Article pubs.acs.org/JAFC
Determination of Melatonin in Rice (Oryza sativa) Grains by Pressurized Liquid Extraction Widiastuti Setyaningsih,†,‡ Irfan Estiono Saputro,‡ Gerardo Fernández Barbero,‡ Miguel Palma,*,‡ and Carmelo García Barroso‡ †
Department of Food and Agricultural Product Technology, Faculty of Agricultural Technology, Gadjah Mada University, Jalan Flora 55281, Yogyakarta, Indonesia ‡ Department of Analytical Chemistry, Faculty of Sciences, University of Cadiz, Campus de Excelencia Internacional Agroalimentario (CeiA3), Campus del Rio San Pedro, 11510 Puerto Real, Cádiz, Spain ABSTRACT: Melatonin provides a number of benefits for human health. The study reported here concerns the optimization, validation, and application of analytical pressurized liquid extraction and high-performance liquid chromatography coupled to a fluorescence detector for the determination of melatonin in rice grains. The factors that are most likely to affect the extraction efficiency were optimized with a 27−2 IV fractional factorial design. The optimum extraction conditions were achieved by applying 70% EtOAc in MeOH at 200 °C and 200 atm for a static time of 5 min in two cycles with 50% flushing and a 60 s purge to extract a 2.5 g rice sample. The method validation ensured excellent linearity, limit of detection, limit of quantification, precision, and recovery. Furthermore, the method was applied to various rice products composed of polished, whole grain, aromatic, black, black glutinous, red, and parboiled rice. All kinds of pigmented rice grains showed high levels of melatonin (>100 μg kg −1), and the highest levels were found in red rice. KEYWORDS: melatonin, pressurized liquid extraction, HPLC-FD, rice grains
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INTRODUCTION As a grain of life, cultivated rice (Oryza sativa) serves as the foremost food for more than half of the world’s population.1 Moreover, rice is essential because it contains naturally occurring bioactive compounds such as melatonin [N-acetyl3-(2-aminoethyl)-5-methoxyindole] (Figure 1).2,3 Recent re-
rice, which contains a diverse range of primary and secondary metabolites. The extraction of melatonin from food is associated with certain difficulties due to its powerful antioxidant activity, which often leads to rapid reaction with other components in the matrix or in the environment.9 Pressurized liquid extraction (PLE) offers a solution to this problem because it is an advanced extraction technique that can enhance the diffusion rate of solvents, as the extraction is carried out with solvents at a high pressures (50−200 atm) and temperatures (50−200 °C) without the critical point being reached.10 This approach leads to a more rapid mass transfer of analytes from the matrix into solvent, and this increases the rate of the extraction. Furthermore, because PLE can operate under a nitrogen atmosphere, oxidation or degradation of melatonin would be minimized. Recent studies have shown that PLE is an important sample preparation technique, and this approach has been used to increase the extraction efficiency for various bioactive compounds.11,12 The most important feature of the PLE technique is that the composition of the extract can be manipulated by adjusting the extraction factors such as temperature, pressure, time, extraction cycles, and solvent. As a consequence, the chemical composition of the extract obtained can be altered by changing the PLE conditions.13 A comprehensive review of PLE has highlighted the factors that affect the yield of the extraction, and these are
Figure 1. Chemical structure of melatonin.
