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One of the analytical method aims is to develop a simpler, faster and reliable method than the existing ones. Therefore, a simple sample preparation technique coupled with reversed phase high-performance liquid chromatography was developed for the determination of tocopherols and tocotrienols (tocols) in cereals. The sample preparation procedure involved a miniaturized hydrolysis of 0.5 g cereal sample by saponification, followed by the extraction and pre-concentration of tocols from saponified extract using dispersive liquid-liquid microextraction (DLLME). Parameters affecting the DLLME performance, such as type and volume of extraction and dispersive solvent, salt addition, extraction and centrifuge time, were optimized to achieve the optimum extraction efficiency. Under optimum conditions, the performance of the developed DLLME method was evaluated. Good linearity was observed over the range assayed (0.0313-4.0 µg/mL) with regression coefficients greater than 0.9989 for all tocols. Limit of detection and enrichment factor were ranged from 0.011 to 0.106 µg/mL and 50 to 73, respectively. Recoveries, performed by extraction of added tocols at spiking levels of 1, 3, 6 µg/mL in the rice samples, were between 87 and 123 %. Intra- and inter-day precisions expressed in relative standard deviations were lower than 9.7%. Tocols content of a rice sample analysed using the developed DLLME method was comparable with the results obtained using conventional extraction methods. This developed DLLME method was successfully applied to cereals: rice, barley, oat, wheat, corn and millet. The advantages of this developed DLLME method are the usage of extraction solvent in micro-litre level, simple step of extract cleanup and pre-concentration, rapid, precise with high enrichment factor and recovery. This study demonstrated that the developed extraction method can be used as an alternative to conventional extraction method for the determination of tocols in cereals.
Keywords: tocopherol; tocotrienol; analysis; HPLC; microextraction; cereal
Vitamin E or tocols is a collective term for a group of eight naturally occurring chemical isomers: α-, β-, γ-, δ-tocopherols (α-, β-, γ-, δ-T); and their four respective unsaturated tocotrienols (α-, β-, γ-, δ-T3). Tocols is synthesized only by photosynthetic organisms and known to have a protective effect against various disease conditions [1-3] such as cancer, cardiovascular diseases, oxidation of LDL, and cell membrane and DNA damage by free radicals.
As the interest on tocols was increased due to the awareness of its health benefits, a high number of tocols analysis procedures using high-performance liquid chromatography (HPLC) was published especially for cereal samples [4-10]. Cereal is regarded as the richest source of tocols. However, sample preparation remains as the most demanding and challenging part in tocols analysis for most samples including cereals. Relative low abundance of tocols that usually associated with lipid components of the cereal matrix and the presence of a range of potential interference compounds emphasize the necessity for a sample preparation step that eliminates impurities, extract and concentrate the tocols. Traditionally, saponification assisted extraction [5-7] and direct solvent extraction [5, 7, 8] are the two popular techniques used for the analysis of tocols in cereals, besides the soxhlet extraction , solid-phase extraction , supercritical-fluid extraction  and pressurized liquid extraction  that were reported.
Saponification is employed prior to tocols extraction to liberate the attached tocols from the sample matrix by hydrolysing the matrix and to reduce the load of material extracted into the organic phase by converting the lipid esters into hydrophilic compounds [4, 13-15]. After saponification, the unsaponifiable compounds including tocols are extracted using organic solvents, which is commonly hexane. Saponification results in a much clean extract  with good tocols separation and detection selectivity [4, 14]. However, the use of organic solvents that are not environmentally-friendly, long operating time, tedious extraction procedure and the tendency of emulsion layer formation in liquid-liquid extraction step are the drawbacks of this extraction method. On the other hand, direct solvent extraction overcomes these drawbacks with the advantages of rapid, simple, consumption of less toxic solvent with less destruction of tocols. However, this extraction was reported to yield lower amount of tocols in cereals than saponification assisted extraction [5, 7, 16]. Moreover, high load of extracted materials could decrease the selectivity of tocols detection  and shelf-life of the column. Nevertheless, these two extractions require a considerable amount of extraction solvents and frequently a pre-concentration step to increase the tocols detection sensitivity. These methods generate a large amount of laboratory waste, consequently implies additional environmental hazard and cost for the disposal of waste solvents. Moreover, multiple steps in sample preparation, especially the solvent evaporation step that is time consuming, limit the possibility to analyse more samples and could introduce error in the results.
