INTRODUCTION:

Levocloperastine fendizoate (LCP) is chemically 1-{2-[(4 Chlorophenyl)phenylmethoxy]ethyl} piperidine, 2- (4-hydroxy- 3- phenylbenzoyl) benzoic acid. Levocloperastine, a novel antitussive, which acts centrally on the cough center and on the peripheral receptors in the tracheobronchial tree in treating chronic cough. It reduced nighttime awakenings and irritability in children while also effective in treating cough induced by angiotensin-converting enzyme inhibitors.

The drug is available in suspension form. It is not official in any of the pharmacopoeia and also not available in Indian market. So far no analytical method neither spectrophotometric nor chromatographic method has been reported for the estimation of LCP. The aim of the present investigation is to develop a simple, sensitive, reproducible, statistically valid, rapid and cost effective UV spectrophotometric methods for the determination of LCP in bulk and pharmaceutical dosage form.

MATERIALS AND METHODS:

Reagents and Materials:

LCP working standard was supplied by Ajanta Pharma Ltd, Mumbai. LCP (Label claim: 708mg/100ml) PRIVITUSS suspension was manufactured by Aesculapius pharmaceuticals, Italy. All other chemicals used in the analysis were AR grade.

Apparatus:

A double–beam spectrophotometer (Jasco UV-630 and Shimadzu^® UV 1700) was used for the detection of absorbance, Shimadzu^® (weighing balance) and sonicator were used for experimental purpose.

Method:

I) Preparation of stock solution:

25mg of the pure drug was weighed and transferred to a 50ml volumetric flask, 25ml methanol was added to the above flask and dissolved; the volume was made up with methanol. Different aliquots were taken from stock solution and diluted with 0.1N NaOH separately to prepare series of concentrations from 2-10 μg/ml. For Absorbance, the solution was scanned in spectrophotometer and λmax was found to be 350 nm.

In Area under Curve method, the sampling wavelength ranges selected for estimation of LCP are 355-345nm. Mixed standard were prepared and their Area Under the Curve were measured at the selected wavelength ranges. These were used to calculate the concentration of drugs in standard and the sample solution. Absorbance and AUC was measured at 350 nm and 355-345nm respectively against 0.1N NaOH as blank. The calibration curve was prepared by plotting absorbance versus concentration (μg/ml) of levocloperastine fendizoate.

Fig 1: Structure of Levocloperastine fendizoate

Fig. II Spectrum of LCP in 0.1M NaOH showing λmax and AUC

Fig.III Calibration Curve for Absorptivity and Area Under Curve

II) Determination of Absorptivity value at selected wavelength:-

5.0 mL of Standard stock solution was taken in a 50.0 mL volumetric flask and the volume was made upto mark with solvent to get the concentration of 10.0μg/mL which was further diluted in range 0.4 mL to 2.0 mL in a series of 10.0 mL volumetric flasks with solvent and volumes were made up to mark with 0.1M NaOH to obtain the concentration range from 2.0- 10.0 μg/mL for levocloperastine fendizoate.

Absorbance of each final dilution was recorded at 350nm (λmax). A(1%, 1cm) values were calculated using formula as given below. The results are shown in Table I

Absorbance

A (1%, 1cm) = -----------------------------------

Concentration (g/100 mL)

Table I: Study of Absorptivity value of LCP:-

Sr. No.	Concentration (g/100mL)	Absorbance	(A1%,1cm)
1	0.00198	0.0789	398.48
2	0.00397	0.1565	394.20
3	0.00594	0.2341	394.10
4	0.00796	0.3169	398.11
5	0.00996	0.3927	394.27
		Mean	395.83
		±S.D.	2.2530
		R.S.D.	0.00569

III) Application of the Proposed Method for the Determination in Suspension:

