Development of a Dispersive Liquid–Liquid MicroextractionMethod for the Determination of α-Tocopherol in PigmentedWheat by High-Performance Liquid Chromatography

Qilong Xie & Shuhui Liu & Yingying Fan & Xiaoke Zhang

Received: 27 December 2012 /Accepted: 25 February 2013# Springer Science+Business Media New York 2013

Abstract A dispersive liquid–liquid microextraction(DLLME) method coupled to high-performance liquid chro-matography was developed for the analysis of α-tocopherolin grain samples. The DLLME parameters including thetype and volume of extractants, the volume of disperserand the addition of salt were examined. The optimizedDLLME procedure consisted in the formation of a cloudysolution promoted by the fast addition to the sample (5 mLof saponified sample solution diluted with 5 mL of water) ofa mixture of carbon tetrachloride (extraction solvent, 80 μL)and ethanol (dispersive solvent, 200 μL) without the addi-tion of salt, followed by shaking for 5 min and centrifugingfor 3 min at 5,000 rpm. Intra- and inter-day repeatabilityexpressed as % RSD were 3.5 and 7.6 %, respectively. Thelimit of detection and the limit of quantification were 1.9and 6.3 μgL−1. The comparison of this method with thenational standardized extraction method, supercritical car-bon dioxide extraction, accelerated solvent extraction, andconventional heat-reflux extraction indicates that theDLLME was accurate (no significant differences at the0.05 % probability level), high efficient, low organicsolvent-consuming, and low cost. This procedure was suc-cessfully applied to 42 samples of 14 types of purple wheat,

for which the content of α-tocopherol exhibited a signifi-cantly negative correlation with the pigment content mea-sured by a spectrophotometer. The recovery rates rangedfrom 90.5 to 103.7 %.

Keywords Dispersive liquid–liquid microextraction .

Micronutrients .α-tocopherol . Pigment content .

Pigmented wheat

Introduction

Vitamin E is also called tocopherol. As an effective antiox-idant, this vitamin plays an important role in preventing theoxidation of unsaturated fatty acids, lowering cholesterollevel, improving blood circulation, and preventing aging(Theriault et al. 1999). It has some analogues, includingthe α, β, g, and δ forms of tocopherol and the four isomersof the three alkene forms of tocopherol, and among theseanalogues, α-tocopherol has shown the highest biologicalactivity (Hosomi et al. 1997).

The main sources of α-tocopherol in the human diet arevegetable fats and oils. Cereal grains can also contributesignificantly to the dietary intake of this micronutrient. Toaid consumers in maintaining healthy diets, it is important todetermine α-tocopherol content in various cereal kernels,for which the extraction of the target analyte from samplematrix is a crucial step. Direct liquid–liquid extraction wasused for vegetable oils and fortified foods (Chávez-Servín etal. 2008); for cereal grains, a saponification process wasrequired followed by liquid–liquid extraction (Lanina et al.2007; Pinheiro-Sant’Ana et al. 2011; Ryynanen et al. 2004).Although this approach produces less interference fromimpurities during the chromatographic separation, the te-dious operation steps involved in the extraction, rinsing,

Q. Xie : S. Liu (*) :Y. FanCollege of Science, Northwest A&F University,No. 3 Taicheng Road, Yangling,Xianyang, Shaanxi 712100, Chinae-mail: shliu815@126.com

S. LiuState Key Laboratory of Crop Stress Biology in Arid Areas,Yangling, Xianyang, China

X. ZhangCollege of Agronomy, Northwest A&F University, Yangling,Xianyang, China

Food Anal. MethodsDOI 10.1007/s12161-013-9592-x

drying and evaporation make the traditional procedure time-and solvent-consuming as well as labor-intensive.

In recent years, researchers have attempted to develop newextraction techniques of vitamin E from grains, such as super-critical carbon dioxide extraction (SFE-CO2) (Ge et al. 2002)and accelerated solvent extraction (ASE) (Bustamante-Rangelet al. 2007). Compared with conventional extraction methods,these procedures possess advantages including high extractionefficiency and reduced solvent consumption; however, largeinvestments in equipment and high operating costs are re-quired, which is not convenient for the routine analysis work.