Recent research in the analytical chemistry has focused on miniaturized, simplified, efficient and particularly environmental friendly extraction technique that inspires toward the development of microextraction method. Low consumption of solvent, simple, rapid with high enrichment factor and recovery are the advantages of microextraction method. As cereals contain modest amounts of lipid, saponification coupled with microextraction technique is highly essential in accordance with the current analytical chemistry trend. Dispersive liquid-liquid microextraction (DLLME), one variant of microextraction developed by Assadi and coworkers , showed a promising possibility to be used for tocols extraction after saponification. DLLME is mainly used on clean samples such as water samples, and the extend application on complex matrix samples are limited . Therefore, we proposed the application of DLLME for the determination of tocols in cereals.
In this paper, the application of DLLME for the extraction of tocols after saponification and the influence of different factors (e.g., type and volumes of extraction and dispersive solvents, extraction and centrifuge time) on the extraction efficiency were evaluated. The DLLME was further optimized, validated and applied for the determination of tocols in cereals. The advantages of this developed extraction method are the use of extremely small extraction solvent volume, inexpensive, simple, fast, with high recovery and enrichment factor. To the best of our knowledge, this report describes the first application of microextraction for the determination of tocols either in cereals or generally in food samples.
2. Materials and methods
Tocopherols (purity ≥ 95%) and tocotrienols (purity ≥ 99.5%) were purchased as isomer kits from Calbiochem (San Diego, CA, USA) and Chromadex (Santa Ana, CA, USA), respectively. HPLC-grade ethanol, methanol and acetonitrile were obtained from Merck (Darmstadt, Germany), and pyrogallol (purity >98%) was purchased from Fluka (Seelze, Germany). All other reagents including tetrachloromethane (CCI4) used were of analytical grade.
2.2 Standard solutions and cereals samples
All standard stock solutions of tocols were prepared in absolute ethanol with an approximate concentration of 50 µg/mL and stored at -20°C. The concentration of each tocol standard stock solution was confirmed spectrophotometrically using the known absorption coefficient of each isomer in ethanol () . From these stock solutions, working standard mixtures consisted of all eight isomers in the concentration of 0.03125 to 8.0 µg/mL of each isomer were prepared. Rice samples were obtained from PadiBeras Nasional Berhad (Malaysia) and cereals samples (rice, barley, oat, wheat, corn, and millet) were obtained from local stores. All samples were ground, sieved through a 500-µm sieve and stored at 4°C prior to analysis.
2.3 High-performance liquid chromatography
An Agilent Technologies 1200 system chromatograph equipped with a degasser, a quaternary pump, an autosampler and a fluorescence detector was used. Separation of tocols at temperature of 25°C was accomplished using a COSMOSIL π-NAP column (250 mm x 46 mm; 5 µm) (Nacalai Tesque, Kyoto, Japan) with a 20-µL injection volume. The mobile phase at a proportion of water: methanol: acetonitrile (13: 80: 7) delivered isocratically at a flow rate of 1.0 mL/min and the fluorescence detection wavelength was set at Ex/Em= 295/330 nm. The column was conditioned with the mobile phase for at least 30 min until a linear baseline was obtained before chromatographic runs were made.
Cereal sample was saponified according to the method reported by Fratianni et al ; however, the amount of sample and chemicals were scaled down to 25% from its original amount. Briefly, a mixture, consisting of 0.5 g of sample, 0.5 mL of 60% potassium hydroxide, 0.5 mL of 95% ethanol, 0.5 mL of 1% aqueous sodium chloride, 1.25 mL of 6% ethanolic pyrogallol and an additional 2.25 mL of 95% ethanol, in screw cap glass test tube was flushed with nitrogen gas and incubated in a shaking water bath at 70°C for 45 min. After that, the tube was cooled to room temperature, vortex for 10 s, and centrifuged at 3000 rpm for 5 min to sediment the floating particles. A 1.0 mL of clear saponified extract containing tocols was subjected to the DLLME. Meanwhile, 0.5 mL of 95% ethanol was replaced with 0.5 mL of working standard mixtures at different concentrations for calibration curve preparation either with sample (matrix-standard calibration) or without sample (blank calibration).
