 μg/ml and 16-80 μg/ml, respectively

The mean recoveries obtained for Atorvastatin

calcium and fenofibrate were 101.76% and

100.06%, respectively Developed method was

found to be accurate, precise, selective and rapid

for simultaneous estimation of atorvastatin

calcium and fenofibrate in tablets (Jain N, 2008)

An isocratic RP-HPLC method was

developed and validated for the quantitation of

Atorvastatincalcium and Amlodipine besylate in

combined tablet dosage forms Quantitation was

achieved using a reversed-phase Hypersil silica

BDS (250x4.6mm with 5+ particle size) column

at ambient temperature with mobile phase

consisting of 0.05M ammonium acetate buffer

(pH-4) and acetonitrilein the ratio (40 + 60, v/v)

The flow rate was 1.0 ml/min Measurements

were made at a wavelength of 240.0nm The

proposed method was validated for selectivity,

precision, linearity and accuracy The assay

method was found to be linear from

30.0-70.0µg/ml for Amlodipine besylate and

60.0-140.0 µg/ml for Atorvastatin calcium All

validation parameters were within the acceptable

range The developed method was successfully

applied to estimate the amount of Atorvastatin

calcium and Amlodipine besylate in combined

dosage forms (Mishra P, 2007)

A stability indicating UPLC method was

developed and validated for the simultaneous

determination of atorvastatin, fenofibrate and

their impurities in tablets The chromatographic

separation was performed on acquity UPLC BEH

C18 column (1.7 microm, 2.1 mmx100 mm)

using gradient elution of acetonitrile and

ammonium acetate buffer (pH 4.7; 0.01 M) at

flow rate of 0.5 ml/min UV detection was

performed at 247 nm Total run time was 3 min

within which main compounds and six other

known and major unknown impurities were

separated Stability indicating capability was

established by forced degradation experiments

and separation of known degradation products

The method was validated for accuracy,

repeatability, reproducibility and robustness

Linearity, loss of drying (LOD) and loss of quality (LOQ) was established (Kadav AA, 2008)

A simple, accurate, rapid and precise isocratic reversed-phase high-performance liquid chromatographic method has been developed and validated for simultaneous determination of aspirin, atorvastatin calcium and clopidogrel bisulphate in capsules The chromatographic separation was carried out on an Inertsil ODS analytical column (150×4.6 mm; 5 μm) with a mixture of acetonitrile: phosphate buffer pH 3.0

adjusted with o-phosphoric acid (50:50, v/v) as

mobile phase; at a flow rate of 1.2 ml/min UV detection was performed at 235 nm The retention times were 1.89, 6.6 and 19.8 min for aspirin, atorvastatin calcium and clopidogrel bisulphate, respectively Calibration plots were linear (r 2

>0.998) over the concentration range 5-30 μg/ml for atorvastatin calcium and 30-105 μg/ml for aspirin and clopidogrel bisulphate The method was validated for accuracy, precision, specificity, linearity, and sensitivity The proposed method was successfully used for quantitative analysis of capsules No interference from any component of pharmaceutical dosage form was observed Validation studies revealed that method is specific, rapid, reliable, and reproducible The high recovery and low relative standard deviation confirm the suitability of the method for routine determination of aspirin, atorvastatin calcium and clopidogrel bisulphate in bulk drug and capsule dosage form (Londhe SV, 2011)

A UV spectrophotometric method was developed for the estimation of atorvastatin calcium & fenofibrate in table dosage form by using simultaneous equation method The drug obeyed Beer’s law & showed good correlation near to 0.999 Absorption maxima of atorvastatin calcium & fenofibrate were found to be at 246 and 286nm respectively Beer’s law was obeyed

in concentration rang of 1-10 μg/ml for atorvastatin calcium & 2-20μg/ml for fenofibrate The method has been validated for linearity, accuracy & precision The recovery was more than 99% The developed method was found to be accurate, simple, precise, economical, and

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atorvastatin calcium &fenofibrate in tablet dosage

form &fenofibrate in tablet dosage form (Rupali

Hiravr, 2010)

