INTRODUCTION:

Levocetirizine dihydrochloride (LEVO), is R-2(2(4(4Chlorophenyl)phenylmethyl)1piperazinyl) ethoxy)acetic acid hydrochloride.(Fig.1) It is indiacted for the relief of symptoms associated with seasonal allergic rhinitis (SAR) and perennial allergic rhinitis (PAR), and for the treatment of the uncomplicated skin manifestations of chronic idiopathic urtcaria (CIU). It is official in Indian Pharmacopoeia(1,2.) Similarly, Phenylephrine hydrochloride (PHE) is R-2Methylamino(3-hydroxyphenyl) ethanol hydrochloride(3). Levocetirizine was officially assayed by Liquid chromatography using stainless steel column 25mm x 4. Phenylephrine was also assayed by potntiometrically(4).Mobile phase was used as 0.05 m potassium dihydrogen phosphate and acetonitrile(60:40v/v with pH adjusted with and 10% w/v sodium hydroxide, packed with octa desyl silane bonded with silica. Phenylephrine hydrochloride was assayed (B.P) by potntiometrically using 0.1 M HCl and 50 ml ethanol (95%) and titrate with 0.1 M ethanolic NaOH(5). Many researchers have dealt with the development of methods that quantify LEVO in pure and in tablets(6).

Methods that include validation of UV Spectrophotometric methods for simultaneous estimation of Levocetrizine dihydrochloride in bulk and pharmaceutical formulation when present alone or in combination with Ambroxol hydrochloride.(7,8) Levocetirizine (CAS—130018-77-8)is the R-enantiomer of racemic cetirizine, is aselective, potent, H1-antihistamine compound indicated for the treatment of allergic rhinitis and chronic idiopathic urticaria (9).The recommended dosing of Levocetirizineis 5mg per day. It has a rapid onset, achieving maximum plasma concentration (tmax)in 0.9 h , with peak serum levels (Cmax)of approximately 270 ng/mL (10). Levocetirizine is generally well tolerated in adults, adolescents and children with allergic conditions (11).

.HCl

(A)

.2HCl

(B)

Figure 1: Sructure of Levocetirizine dihydrochloride(A), Phenylephrine hydrochloride (B)

Liquid chromatographic method for simultaneous estimation of Paracetamol and Phenylephrine in pharmaceutical formulation(12) , RP- HPLC method for simultaneous estimation of Ebastine and Phenylephrine in tablet formulation(13) ,RP-HPLC method for simultaneous estimation of Montelukast and Levocetirizne in tablet formulation were established(14). In the present study, we describe a simultaneous estimation of Levocetirizine and Phenylephrine in tablet formulations by a simple RP-HPLC method which does not need any special extractions and time consuming procedures.

MATERIALS AND METHODS:

Levocetirizine dihydrochloride and phenylephrine hydrochloride were obtained from Zim Laboratory, Kalmeshwar, Nagpur as a gift sample and were used as working standards. Methanol of analytical grade and double distilled water were used throughout the analysis. The pharmaceutical formulation of Levo and Phe that is levocet d⁺(Heteo Health Care, Mumbai) the commercial formulation of levocetirizine and phenylephrine are available in ratio of 1:2 as tablet.

Chromatographic conditions

The mobile phase was prepared by adding 80 ml of methanol to 20 ml of 10mM phosphate buffer, pH adjusted to 5.5.The mobile phase was filtered through 0.45μm membrane filter and was degassed before use, then delivered at a flow rate of 1.0 ml/min.

Solution preparation

The standard and sample solutions were prepared in mobile phase. The working concentration for the determination of Phenylephrine was 10μg/ml and 5µg/ml for Levocetirizine. All the solution preparation was carried out in amber coloured volumetric flask with minimal exposure to light.

RESULT AND DISCUSSION:

Method Development

Initially various mobile phases were tried in attempt to obtain the best separation and resolution between Levocetirizine and Phenylephrine. The mobile phase of 10mM phosphate buffer adjusted with pH 5.5 in the ratio of 80:20 was found to be an appropriate mobile phase allowing the adequate separation of both the compounds using a Hypersil gold C18, 250mm column at a flow rate of 1.0ml/min. a typical chromatogram of separation of the two components is shown in fig.2. As the Levocetirizine and Phenylephrine exhibit significant absorbance at wavelength 268 nm, it was selected as detection wavelength for the simultaneous determination of Levocetirizine and Phenylephrine in pharmaceutical dosage forms. The retention time of Levocetirizine and Phenylephrine were found to be 4.930 and 2.792 respectively. The resolution, theoretical pate and tailing factors are prescribed in table.1.

