Research Article Article
Magnetic Stirring-Assisted Dispersive Liquid? Liquid Microextraction of Naringenin from Grapefruit and Its Determination by High Performance Liquid Chromatography
Seyed Elias Pourghorban, Mohammad Reza Hadjmohammadi*, Elias Ranjbari
Department of chemistry, University of Mazandaran, Babolsar, Iran
*Corresponding author: E-mail: email@example.com (M.R.H)
Citation: Pourghorban SE, Hadjmohammadi MR, Ranjbari E. Magnetic Stirring-Assisted Dispersive Liquid–Liquid Microextraction of Naringenin from Grapefruit and Its Determination by High Performance Liquid Chromatography. J Res Anal. 2017; 3(3)
Naringenin is one of the most important flavonoids with antioxidant property which exists in abundance in grapefruit. Regarding to the effective role of naringenin in prevention of diseases such as cancer and hematite C, extraction and determination of this compound is very important. In this work, a sensitive, rapid and efficient method for the extraction of naringenin as well as its determination in grapefruit sample was developed using magnetic stirring assisted dispersive liquid–liquid microextraction (MSA-DLLME) followed by HPLC-UV. The extraction method is based on the application of an extraction solvent lighter than water in the ternary component solvent (aqueous solution: extraction solvent: disperser solvent) system. The experimental factors affecting the extraction recovery including type and volume of extraction and disperser solvents, pH of sample solution, salt addition and extraction time on extraction recovery were optimized. Under the optimum extraction condition (100 μL 1-octanol as the extraction solvent, 300 μL methanol as the disperser solvent, pH of the sample solution = 4.5 and no addition of NaCl to the sample solution), limit of detection, the extraction recovery and enrichment factor were 0.3 ng mL-1, 86% and 62, respectively; and the obtained linear range was between 1 ng mL-1 and 700 ng mL-1 (R2 = 0.9993). The method was successfully applied for the preconcentration and determination of naringenin in grapefruit sample.
Naringenin (Figure 1) is one of the most important flavonoids with antioxidant property which is the main cause of the bitter taste of citrus fruits [1,2]. It has several biological activities such as antioxidant, anti-carcinogenic, anti-inflammatory, and anti-estrogenic effects [3,4]. Naringenin can pervent the damage of DNA in the exposure of UV-B , inhibits the aggregation and production of infections hepatitis c virus , and induce DNA repair in prostate cancer cells . Grapefruit is replete with niaringenin and among the citrus fruits, grapefruit (Citrus paradisi) has the most amount of this anti-carcinogenic compound. This compound determines the quality of grapefruit and grapefruit products. The consumption of grapefruit leads to increase the oral bioavailability of a number of drugs including cyclosporin, midazolam, triazolam, felodipine and nifedipine . This phenomenon is attributed to the inhibitory effect of naringenin on the intestinal CYP3A4 .
Regarding to the susceptibility of naringenin in treatment of diseases, quantification of naringenin with the aim of determination of the quality of different kinds of grapefruits and different brands of grapefruit products as well as study on its drug interactions is very important. Until now, numerous analytical methods such as capillary electrophoresis (CE) with electrochemical detection , high performance liquid chromatography (HPLC) with UV , photo-diode array (PDA)  and mass spectrometric (MS) detections  have been employed for the quantification of naringenin concentration in the grapefruit samples. However, due to the complex matrix of the real samples and the low concentration of naringenin, making efforts to develop a simple and reliable method for isolation, preconcentration and determination of the naringenin is the main challenge and a very important step for its analysis. The preconcentration technique, which is commonly used to monitor naringenin is liquid-liquid extraction [10–12]. This traditional method is expensive, time consuming and labor intensive. To the best of our knowledge, despite the development of liquid phase microextraction methods, there isn’t any report about the extraction of naringenin from grapefruit using new methods. In the last decade, dispersive liquid-liquid microextraction (DLLME) method has attracted the attention of many scientists. DLLME has been developed by Assadi and co-workers  in 2006. DLLME is a new mode of LLE in miniaturized levels, which in comparison with the older classical methods, consumption of organic extracting solvent in DLLME is significantly lower and the enrichment factor is much higher. DLLME employs a mixture of a high density extracting solvent and a water-miscible polar disperser solvent. After a rapid injection of an appropriate mixture of extracting and disperser solvents into the aqueous sample, a cloudy solution is formed. The contact area between the extracting solvent and the sample solution is extremely large; thus, the extraction equilibrium is obtained rapidly. After centrifugation, the sedimented phase is settled at the bottom of the conical test tube and it can be analyzed by analytical instruments.