2.5 Dispersive liquid-liquid microextraction
A 1.1 mL of a mixture of extraction: dispersive solvent (1: 10, v/v), followed by 1.0 mL of saponified extract were placed in a 10.0 mL glass tube with conical bottom. Subsequently, 3.0 mL of purified water was injected rapidly into the glass tube using 5.0 mL auto pipette. The formed cloudy solution was shaken gently and allowed to stand for 5 min. Then, the tube was vortex for 10 sec and centrifuged at 3000 rpm for 5 min. The sediment of CCI4 (about 75±3 µL) in the bottom of glass tube was collected using 50-µL micro-syringe and transferred into vial-insert for the HPLC analysis. All experiments in DLLME optimization stage were performed in duplicate.
2.6 Enrichment factor and recovery
Enrichment factor (EF) and recovery (R%) are the two important parameters for the DLLME optimization. The EF was defined as the ratio between the analyte concentration in the sedimented phase (Csed) and the initial concentration of analyte (Csam) within the sample. Meanwhile, R% was defined as the percentage of the total analyte amount that was extracted to the sedimented phase. The following equations EF = Csed/ Csam and R% = 100 x (Csed x Vsed)/ (Csam x Vsam) were used to calculate EF and R%, respectively. Vsed and Vsam are the volume of sediment phase and volume of aqueous sample, respectively. Csed was calculated from calibration curves obtained by direct injection of working standard mixtures in the range of 0.5 to 20 µg/mL.
3. Results and discussion
3.1 Optimization of dispersive liquid-liquid microextraction
The type and volume of extraction and dispersive solvent, salt addition, extraction time and centrifuge time are the DLLME parameters that affecting the EF and R% of tocols. A series of experiments using one variable at one time was designed to achieve optimum R%.
3.1.1 Extraction solvent selection
Dichloromethane, chloroform and tetrachloromethane (CCl4) were evaluated to select a suitable extraction solvent for DLLME system based on these properties: (a) higher density than water, (b) lower solubility in water (c) strong capability to extract tocols and (d) good chromatographic compatibility . Meanwhile, ethanol, acetonitrile and acetone were chosen as dispersive solvents. All possible combinations of selected extraction and dispersive solvents were tested for the formation of cloudy solution after addition of water and formation of sediment in DLLME system after centrifugation. A 1.1 mL of a mixture of extraction: dispersive solvent (1: 10, v/v) was added to the tube containing 1.0 mL of saponified extract, and subsequently 3.0 mL of water was rapidly injected. A cloudy solution was formed only with CCl4 as extraction solvent in all tested dispersive solvents, meanwhile a two-phase system was observed in all combinations after the mixtures were centrifuged. Cloudy solution was formed because of the fine particles of extraction solvent, which is dispersed entirely in aqueous phase. Formation of fine particles is essential for extraction in DLLME, thus CCl4 was selected as an extraction solvent.
3.1.2 Dispersive solvent selection
Acetone, acetonitrile and ethanol were selected as dispersive solvents because of their good miscibility in both water and extraction solvent. As each dispersive solvent has different affinity on tocols, the effect of different dispersive solvents on EF and R% of tocols were studied by using 1.1 mL of each dispersive solvent containing 100 µL CCl4. The results (Fig. 1) indicated that the maximum R% and EF were achieved using acetonitrile-CCI4 as dispersive and extraction solvents pair compared to acetone and ethanol. Therefore, acetonitrile was selected as dispersive solvent. Although almost similar R% was obtained using acetone and ethanol, lower EF was obtained for acetone due to the high volume of sediment phase.
3.1.3 Extraction solvent volume
Different volumes of CCl4 (60, 80, 100, 120 and 140 µL) were subjected to the DLLME procedure to evaluate its effect on EF and R% of tocols. As in Fig. 2, an increase of CCl4 volume from 60 to 100 µL increased the R%. However, further increase of CCl4 volume decreased the R%. The sediment volume of CCl4 at 60 µL and 140 µL were 33 µL and 131.5 µL, respectively. It is clear that an increase in volume of CCl4 increased the sediment phase volume and subsequently reduced the EF. Thus, 100 µL of CCl4 was selected in order to obtain high R% with good EF.