Since DLLME was proposed by Rezaee et al. (2006), thismethod has quickly gained popularity, as evidenced by itsappearance in a continually growing numbers of originalpapers (Chen et al. 2009; Shu et al. 2012) and reviews(Rezaee et al. 2010; Zang et al. 2009). Because of its signif-icant advantages over traditional liquid–liquid extraction,DLLME has been widely used for the extraction of organicpollutants in environmental samples (Farahani et al. 2007;Liang et al. 2008). By contrast, this technique has not beenutilized for the analysis of micronutrients in food products.

Although widely consumed as white wheat, there aresome special cultivars of wheat that contains color pigment,such as black, blue and purple wheat (Hu et al. 2007;Knievel et al. 2009). Chinese blue-grained wheat was bredby Li (Li et al. 1982; Mu et al. 1986) from the progenies ofthe hybrids between common wheat and Agropyronelongatum. The purple-grained wheat was bred by Sun etal. from the distant hybridization among common wheat,Agropyron glaucum and Elymus dasystachys (Sun et al.1999; Bai et al. 2002). It is well documented that thecolored-grain skin is rich in the natural pigments, which isa substance that belongs to the anthocyanin class and pos-sess excellent benefits for our bodies including eliminatingfree radicals, increasing immunity and improving myocar-dial nutrition (Hu et al. 2003; Li et al. 2005; Zhou et al.2004; De Pascual-Teresa et al. 2002; Escribano-Bailón et al.2004). By contrast, very little amount of natural anthocya-nins is present in normal wheat. The colored wheat is also arich source of proteins, amino acids, and other essential traceelements (Li et al. 2002; Liu et al. 2002; Gao et al. 2006).Thus, the colored wheat is a precious resource for the cultiva-tion of new wheat varieties (Ortiz-Monasterio et al. 2007).

To date, very few studies have reported on the assay ofthe vitamin E content in the colored wheat (Xiu et al. 2009).Furthermore, exploring the correlation between the naturalpigment and vitamin E content in colored wheat could providemeaningful information for future wheat cultivation.

The objective of this study was to establish an accurate,highly efficient, low organic solvent-consuming and low-cost extraction method of vitamin E in cereal grains bycoupling DLLME to saponification process and to deter-mine the contents of α-tocopherol in 42 samples of 14 types

of purple wheat. Additionally, the relationship between thegrain pigment content and the α-tocopherol content wouldbe investigated.

Materials and Methods

Reagents and Materials

DL-α-Tocopherol (99.5 % pure) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-butyl-3-methylimidazoliumhexafluorophosphate [C4MIM] [PF6], and 1-hexyl-3-methylimidazolium hexafluorophosphate [C6MIM] [PF6]was provided by Aladdin reagent company (Shanghai,China). Analytical-grade carbon tetrachloride, chloroform,absolute alcohol, ascorbic acid, and sodium sulfate and potas-sium hydroxide were obtained from Tianjin BODI ChemicalReagent Co. Ltd. (Tianjing, China). HPLC grade ethanol andmethanol were purchased from Luomiou Chemical ReagentCo. Ltd. (Tianjing, China). To prepare a stock standard solu-tion (1 mg/mL), 10 mg of α-tocopherol was accuratelyweighted and dissolved in 10 mL of ethanol, and stored at4 °C until use. Further dilutions were later made using thesame solvent.

Fourteen purple wheat varieties including Luo Zhen-1(LZ-1), Hei Mai (HM), Kei Zi (KZ), Hei You-1(HY-1),Hei You-2 (HY-2), Hei You-3(HY-3)Hei Li-76 (HL-76),YZ-1, YZ-2, YZ-4, YZ-5, HR-2, HR-1-2 and HB-2 wereprovided by the College of Agronomy, NorthwesternAgricultural and Forestry Technology University (NAFTU).With randomized block design with a block size of 6 m2

(2 m×3 m), the 14 lines were grown in triplicate at theExperiment Station of NAFTU in 2010–2011. Intensive fer-tilizer and water management was used during the growingseason without lodging to prevent plant disease. After harvest,the grain samples were stored in a seed bank and were subse-quently milled with a Taisite Laboratory Mill FW100 toproduce whole-wheat samples (the samples for supercriticalcarbon dioxide extraction were prepared separately). The drymatter content of each sample was determined by drying a 3 gof sample in duplicate at 103±2 °C for 4 h. All the contents ofvitamins E given in this study were systematically moistureblank corrected.