A UV spectrophotometric method using

simultaneous equation was developed for the

simultaneous determination of Telmisartan and

Atorvastatin calcium in a binary mixture In the

proposed method, the signals were measured at

296.0 nm and 247.0 nm corresponding to

Atorvastatin Calcium in methanol respectively

Linearity range was observed in the concentration

range of 5-30 µg/ml for both the drugs

Concentration of each drug was obtained by using

the absorptivity values calculated for both drugs

at two wavelengths, 296.0 nm and 247.0 nm and

solving the simultaneous equation Developed

method was applied to laboratory mixture and its

pharmaceutical formulation The method was

validated statistically and recovery study was

performed to confirm the accuracy of the method

(Chaudhari KU, 2010)

Atorvastatin calcium(ATC) is the active

pharmaceutical ingredient (API) of the best

Twelve ATC crystal forms are known and several

pharmaceutical companies are developing or have

developed generic drug formulations based on

different ATC polymorphs The strong overlap of

the X-ray diffraction patterns (XRD) of the

polymorphs with the respective patterns of the

excipients, the presence of small API quantities in

the tablet and the similarity of the crystal phase

VIII XRD pattern used in the tablet examined in

this work to that of phases IV and IX made

identification difficult Quantitative determination

of Atorvastatin was attempted using Raman

spectroscopy (RS), IR spectroscopy and X-ray

powder diffraction It was found that RS

exhibited lower detection limit and a calibration

model was constructed Its application on

commercial ATC tablets with 40mg strength

yielded an error of 1.25% (Skoda D, 2008)

The aim of the proposed work was to

develop and validate a simple and sensitive assay

for the analysis of atorvastatin, ortho- and

hydroxy-ATC, ATC lactone, and ortho- and

Para-hydroxy-ATC lactone in human plasma using

spectrometry All six analytes and corresponding deuterium (d5)-labeled internal standards were extracted from 50 μL of human plasma by protein precipitation The chromatographic separation of analytes was achieved using a Zorbax-SB Phenyl column (2.1 mm×100 mm, 3.5 μm) The mobile phase consisted of a gradient mixture of 0.1% v/v glacial acetic acid in10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B) All analytes including ortho- and Para-hydroxy metabolites were baseline separated within 7.0 min using a flow rate of 0.35 ml/min Mass spectrometry detection was carried out in positive electro spray ionization mode, with

calibration curves for all analytes were linear (R2≥0.9975, n=3) over the concentration range of 0.05–100 ng/ml and with lower limit of quantitation of 0.05 ng/ml Mean extraction recoveries ranged between 88.6–111% Intra- and inter-run mean percent accuracy was between 85– 115% and percent imprecision was≤15% Stability studies revealed that ATV acid and lactone forms were stable in plasma during bench top (6 h on ice-water slurry), at the end of three successive freeze and thaw cycles and at −80 °C for 3 months The method was successfully applied in a clinical study to determine concentrations of ATV and its metabolites over12

h post-dose in patients receiving atorvastatin (Macwan JS, 2011)

A rapid, simple, sensitive and specific LC-MS/MS method has been developed and validated for the simultaneous estimation of atorvastatin, amlodipine, ramipril and benazepril using nevirapine as an internal standard The API-4000 LC-MS/MS was operated under the multiple-reaction monitoring mode using electro spray ionization Analytes and IS were extracted from plasma by simple liquid-liquid extraction technique using ethyl acetate The reconstituted samples were chromatographed on C 18 column

by pumping 0.1% formic acid-acetonitrile (15:85, v/v) at a flow rate of 1 ml/min A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 0.26-210 ng/ml for ATO; 0.05-20.5 ng/ml for amlodipine( AML);

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0.25-208 ng/ml for RAM and 0.74-607 ng/ml

mean correlation coefficient of ≥0.99 for each

analyte The intra-day and inter-day precision and

accuracy results were well within the acceptable

limits A run time of 2.5 min for each sample

made it possible to analyze more than 400 human

plasma samples per day The developed assay

pharmacokinetic study in human male volunteers

(Pilli NR, 2011)

