INTRODUCTION:

Eugenol is an allyl chain-substituted guaiacol (C₁₀H₁₂O₂; 2-methoxy-4-(2-propenyl) phenol) (Fig. 1). Eugenol oil possesses significant pharmacological actions on various systems¹ and recently many studies have been reported on eugenol delivery systems.^2-8 Drug release or dissolution studies are essential part of product development is considered as one of the major quality control tool for dosage forms. Thus the development of a validated UV spectrophotometric method for in vitro eugenol release studies will always be valuable as the method is very easy and quick.

Fig. 1. Chemical structure of eugenol

Aqueous Polysorbate 80 (Tween 80^®) solutions at a concentration of 0.5% is a widely employed release testing medium for lipohilic drugs for a variety of drug delivery systems such as nanoemulsions and nanoparticles.^9,10 Moreover, 0.5% Tween 80 is a commonly used media for solubilizing eugenol.¹¹ Thus 0.5% w/v aqueous Tween 80 could be employed as the in vitro release studies of eugenol from its dosage forms. The mechanism of action of eugenol solubilization is presumably by micelle formation by the Tween 80 (polysorbate 80) molecules. This provides a sink condition and act as an efficient drug release testing medium for eugenol loaded formulations. Since Tween 80 is a non-ionic surfactant, 0.5% Tween 80 can be incorporated in any dissolution media of any desired pH, for dissolution studies of the eugenol loaded drug delivery system, without affecting the pH.

Till date no studies have been reported a validated UV spectrophotometric assay method for the estimation of eugenol in dissolution media containing Tween 80 as solubilizer. Towards this objective of quantification of eugenol efforts have been made towards the development and validation analytical method by UV spectrophotometry.

Materials and methods:

Materials:

Eugenol (pure) was purchased from Central Drug House, Delhi, India. Tween 80 was purchased from S D Fine-Chem ltd, Mumbai, India. Reagent grade I water (Millipore, Molsheim, France) was used for the study. All other chemicals and reagents used for the study were of analytical grade.

UV spectrophotometric method in 0.5% w/v aqueous Tween 80:

For the purpose of quantification of eugenol in the aqueous phases during the various in vitro evaluation of the developed nanoemulsion gel and nanoparticles of eugenol, development and validation of a UV spectrophotometric method was carried out in 0.5% w/v aqueous Tween 80 solution. The method was validated according to ICH guidelines, Q2 (R1) with respect to linearity and range, precision, accuracy, detection limit (DL) and quantitation limit (QL).¹²

Preparation of standard solutions:

For the preparation of standard plot different known concentrations of 100 mg of eugenol was accurately weighed and mixed well with 2.5 g of Tween 80. The mix was dissolved in 50 mL of distilled water. The solution was completely transferred into a 100 mL volumetric flask and the volume was made up to mark with distilled water to obtain a primary stock concentration of 1000 µg mL^-1. From this stock solution 10 mL was transferred into a 100 mL volumetric flask and was made up to volume with 0.25% w/v Tween 80. The resultant secondary stock had a eugenol concentration of 100 µg mL^-1 and a Tween 80 concentration of 0.5 % w/v. The dilutions of this secondary stock solution were made by diluting the required aliquot with 0.5 % w/v aqueous Tween 80 solution to obtain standard solutions in the range of 5- 50 µg mL^-1. The absorbance of the resultant solutions was determined at a wavelength of 282 nm (λ_max = 282.5 nm).

Linearity and range:

The calibration curve was plotted using the concentration range of 5 - 50 µg mL^-1. The absorbance of the solutions was determined at 282 nm. A calibration curve was constructed by plotting absorbance vs. concentration of standard solution and the regression equation was determined. The experiment was carried out in triplicate.

Accuracy as recovery:

Accuracy was determined by recovery studies using standard addition method. The pre-analyzed samples were spiked with extra 50, 100 and 150% of the standard eugenol and the mixtures were analyzed by the proposed method. The experiment was conducted in triplicate.

Precision:

Three concentrations of eugenol solution (10, 25 and 40 µg mL^-1) were prepared. The precision of the method was assessed by analyzing eugenol for repeatability and intermediate precision.

(a) Repeatability:

Repeatability (intraday) was assessed by analyzing eugenol in three different concentrations (10, 25 and 40 µg mL^-1) of three times a day. The % RSD was calculated for absorbance thus obtained, to obtain the intra-day variation.

(b) Intermediate precision:

Intermediate precision (inter-day) was established by analyzing three different concentrations (10, 25 and 40 µg mL^-1) of eugenol for three different days. The % RSD was calculated for absorbance thus obtained, to obtain the inter-day variation.

Detection and quantitation limits:

The detection limit (DL) is the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated. The quantitation limit (QL) is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. The limit of quantification and limit of detection were determined based on the technique of signal-to-noise ratio using the equations (1) and (2).¹²

QL = 10 σ / S --------------- (1)

DL = 3.3 σ / S --------------- (2)

Where, σ is the standard deviation of the intercept of the calibration plot and S is the slope of the calibration curve.