Instrumentation

The quantitative analysis was performed on a ShimadzuProminence LC-20A (Kyoto, Japan) equipped with twoLC-20AT pumps, a 7725i manual sample injector and aSPD-M20A photodiode array detector. Separations werecarried out on a Waters Xterra C18 column (15 cm×4.6 mm, with 5 μm particle size). A mixture of water andmethanol (5:95) at a flow rate of 1 mL/min was used as a

Food Anal. Methods

mobile phase in isocratic elution mode. The injection vol-ume was 20 μL for all the solutions and the detection wasperformed at the wavelength of 290 nm.

An ultrasonic cleaning machine (Model KQ-5200E,Kunshan City Ultrasonic Instruments Inc., China) was used.Centrifugation was done with a Beckman (Allegra X-12)system. A microsyringe (Hamilton, 100 μL) was used tocollect and measure the volume of extraction solvent. pHmeter (Model 213, Hanna, Italy) was used. Water waspurified using a Millipore Direct-Q 3 system (MilliporeCorporation, Bedford, MA, USA). A Shimadzu spectropho-tometer UV-2600/2700 (Kyoto, Japan) was used to measurethe total pigment content in the purple wheat.

Sample Preparation

The National Standardized Extraction Method

This procedure was carried out based on the nationalstandard (National Standard of the People’s Republic ofChina, GB/T 5009.82-2003).

Saponification: In a round-bottomed flask, 5.0 g of thegroundedwheat sample that was weighted accurately wascombined with 50 mL of absolute ethanol and stirreduntil the particles were well-dispersed. Then, 0.5 g ofascorbic acid was added and mixed, and 10 mL of potas-sium hydroxide solution (1:1 KOH/water,w/v) was addedthereafter. The flask was next placed into a water bathwith boiling water and refluxed for 30 min. Afterward,the flask was immediately cooled with cold water.Extraction: The supernatant from the saponified samplewas transferred to a separatory funnel, and the residuewas subsequently washed two to three times with a totalof 50 mL water, which was added to the funnel. Theresidue was further rinsed twice with a total of 100 mLether, which was also transferred to the funnel. Aftershaking gently for 2 min and standing for 30 min, thetwo phases separated, and aqueous phase was drained.Rinsing: The organic layer was rinsed with a total of150 mL water until the pH value of the washing solu-tion reached pH7.0.Evaporation: The ether phase was filtered through 5 gof anhydrous sodium sulfate from top of the separatoryfunnel into a round-bottomed flask. The separatoryfunnel was rinsed three times with a total of 100 mLether, which was also added to the flask by filteringthrough anhydrous sodium sulfate. The ether extractantwas concentrated to 2 mL with a rotary evaporator at30 °C and further dried with flowing nitrogen to re-move the residual ether. Afterwards, 10 mL of ethanolwas added to the flask, and the sample was injected intothe HPLC system.

DLLME Method

Saponification: The saponification procedure wasperformed as described in the national standardizedmethod.Extraction: The supernatant from the saponified samplewas transferred to a 100-mL of volumetric flask, andthe residue was rinsed for three times with ca. a total of50 mL water, which was combined with the superna-tant. The flask was subsequently made up to the markwith water, and the solution was later transferred to asample bottle. Next, the solution pH was adjusted to pH7.0 with 6 M hydrochloric acid, volume of whichconsumed was accurately measured (ca. a few of milli-liter). Afterwards, 5 mL of the saponified solution wasdiluted to 10 mL with water in a conical centrifugetube, into which the extractant mixture containing80 μL of carbon tetrachloride and 200 μL of ethanolwas rapidly injected with a microsyringe. After shakingfor 5 min and centrifuging for 3 min at 5,000 rpm, thesedimented liquid phase was completely transferredinto a sample vial using a microsyringe (60–65 μL)and was injected into the HPLC system.

Supercritical Carbon Dioxide Extraction

This procedure was carried out according to Ge’s report(Ge et al. 2002).

Sample pretreatment: The wheat kernel was crushedand passed through a sieve, and the resultant particlesizes were between 20 and 40 mesh. Then the sampleswere heated at 105 °C in an oven for 15 min to maintainthe moisture content at approximately 5 %.Extraction: 5.0 g of the treated wheat sample weightedaccurately was placed in the extraction vessel. Theextraction pressure and temperature were set to 300 barsand 45 °C, respectively, and the extraction time was90 min. The collected light yellow extract was quanti-tatively transferred to a 5 mL volumetric flask, andethanol was used to create a constant volume. Thesamples were then centrifuged for 5 min (10,000 rpm)and directly injected for chromatographic analysis.