CONCLUSION

This review is targeted at out lining the

various analytical methods and other related

aspects of atorvastatin it is however pertinent to

state that newer analytical methods are being

developed with respect to advancing technology

and this may necessitate a future review

REFERENCES

Bisulphate in Capsules, Indian J Pharm Sci,

73(1), 2011, 23-9

Chaudhari KU P.D Gaikwad, V.H Bankar and

S.P Pawar, Development and validation of

uv-spectrophotometric method for simultaneous

estimation of telmisartan and atorvastatin calcium

Inj.Jour.PharmTech, 2(1), 2010, 255-264

Jain N, Raghuwanshi R, Jain D

Development and Validation of

estimation of Atorvastatin Calcium and

Fenofibrate in tablet dosage forms,

Indian J Pharm Sci, 70, 2008, 263-5

Joseph L, George M, Rao B VR, Simultaneous

estimation of atorvastatin and ramipril by

RP-HPLC and spectroscopy, Pak J Pharm Sci, 21(3),

2008, 282-4

Kadav AA, Vora DN Stability indicating UPLC

method for simultaneous determination of

atorvastatin, fenofibrate and their degradation

products in tablets, J Pharm Biomed Anal, 48(1),

2008, 120-6

Londhe SV, Deshmukh RS, Mulgund SV, Jain

KS, Development and Validation of a

Determination of Aspirin, Atorvastatin Calcium and Clopidogrel

Macwan JS, Ionita IA, Dostalek M, Akhlaghi F Development and validation of a sensitive, simple, and rapid method for simultaneous quantitation of atorvastatin and its acid and lactone metabolites by liquid chromatography- tandem mass spectrometry (LC-MS/MS), Anal Bioanal Chem, 400(2), 2011, 423-33

Mishra P Alkagupta and K Shah, Simultaneous estimation of atorvastatin calcium and amlodipine

pharmaceutical sciences, 69(6), 2007, 831-833 Nováková L, Lopéz SA, Solichová D, Satínský

D, Kulichová B, Horna A, Solich P, Comparison

of UV and charged aerosol detection approach in pharmaceutical analysis of statins, Talanta, 78(3),

2009, 834-9

determination of atorvastatin calcium and ramipril in capsule dosage forms by high- performance liquid chromatography and high- performance thin layer chromatography, J AOAC Int, 93(5), 2010, 1450-7

Patil UP S V Gandhi M R Sengar, V S Rajmane A, Validated densitometric method for analysis of telmisartan and atorvastatin calcium in fixed dose combination, J Chil Chem Soc., 55,2010,94-96

Patole S, Khodke A, Potale L, Damle M, A validated densitometric method for analysis of atorvastatin calcium and Metoprolol Tartarate as bulk drugs and in combined capsule dosage forms, J Young Pharm, 3(1), 2011, 55-9

Pilli NR, Inamadugu JK, Mullangi R, Karra VK, Vaidya JR, Rao JV, Simultaneous determination

benazepril in human plasma by LC-MS/MS and its application to a human pharmacokinetic study, Biomed Chromatogr, 25(4), 2011, 439-49

Rupali Hirave, Ravindra Bendagude, Manish Kondawar, RP-HPLC method for simultaneous

Fenofibrate in tablet dosage forms, Journal of Pharmacy Research, 3(10), 2010, 2400-2401

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Shah DA, K.K Bhatt, R.S Mehta, S.L Baldania

and T.R Gandhi, Stability indicating RP HPLC

estimation of atorvastatin calcium and amlodipine

besylate in pharmaceutical formulations Indian

journal of pharmaceutical sciences, 70(6), 2008,

754-760

Shah Y, Iqbal Z, Ahmad L, Khan A, Khan MI,

Nazir S, Nasir F, Simultaneous determination of

rosuvastatin and atorvastatin in human serum

development, validation and optimization of

various experimental parameters, J Chromatogr B

Analyt Technol Biomed Life Sci, 879(9-10),

2011, 557-63

Skoda D, Kontoyannis CG, Identification and

quantitative determination of atorvastatin calcium

spectroscopy, Talanta, 74(4), 2008, 1066-70

Stanisz B, Kania L, Validation of HPLC method

for determination of atorvastatin in tablets and for

monitoring stability in solid phase, Acta Pol

Pharm, 63(6), 2006, 471-6

Chandrashekar SM, Development and validation

of reversed-phase HPLC method for simultaneous

Telmisartan in tablet dosage form, International

Journal of Pharma Tech Research, 2(1), 2010,

463-470

pharmacology, sixth edition, Jaypee publication,

2008, 614-615

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A review on the use of Bleomycin-Cisplatin-Vinblastine combinations in therapy of

testicular cancer

Praveen D*, Ranadheer Chowdary P

School of Pharmaceutical Sciences, Vels University

*Corresponding author: Email.id: praveennandan.1993@gmail.com, +919940510419

ABSTRACT

Combination chemotherapy with vinblastine plus bleomycin in disseminated testicular cancer was pioneered by Samuels and represented a major therapeutic advance (Samuels ML, 1976) Another milestone in the chemotherapy of testicular cancer was the discovery of the activity of cis-diammine

