Development and validation of UV spectrophotometric method for the quantitative estimation of eugenol

 

K. Pramod1, Shahid H. Ansari2, Javed Ali1*

1Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India.

2Department of Pharmacognosy & Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India.

*Corresponding Author E-mail: javedaali@yahoo.com

 

ABSTRACT:

Eugenol is an allyl chain-substituted guaiacol and finds variety of applications. Thus development of a validated UV spectrophotometric method will always be advantageous as the method is simple and rapid. The method was validated according to International Conference on Harmonisation (ICH) guidelines Q2(R1) with respect to linearity and range, precision, accuracy, detection limit (DL) and quantitation limit (QL). 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.

 

KEYWORDS: ICH, linearity, range, precision, accuracy, detection limit, quantitation limit

 


 

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 g mL-1. The absorbance of the resultant solutions was determined at the λmax of 282 nm.

 

Linearity and range

The calibration curve was plotted using the concentration range of 5 - 50 g 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 g 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 g 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 g 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 g 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-Lamberts 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 g mL-1 of eugenol.

 

(a) Repeatability

Repeatability (intra-day) was assessed by analyzing eugenol in three different concentrations (10, 25 and 40 g 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

Intermediate precision (inter-day) was established by analyzing three different concentrations (10, 25 and 40 g mL-1) of eugenol for three different days. The % RSD was calculated for absorbance thus obtained, to obtain the inter-day variation and is given in Table 2.

 

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

29.990.48

99.96 1.60

1.60

100

20

40

40.700.56

101.74 1.39

1.37

150

20

50

49.950.54

99.89 1.07

1.08

 

Table 2 Repeatability and intermediate precision

Concentration

(g mL-1)

Repeatability (n=3)

Intermediate precision (n=3)

Mean absorbance

at 282 nm SD

% RSD

Mean absorbance

at 282 nm SD

% RSD

10

0.21190.0039

1.83

0.21250.0033

1.56

25

0.51830.0043

0.82

0.51450.0053

1.03

40

0.83430.0078

0.93

0.83310.0061

0.73

 

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

Parameter

Data (Mean SD)

Optical characteristics

E1%, 1cm

 

204.642.54

Regression analysis

Slope

Intercept

Regression coefficient (R2)

 

0.02060.0003

0.00630.0051

0.99870.0005

Validation

Range (g mL-1)

Detection limit (DL) (g mL-1)

Quantitation limit (QL) (g mL-1)

 

5 50

0.82

2.48

 

References:

1.        Pramod K, Ansari SH and Ali J. Eugenol: A natural compound with versatile pharmacological actions. Nat Prod Commun. 5 (12); 2010: 1999-2006.

2.        Kriegel C, Kit KM, McClements DJ, Weiss J. Nanofibers as carrier systems for antimicrobial microemulsions. II. Release characteristics and antimicrobial activity. J Appl Polym Sci. 118; 2010: 28592868.

3.        Pokharkar VB, Shekhawat PB, Dhapte VV, Mandpe LP. Development and optimization of eugenol loaded nanostructured lipid carriers for periodontal delivery. Int J Pharm Pharm Sci. 3; 2011: 138-143.

4.        Gomes C, Moreira RG, Castell-Perez E. Poly (DL-lactide-co-glycolide) (PLGA) nanoparticles with entrapped trans-cinnamaldehyde and eugenol for antimicrobial delivery applications. J Food Sci. 76 (2); 2011: N16-N24.

5.        Shu-Ya J. 2010. Encapsulation of active substance in nanocapsules by emulsion-diffusion method [Accessed 12 February 2011]. Available from: URL: http://140.121.155.217/ seminar/19932033-1.pdf.

6.        Shah A, Garg A, Sairam K, Singh S. 2010. Pharmacological evaluation of eugenol loaded solid lipid nanoparticles in irritable bowel syndrome [Accessed 10 March 2011]. Available from: URL: www.scientificipca.org/paper/2009/09/25/20090925 1910570A.doc.

7.        Chen F, Shi Z, Neoh KG, Kang ET. Antioxidant and antibacterial activities of eugenol and carvacrol-grafted chitosan nanoparticles. Biotechnol Bioeng. 104 (1); 2009: 30-39.

8.        Jadhav BK, Khandelwal KR, Ketkar AR, Pisal SS. Formulation and evaluation of mucoadhesive nanocapsules containing eugenol for the treatment of periodontal diseases. Drug Dev. Ind. Pharm. 30; 2004: 195-203.

9.        Chaieb K, Hajlaoui H, Zmantar T, Kahla-Nakbi AB, Rouabhia M, Mahdouani K, Bakhrouf A. The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother. Res. 21; 2007: 501-506.

10.     ICH-Guidelines Q2(R1). Validation of Analytical Procedures: Text and Methodology. Geneva, Switzerland: 2005.

 

 

Received on 18.04.2013 Accepted on 25.05.2013

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