Accelerated Solvent Extraction

This procedure was performed following Bustamante-Rangel’s work (Bustamante-Rangel et al. 2007).

A Dionex ASE 200 accelerated solvent extractor wasperformed to extract the target analyte. First, 5.0 g of thewheat sample was accurately weighed, mixed thoroughlywith 1.0 g of diatomite and then transferred to an 11-mLstainless steel extraction thimble (a filter paper on the

Food Anal. Methods

bottom). Methanol was used as an extraction solvent, andthe extraction pressure was set at 1,500 psi, temperature at50 °C, and a static extraction time for 5 min. After extrac-tion, the thimble was rinsed with fresh extraction solvent(60 % of the extraction cell volume) and purged with a flowof nitrogen (150 psi during 90 s). The extract was collectedin a 40 mL vial and then transferred to a 25 mL volumetricflask, then which was made up to the mark with methanol. Asmall amount of the solution was transferred to a centrifugetube. After centrifugation for 5 min (10,000 rpm), thesample was then used for chromatographic analysis.

Conventional Heat-Reflux Extraction

In a round-bottomed flask, 5.0 g of the grounded wheatsample that was weighted accurately was combined with50 mL of absolute ethanol and stirred until the particles werewell-dispersed. The flask was next placed into a water bathwith boiling water and refluxing for 2 h. Afterward, the flaskwas immediately cooled with cold water. The supernatant wascollected, and the residue was washed twice using 20 mL ofethanol, which was then added to the extracted supernatant.The ethanol solution was then recycled via rotary evaporationat 30 °C until 2 mL of ethanol remained, which was thenquantitatively transferred to a 5 mL volumetric flask, and aconstant volumewas obtained through the addition of ethanol.A small quantity of this solution was then transferred to acentrifuge tube and centrifuged for 5 min (10,000 rpm), and itwas then used for chromatographic analysis.

Validation

Linearity was studied by spiking 25, 50, 100, 200, 500,1000 μL of α-tocopherol standard solution (100 μg/mL)into the blank sample solution containing 50 mL of absoluteethanol and 0.5 g of ascorbic acid and 10 mL of potassiumhydroxide solution (1:1 KOH/water, w/v) (free of cereal sam-ple), respectively. The resulting samples were pretreated by theprocedure as represented in section of DLLME including sa-ponification and DLLME. After that, 20 μL of the sedimentedphase was injected in the HPLC system. Calibration curveswere constructed by plotting peak areas versus the concentra-tions of the analytes in the blank samples before DLLME. Thecurves were applied to evaluating the performances of theproposed method in evaluation of the method.

Three real samples were chosen to determine therecovery capacity of the method; 200, 400 and 600 μL ofα-tocopherol standard solution (100 μg/mL) were spikedinto cereal samples, which were subjected to saponificationand DLLME. Every spiked sample was extracted intriplicate, and each was injected to the HPLC system threetimes. Recovery rates were calculated by the equationR%=[(Cs−Cp)/Ca]×100, where R (%) is the percent

recovery of added α-tocopherol, Cs is total α-tocopherolcontent in the spiked sample, Cp is endogenous α-tocopherolcontent in the sample, and Ca is the amount of α-tocopherolstandard added to the sample.

Statistical Analysis

The statistical analysis software package, version 9.1 (SASInstitute, Cary, NC, USA), was used. To identify individualdifferences, ANOVA and Tukey’s studentized range testwere performed. A p value of less than 0.05 was consideredsignificant.

Assays for Pigment Amount

The pigment amount was assayed according to the methodsused by Abdel-Aal and Hucl (Abdel-Aal ES and Hucl 1999).In a vial, 1 g of the whole-wheat flour was accurately mea-sured. After 10 mL of 95 % ethanol and 1.5 M HCl solution(85:15, v/v) was added, the vial was oscillated in a water bathat 60 °C for 5 h. The absorbance value (A) was measured at535 nm in triplicate. An A value of 1 was designated as oneunit of pigment (U), and the number of pigment units in 1 g ofwheat represented the pigment amount (expressed as U/g).Additionally, the pigment amount was defined as A×10/W,where W represents the mass of the sample.