However, despite several advantages of DLLME, this conventional method employs extraction solvents with higher density than water; therefore, withdrawing the extraction product is carried out from the bottom of the conical test tube which is associated with some problems in the real samples, owing to the presence of the interfering materials. Also, chloroform, carbon tetrachloride, and dichloromethane which are usually used in conventional DLLME, show bad chromatographic behavior in HPLC, and should be evaporated before the injection to the HPLC column . On the other side, the extraction solvents that are typically used in the conventional DLLME are chlorobenzene, chloroform, carbon tetrachloride, and carbon disulfide which they are toxic and environmentally unfriendly. To overcome these problems, Farajzadeh et al.  introduced DLLME with the extraction solvents lighter than water which are settled at the top of the sample solution, after centrifugation. Furthermore, because of the good chromatographic behavior of low density solvents in comparison with chlorinated solvents, the evaporation step is eliminated [16-18]. However, our previous investigations [17,18] indicated that injection of the mixture of disperser and extracting solvents into the vortex of sample solution, makes more stable cloudy solution; hence, in this work, extraction and preconcentration of naringenin in the grapefruit samples was performed by magnetic stirring-assisted dispersive liquid–liquid microextraction (MSA-DLLME). Then, it was followed by HPLC-UV for determination of naringenin. Effect of several experimental factors consist of type and volume of extraction and disperser solvents, pH of sample solution, stirring rate, extraction time and salt addition on the extraction recovery were studied and optimized.
2.1. Chemicals and stock solutions
Naringenin was purchased from Sigma–Aldrich (Steinheim, Germany). Methanol, acetonitrile, and acetone (HPLC-grade), were obtained from Merck (Darmstadt, Germany). Xylene, n-hexane, toluene, dodecane, 2-ethylhexanol and 1-octanol were obtained from Aldrich (Milwaukee, WI, USA). The water used for mobile phase was double distilled deionized. A stock standard solution of naringenin (100 mg L-1) was prepared in methanol. The working solutions were prepared by appropriate dilution of the stock solution with double distilled/deionized water. All of the standard solutions were stored in the dark at 4°C.
2.2. Instrumentation and operating condition
Chromatographic measurements were carried out using an HPLC system equipped with a series 10-LC pump, UV detector model LC-95 set at 290 nm and model 7725i manual injector with a 20 µL sample loop (Perkin-Elmer, Norwalk, CT, USA). Column used was C18 (250 × 4.6 mm, 10 µm particle size) from Dr. Maisch GmbH (Ammerbuch-Entringen, Germany). Mobile phase was a mixture of acetonitrile and water (35:65, v/v) with flow rate of 1.0 mL min-1. Adjustment of pH was done by model 3030 Jenway pH meter (Leeds, UK). A Hettich Rotanta centrifuge model MIKRO 22R (Kirchlengern, Germany) was used to accelerate the phase separation.
2.3 Magnetic stirring–assisted dispersive liquid–liquid microextraction procedure
A 10 mL of sample solution containing 10 ng mL-1 of naringenin was placed in the handmade centrifuge tube with narrow neck (4 mm i.d.) which was specially designed for ease of withdrawing supernatant phase. A mixture of disperser solvent (300 µL methanol) and extracting solvent (100 µL 1-octanol) was injected into the sample solution using 1.0 mL syringe, rapidly and followed by vortex mixing at 1000 rpm stirring rate to achieve cloudy mixture. The cloudy solution was centrifuged for 5 min at 3500 rpm. Then the extraction product as supernatant was collected at the top of handmade centrifuge tube (about 80 ± 2 µL) and it was injected to the HPLC for the quantification analysis. All the experiments were performed in triplicates and average of the results was reported.
3. Results and Discussion
In order to achieve good sensivity and precision for extraction and determination of naringenin, several experimental factors consist of type and volume of extraction and disperser solvents, pH of sample solution, stirring rate, extraction time and salt addition affecting on the extraction recovery were studied and optimized by one variable-at-a-time optimization method.
