INTRODUCTION:

Cavinton is a semisynthetic derivative alkaloid of vincamine an extract from the periwinkle (plant) Vinca minor¹. Vinpocetine is reported to have cerebral blood-flow enhancing² and neuroprotective effects³, and is used as a drug in Eastern Europe for the treatment of cerebrovascular disorders and age-related memory impairment⁴. Vinpocetine has been shown to selectively inhibit voltage-sensitive Na+ channels, resulting in a dose-dependent decrease in evoked extracellular Ca+ ions in striatal nerve endings⁵. Cavinton is unique memory enhancer drug, which increases cerebral metabolism by increasing glucose and oxygen utilization. Cavinton has half-life 2.5 ± 0.48⁶. So far no HPTLC method has been reported for the estimation of Cavinton. Now a day HPTLC is becoming a routine analytical technique due to its advantages of low operating cost, high sample throughputs, and need of minimum sample clean up. Our study made an attempt to develop a simple, accurate, precise, specific and reproducible HPTLC method for routine analysis of Cavinton in bulk drug and in ophthalmic dosage forms.

MATERIALS AND METHODS:

Reagents and other materials:

Silica gel 60 f 254 on aluminium sheets layer thickness 0.2 mm was used as stationary phase. All the chemicals and reagents used were analytical grade. Tablets formulation containing Cavinton was procured from local market. Camag HPTLC system comprising of Camag Linnomate V semiautomatic sample applicator, Hamilton syringe, Camag TLC scanner 3. Camag win CAT S4 software, Camag Twin-through chamber (20 x 10 cm) and ultrasonicator were used during study.

Preparation of standard solution of Cavinton:

Cavinton (10 mg) was accurately weighed and dissolved and diluted with Petroleum ether: Ethyl acetate: glacial acetic acid to obtained final concentration of 100 μg/ml of each drug.

Preparation of sample solution:

The tablets dosage form equivalent to 5 mg of Cavinton was accurately transferred and mixed with Petroleum ether: Ethyl acetate: Glacial acetic acid (2.5:2.5:0.5) and sonicated for 10 mins. Then the volume is made up to 10 ml.

HPTLC METHOD AND CHROMATOGRAPHIC CONDITIONS:

The chromatographic estimations were performed using following conditions; stationary phase, precoated silica gel 60F 254 aluminium sheets (0.2 mm thickness) with solvent system of Petroleum ether: Ethyl acetate: Glacial acetic acid (2.5:2.5:0.5). Linear ascending development was carried out in 20 X 10 cm twin trough glass chamber at room temperature (25 °± 2), no chamber saturation, migration distance 80 mm; the plates were dried in a current of air with the help of an air dryer in wooden chamber with adequate ventilation. Wavelength of detection at 268 nm in absorbance mode, slit dimension 6 x 0.45 mm. A deuterium lamp is the source of radiation. Cavinton (2μl) was spotted and developed at constant temperature and wavelength was selected by scanning standard solution of the drug over 400 nm to 200 nm wavelength. Cavinton show maximum absorbance at 268nm.

Calibration curves of Cavinton:

Standard Cavinton 2-7µl was spotted on precoated separate TLC plate, using semiautomatic spotter under nitrogen stream. The TLC plates were developed and photometrically analyzed as described under chromatographic separation. The calibration curves were prepared by plotting peak area verses concentration.

Quantification of dosage form:

A sample solution 10 µl was applied on TLC plate, developed and scanned as described in chromatographic separation. The amount of Cavinton present in sample was determined by fitting area values for peaks into the equation of line representing calibration curve for Cavinton.

RESULT AND DISCUSSION:

In present work HPTLC method was developed for estimation of cavinton in bulk and marketed formulation. HPTLC method is cost effective and less time consuming. Mobile phase comprising of Petroleum ether: Ethyl acetate (4:1) gave comparatively best resolution, so it was further developed to optimize ratio of solvents. Different ratios of Petroleum ether: Ethyl acetate was tried out. The mobile phase containing (2.5:2.5) ratio of solvent gave best resolution of Cavinton but some tailing was observed. In order to minimize tailing varying concentrations of Glacial acetic acid were incorporated in the mobile phase. Mobile phase containing Petroleum ether: Ethyl acetate: Glacial acetic acid (2.5:2.5: 0.5ml) gave best resolution without tailing so it was finalized as mobile phase for TLC analysis of Cavinton.