search has led to an increase in the production of melatonin in transgenic rice seeds, and melatonin-rich rice plants have been successfully generated.4 Functional rice products have also become more widely appreciated in the current market.5 Research into melatonin was started almost a century ago by Heinlein,6 and this has continued to be an active field to the present day, with many results revealing the utility of this compound. Melatonin diminishes neuro-degenerative diseases, such as Parkinson’s and Alzheimer’s diseases,7 and it also acts as an anticancer agent.8 However, further research into the role of melatonin in biological systems has been limited by encountering difficulties due to the low concentrations of this compound in the matrices, the limited number of reliable analytical methods, and the complexity of the biological matrices. Hence, it is of great interest to extract and quantify accurately melatonin contents, especially in plant tissues such as © 2015 American Chemical Society
Received: Revised: Accepted: Published: 1107
October 23, 2014 December 6, 2014 January 8, 2015 January 8, 2015 DOI: 10.1021/jf505106m J. Agric. Food Chem. 2015, 63, 1107−1115
Article
Journal of Agricultural and Food Chemistry predominantly pressure, temperature, and solvent type.10 As a number of factors can influence the course of the extraction, the screening and optimization of the significant factors must be carried out to establish a reliable analytical PLE method. In the study described here, seven extraction factors were evaluated, namely, solvent composition, extraction temperature, pressure, flushing, static time, solvent purge, and sample weight. In PLE the pressure generally acts by forcing liquid into the solid matrix.14 In addition, an increase in pressure leads to an increase in the extraction rate because, if sufficient pressure is exerted on the solvent during extractions, temperatures above the boiling point can be reached while the solvent is maintained in the liquid state.15 Temperature plays a role in enhancing the extraction yield because higher temperatures can decrease the viscosity of liquid solvents, thus permitting faster diffusion rates and better penetration into the matrix. Furthermore, thermal energy can disrupt the strong interactions between the solute molecules and active sites on the matrix, and this can aid the release of target molecules. However, as melatonin is sensitive to temperature,16 a preliminary study on its stability under different PLE temperatures was required to define the working temperature range for the optimization. The extraction yields from plant samples are highly dependent on the nature of the solvent employed17 because of the effects of both the solvent diffusivity and the solvent polar properties. The polarity of the solvent will affect the extraction depending on the polar properties of the target compound. On the other hand, the enhanced diffusivity of the solvent leads to an increase in extraction speed and efficiency.18 The amphipathic structure of the melatonin molecule, which is composed of polar and nonpolar moieties, makes it difficult to select a solvent that yields complete recovery.19 In the study described here, this constraint was therefore taken into account in a preliminary study by screening several solvents, including water, methanol, ethanol, and ethyl acetate, prior to the selection of the extraction solvents to be used in the optimization experiments. A factorial design with a reduced number of runs is preferable to a full factorial design for more than four factors, particularly when the available resources are limited. Therefore, a chemometrical approach based on a fractional factorial design (FFD) is a reasonable option to evaluate the significance of the studied factors prior to optimization of the PLE conditions.20,21 The particular focus of this study was on the optimization and validation of PLE for the extraction of melatonin from rice grains using an FFD.
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extraction method was applied to a wide range of rice products available in the market, and these covered the varieties of polished (short and long) grain, whole (short and semilong) grain, aromatic rice, black rice, black glutinous rice, red rice, and parboiled rice products. Samples were acquired in several different markets in Spain. Extraction of Melatonin. Pressurized liquid extraction (PLE) of melatonin was performed in a Dionex ASE 200 extractor (Dionex, Sunnyvale, CA, USA) equipped with stainless steel extraction cells (11 mL volume) and collection vials (60 mL capacity). A cellulose filter (Dionex) was inserted at the outlet end of the extraction cell. Rice powder was accurately weighed and loaded into the extraction cell, and washed sea sand was used to complete the extraction cell. The extraction cell was then filled with the extraction solvent (80−100% EtOAc in MeOH), pressurized (100−200 atm), and heated (100−200 °C). The cell was heated for a fixed time to guarantee that the sample reached thermal equilibrium. The sample was then extracted in a static extraction cycle (5−10 min) with the solvent at the experimental temperature and pressure. After the extraction, the cell was rinsed with fresh solvent (50−100% of the extraction cell volume) and purged with a flow of nitrogen for 60−120 s. The extract was dried under vacuum in a rotary evaporator. The residue was reconstituted with methanol and adjusted to a final volume of 5 mL. The liquid was then passed through a 0.45 μm nylon filter prior to injection in the HPLCFD system. Determination of Melatonin. High-performance liquid chromatography (HPLC) analyses were carried out on an Alliance HPLC 2695 system with a fluorescence detector (Waters 474 fluorescence detector), controlled by an Empower Pro 2002 data station (Waters, Milford, MA, USA). Separations were performed in a reverse phase RP 18 Lichrospher Column [LiChroCART 250 × 4 (5 μm)] from Merck at a temperature of 25 °C. The mobile phase was a binary solvent system consisting of phase A (2% acetic acid and 5% methanol in water) and phase B (2% acetic acid and 88% methanol in water) with a flow rate of 0.5 mL min−1. The mobile phase was filtered through a 0.45 μm membrane filter (Millipore) and degassed for 15 min prior to use. The 25 min programmed gradient was as follows: (time, solvent B): 0 min, 0%; 5 min, 35%; 12 min, 40%; 15 min, 40%; 20 min, 45%; 25 min, 50%. After each extraction, the column was washed with 100% B for 5 min and equilibrated with 0% B for 5 min. The established conditions for the fluorescence detector were as follows: excitation wavelength, 290 nm; emission wavelength, 330 nm; gain, 1000; attenuation, 16; injection volume, 10 μL. Performance of the Method. The chromatographic method used to determine melatonin was performed according to the suggestions of ISO 17025 and the recommendations of ICH Guideline Q2 (R1).22,23 The range, linearity, recovery, precision, detection, and quantification limits of the method were evaluated. Linearity was estimated to express how the method provides test results that are directly proportional to the melatonin concentration within the studied range. A series of dilutions from a stock solution of melatonin were carried out to give concentrations ranging from 0.75 to 15 μg L−1 and from 15 to 750 μg L−1. Gnumeric 1.12.17 was used to generate the regression analysis of the calibration curves. The standard deviation estimated for the response and the slope from the regression were then used to calculate the limit of detection (LOD) and the limit of quantification (LOQ). A certified reference material was not available for melatonin in rice matrices and, as a consequence, definitive statements cannot be made with regard to accuracy. Nonetheless, the extraction recovery (%R) was determined by comparing the absolute response of the analytes spiked in control samples before and after the extraction procedure. The precision of the method was evaluated by studying the repeatability (intraday) and intermediate precision (extraday). Repeatability was assessed by nine independent analyses of the same samples on the same day, whereas intermediate precision was determined by three independent analyses on three consecutive days. Precision was expressed as the coefficient of variance (CV) of the retention time and peak height. The acceptable CV limit is ±10% according to the AOAC manual for the Peer-Verified Methods
MATERIALS AND METHODS
Chemicals and Reagents. HPLC grade methanol (MeOH), ethanol (EtOH), ethyl acetate (EtOAc), and acetic acid were purchased from Merck (Darmstadt, Germany). Melatonin standard was obtained from Sigma-Aldrich (St. Louis, MO, USA). Water was purified with a Milli-Q purification system (Millipore, Billerica, MA, USA). Rice Sample Preparation. Red rice samples from Thailand were used in this study. A rice sample (20 g) was placed in a plastic cylinder, and the rice grains were milled with an Ultraturrax homogenizer (IKA T25 Digital, Germany) for 10 min prior to extraction. The milling process was stopped every minute to avoid excessive heating of the sample. The fine powder of rice grain was then homogenized by stirring, and the samples were stored at room temperature in a closed container until analysis (1−5 weeks depending on the sample). No specific humidity conditions were applied during storage. The final 1108
DOI: 10.1021/jf505106m J. Agric. Food Chem. 2015, 63, 1107−1115
Article
Journal of Agricultural and Food Chemistry Table 1. Analytical Characteristics for the Determination of Melatonin by HPLC-FD intraday, CV (%)
n=3×3
n=9 −1
−1
2
linear range (μg L )
observations
linear eq
R
0.75−15 15−750
6 6
y = 580.79x − 1806.8 y = 615.11x − 961.13
0.996 0.999
program.24 The CV values with reference to the peak height for both repeatability and intermediate precision were