Results and Discussion

Optimization of the DLLME

A purple wheat sample was selected to perform the optimi-zation experiment. Firstly, the content of α-tocopherol in thewheat sample was determined by the national standardizedmethod represented in section of materials and methods.Afterwards, DLLME was applied to the wheat sample.Extraction rate (ER) was chosen as the indicator, whichwas calculated by equation ER%=(Co×Vo)×100/w, whereCo and Vo were the target analyte concentration and volumein the sedimented phase when DLLME was used, respec-tively; w is the amount of the target analyte measured by thestandardized method. The influences of different DLLMEexperimental parameters including the type and volume ofextraction solvent, the volume of dispersers, salt concentra-tion, extraction and centrifuge time on the ER% of α-tocopherol were examined and the optimal conditions wereselected.

Selection of Extractant Solvent and Volume

The sample solution showed strong alkalinity after saponi-fication, in which the extraction solvent was not easily

Food Anal. Methods

dispersed. Based on the rinsing step in the standardizedextraction method, the pH of the saponified solution wasadjusted to pH7.0.

The selection of an extractant is essential to achieve ahigh extraction efficiency of DLLME. Mainly halogenatedhydrocarbons such as chlorobenzene, chloroform, carbontetrachloride, tetrachloroethylene, and chlorobenzene areused because of easy separation of the extractant from thebulk solution via centrifugation. However, these extractantsolvents are high toxic, and some are even possible humancarcinogen. Recently researchers have attempted to use ion-ic liquid as extractants to overcome this drawback (Guo andLee, 2011; Zhou et al. 2009; 2012).

In this study, combinations of 300 μL of different extractionmixtures (100 μL extract and 200 μL of ethanol), namely,chloroform, carbon tetrachloride, 1-butyl-3-methylimidazoliumhexafluorophosphate ([C4MIM] [PF6]), 1-hexyl-3-methylimidazolium hexafluorophosphate ([C6MIM] [PF6])with 10 mL of the sample containing 3 mL of the saponifiedsolution and 7 mL of water were examined in order to find themost suitable extractant for the DLLME. Having the two kindsof ionic liquid as extraction solvents, although the emulsifiedDLLME solution was formed, the two-phase system washardly appeared and the sedimented phase volume was verysmall after centrifugation. For the halogenated solvents, theER% of the target analyte achieved by carbon tetrachlorideand chloroform were 93.2 and 74.3 %, respectively, showingthe former was an ideal extractant.

To assess the effect of extractant solvent volume on theER%, extractant mixture containing different volumes ofcarbon tetrachloride (60, 80, 100 μL) and fixed volume ofethanol (200 μL) were used for DLLME procedures. Byincreasing the volume of carbon tetrachloride from 60 to100 μL, the volume of the sedimented phase increasedfrom ca. 45 to 80 μL. Also, according to Fig. 1, the ER%

of α-tocopherol increased as the extractant volume wasincreased from 60 to 80 μL, and then leveled off with thevolume varying from 80 to 100 μL, illustrating that 80 μL ofthe extractant was sufficient for this sample solution and wasselected as an optimal volume of the extractant solvent.

Selection of Disperser and Volume

Because ethanol was used in the saponification process, thesaponified solution contained ca.50 % ethanol, which couldreadily act as a disperser because of its miscibility with mostextractants.

Aliquots of 1, 3, 5 and 7 mL of the saponified solutionwere diluted to 10 mL with water, from which α-tocopherolwas extracted to investigate the influences of the disperservolume (the sample solution contained a certain amount saltbecause of the introduction of the potassium hydroxide andhydrochloric acid in saponification process and the subse-quent pH adjustment, respectively) on the ER%. As shownin Fig. 2, when the volume of the saponified solution wasincreased from 1 to 3 mL, the ER% of α-tocopherol in-creased significantly, which could be explained by the factthat increasing disperser volume enhanced extraction effi-ciency. By increasing the volume from 3 to 5 mL, the ER%of α-tocopherol increased slightly; above 5 mL, it leveledoff at a maximum value. Therefore, diluting 5 mL of thesaponified solution with 5 mL of water possessed an appro-priate disperser volume for the sample solution.