3.1. Optimization of MSA-DLLME
In order to obtain the optimized extraction condition, extraction recovery percent (ER%) and preconcentration factor (PF) for naringenin were calculated by the following equations .
In these equations n0 and nsup, are the number of moles of analyte in the aqueous sample and supernatant phase, respectively. Also, C0, Csup, V0 and Vsup show the initial concentration of the analyte in sample solution, concentration analyte in the supernatant phase, volume of aqueous sample and supernatant phase, respectively.
3.1.1. Selection of extraction and disperser solvents
Selection of an appropriate extraction and disperser solvents is a necessery factor to obtain a good extraction recovery of MSA-DLLME of naringenin. In order to extract the analytes efficiently, the extraction solvent must have lower density than water and low solubility in water. Hence, 1-octanol (density, 0.82 g mL-1), 2-ethylhexanol (density, 0.83 g mL-1), n-hexan (density, 0.65 g mL-1), dodecane (density, 0.75 g mL-1), toluene (density, 0.87 g mL-1) and xylene (density, 0.86 g mL-1) were tested to find the most suitable extraction solvent for naringenin.
The miscibility of disperser solvent with extraction solvent and sample solution is crucial. For extraction of naringenin acetonitrile, methanol and acetone were tested as the disperser solvent. To obtain a high extraction recovery, all combinations of 250 µL of different extraction solvents were tried with 400 µL different disperser solvents. Figure 2 indicates that the methanol as disperser solvent and 1-octanol as extraction solvent are resulted in maximum extraction recovery, 82%. Thus, 1-octanol/methanol combination was selected for subsequent experiments.
3.1.2. Influence of extraction solvent volume on the extraction recovery
To check influence of extraction solvent volume, the volume of methanol as disperser solvent fixed at 400 µL and different volumes of 1-octanol were examinated for extraction of naringenin from standard sample solution (100 ng mL-1). The volume of 1-octanol was changed in the range of 50–350 µL because at volumes lower than 50 µL supernatant organic phase was not formed at the top of aqueous phase and volume upper than 350 µL were not been considered for saving and environment protection. The results shown in table 1 indicates that by increasing the volume of 1-octanol as extraction solvent up to 100 µL the extraction recovery and preconcentration factor will enhance; however, after this volume (100 µL), we can see a depression in these factors. It can be explained by the fact that in the volumes more than 100 µL the cloudy solution rejects the excess amount of 1-octanol; the surplus solvent will be stranded in the narrow neck of the extraction cell and will not be involved in the extraction process; as a result, after centrifugation the analytes enriched in the extraction solvent involved in the extraction process will be diluted by the non-involved ones in supernatant thus EF and ER will be decreased.
3.1.3. Influence of disperser solvent volume on the extraction recovery
To obtain appropriate volume of methanol as disperser solvent, extractions were carried out by changing the volume of methanol in the range of 100–500 µL. The peresented results in figure 3 indicate that with increasing the volume of methanol up to 300 µL the recovery of extraction increased and then decreased after this volume. Utilization of lower volume of methanol caused poor extraction recovery; because the fine droplets of 1-octanol could not be well dispersed among aqueous solution and cloudy state was not formed well. Therefore for the following experiments, 300 µL methanol was used as optimal disperser solvent volume.
3.1.4. Influence of sample pH
pH of the sample solution for the extraction of compounds with ionization capability is a key factor. The ionic form of a neutral molecule normally does not transfer from the sample solution into the organic solvent as well as its neutral form does. Hence, pH of sample solution should be adjusted to be sure that neutral molecular forms of the analytes are present prior the microextraction step. For this reason, effect of pH of the solution on the amount of extracted naringenin was examined in the range of 3–7. Figure 4 indicates that, the highest extraction recovery obtained at pH=4.5. So, subsequent experiments were performed at this pH.
3.1.5. Influence of stirring rate
In order to obtain a stable cloudy solution, the sample solution was stirred by a magnetic stirrer during and after injection of the mixture of extraction and disperser solvents into the sample solution. The effect of stirring rate on the extraction of naringenin was investigated in the range of 0–1000 rpm (regarding to the stirrer limitation). As can be seen in figure 5, enhancement of the stirring rate up to 600 rpm led to acceleration of mass transfer of analytes from the sample solution to the extraction solvent. However, the higher rates had not the significant effect on the extraction recovery. Therefore, 600 rpm stirring rate were applied for all the extraction experiments.