dichloro platinum (CDDP) in germinal neoplasm Complete response rates between 60-80% have been

reported for patients with advanced disease and the vast majority of these patients are cured of their

disease as the relapse rates are less than 15% (Robert F, 1983) This discovery has led to a new and better

chemotherapeutic combination in the treatment of testicular cancer For the first time, in a random prospective cooperative group clinical trial evaluating the effectiveness of three Chemotherapy combinations in the treatment of a solid tumor, in which the dose of one drug is the only treatment variable, a clear-cut relationship has been shown for dose of therapy, not only with response and survival, but with an increased potential for cure as well Their plasma half-lives, pharmacokinetic parameters, toxicities, adverse drug effects were also evaluated in various studies This combination have been proved better than Bleomycin-cisplatin-etoposide as the former combination masks the toxic effects of bleomycin such as mucositis etc Thus we conclude that this combination is a safer one with a good therapeutic efficacy and reduced toxicity

Key Words: Combination chemotherapy, bleomycin, cisplatin, testicular cancer

INTRODUCTION

Testicular cancer is a cancer that develops in

the testicles, a part of the male reproductive system

Testicular cancer has one of the highest cure rates of

all cancers: a five-year survival rate in excess of 90 %

overall, and almost 100 % if it has not spread

(metastasized) Even for the relatively few cases in

which malignant cancer has spread widely,

modern chemotherapy offers a cure rate of at least

80% One of the first signs of testicular cancer is often

a lump or swelling in the testes The three basic types

of treatment are surgery, radiation therapy,

and chemotherapy Surgery is performed

by urologists; radiation therapy is administered

by radiation oncologists; and chemotherapy is the

work of medical oncologists In most patients with

testicular cancer, the disease is cured readily with

minimal long-term morbidity While treatment

success depends on the stage, the average survival rate

after five years is around 95%, and stage 1 cancers

cases (if monitored properly) have essentially a 100%

survival rate (which is why prompt action, when

testicular cancer is a possibility, is extremely

important)

Testicular cancer: Testicular cancer is a disease in

which cells become malignant (cancerous) in one or

both testicles The testicles (also called testes or

gonads) are a pair of male sex glands They produce

and store sperm and are the main source of

testosterone (male hormones) in men These hormones control the development of the reproductive organs and other male physical characteristics The testicles are located under the penis in a sac-like pouch called the scrotum Based on the characteristics of the cells

in the tumor, testicular cancers are classified as seminomas or nonseminomas Other types of cancer that arise in the testicles are rare and are not described here Seminomas may be one of three types: classic, anaplastic, or spermatocytic Types of nonseminomas include choriocarcinoma, embryonal carcinoma, teratoma, and yolk sac tumors Testicular tumors may contain both seminoma and nonseminoma cells

Epidemiology: Testicular cancer accounts for only

1% of all cancers in men in the United States About 8,000 men are diagnosed with testicular cancer, and about 390 men die of this disease each year Testicular cancer occurs most often in men between the ages of

20 and 39, and is the most common form of cancer in men between the ages of 15 and 34 It is most common in white men, especially those of Scandinavian descent The testicular cancer rate has more than doubled among white men in the past 40 years, but has only recently begun to increase among black men The reason for the racial differences in incidence is not known

Risk Factors:

Undescended testicle (cryptorchidism): Normally,

the testicles descend from inside the abdomen into the

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

scrotum before birth The risk of testicular cancer is

increased in males with a testicle that does not move

down into the scrotum This risk does not change even

after surgery to move the testicle into the scrotum

The increased risk applies to both testicles

abnormalities of the testicles, penis, or kidneys, as

well as those with inguinal hernia (hernia in the groin

area, where the thigh meets the abdomen), may be at

increased risk

History of testicular cancer: Men who have had

testicular cancer are at increased risk of developing

cancer in the other testicle

Family history of testicular cancer: The risk for

testicular cancer is greater in men whose brother or

father has had the disease

Symptoms: Common symptoms are painless lump or

swelling in a testicle, pain or discomfort in a testicle

or in the scrotum, any enlargement of a testicle or

change in the way it feels, a feeling of heaviness in the

scrotum, a dull ache in the lower abdomen, back, or

groin, a sudden collection of fluid in the scrotum

Diagnosis: Blood tests that measure the levels of

tumour markers Tumour markers are substances often

found in higher-than-normal amounts when cancer is

present Tumour markers such as alpha-fetoprotein

(AFP), Beta-human chorionic gonadotropin (HCG),

and lactate dehydrogenase (LDH) may suggest the

presence of a testicular tumor, even if it is too small to

be detected by physical exams or imaging tests

Ultrasound: A test in which high-frequency sound

waves are bounced off internal organs and tissues

Their echoes produce a picture called a sonogram

Ultrasound of the scrotum can show the presence and

size of a mass in the testicle It is also helpful in ruling

out other conditions, such as swelling due to infection

or a collection of fluid unrelated to cancer

Biopsy: Biopsy (microscopic examination of

testicular tissue by a pathologist) to determine whether

cancer is present In nearly all cases of suspected

cancer, the entire affected testicle is removed through

an incision in the groin This procedure is called

radical inguinal orchiectomy

Treatment: Chemotherapy is the use of anticancer

drugs to kill cancer cells When chemotherapy is

given to testicular cancer patients, it is usually given

as adjuvant therapy (after surgery) to destroy

cancerous cells that may remain in the body

Chemotherapy may also be the initial treatment if the

cancer is advanced; that is, if it has spread outside the

testicle at the time of the diagnosis Most anticancer drugs are given by injection into a vein Chemotherapy is a systemic therapy, meaning drugs travel through the bloodstream and affect normal as well as cancerous cells throughout the body The side effects depend largely on the specific drugs and the doses Common side effects include nausea, hair loss, fatigue, diarrhea, vomiting, fever, chills, coughing/shortness of breath, mouth sores, or skin rash Other side effects include dizziness, numbness, loss of reflexes, or difficulty hearing Some anticancer drugs also interfere with sperm production Although the reduction in sperm count is permanent for some patients, many others recover their fertility Some men with advanced or recurrent testicular cancer may undergo treatment with very high doses of chemotherapy These high doses of chemotherapy kill cancer cells, but they also destroy the bone marrow, which makes and stores blood cells Such treatment can be given only if patients undergo a bone marrow transplant In a transplant, bone marrow stem cells are removed from the patient before chemotherapy is administered These cells are frozen temporarily and then thawed and returned to the patient through a needle (like a blood transfusion) after the high-dose chemotherapy has been administered

glycopeptide antibiotic with a unique mechanism of antitumor activity The drug binds to guanosine-cytosine-rich portions of DNA via association of the

"S" tripeptide and by partial intercalation of the bithiazole rings A group of five nitrogen atoms arranged in a square-pyramidal conformation binds divalent metals including iron, the active ligand, and copper, an inactive ligand Molecular oxygen, bound

by the iron, can produce highly reactive free radicals and Fe(III) The free radicals produce DNA single-strand breaks at 3'-4' bonds in deoxyribose This yields free base propenals, especially of thymine: cytotoxicity is cell-cycle-phase specific for G2 phase

In humans, bleomycin is rapidly eliminated primarily

by renal excretion This accounts for approximately half of a dose In patients with renal compromise or extensive prior cisplatin therapy, the drug half-life can extend from 2 to 4 hours up to 21 hours Thus, dose adjustments are needed when creatinine clearance is less than or equal to 3N mL/min (Dorr RT, 1992) Platinum complexes are clinically used as adjuvant therapy of cancers aiming to induce tumor cell death Depending on cell type and concentration, cisplatin induces cytotoxicity, e.g., by interference with transcription and/or DNA replication mechanisms Additionally, cisplatin damages tumors via induction

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of apoptosis, mediated by the activation of various

signal transduction pathways, including calcium

signalling, death receptor signalling, and the

activation of mitochondrial pathways Unfortunately,

neither cytotoxicity nor apoptosis are exclusively

induced in cancer cells, thus, cisplatin might also lead

to diverse side-effects such as neuro- and/or

renal-toxicity or bone marrow-suppression Moreover, the

binding of cisplatin to proteins and enzymes may

modulate its biochemical mechanism of action (

Ana-Maria Florea 2011)