The extractant mixtures containing 80 μL of carbontetrachloride and different volume of ethanol (0, 200, 500,800 μL) were injected into the sample solution with theoptimal dilution factor to examine the effect of the amountof ethanol on the ER%. The results showed the ER% wasslightly higher (ca. by 5–7 %) with ethanol in the extractantmixture than without ethanol; however, the amount of

Fig. 1 Effect of the extractant volume on the extraction rate ofα-tocopherol (n=3)

Fig. 2 Effect of the disperser ratio on the extraction rate of α-tocopherol(n=3)

Food Anal. Methods

ethanol varying in the range of 200–800 μL did not causethe ER% shift. In the following study, the extractant mix-tures were prepared by mixing an 80 μL of carbon tetra-chloride and 200 μL of ethanol.

Addition of Salt

To evaluate the effect of salt addition on the performance ofthe DLLME procedure, 5 % or 10 % sodium chloride wasadded to the sample solution with the optimal dilution factorand subjected to the procedure. The findings revealed thatthe addition of salt did not influence the ER% of the targetanalyte, demonstrating that extra salt was not required forthe DLLME.

Shaking and Centrifuging Time

In this study the extraction time was evaluated in the rangeof 1–10 min, remaining constant all the experimental con-ditions. When shaking time was increased from 1 to 3 min,the ER% increased moderately. With shaking time varyingfrom 3 to 5 min, the ER% was enhanced slightly; above5 min, negligible effect on the ER% was observed.Therefore, a shaking time of 5 min was adopted in thesubsequent experiment.

The effect of centrifuging time was studied in the rangeof 1–10 min at 5,000 rpm. With centrifugation time increas-ing from 1 to 2 min, the ER% was increased slightly; longercentrifugation time did not increase the ER% further. Thisphenomenon could be explained by the fact that aftermixing of the three solvent components (sample, extractionsolvent and dispersive solvent) the mass transfer equilibriumof the target analyte was achieved in short period becauseof the large contact surface between the tiny drops of

extraction solvent and sample. Thus, centrifugation wasonly utilized to help separating two phases. A centrifuga-tion time of 3 min was chosen to ensure that the transferof droplet to bottom of a centrifuge tube.

Performance Characteristics for the Validated Method

Linearity and Repeatability and Detection and QuantitationLimits

Under optimum conditions, a series of experiments wereperformed to obtain linear range, repeatability and detectionlimit and quantitation limits.

The calibration curve was established by plotting peakarea versus the target analyte concentration. A good linearcorrelation between the compound concentration in a rangeof 10–500 ng/mL and peak areas was found with a correla-tion coefficient (r) over 0.998. The α-tocopherol amount inthe wheat sample was determined over six replicates duringone day and over six different days to obtain the intra-dayand inter-day precisions with RSD values of 3.6 and 7.8 %,respectively. All of the RSDs were adequate for the levels ofanalytes in samples.

The limit of quantification (LOQ, based on S/N=10) wasdetermined to be 6.3 μgL−1. The limit of detection (LOD,based on S/N=3) was found to be 1.9 μgL−1. Table 1compares the proposed DLLME method with other previ-ously reported methods dealing with the extraction anddetermination of α-tocopherol in food samples. It can beobserved that, though the recoveries and intra-day repeat-ability resulted from different methods are quite similar, themethod we developed has some obvious advantages overpreviously reported methods in terms of the sensitivity andlow cost.

Table 1 Comparison of the proposed methods with other previously reported methods

Methods Analyte Sample Linear range(μgL−1)

LOD (μgL−1) Intra-dayRSD (%)

Recovery (%) References

ASE-LC-ESI-MS α-tocopherol Wheat 25–4000 15 7.5 92 Bustamante-Rangel et al. 2007

Solvent extraction-LC-MS-ESI

α-tocopherol Sunflower oil 60–3100 20 1.9 106 Lanina et al. 2007

SPE-HPLC α-tocopherol Cereals 100–40000 460 2.1–4.6 95.4–113.2 Irakli et al. 2012