3.1.4 Dispersive solvent volume
Different volumes of acetonitrile in the range of 0.70 to 1.75 mL in 0.15-mL interval were used to evaluate its effect on EF and R% of tocols. Fig. 3 shows that no obvious variation in R% was observed with increasing volume of acetonitrile from 1.0 to 1.45 mL. However, lower R% was obtained at 0.70, 0.85 and 1.75 mL of acetonitrile. It seems that at lower volumes of acetonitrile, the formation of tiny extraction droplet is not effective which resulted low R%. Moreover, at 0.70 mL of acetonitrile, white precipitation was formed on the bottom of centrifuge tube that caused a poor separation between sediment and aqueous phases. Meanwhile, the solubility of the tocols in aqueous phase increases at higher volumes of acetonitrile. It reduces the tocols partition with extracting droplets and consequently the R%. An increase in the volume of acetonitrile from 1.0 to 1.75 mL reduced the volume of the sediment phase from 81.5 to 39 µL, thereby increased the EF (Fig. 3). As the R% of tocols was similar in the range of 1.0 and 1.45 of acetonitrile, a 1.0 mL was selected to achieve suitable sediment volume for HPLC analysis with good EF.
3.1.5 Salt addition
The effect of salt addition on the EF and R% of tocols were evaluated by adding different amount of NaCI (0, 1, 2, 3%, w/v). Addition of 1% of NaCI slightly improved the R% and further increase of NaCI decreased slightly the R% and EF (supplement data: Fig. 5). Addition of NaCI caused flocculation precipitated at the bottom of the aqueous phase in a centrifuge tube. It was due to the precipitation of hydrolysed cereal sample matrixes under salting effect. This precipitation caused difficulties in the collection of extraction solution after centrifugation. Incomplete collection of sediment phase resulted lower R%
Therefore, DLLME was carried out without the addition of salt as the sample extract has NaCI and KOH ions, added in saponification step, which is sufficient to enhance the extraction.
3.1.6 Extraction and centrifuge time
The influence of extraction time (0, 2.5, 5 and 10 min) on EF, R% and peak area of each tocol isomers were evaluated. Extraction time is defined as the interval time between the injection of the mixture of dispersive and extraction solvents into sample, and starting to centrifuge. The variations of peak area of tocols over tested time has no obvious effect, which indicates that the DLLME was time-independent (supplement data: Fig. 6). The EF and R% were almost similar for the tested time. Effect of different centrifuge time (2.5, 5, 7.5 and 10 min) at 3000 rpm on R%, EF and peak area of each tocol isomer were evaluated and the results obtained were similar to results of extraction time (supplement data: Fig. 7). Although, extraction and centrifuge time had less effect on R%, EF and peak area, 5 min of extraction time and 5 min of centrifuge time were chosen to achieve complete extraction in order to obtain maximum EF and R%.
3.2 Analytical performance
A series of experiments were performed under the optimized DLLME conditions to obtain method linearity, limit of detection (LOD), limit of detection (LOD), enrichment factor (EF), precision and accuracy. For linearity, LOD and LOQ determination, 1.0 mL of saponified extract containing different concentrations of standard mixture was subjected to the DLLME. The results of linearity, LOD, LOQ and EF are shown in Table 1. The developed DLLME method demonstrated good linearity in the tested concentration range at six different levels with the regression coefficient (R2) ≥0.9989 for all tocol isomers. LOD and LOQ for each tocol, based on signal-to-noise of 3 and 10, were low and ranged from 0.011 to 0.106 µg/mL and 0.052 to 0.303 µg/mL, respectively. EFs were high because of high tocols pre-concentration in small volume of CCI4. Fig. 4a illustrates chromatograms obtained for a standard mixture after 1 mL of standard mixture at 0.6 µg/mL subjected to DLLME and for direct injection of standard mixture at 6 µg/mL. The DLLME gave a higher detection sensitivity (Fig. 4a-i) than direct injection (Fig. 4a-ii); although, the concentration of standard mixture used for DLLME was 10 fold lower than in direct injection. Intra- and inter-day precisions of the method were evaluated by successive triplicate analyses of a sample at three spiked concentration levels on the same day and on three different days, respectively. The RSDs for intra- and inter-day precision were less than 9.7 and 8.4% indicating that this method has an acceptable repeatability (Table 2). Meanwhile, standard addition procedure was used to evaluate the recovery of the DLLME method due to unavailability of blank cereal sample. A sample in triplicate was spiked at three concentration levels with known amount of tocols standard (1, 3 and 6 µg/mL) prior to saponification and subjected to the entire extraction method. The proposed method gave satisfactory recoveries of the tocols that were in the range of 87 and 123 % (Table 2). The selectivity of the method was demonstrated by good separations of tocols with no interfering peaks in the elution region of the analytes for cereal matrices (Fig. 4a-h).