There are two high affinity

vinblastine-binding sites per mole of embryonic chick brain

tubulin (KA = 3-5 X 10(5) l./mol) Binding to these

sites was rapid, and relatively independent of

temperature between 37 and 0degreeC Vincristin

sulfate and desacetylvinblastine sulfate, two other

active vinca alkaloid derivatives, competitively

inhibited the binding of vinblastine The inhibition

constant for vincristine was 1.7 X 10(-5) M; and for

desacetylvinblastine, 2 X 10(-5) M The vinblastine

binding activity of tubulin decayed upon aging, but

this property was not studied in detail Vinblastine did

not depolymerize stable sea urchin sperm tail outer

doublet microtubules, nor did it bind to these

microtubules (Wilson L , 1975) Since 1974, with the

introduction of platinum-based combination

chemotherapy, the complete response rate of patients

with metastatic germ cell neoplasms of the testis has

improved from 10%-30% to 50%-75% Furthermore,

a significant proportion of patients entering complete

remission are relapse free after 2 years, and may be

considered cured of their disease Important

prognostic factors predicting for complete response

include initial performance status, tumor histology,

and tumor burden at presentation

Numerous in vivo studies of antitumor agents

exposed to a variety of transplantable experimental

tumors have indicated a steep response and

dose-toxicity These observations apply to both cell cycle

specific as well as non-cell-specific agents In tumors

composed of a heterogenous cell population with both

drug-sensitive and drug-resistant subpopulations the

dose response curve is modified, depending on the

ratio of resistant to sensitive fraction While there are

few prospective randomized clinical trials addressing

dose as a treatment variable, there appears to be a

confirmation of the fore mentioned concepts in

Hodgkin's and certain non-Hodgkin's lymphoma,

acute lymphocytic leukaemia, and oat cell carcinoma

of the lung, all regarded as chemotherapy-"sensitive"

turners (Michael K, 1984) Previous clinical trials in

testicular cancer incorporating cisplatin (DDP) have

employed either “high-dose’’ (100- 120 mg/m2) or

“low dose” (75 mg/m2) schedules without a clear

definition of dose response testicular cancer is regarded as a “sensitive” tumor, the Southwest Oncology Group (SWOG) initiated, in 1978, a prospective randomized Phase III comparative trial in which the primary objective was to determine whether

a high dose of DDP (cisplatin) when combined with vinblastine and bleomycin resulted in a superior response and survival experience than a lower dose of DDP with the same combination of vinblastine and bleomycin (Lawrence H Einhorn, 1980)

CONCLUSION

Platinum, vinblastine, bleomycin combination chemotherapy consistently produces 70% complete

remissions, and a further 10% of patients will be

rendered disease free following surgical excision of residual disease For the first time, in a random prospective cooperative group clinical trial evaluating the effectiveness of two chemotherapy combinations

in the treatment of a solid tumour, in which the dose

of one drug is the only treatment variable, a clear-cut relationship has been shown for dose of therapy, not only with response and survival, but with an increased potential for cure as well Historically, testicular cancer was one of the first malignancies of non-hematologic origin in which long-term survival, and cure, could be achieved with combination chemotherapy (Lawrence H Einhorn, 1980; Robert F Ozols, 1983; Michael K Samson, 1984)

REFERENCES

Ana-Maria Florea and Dietrich Büsselberg, Cisplatin

as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance and Induced Side Effects,

Cancers, 2011, 3, 1351-1371

Dorr RT, Bleomycin pharmacology: mechanism of action and resistance, and clinical pharmacokinetics Semin Oncology, 1992, 19 (2 Suppl 5), 3-8

Lawrence H Einhorn, and Stephen D, Chemotherapy

of disseminated testicular cancer -a random prospective study, Cancer, 46, 1980, 1339- 1344 Michael K Samson, Dose-response and dose-survival advantage for high versus low-dose cisplatin combined with vinblastine and bleomycin in disseminated testicular cancer, Cancer, 53, 1029-1035,

1984

Robert F Ozols, Treatment of poor prognosis nonseminomatous testicular cancer with a "high-dose" platinum combination chemotherapy regimen, Cancer,