Results and discussion:

UV spectrophotometric method in 0.5% w/v aqueous Tween 80:

Fig. 2 shows the UV spectrum of eugenol in 0.5% aqueous Tween 80 with a λ_max of 282.5 nm.

Fig. 2 UV spectrum of eugenol in 0.5% w/v aqueous Tween 80

Linearity and range:

The absorbance of the prepared standard solutions (5-50 µg mL^-1) was determined at 282 nm. The mean absorbance was found to be 0.1217 – 1.2651. The plot of absorbance versus concentration (Fig. 3) obeyed Beer-Lambert’s law in above concentration range with regression coefficient of 0.9978.

Fig. 3. Calibration curve of eugenol in 0.5% w/v aqueous Tween 80 by UV spectrophotometry

Accuracy as recovery:

Accuracy was investigated by analyzing three concentrations of standard drug solution previously analyzed using standard addition technique. The recovery studies were carried out to check the sensitivity of the method to estimate eugenol. The standard addition technique was carried out by adding 50, 100 and 150% of the eugenol concentration in the sample. The % recoveries of the three concentrations were found to be 99.96 – 101.74 %, indicative of high accuracy. The values of % recovery and % RSD are displayed in Table 1. The mean % recovery values, close to 100%, and their low % RSD values indicated high accuracy of the analytical method.

Precision:

The precision method was assessed by analyzing eugenol in three different concentrations as 10, 25 and 40 µg mL^-1of eugenol.

(a) Repeatability:

Repeatability (intra-day) was assessed by analyzing eugenol in three different concentrations (10, 25 and 40 µg mL^-1) of eugenol three times a day. The % RSD was calculated for absorbance thus obtained, to obtain the intraday variation and is displayed in Table 2.

(b) Intermediate precision:

The low values of % RSD for repeatability and intermediate precision suggested an excellent precision of the developed UV spectrophotometric method.

Table 1 Recovery data for the accuracy of the UV method in 0.5% w/v aq. Tween 80

Excess of eugenol added (%)	Concentration of sample (μg mL^-1)	Theoretical concentration of spiked sample (μg mL^-1)	Concentration of spiked sample ± SD (μg mL^-1) (n=3)	Recovery ± SD (%)	% RSD of recovery
50	20	30	29.90±0.17	99.66±0.57	0.57
100	20	40	39.56±0.67	98.90±1.68	1.70
150	20	50	49.40±0.33	98.81±0.65	0.66

Table 2 Repeatability and intermediate precision

Concentration (µg mL^-1)	Repeatability (n=3)		Intermediate precision (n=3)
Concentration (µg mL^-1)	Mean absorbance at 282 nm ±SD	% RSD	Mean absorbance at 282 nm ±SD	% RSD
10	0.2676±0.0023	0.85	0.2644±0.0029	1.10
25	0.6474±0.0059	0.91	0.6444±0.0058	0.90
40	1.0611±0.0070	0.66	1.0545±0.0076	0.72

Table 3 Optical, linear regression and validation data (n = 3)

Parameter	Data (Mean ± SD)
Optical characteristics E_{1%, 1cm}	257.79±2.47
Regression analysis Slope Intercept Regression coefficient (R²)	0.0258±0.0002 0.0036±0.0049 0.9978±0.0004
Validation Range (µg mL^-1) Detection limit (DL) (µg mL^-1) Quantitation limit (QL) (µg mL^-1)	5 – 50 0.62 1.88

Detection and quantitation limits:

The detection limit (DL) and quantitation limit (QL) were determined as per the ICH guidelines and were found to be 0.62 and 1.88 μg mL^-1 respectively.

The summary of optical, linear regression and validation data are displayed in Table 3. The method was valid with respect to linearity and range, accuracy, precision, detection limit and quantitation limit.

Conclusions:

The UV spectrophotometric method in 0.5% w/v aqueous Tween 80 for the quantification of eugenol was successfully developed and validated. The method was validated in terms of linearity and range, accuracy and precision. The detection limit and quantitation limit were determined as per the ICH guidelines and were found to be 0.62 and 1.88 μg mL^-1 respectively. Thus it was confirmed that the developed method could be employed for the quantification of eugenol from 0.5% w/v aqueous Tween 80 solutions used as aqueous phase for in vitro release studies of eugenol loaded drug delivery systems. The present study could be extrapolated for the estimation of eugenol in any aqueous in vitro release media which contains 0.5% w/v Tween 80 as solubilizer provided we establish the linear regression equation.

Acknowledgements:

Pramod K. gratefully acknowledges Indian Council of Medical Research (ICMR), New Delhi, India, for providing Senior Research Fellowship (No. 35/3/10/NAN/BMS).

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Received on 08.07.2013 Accepted on 30.07.2013

Asian J. Pharm. Ana. 3(3): July-Sept. 2013; Page 86-89