Determination of Safrole in Ethanol Extract of Nutmeg ( Myristica fragrans Houtt ) Using Reversed-Phase High Performance Liquid Chromatography

Febrina Amelia Saputri, Mutakin, Keri Lestari & Jutti Levita 1 Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Jl. Raya Bandung-Sumedang Km. 21, Jatinangor, West Java, Indonesia 2 Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Jl. Raya Bandung-Sumedang Km. 21, Jatinangor, West Java, Indonesia


Introduction
Dehydrodiisoeugenol (DDIE), myristicin, and safrole are chemical compounds contained in fruit and seed of nutmeg (Myristica fragrans Houtt).DDIE shows antidiabetic activity on PPARγ receptor, while myristicin is hallucinogenic agent.Of the three compounds, safrole is the most toxic substance due to its carcinogenic activity (Lestari, 2010;Li & Yang, 2012).Safrole and isosafrole (0.5%) has been shown to increase the occurance rate of malignant tumours in mice (Benedetti, Malnoe, & Broillet, 1977).The major toxicity of safrole is caused by its metabolite character.Safrole is oxidized into 1-hydroxysafrole in human body, which is carcinogenic (Peele Jr. & Oswald, 1978).The maximum dose of safrole as stated by UK and French governments is 1 mg/day (European Commission, 2002).
Currently Indonesian pharmaceutical industry is developing a formula of nutmeg extract as antidiabetic drug, based on its DDIE activity which inhibits PPARγ receptor, therefore a rapid and accurate method for the quantification of safrole in nutmeg extract is interesting to be developed.At present, methods of analysis of safrole are HPLC and GC (Archer, 1988;France, Association Francaise de Normalisation, 1986;Choong & Lin, 2001).AOAC described a procedure to determine safrole and isosafrole in soft drinks, where both compounds have to be distilled with steam, extracted with organic solvent, e.g CHCl 3 , and then injected into GC column for separating and analyzing steps.HPLC using small particles with a high-pressure pump system and sensitive detector.Advantage of HPLC is to provide high-resolution, efficient and fast separation (Skoog, Holler, & Nieman, 1992;Willard, Merrit Jr., Dean, & Settle Jr., 1988;Nagore, Vinod, Pankaj, & Tushar, 2013;Chan, Herman, Lee, & Zhang, 2004).The aim of this study was to determine the concentration of safrole in ethanol extract of nutmeg.

Preparation of Standard Solution
Standard solution was prepared by diluting safrole in methanol (16 μg/mL).

Preparation of Extract Solution
Nutmeg extract was accurately weighed 100 mg and dissolved in 10 mL methanol (Chan, Herman, Lee, & Zhang, 2004).The concentration of the extract solution was 10000 μg/mL.

Preparation of Mobile Phase
Mobile phase was chosen based on the solubility of safrole.In this study, the mobile phase was a mixture of methanol:water (73:27).This composition was selected based on the result of resolution value (> 1.5) in optimization steps, as shown in Table 1.

HPLC Conditions
Analysis of safrole was performed at room temperature on isocratic conditions at a flow rate 1 mL/min, and was detected at 282 nm using both UV and PDA detectors.

Validation of Analytical Method [According to ICH Guidelines]
2.6.1 Linearity Standard solution was pipetted 6.25; 12.5, 25, 50, and 100 mL, respectively, and inserted into the eppendorf tube.The solutions were adjusted using methanol to obtain solutions of 1, 2, 4, 8, and 16 µg/mL, respectively.The solutions were injected into the chromatograph injection gate.Linearity is determined based on the correlation coefficient (r) in the linear regression y = bx + a from the curve of the relationship between area with concentration (Archer, 1988).

Precision
Precision is measured by determining three concentrations of analyte with three times replication or minimum six times replication at 100% concentration of analyte (ICH Harmonised Tripartite Guideline, 1996).Standard www.ccsen solution w solutions.

Limit o
LOD and following SD: Stand

Determ
Nutmeg ex 20 µL ext extract wa  Safrole was detected at 10.45 minute (Figure 2), indicating that this compound retained in the nonpolar stationary phase due to its lipophilic character, while myristicin showed a double peak at 7.5 minute.This double peak probably was caused by poor separation of myristicin and its analogue, elemicin (Figure 3) due to their similar polarity.
(a) (b) (c) Figure 4. Linear responses of peak area against the concentrations of DDIE (a), myristicin (b) and safrole (c) All three curves indicated that the instrument response is proportional with the concentration.These data showed that Lambert-Beer law was obeyed by the condition of analytical method for all three compounds within the interval of concentrations selected.

Accuracy and Precision
Determination of accuracy and precision obtained from the calculation of six times measurements at concentration 4 µg/mL.The measurement results are shown in Table 2. Instrument response (area) entered into the linear regression equation which has been previously calculated, so that the measured concentration was obtained.Then the recovery and coefficient of variance were calculated, i.e. 101.421% and 0.838%.The values show that the method used for safrole quantification presents high accuracy and precision.LOD and LOQ were calculated with the formula previously described, the SD values was obtained by the formula: SD = ∑ .In Table 3, calculations have been described to obtain the value of (y-y 1 ) 2 .By inserting these values into the formula, with n = 5, obtained SD is 0.0257.LOD and LOQ of safrole with this method are 0.668 µg/mL and 2.023 mg/mL, respectively.LOD and LOQ indicate safrole concentration limit that can still be detected and quantized using this method.

Determination of Safrole in Nutmeg Seed Extract
Determination of safrole concentration in the extract was calculated by entering the instrument's response (area) to the linear regression equation.Safrole concentration from three measurements are shown in Table 4. Average concentration of safrole contained in nutmeg seed extract is 10.979%.Standard deviation and coefficient of variance, respectively are 0.005 and 0.045, respectively.

Table 1 .
Determination of Resolution of Analytes in different mobile phase composition

Table 2 .
Accuracy and precision of safrole

Table 3 .
Determination of LOD and LOQ

Table 4 .
Determination of safrole in the extract4.ConclusionOur HPLC method can be used to determine the concentration of safrole in nutmeg seed extract with methanol: water (73:27) as mobile phase, flow rate 1 mL/min.Detection wavelength was at 282 nm.This method fulfilled validation criterias, excluding specifity and robustness.Mean concentration of safrole in the nutmeg seed extract is 10.979%.