3.1.6. Influence of extraction time
In MSA-DLLME, the time of extraction is defined as interval time between injection of the mixture of extraction and disperser solvents into the aqueous sample and before starting to centrifuge. In this work, effect of the extraction time was investigated from 0 to 30 min. Results in figure 6 shows that, extraction recovery versus the variations of extraction time is almost fixed. It can be explained by the fact that, transfer of the analyte from aqueous phase to the extraction solvent is carried out very fast and after injection the cloudy solution was formed quickly, because the surface area between the extraction solvent and the aqueous phase is very large; hence, equilibrium state was obtained rapidly. Therefore effect of extraction time on the extraction recovery is not significant.
3.1.7. Influence of salt concentration on the extraction recovery
Some researchers reported that in microextraction procedures salt addition can improve the extraction recovery [20, 21]. It is well known that the addition of salt to the aqueous sample in LLE technique usually increases the ionic strength and reduces the solubility of the analytes in aqueous solution and causes the migration of analytes to extraction solvent and improves the extraction of analytes . In this work, the effect of salt addition on the extraction recovery was examined in the range of 0.0 to 5.0 M of NaCl. Results in figure 7 indicates that the recovery decreases by increasing NaCl concentration. It can be explained by the fact that, interaction may take place between the analyte and the salt .
3.2. Analytical performance of the MSA−DLLME−HPLC for determination of naringenin
In order to evaluate the analytical performance of the MSA-DLLME technique, quantitative characteristics of the this method consisting linear range, limit of detection (LOD), extraction recovery and preconcentration factor for extraction of naringenin from standard aqueous solutions under the optimal conditions (disperser solvent: 300 µL methanol, extraction solvent: 100 µL 1-octanol, pH of sample: 4.5, stirring rate: 600 rpm and no addition of NaCl) were studied. While the LOD, defined as 3Sb/m (where Sb is the standard deviation of blank and m is the slope of calibration graph after preconcentration), was 0.30 ng mL-1, the LOQ, defined as 10Sb/m was 1.0 ng mL-1. Linear range was 1.0 to 700 ng mL-1 with determination coefficient of R2 = 0.9993. The extraction recovery and preconcentration factor were 86% and 62, respectively.
4. Analysis of real sample using MSA-DLLME
To evaluate the practicality of the proposed method in real samples, a fresh grapefruit was tested using the MSA-DLLME. The fresh grapefruit squeezed with a squeezer and the juice was collected. The grapefruit juice was centrifuged at 3000 rpm for 15 min . The supernatant was filtered  and was used for the analysis. The extraction procedure (MSA-DLLME) was done under the optimized conditions. The sample was spiked with naringenin standard at six levels; subsequently, they were extracted using the MSA-DLLME technique and finally the extracts were analyzed by HPLC. The chromatograms of the naringenin standard, blank fresh grapefruit juice, and the spiked sample were shown in Figure 8. Using standard addition method, the obtained concentration of naringenin in grapefruit juice was 241 ng mL-1.
In this work, a special mode of DLLME technique, i.e., MSA-DLLME followed by HPLC-UV applied for the extraction and determination of naringenin in grapefruit. Only one LLE method is available for determination of this important compound . In comparison with previously reported LLE procedure , the proposed method shows lower LOD (0.3 ng mL-1 in MSA-DLLME (present work) respect to 1.0 ng mL-1 in LLE ) and wider linear rang (1-700 ng mL-1 in MSA-DLLME (present work) versus 2-400 ng mL-1 in LLE ) for quantitative analysis of naringenin. In this work, 1-octanol was used as the extraction solvent with lower density than water which has a good chromatographic behavior in comparison with chlorinated solvents and the evaporation step is eliminated. Overall, the results revealed that MSA-DLLME- HPLC-UV method beyond to the good extraction recovery and preconcentration factor, provides a simple, easy to use, inexpensive and environmentally friendly procedure for the extraction of naringenin from the complex matrix of grapefruit.
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