SIMULTANEOUS DETERMINATION OF LEVOCETIRIZINE AND AMBROXOL BY NEW ANALYTICAL METHODS.

MASTER OF PHARMACY DISSERTATION PROTOCOL,

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE.

BY

FADADU TRUSHANT DIPAKBHAI

Under The Guidance Of

MRS. PADMAVATHI P. PRABHU

DEPARTMENT OF QUALITY ASSURANCE.

SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143.

2010 – 2012

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.

ANNEXURE-II

.PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / NAME OF THE CANDIDATE
AND ADDRESS (IN BLOCK LETTERS) / FADADU TRUSHANT DIPAKBHAI
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL,
POST-FARENGIPETE
MANGALORE-574143,
KARNATAKA.
2. /

NAME OF THE INSTITUTION

/ SRINIVAS COLLEGE OF PHARMACY,
VALACHIL,
POST- FARANGIPETE
MANGALORE-574143,
KARNATAKA
3. /

COURSE OF STUDY AND SUBJECT

/ MASTER OF PHARMACY
(QUALITY ASSURANCE)
4. / DATE OF ADMISSION OF COURSE / 31st may2010.
5. /

TITLE OF TOPIC

/ SIMULTANEOUS DETERMINATION OF LEVOCETIRIZINE AND AMBROXOL BY NEW ANALYTICAL METHODS.
6.0
7.0
8.0 / BRIEF RESUME OF THE INTENDED WORK:
6.1NEED FOR THE SYUDY:
Several spectrophtometric methods have been used for quantitative determination of levocetirizine and ambroxol in pharmaceutical formulations and in human plasma.
Levocetirizine and ambroxol is novel in market and no much data is available on Analytical methods by UV and HPLC method. Our intension is to developeconomic, less time consuming method for the estimation oflevocetirizine and ambroxol by different analytical methods such as UV and HPLC.
Levocetirizine (as levocetirizine dihydrochloride) is a third-generation non-sedative antihistamine, developed from the second-generation antihistamine cetirizine. Chemically, levocetirizine is the active enantiomer of cetirizine. It is the L-enantiomer of the cetirizineracemate. Levocetirizine works by blocking histamine receptors. It does not prevent the actual release of histamine from mast cells, but prevents it binding to its receptors. This in turn prevents the release of other allergy chemicals and increased blood supply to the area, and provides relief from the typical symptoms of hayfever.
Levocetirizine is called a non-sedating antihistamine as it does not enter the brain in significant amounts, and is therefore unlikely to cause drowsiness. However, some people may experience some slight sleepiness, headache, mouth dryness, vision problems.
6.2 REVIEW OF LITERATURE:
6.2.1 LEVOCETIRIZINE
Molecular Structure:

Name: / Levocetirizine
Systematic (IUPAC) name: / 2-[2-[4-[(R)-(4-chlorophenyl)-phenyl-methyl]
piperazin-1-yl]ethoxy]acetic acid
Molecular Formula: / C21H25ClN2O3
Molecular Weight: 388.888
Bioavailability: high
Protein binding:90%
Half life:6 to10 hours
  • Shaikh KA and Patil AT1, has given Stability indicating reverse phase high performance liquid chromatographic method for the simultaneous determination of Levocetirizine dihydrochloride and Pseudoephedrine sulfate in pharmaceuticalformulation. The mobile phase A consisted of Potassium dihydrogen phosphate Buffer 0.05M and 1-Ocatne sulphonicacid sodium salt 0.25%, pH adjusted to 3.0 with orthophospheric acid. Mobile Phase B: Acetonitrile, Gradient elution atflow rate of 1 ml/min and Column temperature at 40oC. Detector wavelength of 242 nm using a photodiode arraydetector. The described method shows excellent linearity over a range of 200–100 μg/ml for Levocetirizinedihydrochloride and 7200-360 μg/ml.1 for Pseudoephedrine sulfate. The correlation coefficient for Levocetirizinedihydrochloride and Pseudoephedrine sulfate are 0.9999.
  • Ashokkumar S, Senthil RM, Perumal P.2In this study, reverse phase high performance liquid chromatographic method have beendeveloped and validated for the simultaneous determination of Montelukast and Levocetirizine combined pharmaceutical formulation. HPLC separation was achieved with a Phenomenex Luna, C18 (250 x 4.6 mm i.d.,5µ) as stationary phase and phosphate buffer (pH adjustedto 5.5 with dil. KOH): Acetonitrile (65:35, v/v) as eluent, at a flow rate of 1.5ml/min.UV detection was performed at 230 nm. The retention time of Montelukast and Levocetirizine were found to 3.7 and 6.1 min respectively. Results of the analysis were validated statistically by recovery studies.
  • Prashant S, Virag S, Dhananjay G, Rohit S, Shital kumar P, Dhanya Kumar C.,3 given selective, simple, accurate and reproducible spectrophotometric method for the estimation of levocetirizine dihydrochloride in bulk and pharmaceutical formulation. The drug obeyed the Beer’s law and showed good correlation. It showed absorption maxima at 231nm; in Phosphate Buffer Solution pH 7.0. The developed method was validated with respect to linearity, accuracy and precision.
  • Lakshmana SP, Shirwalkar AA, Anie S, Dinesh Kumar C, and Arvind Kumar4 givena spectrophotometric method that is developed for simultaneous estimation of ambroxol hydrochloride and levocetirizine dihydrochloride. The method involved solving simultaneous equations based on measurement of absorbance at two wavelengths 242 nm and 231 nm, the λmax of ambroxol hydrochloride and levocetirizine dihydrochloride, respectively. Beer's law was obeyed in the concentration range 10–50 μg/ml and 8–24 μg/ml for Ambroxol hydrochloride and Levocetirizine dihydrochloride respectively. Results of the method were validated statistically and by recovery studies.
AMBROXOL
Ambroxol is a secretolytic agent used in the treatment of respiratory diseases associated with viscid or excessive mucus. Ambroxol is available in many formulations such as syrup,dry powder,drops,ampoules etc.
6.2.2 REVIEW OF LITERATURE:
Molecular structure: /
Name: /