Method optimization⁷:

The method was validated in terms of linearity, interday and intraday precision, accuracy and specificity. The limit of detection (LOD) and limit of quantification (LOQ) were also determined. The R_f value of Cavinton was 0.411 (fig 1). A representative calibration curve of Cavinton was obtained by plotting the mean peak area of Cavinton against the concentration over the range of 2000-7000 ng /spot. A correlation coefficient was found to be 0.998 (Fig.2). The average linear regression equation was represented as y= 0.00001x +0.0116 Where y = peak area, x = concentration of Cavinton.

Fig. 1: HPTLC chromatogram of Cavinton

Fig. 2: Calibration Curve of Cavinton

The Limit of detection was found to be 200 ng/spot and Limit of quantitation was found to be 1000 ng/spot for Cavinton.

Intra-day assay precision was found by analysis of standard drug at three times on the same day. Inter-day assay precision was carried out using at three different days, and percentage relative standard deviation (% RSD) was calculated. Intraday and interday variation was found to be 0.2148 -0.5279 and 0.1919 -0.2896 for Cavinton.

Accuracy of the method was evaluated by calculating recovery of the drugs by standards addition method at 3 levels of calibration curves (n=3). The % recovery was found to be 99.60-100.05% for Cavinton ensuring that this method is accurate. The method is found to be specific for Cavinton.

Different validation parameters for proposed HPTLC method for determination of Cavinton content was summarized in Table 1.This method was applied to determine the content of Cavinton label claim 5 mg and was found to be 4.98mg and ; 99.56% (n=6). The result indicated that the proposed HPTLC method was found to be simple, accurate, precise and reproducible for estimation of Cavinton in marketed formulation.

Table 1: Summary of validation parameters

Sr. No.	Parameters	Cavinton
1.	Linearity range(ng /spot)	2000-7000
2.	Correlation coefficient (r²)	0.998
3.	Precision(% RSD)
	Intraday	0.2148 -0.5279
	Interday	0.1919 -0.4896
4.	Slope(m)	0.0001
5.	Intercept(c)	0.0116
6.	LOD	200
7.	LOQ	1000
8.	% Recovery	99.60-100.05%

CONCLUSION:

The results indicate that the proposed method is simple, accurate, precise and reproducible for estimation of Cavinton in bulk and marketed formulation.

REFERENCE:

1. Lorincz C, Szasz K, Kisfaludy L (1976). "The synthesis of ethyl apovincaminate". Arzneimittel-Forschung 26 (10a): 1907.

2. Szilagyi G, Nagy Z, Balkay L, et al. (2005). "Effects of vinpocetine on the redistribution of cerebral blood flow and glucose metabolism in chronic ischemic stroke patients: a PET study". Journal of the Neurological Sciences 229-230: 275–84.

3. Dezsi L, Kis-Varga I, Nagy J, Komlodi Z, Kárpáti E (2002). "Neuroprotective effects of vinpocetine in vivo and in vitro. Apovincaminic acid derivatives as potential therapeutic tools.

4. "Vinpocetine. Monograph". Alternative Medicine Review 7 (3): 240–3. 2002.

5. Sitges M, Galván E, Nekrassov V (2005). "Vinpocetine blockade of sodium channels inhibits the rise in sodium and calcium induced by 4-aminopyridine in synaptosomes". Neurochemistry International 46 (7): 533–40

6. Roghieh, Nanotechnology approaches to solving the problems of poorly water soluble drugs, Drug discovery world summer, 2005, 71-76

7. ICH, Q2A, Text on validation of analytical procedures, ICH, Geneva, Oct.- 1994 1-5.

Received on 21.06.2011 Modified on 21.07.2011

Asian J. Research Chem. 4(10): Oct., 2011; Page 1505-1507