The proposed method was applied in order to determine the levocloperastine fendizoate in suspension formulation. The suspension equivalent to 25mg of levocloperastine fendizoate was transferred in 50 ml volumetric flask and dissolved in methanol by shaking. The volume was made up to mark to get final concentration of 0.5mg/ml. The solution was then filtered through Whattman filter paper #41.This filtrate was further diluted suitably with 0.1N NaOH to get the solution of 2-10μg/ml concentration. The working solution of drug (10μg/ml) was prepared from standard stock solution in 0.1N NaOH. The absorbance of this solution was measured and amount of levocloperastine fendizoate was calculated from the calibration curve. The readings were taken in triplicate. To examine the absence of either positive or negative interference of excipients used in formulation, recovery studies were carried out at five different levels by adding diluted pure drug solution equivalent to 0, 0.05, 0.10, 10 and 20 mg to five samples of suspension solution. The determination was carried out for five replicates at each interval.

Method Validation:

Various methods for analysis of LCP in bulk and pharmaceutical formulation was carried out as per ICH guideline.

I) Linearity:

The method was validated according to ICH Q2B guidelines for validation of analytical procedures in order to determine the linearity, sensitivity, precision and accuracy of the analyte. For LCP, five point calibration curves were generated with the appropriate volumes of the working standard solutions for UV methods. The linearity was evaluated by the least-square regression method using unweighed data.

II) Precision and accuracy:

Precision is the degree of repeatability of an analytical method under normal operational conditions. The precision and accuracy were determined with standard quality control samples (in addition to calibration standards) prepared in triplicate at different concentration levels covering the entire linearity range. The precision of the assay was determined by repeatability (intraday) and intermediate precision (inter-day) and reported as %RSD for a statistically significant number of replicate measurements. The intermediate precision was studied by comparing the assays on three different days and the results are documented as the standard deviation and %RSD. Accuracy is the percent of analyte recovered by assay from a known added amount. Data from nine determinations over three concentration levels covering the specified range were obtained.

III) LOD and LOQ:

The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from back-ground levels. In this study, LOD and LOQ were based on the standard deviation of the response and the slope of the corresponding curve using the following equations-

LOD = 3 s/m; LOQ = 10 s/m

Where s noise of estimate is the standard deviation of the absorbance of the sample and m is the slope of the related calibrations graphs.

The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with an acceptable accuracy, precision and variability. The values of LOD and LOQ are given in Table II.

Table II: Linearity study of LCP:-

Concentration	Absorbance	Area Under Curve
2	0.0789	0.7789
4	0.1565	1.5641
6	0.2341	2.3231
8	0.3169	3.0892
10	0.3927	3.9267

IV) Recovery study:

Recovery of the analyte of interest from a given matrix can be used as a measure of the accuracy or the bias of the method. The same range of concentrations, as employed in the linearity studies was used. To study the accuracy, precision and reproducibility of the proposed method and dosage forms, recovery experiments were carried out using the standard addition method. These studies were performed by the addition of known amounts of pure LCP to the pre-analyzed suspension formulation and the mixtures were analyzed using the proposed techniques. After parallel analyses, the recovery results were calculated using the related calibration equations

RESULTS AND DISCUSSION:

The development of a simple, rapid, sensitive and accurate analytical method for the routine quantitative determination of samples will reduce unnecessary tedious sample preparations and the cost of materials and labor. LCP is a UV-absorbing molecule with specific chromophores in the structure that absorb at a particular wavelength and this fact was successfully employed for their quantitative determinations using the UV spectrophotometric method. The λmax of the drug for analysis was determined by taking scans of the drug sample solutions in the entire UV region. It was found to be that only one peak was observed in this method at the wavelength of 350nm.

Calibration curves:

Calibration curve data were constructed in the range of the expected concentrations of 2 µg/mL to 10 µg/mL. Beer’s law was obeyed over this concentration range. The regression equation was found to be Y =0.039x - 0.001. The correlation coefficient (r) of the standard curve was found to be greater than 0.999. The stock solutions were made in methanol and working standards were made in 0.1N NaOH. Calibration curve was presented in above fig.