Ambroxol

IUPACName: / trans-4-(2-Amino-3,5-dibrombenzylamino)-cyclohexanol
Molecular Formula: / C13H18Br2N2O
Molecular Weight: / 378.10

Neela MB, Santosh KG, and Harinath NM5, gavespectrophotometric method for the simultaneous analysis of ambroxol hydrochloride and cetirizine hydrochloride in their tablet formulation. The chromatographic methods were standardized using a HIQ SIL-C 18 column (250x4.6 mm i.d., 10 µm particle size) with UV detection at 229 nm and mobile phase consisting of methanol-acetonitrile-water (40:40:20, v/v/v). Ambroxol hydrochloride and cetirizine hydrochloride have absorbance maxima at 243 nm and 229 nm, respectively. The isoabsorptive wavelength for both the drugs was 236 nm. For absorbance ratio method developed, wavelengths selected were 243 nm and 236 nm.

  • Heinanen M, Barbas C6gives the method for estimation of compounds by HPLC with UV detection at 247 nm in syrup as pharmaceutical presentation. Optimal conditions were: Column Symmetry Shield RPC8, 5 micron 250 x 4.6 mm, and methano1/(H(3)PO(4) 8.5 mM/triethylamine pH=2.8) 40:60 v/v. Validation was performed using standards and the pharmaceutical preparation which contains the compounds described above. Results from both standards and samples show suitable validation parameters. The pharmaceutical grade substances were tested by factors that could influence the chemicals.
  • Krupa MK, Balasundaram J, Amit P. Khandhar, Rajnish KM7, developed and validated analytical method for quantitative determination of Levofloxacin and Ambroxol hydrochloride in a new tablet formulation. Chromatographic separations of the two drugs were analyzed on a Hypersil BDS C18 column (25cm X 4.6mm, 5µm). The mobile phase constituted of Buffer: Acetonitirile: Methanol (650:250:100) with triethylamine and pH adjusted to 5.2 with dilute orthophosphoric acid was delivered at the flow rate 1.0 mL/min. Detection was performed at 220 nm. Separation was completed within 10 min. Calibration curves were linear with coefficient correlation between 0.99 to 1.0 over a concentration range of 7 to 22 µg/mLof Levofloxacin and 50 to 150µg/mL for Ambroxol hydrochloride respectively. The relative standard deviation (R.S.D) was found <2.0%.
6.3 OBJECTIVES OF THE STUDY:
  • To determine λmax in different solvent and pH condition for levocetirizine and ambroxol.
  • To develop simple, selective, rapid, precise and economical analytical method for the estimation of levocetirizine and ambroxol.
  • To validate method in terms of accuracy, precision, linearity, limit of detection, limit of quantitation etc.
MATERIALS AND METHODS:
The standard drug will be requested from the industries in India.Depending on the availability of facilities, one of the following methods will be applied for the estimation of levocetirizine and ambroxol.
  1. UV Method:
Standard stock solution can be prepared by dissolving 100mg of levocetirizine and 100mg of ambroxol in 100 ml of waterseparately and then final volume of both solutions made upto 100 µg/ml of levocetirizine and ambroxol in volumetric flask. Solutions containing 20 µg/ml of both Levocetirizine and Ambroxol can be prepared and scanned in UV region separately for λmax.
For the compound levocetirizine and ambroxol λmax can be obtained, By dilution of Standard drug in 5,10,15,20 ….50 µg/ml of each drug can be prepared and scanned at 200-400 nm. The values of absorbances can be recorded. From this data absorptivity and molar absorbance can be determined forlevocetirizine and ambroxol. Finally, drug formulations can be estimated from the available data.
  1. HPLC Method:
Mixed solutions consisting of 100 µg/ml of levocetirizine and ambroxol can be prepared in different combinations of solvents using acetonitrile, water, acetone, methanol. Then, it is passed in HPLC column using different mobile phases. From HPLC data Retention time, Peak area can be obtained which is useful for estimation of drug in formulations.