DLLME -HPLC α-tocopherol Pigmented wheat 10–500 1.9 3.6 90.5–103.7 This study

Table 2 The contents ofα-tocopherol extracted bydifferent methods (μg/g±RSD%n=3)

aIndicate the national standardextraction method. y Notdetectable

Samples DLLME Standarda SFE-CO2 ASE Heat-reflux

HM-1(1) 16.34ab±0.33 16.71a±0.99 5.79e±6.65 15.16ab±0.93 ndy

HL-76(2) 14.71ab±1.48 14.49ab±4.29 4.98e±6.78 13.33bc±2.34 nd

HR-2(1) 13.90ab±3.64 13.0bc±4.66 nd

HR-2(2) 10.30cd±3.25 10.16cd±6.52 5.22e±2.11 10.23d±4.84 nd

HR-2(3) 13.78ab±2.19 12.63bcd ±2.45 nd

Food Anal. Methods

According to equation PF=Csed/C0, where Csed was ana-lyte concentration in the sedimented organic phase, and C0

was the initial concentration of analyte in the aqueous solu-tion before DLLME, a preconcentration factor (PF) of 120was achieved when measuring the spiked blank samples bythe DLLME developed.

Comparison of Different Extraction Methods

Five wheat samples were extracted using the proposedDLLME method, the national standardized extract method,SFE-CO2, ASE, and conventional heat-reflux extraction.

After that, all the samples were then analyzed usingHPLC. The results are summarized in Table 2 and Fig. 3.

When using the conventional heat-reflux extractionmethod, because the saponification process was notemployed, the interference from the sample matrix duringthe chromatographic process resulted in a complex andconvoluted peak signal that prevented identification of thetarget compound, which caused a measurable amount of α-tocopherol was not observed.

The α-tocopherol content extracted using SFE-CO2 wascomparatively low. The statistical analysis manifested in thisextraction method exhibited a significant difference com-pared to the other extraction methods. This result could beattributed to the fact that the samples used in the studyconsisted of wheat flour, which contained a very smallamount of germ oil and might be not suitable for usingSFE-CO2.

The extracted α-tocopherol content using the nationalstandardized extraction method, ASE, and DLLME did notvary significantly (p<0.05). Among these three methods,the national standardized extraction process was the mostcumbersome and required numerous steps, and the totalextraction time for one sample was ca. 4 h. The DLLMEprocess was based on reducing four steps in the nationalstandardized method (extraction, washing, drying, and con-centrating via rotary evaporation) to two main steps, extrac-tion and centrifugation, which shortened the extraction timeto ca. 40 min. Additionally, the quantity of required organicsolvent was decreased from 200 mL to 80 μL for oneextraction.

Although the extraction rate of α-tocopherol for thewheat samples using ASE was comparable to those ofDLLME and the national standardized method, the

Fig. 3 Chromatograms of a blank sample spiked with 80 ng/mLstandard extracted by DLLME (a) and a real sample extracted byDLLME (b) and by the national standardized method (c) and byaccelerated solvent extraction (d) and by supercritical carbon dioxideextraction (e)

Table 3 The contents ofpigment and α-tocopherol forthe 42 samples of 14 types ofpurple wheat

a(1), (2) and (3) indicate thewheat samples grown intriplicate

Contents of total pigment (U/g) α-Tocopherol (μg/g ± %RSD, n=3)