3.3 Matrix effect
As blank calibration curve was prepared without the addition of sample, the possibility of matrix effect on calibration curve was investigated by comparing the slope of matrix-standard calibration curve with the slope of the blank calibration curve . Since it was not possible to find a blank cereal, matrix-standard calibration curve was constructed by analysing cereal samples (milled rice and brown rice) which were spiked with standard solutions in the range of 0.75 to 6.0 µg/mL. Regression equations were calculated by least-squares linear regression analysis of the peak area versus tocol concentration. Table 5 (supplement data) shows regression equations and the slope ratio of the matrix to the blank for each tocols. The slope ratios, in the range of 0.84 to 1.10, were approximately 1.0 indicating that there was no significant matrix effect on the blank calibration curve. As saponification hydrolyse the sample into smaller fraction, the saponified extract is expected to be similar for all samples that contributes to the no significant matrix effect. However, quantitation of analytes in the cereals samples using matrix-standard calibration is recommended to obtain results that are more accurate.
3.4 Comparison of developed DLLME-, saponification assisted- and direct extraction-method
The performance of this developed DLLME method to extract tocols from brown rice sample was compared with saponification assisted-  and direct solvent-extraction . The amount of tocols obtained using this developed DLLME method was statistically not different (ANOVA, p>0.05) with the results of the latter extraction methods (Table 3). This result demonstrated that the developed DLLME method has an equivalent extraction performance with conventional extraction methods for the extraction of tocols in rice sample. Moreover, the characteristics of these three extraction methods were also compared. In this developed DLLME method, only 1.0 mL of acetonitrile and 0.1 mL of CCl4 are required for the extraction of tocols after saponification instead of approximately 10.0 ml of extraction solvent for conventional extraction methods. Significant reduction in the volume of extraction solvent not only saves cost, but also reduces the risk on human health and the environment. It is also possible to scale-down the saponification sample size as low as 0.2 g because only 1.0 mL of saponified extract is required for DLLME. The DLLME procedure after saponification is simple, rapid and the handling of the sample is reduced with the elimination of double extraction and solvent evaporation step. The elimination of these two steps reduces the time needed for each analysis and more samples can be analysed in short time. Although this developed DLLME method is not as simple as the direct extraction method due to the present of saponification step, the latter step is highly required for cereal samples to release the tocols from its matrix and hydrolyse the lipid compounds . It can be seen that a large impurity peak observed in the direct extraction method chromatogram (Fig. 4b-i) was not found in the chromatograms of the other two methods that include saponification step (Fig. 4b-ii and Fig. 4c).
3.3 Application to cereals samples
Tocols content of cereals (rice, barley, oat, wheat, corn and millet) was determined using the developed DLLME method. As in Table 4, the amount and composition of tocols were varied from cereal to cereal. Among the cereals samples analysed, millet has the highest amount of total tocols content followed by wheat, black rice, brown rice, corn and oat. The composition of tocols in this study is similar to the previous studies [5, 10, 12]; however, the amount of tocols was different due to the factors of genotype and location. In addition, these cereals samples were spiked with standard tocol isomers to investigate the recovery. Good recoveries in the range 99 to 125% were obtained and it demonstrates the ability of this DLLME method in extracting tocols from cereals samples. Fig. 4c-h show chromatograms of cereals analysed using the developed DLLME method. As can be seen, good separation of tocols with no interference of impurity peaks was observed.
This paper outlined the successful development and application of dispersive liquid-liquid microextraction (DLLME) coupled with RP-HPLC for the analysis of tocopherols and tocotrienols in cereals. The parameters that affect the DLLME efficiency in extracting tocols from saponified extract were optimized. Results of validation indicated that the developed DLLME method is precise and accurate in determining the tocols content in cereals. In comparison with conventional methods, this developed DLLME method has advantages such as simple, fast, inexpensive, good accuracy and repeatability, high enrichment factor and micro-litre level extraction solvent volume consumption. Thus, this developed DLLME method is suitable to be used for routine tocols analysis in cereals. It is also possible to use this DLLME procedure as alternative extraction method to extract tocols after saponification for samples other than cereals.