All the validation parameters such as LOD, LOG, Linearity, Precision and Accuracy can be validated using ICH Q2b guidelines.
The above method likely to change depending on the observations recorded time to time.
7.1 Material:
HPLC instrument: Jasco International Co Ltd., Japan. With C18column. Detector: UV-visible. Bulk Drugs: Levocetirizine and Ambroxol,
formulation: combination of LevocetrizineandAmbroxol tablets capsules syrup, drops etc. Solvents: HPLC grade water, hexane, ethyl acetate, dichloromethane, methanol, acetonitrile, ethanol, chloroform, glacial acetic acid, acetone, propanolChemicals for pH adjustment: phosphate buffer, ortho-phosphoric acid, hydrochloric acid, sodium hydroxide, potassium bromide etc.
7.2 Source of data:
Data will be obtained from Pubmed, Science direct, Medline, and other internet facilities, literature search and related articles from library of Srinivas College of Pharmacy, Mangalore.etc.
7.3 Method of Collection of Data (Including Sampling Procedure, If Any)
  1. Procurement of the drug and marketed formulations.
  2. Solubility determination of Levocetirizine and Ambroxol in different solvents and buffers.
  3. Determination of wavelength of maximum absorbance and intensity of absorption in different solvents and buffers for various analytical studies.
  4. Develop and validate analytical method for Levocetirizine and Ambroxol by UV-Visible spectrophotometer using standard absorptivity value, standard calibration curve, single point and double point standardisation.Develop and validate HPLC method to estimate Levocetirizin and Ambroxol in bulk and pharmaceutical formulations.
7.4Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please mention briefly.
--NOT APPLICABLE--
7.5Has ethical clearance been obtained from your institution in case of 7.3?
--NOT APPLICABLE--
LIST OF ReferenceS:
  1. ShaikaKA,Patil AT.Stability indication LC methods for the simultaneous determination of levocetirizine and pseudoephedrine sulphate in tablet dosage forms. Int J ChemTech Res2010;2(1):454-61.
  2. Ashokkumar S, SenthilRM, Perumal P. RP-HPLC method development and validation for simultaneous estimation of montelukast sodium and levocetirizinedihydrochloride. Int J Pharma Res 2009; 1(4):8-12.
  3. Prashant S, Virag S, Dhananjay G, Rohit S, Shital kumarP, Dhanya Kumar C. Validation of UV Spectrophotometric method for estimation of levocetrizine HCl in Bulk and Pharmaceutical Formulation. Ind J Pharma Res2010; 3(10):2386-87.
  4. Lakshmana SP, Shirwalkar AA, Anie S, Dinesh Kumar C, Arvind Kumar. Simultaneous UV Spectrophotometric estimation of ambroxol HCl and Levocetrizine HCl. IndJ Pharma Sci 2008; 70(2):236-38.
  5. Neela MB, Santosh KG, Harinath NM. Spectrophotometric estimation of ambroxol HCl and cetrizine HCl intablets. Asian J Pharm 2008; 2(3):159-62.
  6. Heinanen M, Barbas C. Validation of an HPLC method for the quantification of ambroxol hydrochloride and benzoic acid in syrup as pharmaceutical form stress test for stability evaluation. J Pharm Biomed Ana2001;24:1005–10.
  7. Krupa MK, Balasundaram J, Amit P. Khandhar, Rajnish KM. Quantitative determination of levofloxacin and ambroxol HCl in pharmaceutical dosage form by RP-HPLC. Eura J Anal Chem 2007; 2(1):21-30.

9. / Signature of the candidate / (FADADU TRUSHANT )
10. / Remarks of the Guide / The candidate is working under my direct supervision in laboratories of Srinivas College of Pharmacy, Mangalore-574143.
11. / 11.1 Name and Designation of Guide
11.2 Signature / MRS. PADMAVATHI P. PRABHUM.Pharma (Ph.D)
ASSISTANT PROFESSOR
(DEPARTMENT OF QUALITY ASSURANCE)
SRINIVAS COLLEGE OF PHARMACY
MRS. PADMAVATHI P. PRABHU
11.3 Head of the department
11.4 Signature / PROF. DR. E.V.S SUBRAMANYAM M.Pharma Ph.D
DEPARTMENT OF QUALITY ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY.
DR.E.V.S. SUBRAMANYAM
12. / 12.1 Remarks of Principal
12.2 Signature / FORWARDED AND RECOMMENDED FOR FAVOURABLE CONSIDERATION.
PROF. A.R. SHABRAYA