51, 1983, 1803-1807

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

Samuels ML, Lanzotti VJ, Holoye PY, Boyle LE,

Smith TL, Johnson DE, Combination chemotherapy in

germinal cell tumors, Cancer Treat Rev, 3, 1976,

185-204

Wilson L, Creswell KM, Chin D, The mechanism of

action of vinblastine, Biochemistry, 30;14(26), 1975,

5586-92

Wozniak AJ, A randomized trial of cisplatin,

vinblastine, and bleomycin versus vinblastine,

cisplatin, and etoposide in the treatment of advanced

germ cell tumors of the testis: a Southwest Oncology

Group study, Journal of clinical oncology, 1991, 9(1),

70-76

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Method development and validation for the simultaneous estimation of Ofloxacin and Tinidazole in bulk and pharmaceutical dosage form by reverse phase HPLC

method

Y.Bhargav*, K Haritha Pavani, S Amareswari

Nimra College of Pharmacy, Vijayawada, A.P, India

*Corresponding author: Email: bhargavy8@rediffmail.com ; Phone no: 8466941272

ABSTRACT

A new, simple, accurate, rapid, precise RP-HPLC method was developed for the simultaneous estimation of ofloxacin and tinidazole in bulk and pharmaceutical dosage form A good chromatographic separation was achieved with Intersil ODS C18 (250×4.6mm.5µ) column and ammonium acetate buffer

pH 4.0, acetonitrile, and tetrahydrofuran 60:30:10 was used as mobile phase at a flow rate of 1.0ml/min and the detection was carried out at a wavelength of 304 nm The retention times was found to be 2.350 min for ofloxacin and 3.613 min for tinidazole The linearity of the method was in the concentration range

of 15-35µg/mL for ofloxacin and 45-105µg/mL for tinidazole The developed method was validated for system suitability, specificity, precision, recovery and linearity according to ICH guidelines The method was successfully applied for routine analysis for the determination of oflaxacin and tinidazole in bulk and dosage forms

Key Words: Oflaxacin, Tinidazole, RP-HPLC, Validation

INTRODUCTION

Ofloxacin was chemically (RS) 9-fluoro-2,

3dihydro-3-methyl-10-(4methl-1-piperazinyl)-7-

oxo-7H-prido [1, 2, 3-de]-1, 4benzoxazine-6-carboxylic

acid Ofloxacin belongs to class of drugs called

quinolone antibiotics Ofloxacin is a broad spectum

antibiotic that is active against both Gram-positive and

Gram-negitive It inhibition of topoisomrase enzymes,

which inhibits relaxation of supercoild DNA and

promotes breakage of double stranded DNA It is

used to treat a variety of bacterial infections

Tinidazole was chemically 1-[ethanesulfonyl)

ethyl]-2-methyl-5-nitro-1H-imidazole Tinidazole is

Antiprotozoal, Antibacterial agent The nitro-group of

tinidazole is reduced by cell extracts of trichomonas

The free nitro-radical generated as a result of this

reduction may be responsible for antiprotozoal

activiiaty Chemically reduced Tinidazole was shown

to release nitrites and cause damage to purified

bacterial DNA in vitro

Literature survey revealed that very few

methods have been reported for the analysis of

Ofloxacin and Tinidazole combinational dosage forms

which include UV spectroscopy, Reverse Phase High

performance Liquid Chromatography, HPTLC

methods The present study illustrate development and

validation of simple, economical, selective, accurate,

precise RP-HPLC method for the determination of

Ofloxacin and Tinidazole in bulk and Pharmaceutical

dosage forms as per ICH guidelines

Shimadzu HPLC equipped with spinchrome

software, Elico UV-Spectrophotometer, Intersil ODS

C18 column (250×4.6mm.5µ), polmon pH Meter, Weighing Balance Ax200 Shimadzu, LAB INDIA Sonicator, rheodyne injector

Preparation of mobile phase: 1.925g of Ammonium

acetate was accurately weighed and dissolved in 500ml of distilled water The pH of the buffer was adjusted to 4.0 with ortho phosphoric acid A suitable quantity of degassed mixture of pH4.0 Ammonium acetate buffer, Acetonitrile, Tetra hydro furan in the ratio of 60:30:10 was prepared and filtered through 0.45µ filter under vacuum filtration

Preparation of standard stock solution: Accurately

weighed 10mg of Ofloxacin and 10mg of Tinidazole into a 10ml volumetric flasks and 10ml of diluents

pH4.0:ACN:THF(60:30:10) was added and sonicated for 5min and make up to the volume with diluent From the stock 1ml of the solution was taken and diluted to 10ml to get a concentration of 100µg/ml solution from the above solution 2ml of Ofloxacin and

