Master of Pharmacy Dissertation Protocol

“DEVELOPMENT OF STABILITY INDICATING HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF LEVOCETIRIZINE DIHYDROCHLORIDE AND AMBROXOL HYDROCHLORIDE”

MASTER OF PHARMACY DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BANGALORE

BY

Ms. VENKATESWARI PARIGAPATI

Under The Guidance of

Asst. Prof. SUDHA MALLAPUR

P.G. DEPARTMENT OF PHARMACEUTICAL ANALYSIS

EAST WEST COLLEGE OF PHARMACY, BANGALORE – 560091

2010 – 2012

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE –ІІ

REGISTRATION OF SUBJECT FOR DISSERTATION

1.0 / NAME AND ADDRESS OF
THE CANDIDATE / VENKATESWARI PARIGAPATI
DEPARTMENT OF PHARMACEUTICAL
ANALYSIS
EAST WEST COLLEGE OF PHARMACY
#63,1 PHASE,BEL LAYOUT,BHARATHNAGAR,
VISHWANEEDAM EAST PO.OFF.MAGADI ROAD,
BANGALORE-560091
KARNATAKA
2.0 / NAME OF THE INSTITUTION / EAST WEST COLLEGE OF PHARMACY
MAGADI ROAD,
BANGALORE-560091
KARNATAKA
3.0 / COURSE OF STUDY & SUBJECT / MASTER OF PHARMACY
(PHARMACEUTICAL
ANALYSIS)
4.0 / DATE OF ADMISSION / 8th NOVEMBER 2010
5.0 / TITLE OF THE TOPIC:
“DEVELOPMENT OF STABILITY INDICATING HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF LEVOCETIRIZINE DIHYDROCHLORIDE AND AMBROXOL HYDROCHLORIDE”
6.0 / BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED FOR STUDY:
Stability is defined as the capacity of a drug substance or drug product to remain within established specifications to maintain its identity, strength, quality, and purity throughout the re-test or expiration dating periods. The purpose behind stability testing of a drug is that, to monitor quality of a drug product which may vary with time because of the influence of many of the environmental conditions such as temperature, light, humidity etc. The results are applied in developing manufacturing processes and selecting proper packaging, storage conditions, product's shelf life and expiry dates.
Stress testing of the drug can help to identify the degradation products which can help to establish degradation pathways and the intrinsic stability of the molecule and validate stability indicating power for analytical procedures used.
Validation of a method indicates to establish documented evidence that the system is doing what is purpose to do. Validation is necessary when a method or a procedure is going to be used by a manufacturing company or to be published in any Pharmacopoeias. The validated assay methods will be more accurate, precise and reproducible.
Basic criteria for new method development of stability indicating assay method:
·  The drug or drug combination may not be official in any pharmacopoeias.
·  A proper stability indicating analytical procedure for the drug may not be available in the literature due to patent regulations.
·  The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
·  Stability indicating assay method provides the support to track the quality of the
product from time to time.
Methods for analyzing drugs in dosage forms can be developed, provided one has knowledge about the nature of the sample, its molecular weight, polarity, ionic character and the solubility parameter. Method development involves considerable trial and error procedures. The most difficult problem usually is where to start, what type of column is worth trying with what kind of mobile phase.
The following is a suggested method development scheme for a typical HPLC
related substance method.
1. To define the goals for method development (e.g., what is the intended use of the method), and to understand the chemistry of the analytes and the drug product.
2. To develop preliminary HPLC conditions to achieve minimally acceptable separations. These HPLC conditions will be used for all subsequent method development experiments.
3. To develop a suitable sample preparation scheme for the drug product.
4.To determine the appropriate standardization method and the use of relative response factors in calculations.
5.To identify the “weaknesses” of the method and optimize the method through experimental design. Understand the method performance with different conditions, different instrument set ups and different samples.
6. To complete method validation according to ICH guidelines as mentioned in Q2 (R1).
Levocetirizine dihydrochloride and Ambroxol hydrochloride combination is available as marketed formulation and prescribed by physician for the treatment of allergy.
A few of the brands available in the market are LAZINE PLUS (HETERO HC), BOROX-L (BIOMAX).
7.0
8.0 / DRUG PROFILE:
Ambroxol hydrochloride Levocetirizine dihydrochloride

