INTRODUCTION:

Eugenol (C₁₀H₁₂O₂; 2-methoxy-4-(2-propenyl) phenol), is an allyl chain-substituted guaiacol (Fig. 1). Eugenol oil possesses anti-inflammatory and anesthetic properties along with its recognized antioxidant, antimicrobial, antiviral and antifungal activities. Thus eugenol has been recognized as a potent pharmacologically phytochemical¹ and good number of eugenol delivery systems have been reported for a variety of applications.^2-8 Eugenol has its applications in the fragrance and flavoring industries also.⁹ Thus development of a validated UV spectrophotometric method will always be advantageous as the method is simple and rapid.

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

Fig. 1. Chemical structure of eugenol

Materials and methods:

Materials

Eugenol (pure) was purchased from Central Drug House, Delhi, India. Methanol was purchased from S D Fine-Chem ltd, Mumbai, India. Reagent grade I water (Millipore, Molsheim, France) was used for the study.

UV spectrophotometry

A Shimadzu UV – 1601 (Shimadzu Corp, Kyoto, Japan) spectrophotometer was employed in the study. The method was validated according to ICH guidelines, Q2(R1)¹⁰ with respect to linearity and range, precision, accuracy, detection limit (DL) and quantitation limit (QL).

Preparation of standard solutions

Eugenol (100 mg) was dissolved methanol in a 100 mL volumetric flask and then the volume was made up with methanol. The dilutions of this stock solution were made by diluting the required aliquot with methanol to obtain standard solution in the range of 5- 50 µg mL^-1. The absorbance of the resultant solutions was determined at the λ_max of 282 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 ratio¹⁰ using the equations (1) and (2).

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 spectrophotometry

The UV method for the estimation of eugenol in methanol was validated. Fig. 2 shows the UV spectrum of eugenol in methanol with absorption peaks at 210, 226.0 and 281.5 nm.

Fig. 2. UV spectrum of eugenol in methanol

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 0.1100 – 1.0182. The plot of absorbance versus concentration (Fig. 3) obeyed Beer-Lambert’s law in above concentration range with regression coefficient of 0.9987.

Fig. 3. Calibration curve of eugenol in methanol by UV spectrophotometry (282 nm)

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 given 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.

Detection and quantitation limits

The detection limit (DL) and quantitation limit (QL) were determined as per the ICH guidelines and were found to be 0.82 and 2.48 μg mL^-1 respectively.

Table 3 summarizes the optical, linear regression and validation data of UV spectrophotometry for the quantification of eugenol in methanol.

Conclusions:

The UV spectrophotometric method using methanol as solvent 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 (DL) and quantitation limit (QL) were determined as per the ICH guidelines and were found to be 0.82 and 2.48 μg mL^-1 respectively. The method is expected to be useful in a variety of industries where eugenol finds its application.

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).

Table 1 Recovery data for the accuracy of the UV method in methanol

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
50	20	30	29.99±0.48	99.96± 1.60	1.60
100	20	40	40.70±0.56	101.74± 1.39	1.37
150	20	50	49.95±0.54	99.89± 1.07	1.08

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	0.2119±0.0039	1.83	0.2125±0.0033	1.56
25	0.5183±0.0043	0.82	0.5145±0.0053	1.03
40	0.8343±0.0078	0.93	0.8331±0.0061	0.73

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

Parameter

Data (Mean ± SD)

Optical characteristics

E_{1%,
1cm}

204.64±2.54

Regression analysis

Slope

Intercept

Regression coefficient (R²)

0.0206±0.0003

0.0063±0.0051

0.9987±0.0005

Validation

Range (µg mL^-1)

Detection limit (DL) (µg mL^-1)

Quantitation limit (QL) (µg mL^-1)

5 – 50

0.82

2.48

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Received on 18.04.2013 Accepted on 25.05.2013

Asian J. Pharm. Ana. 3(2): April- June 2013; Page 58-61