Table.1 The resolution, theoretical pate and tailing factors

Parameters \Drugs	Levocetirizine dihydrochloride	Phenylephrine hydrochloride
Tailing factor	1.104	2.039
Theoretical plate	3983	4455
Resolution	4.539

Assay of Formulation

Twenty tablets of the formulation were weighed and the average weight of one tablet was calculated. All twenty tablets were crushed and grounded to a fine powder and powder equivalent to 100mg Phenylephrine (50mg of Levcetirizine) was weighed and transferred to 100ml coloured volumetric flask. It was dissolved in mobile phase and filtered through membrane filter(0.45μ). This solution was suitably diluted and used for analysis. After setting the chromatographic conditions and stabilizing the instrumention to obtain a steady baseline, the sample solution was loaded in the 20μl fixed sample loop of the injection port. The solution was injected and a chromatogram was recorded. The injections were repeated six times and the peak area were recorded. The quantification was done by direct comparison method and the results obtained are shown in the table 2.

Table. 2 Assay Result

Drug	%Assay	Amount Present
Levocetirizine dihydrochloride	99.79	4.978
Phenylephrine hydrochloride	99.65	9.974

Method Validation

The developed method was validated forsimultaneous assay method for determination of Levocetirizine and Phenylephrine presented in the table.3.

Table.3 Validation Parameter

Parameters /Drugs	Levocetirizine	Phenylephrine
Linearity	2-12.5	5-25
R2	0.999	0.999
Intraday
(%RSD)	0.0328	0.0351
Interday
(%RSD)	0.2935	0.0671
Repeatability
(%RSD)	0.1733	0.0339
Accuracy
(%Recovery mean)	99.89	99.75

Recovery studies

The accuracy of the method was studied by recovery studies. Where the known standard drug was added to the assay sample.The amount present was calculated and the assay amount was reduced from it,which gives the amount recovered. This recovery study was conducted in three stages,80,100 and 120% of the assay amount. The results obtained are shown in table.3.

Stability studies

The stability of Levocetirizine in solution was determined. The samples were checked for 3 days of storage and the data were compared with freshly prepared sample.The solution kept in tightly closed amber coloured flask in dark was found to be stable and the RSD values of assay were

well below 2% against freshly prepared sample.

CONCLUSION:

The developed RP-HPLC method was developed and validated for the simultaneous determination of Montelukast and Levocetirizine dihydrochloride in tablet dosage form. The method was found to be simple, precise and rapid. The assay result obtained by this method is in fair agreement. This method can be used for the routine determination of Levocetirizine and Phenylphrine in commercial formulations.

REFERENCE:

1. Budhavari S. The Merk Index. 14^th ed., Merk Research Lab. Division of Merck and Co. Inc. Whitehouse station NJ; 2001. p. 1011.

2. Budhavari S. The Merk Index. 14^th ed. Merk Research Lab. Division of Merck and Co. Inc. Whitehouse station NJ; 2001. p. 1307.

3. Sweetman SC, Martin D. The Complete Drug Reference. 35^th ed. Pharmaceutical Press, London; 2007. p. 526.

4. Indian Pharmacopoiea. Government of India Ministry of Health and Family Welfare;2007. Vol.2. p. 893.

5. British Pharmacopoeia. Int Ed.Published on the recommendation of the medicine commission pursuant to medicine act 1968; 1993.vol.1. p. 509.

6. Shende P, Shah V, Ghodke D. Validation of UV Spectrophotometric method for estimatiion of Levocetrizine dihydrochloride in bulk and pharmaceutical formulation. Journal of Pharmacy 2010; 3(10):2386-2387.

7. Merukar SS, Mhaskar PS, Bavaskar SR, Burade KB, Dhabale PN. Simultaneous spectrophotometric methods for estimation of Levocetrizine and Pseudoephidrine in Pharmaceutical Tablet Dosage Form. Journal of Pharmaceutical Sciences and Research 2009;1(2):3842.

8. Choudhari V, Kale A, Kuchekar B, Gawli V, Patil N. Simultaneous determination of Montelukast sodium and Levocveterizine dihydrochloride in pharmaceutical preparations by Ratio Derivative Spectroscopy. International Journal of Pharm Tech Research. March 2010;2(4):985-987.

9. P.I.Hair, L.J. Scott, Drugs 66(2006) 973.

10. G. Passalacqua, G.W. Canonica, Clin.Ther. 27 (2005) 979.

11. Swarbrick J, Boylan J. Encyclopedia of Pharmaceutical Technology, Vol. I, Marcel Dekker Inc. New York; 1998. p. 217 - 224.

12. Ebru ÇD, Mehmet G,Hale C.Validation of method for simultaneous determination of Paracetamol and Phenylephrine in pharmaceutical formulation by RP-LC method. International Journal of Comprehensive pharmacy 2011;6(09):231-232.

13. Hajare RA, Tated AG, Gadbail PA, Khan FA, Kayal SD.Method development and validation for simultaneous determination of Ebastine and Phenylephrine HCl in tablet formulation by RP-HPLC.International Journal of Pharmaceutical Research and development 2011;3(7):214-220.

14. Ashokkumar S, Senthil Raja M, Perumal P. RP-HPLC Method Development and Validation for Simultaneous Estimation of Montelukast Sodium and Levocetrizine. Journal of Pharmaceutical Research 2009; 1(4): 8-12.

Received on 28.11.2012 Modified on 14.12.2012

Asian J. Research Chem. 6(1): January 2013; Page 01-03