PARAMETERS / LEVOCETIRIZINE1 DIHYDROCHLORIDE / AMBROXOL2 HYDROCHLORIDE
Description / A white powder / White or yellowish crystalline powder
IUPAC Name / (2-(4-[(R)-(4-Chlorophenyl) (phenyl) methyl] piperazin-1-yl) ethoxy) aceticacid dihydrochloride. / Trans-4-[(2-Amino-3,5-dibromobenzyl)amino]cyclohexanol hydrochloride.
Molecular formula / C21H27Cl3N2O3 / C13H18Br2N2O, HCl
Molecular weight / 461.81 / 414.6
Melting point / 215-2200C / 233-234.50C
Solubility / Soluble in water and methanol. / soluble in methanol
Category / H1-histamine receptor antagonist / Expectorant
Route / Oral / Oral
Storage / Store at 20-25°C; excursions permitted to 15-30°C. / Store at a temperature not exceeding 30 degrees Celsius
Levocetirizine dihydrochloride:
Pharmacodynamics
Levocetirizine, the (R) enantiomer of cetirizine, is a potent and selective antagonist of peripheral H1-receptors. Binding studies revealed that levocetirizine has high affinity for human H1 -receptors.
Pharmacokinetics:
Absorption:
The extent of absorption is dose independent and is not altered by food, but the peak concentration is reduced and delayed.
Distribution:
The distribution of levocetirizine is restrictive, as the volume of distribution is 0.4 l/kg.
Metabolism:
Metabolism is less than 14% and by aromatic oxidation, N- and O-dealkylation, and taurine conjugation.
Elimination:
Excreted both by glomerular filtration and active tubular transport.
Ambroxol hydrochloride:
Pharmacodynamics
Ambroxol is an active N-desmethyl metabolite of the mucolytic, bromhexine. It found to play a major role in the lung host defence mechanism, thereby further protecting against lung inflammation and infection.
Pharmacokinetics:
Absorption :
Ambroxol is rapidly absorbed (70-80%) after oral administration.
Distribution:
The distribution half-life of Ambroxol is around 1.3 hours.
Metabolism:
Metabolite is dibromoanthranilic acid (activity unspecified).
Elimination:
Excretion is via the kidneys.
6.2  REVIEW OF LITERATURE :
A literature survey was carried out for the stability indicating assay methods for
the combination of levocetirizine dihydrochloride and ambroxol hydrochloride. It was found that a very few methods have been reported for this combination. The collection of references have been reported below:
1.  Prashant Shende, Virag Shah, Dhananjay Ghodke, Rohit Shah, Shital Kumar Patil, Dhanya Kumar Chougule3, modified and validated a selective simple spectrophotometric method for the estimation of Levocetirizine dihydrochloride in bulk and pharmaceutical formulation using JASCO UV-Visible spectrophotometer model V-550. It showed the λmax at 231nm in phosphate buffer solution pH 7.0. The linearity was observed between 0-36µg/ml. LOQ and LOD were found to be 0.4457 and 0.1470.
2.  Raghad Hommoss, Hind Elzein, Samer Haidar4, have validated a chiral HPLC method and applied for determination of configurational stability of levocetirizine dihydrochloride in tablets using HPLC LA Chrom ELITE, VWR Hitachi, with a 250×4.6mm, 10µm chiral column and programmable detector. Mobile phase consisting a mixture of 0.5mol/L NaClO4 buffer and acetonitrile (60:40), pH adjusted at 2. Flow rate was kept at 0.4ml/min and detection was monitered at 230nm.
3.  Lakshmana Prabhu S, Shirwaikar AA, Shirwaikar Annie, Dinesh Kumar C, Aravind Kumar G5, have developed a spectrophotometric method for simultaneous estimation of ambroxol hydrochloride and levocetirizine dihydrochloride using SHIMADZU UV-Vis spectrophotometer, model-1601. λmax of ambroxol hydrochloride and levocetirizine dihydrochloride were 242nm and 231nm respectively. Beer’s law obeyed in the concentration range of 10-50µg/ml and 8-24µg/ml for ambroxol and levocetirizine respectively. The percentage recovery studies were found to be in the ranges of 99.13 to 99.52% and 98.88 to 99.42% for ambroxol and levocetirizine respectively