6ml of Tinidazole and mixed and injected

Preparation of sample: Twenty tablets were weighed

and powdered the powder equivalent to 25mg of Ofloxacin and 75mg of Tinidazole were weighed and taken into a 200ml volumetric flask To this 50ml of diluents was added and sonicated for 15min to dissolve the drugs then made up the volume to required volume with the diluents From this solution 5ml was taken into a 50ml flask and made up to final volume with diluents to get a concentration of 100ppm filtered through 0.45µ filter under vacuum filtration From this stock solution further dilutions were made for the validation of the method developed

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Bhargav et.al Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online)

experimental conditions were carried out to achieve

the best chromatographic conditions for the

simultaneous determination of the drug substances

Several column types and lengths were tried for better

elution and for chromatographic parameters A good

chromatographic separation was achieved with Intersil

ODS C18 (250×4.6mm.5µ) column and ammonium

acetate buffer pH 4.0, acetonitrile and tetrahydrofuran

60:30:10 was used as mobile phase at a flow rate of

1.0ml/min and the detection was carried out at a

wavelength of 304nm The retention times was found

to be 2.350min for oflaxacin and 3.613min for

tinidazole

System suitability: From the system suitability

studies it was observed that theoretical plates were

found to be more than 2125 for Ofloxacin and 3873

for Tinidazole USP tailing factor was found to be

1.76 for Ofloxacin and 1.72 for Tinidazole All the

parameters were within the limit The results of

system suitability studies were given in Table.1 and

the standard Chromatograms can be were shown in

the Figure.1

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 The results were tabulated in the Table.2 The

chromatograms were shown in the Figures 2,3,4 for

standard, sample, blank and placebo

Linearity: From the Linearity data it was observed

that the method was showing linearity in the

concentration range of 15-35μg/ml for Ofloxacin and

45-105μg/ml for Tinidazole Correlation coefficient

was found to be 0.999 and 0.998 for both the

compounds The linearity data was tabulated in

Table.3 The Chromatograms for the linearity data

were shown in the fig no: and the linearity curve was

plotted and given in the Figures 5,6

Accuracy: The recoveries of pure drug from the

analyzed solution of formulation were 100.81 % for Ofloxacin and 99.15 % for Tinidazole, which shows that the method was accurate The results were

tabulated in the Table.4

The chromatograms were shown in the Figure.7

Precision: The %RSD for the sample chromatograms

of method precision were found to be 0.52 & 0.73 for Ofloxacin and 0.41 &0.35 for Tinidazole Hence it passes method precision The results were tabulated in the Table.5 The chromatograms were shown in the

Figure.8

Assay: The %assay of the Ofloxacin was found to be

99.87% and for Tinidazole 99.23% The results were tabulated in the Table.6 The chromatograms were

shown in the Figures 9,10

Ruggedness: Comparison of both the results obtained

for two different Analysts shows that the method was rugged for Analyst-Analyst variability The system suitability parameters of Ruggedness were found to be within the limits and were tabulated in Table.7 The Chromatograms for ruggedness were shown in Figures 11,12

Robustness: All the system suitability parameters are

within limits for variation in flow rate (±0.2 ml) and for variation in the wavelength (±2nm)

Forced degradation studies: A forced degradation

study is an important step in drug development process to observe the drug products stability An attempt has been made to stress the drug products to acid hydrolysis by using 0.1N HCl, base hydrolysis by using 0.1N NaoH, oxidative degradation by using 1% H2O2, thermal treatment heated at 800c, photolytic degradation for 4hrs to evaluate the stability of the propose method to separate the drugs and its degradation products The results were tabulated in

the Table: 8 The chromatograms were shown in the

Figures 13,14,15,16,17

Table.1.System suitability data for Ofloxacin and Tinidazole

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Table.2 Specificity Data for Ofloxacin and Tinidazole

Standard

Retention time

Plates

Retention time

Table.4 Accuracy Data For Ofloxacin and Tinidazole

added (mcg)

Amount found (mcg)

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Bhargav et.al Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online)

Table.7.Ruggedness data of Ofloxacine and Tinidazole

Area Retention time Area Retention time

Analyst 1 Analyst 2

Table No: 8 Forced degradation data for Ofloxacin and Tinidazole

(hrs)

Time (hrs)

Table.9 Summary of validated parameters of Ofloxacin and Tinidazole

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