The analytical characteristics and necessary validation parameters for the UV techniques for LCP is presented in Table III below.

Table III: Validation parameters:-

Parameters	Values
Linearity range (µg/ml)	2-10
Precision (%)	1.2057 ± 0.01214
Accuracy (%)	99.752 ± 0.6653
LOD (µg/ml)	3.194
LOQ (µg/ml)	0.310
Std Deviation (SD ±)	0.1246

Performing replicate analyses of the standard solutions was used to assess the accuracy, precision and reproducibility of the proposed methods. The selected concentration within the calibration range was prepared in 0.1N NaOH and analyzed with the relevant calibration curves to determine the intra- and inter day variability. The intra- and inter day precision were determined as the %.RSD. The precision, accuracy and reproducibility of the results are given in Table, which demonstrate a good precision, accuracy and reproducibility. The proposed methods can be successfully applied for LCP assay in suspension dosage forms without any interference. The assay showed the drug content of this product to be in accordance with the labeled claim 708mg.

The recovery of the analyte of interest from a given matrix can be used as a measure of the accuracy of the method. In order to check the accuracy and precision of the developed method and to prove the absence of interference by excipients, recovery studies were carried out after the addition of known amounts of the pure drug to various pre-analyzed formulations of all drugs. The application of this procedure is explained in the experimental section. The obtained results demonstrate the validity and accuracy of the proposed method for the determination of all drugs in suspension. These results reveal that the developed method have an adequate precision and accuracy and consequently, can be applied to the determination of LCP in pharmaceuticals suspension without any interference from the excipients.

CONCLUSION:

The developed spectrophotometric method was simple, sensitive, and specific, for the detection of LCP in bulk and pharmaceutical formulation. It could be precisely quantify and LOD was found to be 3.194 and the limit of quantification to be 0.310 . All the calibration curves shows a linear relationship between the absorbance and concentration and coefficient correlation was higher than 0.99. Precision of the method was found to be 1.2057 ± 0.01214 against the label claim of 708mg. The percentage recovery was found to be 99.752 ± 0.6653. The proposed method will be suitable for the analysis of LCP in bulk and pharmaceutical formulation.

ACKNOWLEDGEMENT:
Authors thanks to the Ajanta Pharmaceuticals Ltd., Mumbai for the gift sample of Levocloperastine fendizoate and also thankful to Principal, S.K.B. College of Pharmacy, New Kamptee for providing laboratory facilities and constant encouragement.

REFERENCE:

1. ICH harmonised tripartite guideline; Validation of analytical procedures: text and methodology, Q2 (R1); Current Step 4 version; Parent Guideline dated 27 October 1994.

2. Martindale-The Extra Pharmacopoeia; Edited by Sean C. Sweetman; Pharmaceutical Press, London; 34^thEdition, p.1117.2

3. Japanese Pharmacopoeia; Official monograph of Cloperastine hydrochloride; XV edition, p.530-531

4. http://en.wikipedia. Org/wiki/Cloperastine

5. Scotti L, Borzani M.; Cloperastine fendizoate in the treatment of cough-producing diseases in pediatrics. Minerva Pediatric 1988; 40 (5):p. 283-6

6. P.D.Sethi and Rajat Sethi; HPLC-quantitative analysis of pharmaceutical formulations; vol.II; CBS Publication; New Delhi-2007; 1^st Edition; p.443.

7. Douglas A. Skoog, F. James Holler and Stanley R. Crouch; Principle of Instrumental Analysis; Thomson Publication; 6^th Edition, 2007; p.01-05

8. A. H. Beckett and J. B. Stenlake, Practical Pharmaceutical Chemistry; CBS Publication; 2004- New Delhi; 4^th Edition; p.01-08, 85-17

Received on 08.04.2011 Modified on 13.05.2011

Asian J. Research Chem. 4(8): August, 2011; Page 1231-1234