4.  Senthil Raja M, Shan SH, Perumal P, and Moorthy MTS6, developed and validated a simple reverse phase liquid chromatography for simultaneous estimation of azithromycin and ambroxol hydrochloride in combined dosage form using HPLC SHIMADZU with PDA detector. Mobile phase consisting of acetonitrile and mono basic potassium phosphate buffer in the ratio of 65:35v/v, pH was adjusted to 8.5 using dilute potassium hydroxide. The column used was C-18 phenomenex Gemini with flow rate of 2ml/min and λmax was found to be 220nm. The described method was linear over a concentration range of 96-145µg/ml and 80-125µg/ml for azithromycin and ambroxol respectively. The retention times of ambroxol and azithromycin were found to be 3.7 and 6.1min respectively. The LOQ and LOD for azithromycin and ambroxol HCl were found to be 96.7µg/ml and 8.35µg/ml and 31.91 µg/ml and 2.75 µg/ml respectively.
5.  Mukesh Maithani, Richa Raturi, Vertika Gautam, Dharmendra Kumar, Anand Gaurav and Ranjith Singh7, developed reverse phase high performance liquid chromatographic method for simultaneous estimation of ambroxol hydrochloride and cetirizine hydrochloride in tablet dosage forms using HPLC Perkin-Elmer series 200 instrument with a princeton C-8 (4.6×250mm, 5µm) column and mobile phase containing methanol, potassium dihydrogen phosohate buffer(80:20) v/v, pH 3.5±0.02, adjusted with orthophosphoric acid. The flow rate was 1ml/min with UV-Visible detector and λmax was 276nm. The linearity for both the drugs was in the range of 2-12µg/ml. The retention times of Ambroxol HCl and Cetirizine HCl were 2.7min and 4.2min respectively.
6.4 OBJECTIVES OF THE STUDY :
The objective of the present study is to establish and generate inheriting stability data for levocetiriziine dihydrochloride and ambroxol hydrochloride through stress degradation studies under ICH recommended stress conditions (like temperature, light, solvent system, exposure to UV radiations) and develop stability indicating assay method. ICH recommend a generation of stability data through stress degradation studies for life saving drugs as they are key to understand the fate of drugs exposed under various stringent conditions, right from the production up to their consumption by the patients as medication.
MATERIALS AND METHODS :
Ø  All experiments shall be carried out in the Department Of Pharmaceutical Analysis, East West College of pharmacy, Bangalore.
Ø  Pure sample of levocetirizine dihydrochloride and ambroxol hydrochloride shall be procured from industries involved in bulk manufacturing of these drugs.
Ø  Dosage formulation shall be procured from local market.
Ø  The methods shall be developed and validated in pharmaceutical analysis lab of East West College of Pharmacy.
Ø  The methods shall be first developed, and then validated as per ICH guidelines, and then the method shall be applied to the formulations.
Ø  HPLC Instrument SHIMADZU PROMINENCE LC-20A, BINANG GREDIENT HPLC SYSTEM with LUNA C18 column shall be used.
Ø  UV-VIS spectrophotometer SHIMADZU 1800 with matched quartz shall be used for measuring absorbance for levocetirizine and ambroxol solutions.
7.1 SOURCES OF DATA :
Ø  References from library-East West College of Pharmacy
Ø  www.google.com
Ø  www.sciencedirect.com
Ø  www.drugupdate.com
Ø  www.pubmed.com
Ø  Helinet.jccc.in
SOURCES OF EXPERIMENTAL DATA :
Experimental investigation in the laboratory.
UV-spectrophotometer, HPLC, humidity chamber.
7.2 METHODS:
Degradation studies:
a) Hydrolytic studies : Acid decomposition studies will be performed by heating the solution of drug in 0.1N HCl at 80ºc for 12hrs.The studies in alkaline conditions will be done using 0.1M NaOH followed by heating at 80ºc for 12hrs. For study in neutral conditions, drug will be dissolved in aqueous medium.
b) Oxidative studies :The studies will be performed using initially 3% and than 10% H2O2 and
heating at 80ºc for 12hrs.
c) Photolytic studies: The studies will be performed by keeping the sample in photostability chamber or direct sunlight for 48hrs.
d) Thermal (dry heat) studies: susceptibility of the drug to the dry heat will be studied by exposing the solid drug to 100ºc for 12 to 14hrs.
7.3 Does the study require any investigation to be conducted on patients and animals?
No
7.4 Has the ethical clearance been obtained from your institution in case of 7.3?
Not applicable
REFERENCES:
1.  http://www.globalchems.com/Sources-List/130018-87-0. 2011 May 22: 11.05.
2.  http://www.Sciencelab.com/MSDS-Ambroxol/_Hydrochloride-9922869 2011 May 22 11:00.
3.  Prashant Shende, Virag Shah, Dhananjay Ghodke, Rohit Shah, Shital Kumar Patil, Dhanya Kumar Chougule.Validation of UV spectrophotometric method for estimation of Levocetirizine dihydrochloride in bulk and pharmaceutical formulation. J Pharm Res 2010;3(10):2386-7.
4.  Raghad Hommoss, Hind Elzein, Samer Haidar. Determination of Levocetirizine configurational stability in tablets using chiral HPLC method. Int J Pharm Sci 2011;3(2):103-7.
5.  Lakshmana Prabu S, Shirwaikar AA, Shirwaikar Anne, Dinesh Kumar C, Aravind Kumar G. Simultaneous UV spectrophotometric estimation of ambroxol hydrochloride and levocetirizine dihydrochloride. Indian J Pharm Sci 2008;70(2):236-8.
6.  Senthil Rja M, Sahn SH, Perumal P, Moorthy MTS. RP-HPLC method development and validation for the simultaneous estimation of Azithromycin and Ambroxol hydrochloride in tablets. Int J Pharmtech Res 2010;2:36-9.
7.  Mukesh Maithani, Richa Raturi, Vertika Gautam, Dharmendra Kumar, Anand Gaurav, Ranjit Singh. Simultaneous estimation of Ambroxol hydrochloride and Cetirizine hydrochloride in tablet dosage form by RP-HPLC method. Int J Com Pharm 2010;2(3):1-3.
8.  Ilangovan Ponnilavarasan, Sunil Narendra Kumar Chebrolu, Asha P. Simultaneous estimation of ambroxol hydrochloride and Loratadine in tablet dosage form by using UV spectrophotometric method. Int J Pharm and Bio Sci 2011;2(2):338-44.
9.  Dhiraj Nikam S, Swapnil Aswale C, Stability indicating RP-HPLC method for simultaneous estimation of ambroxol hydrochloride and roxithromycin in bulk and tablet
dosage form. Int J Pharm Res Dev 2010;2(10):87-92.
10.  Sunil Dhaneswar R, Janaki Salunkhe V, Vidhya Bhusari K. Validated HPLC method for simultaneous quantitation of levocetirizine hydrochloride and nimesulide in bulk drug and formulation. Int J Comp Pharm 2011;2(5):1-4.
11.  Mahesh Deshpande M, Veena Kasturi S, Seema Gosavi A. Application of HPLC and HPTLC for the simultaneous determination of cefixime trihydrate and ambroxol hydrochloride in pharmaceutical dosage form. Eurasian J Anal Chem 2010;5(3):227-38.
12.  Kamarapu SK, Vaijayanthi, Bahlul GEA, Venisetty RK. Development of RP-HPLC method for the analysis of levocetirizine dihydrochloride and ambroxol hydrochloride in combination and its application. Int J Pharm Sci and Nanotech 2010;3(1):893-6.
13.  http://www.Pharmaqbd.com/tag/ich-q2-r1/ 20.11 May 22:11:11.
9.0 / SIGNATURE OF THE CANDIDATE / VENKATESWARI PARIGAPATI
10.0 / REMARKS OF THE GUIDE
11.0 / NAME AND DESIGNATION OF GUIDE / SUDHA MALLAPUR
ASST. PROFESSOR,
DEPT. OF PHARMACEUTICAL ANALYSIS,
EAST WEST COLLEGE OF PHARMACY
BANGALORE-560 091
11.1 / SIGNATURE
11.2 / HEAD OF THE DEPARTMENT / Dr. SANGAMESH B. PURANIK
PROFESSOR HOD,
DEPT. OF PHARMACEUTICAL ANALYSIS,
EAST WEST COLLEGE OF PHARMACY
BANGALORE-560 091
11.3 / SIGNATURE
12.0 / REMARKS OF THE PRINCIPAL / PROF. K. A. SRIDHAR
PRINCIPAL
EAST WEST COLLEGE OF PHARMACY
BANGALORE-560 091.
12.1 / SIGNATURE

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