(1)a (2)a (3)a Average (1)a (2)a (3)a Average

LZ-1 13.72 12.12 15.82 13.88 ab 14.92±2.64 13.52±4.57 15.36±2.76 14.59 ab

HM 11.43 14.18 12.21 12.60 abc 16.33±0.33 18.76±2.58 14.51±6.01 16.54 ab

KZ 7.55 7.82 7.33 7.57 c 18.27±3.21 17.04±3.65 14.08±2.21 16.46 ab

HY-1 5.94 11.55 5.43 7.64 c 17.01±2.83 16.39±2.63 18.79±2.59 17.40 ab

HY-2 9.65 6.45 6.52 7.54 c 17.28±2.18 15.20±1.28 16.35±2.15 16.28 ab

HY-3 6.84 6.59 7.61 7.01 c 17.97±0.13 17.68±2.90 13.41±5.96 16.35 ab

HR-2 15.53 18.00 16.35 16.63 a 13.90±3.64 10.30±3.25 13.78±2.19 12.66 b

HR-1-2 11.63 12.59 11.85 12.03 abc 18.07±2.16 14.72±0.27 14.88±3.02 15.89 ab

HB-2 9.12 9.28 6.57 8.32 bc 19.71±5.04 17.69±1.52 19.52±3.77 18.97 a

YZ-1 13.21 12.02 9.91 11.71 abc 14.42±7.03 15.67±0.70 18.92±2.63 16.34 ab

YZ-2 11.30 7.56 10.19 9.68 bc 15.98±3.94 17.32±2.95 14.28±0.95 15.86 ab

YZ-4 7.16 8.08 7.66 7.64 c 15.16±5.63 16.77±3.71 12.94±1.49 14.96 ab

YZ-5 10.92 11.33 6.50 9.58 bc 15.76±7.73 15.28±2.99 16.69±0.65 15.91 ab

HL-76 19.28 16.93 13.54 16.59 a 13.56±3.41 14.71±1.48 10.98±4.70 13.08 b

Food Anal. Methods

extraction volume could only be automatically con-trolled by the ASE instrument, where ca. 20 mL ofthe extraction solution was obtained when using anextraction cell of 11 mL, which resulted in a signifi-cantly smaller sample peak and a lower quantitativeaccuracy compared to the DLLME method. Also, forthe samples that have small amounts of the target analyte,the preconcentration treatment is required before HPLCanalysis.

To summarize, the DLLME method was the mostoptimum method for extracting α-tocopherol from cerealgrains.

Analysis of α-Tocopherol Content in Purple Wheat Samplesand Recovery Rates

The α-tocopherol contents in 42 samples of 14 types ofpurple wheat were measured under the optimized DLLMEconditions, and the results are depicted in Table 3. Theaverage α-tocopherol contents in the 14 types of wheatranged from 13.08 (HL-76) to 18.97 μg/g dry weight(DW) (YZ-1). A number of comparative studies of thecontents of α-tocopherol in wheat lines have been reportedby Lampi et al. (2008), who found a content rang of α-tocopherol of 6.4–19.9 μg/g dw for 175 genotypes ofdifferent wheat types grown under similar conditions;Hejtmánková et al. (2010), who determined a content rangeof 8.5–13.7 μg/g dw for three varieties of spring wheat; andHussain et al. (2012), who investigated forty organicallygrown spring and winter wheat genotypes and obtained acontent range of 5.18–10.3 μg/g dw. The contents of α-tocopherol determined in the current study were therefore inline with Lampi’s reports.

The results indicated that there was a significantdifference in the α-tocopherol content between the HB-2and HR-2 or HL-76 (p<0.05) samples, whereas the othertypes of wheat exhibited negligible differences.

Three real samples were chosen to test the recoveryrate of the method, and the results are given in Table 4,

showing that the recovery rates were in the range of90.5–103.7 % with a relative standard deviation lessthan 4.1 %.

Correlations Between the α-Tocopherol and PigmentContent

The kernel pigment amounts for the 42 samples of 14 purplewheat lines are shown in Table 3, demonstrating the pigmentamounts varied significantly among some of the wheatcultivars.

Correlations between the average contents of α-tocopherol and those of pigment for the 14 purple whatlines were examined, and the corresponding result is shownin Fig. 4. The slope of the curve (−0.3556) is significantlydifferent from zero, which demonstrates the contents ofα-tocopherol and pigment were negatively correlated.Furthermore, the statistical analysis manifested a significantcorrelation between them at the 0.01 level with r (p) value of−0.739(0.003).

Table 4 Recovery rates ofα-tocopherol in real samples

aThe content of α-tocopherol inpigmented wheat samples

Samples Contentsa

(μg/g ± RSD%, n=3)Amount spiked(μg/g)

Total amount found(μg/g ± RSD%, n=3)

Average recovery(% ±RSD%, n=3)

HM(1) 16.36±0.33 4.0 20.22±0.40 96.6±1.29

8.0 24.36±0.99 100.0±2.43

12.0 27.22±1.71 90.5±3.35

4.0 18.60±0.65 97.2±2.86

HL-76(2) 14.71±1.48 8.0 22.10±1.03 92.4±3.50

12.0 25.93±0.22 93.5±0.65

4.0 17.93±0.68 103.7±1.91

HR-2(3) 13.78±2.19 8.0 22.00±1.86 102.8±4.07

12.0 25.60±1.25 98.5±2.19

Fig. 4 The correlation between the average contents of α-tocopheroland pigment

Food Anal. Methods

Conclusions

The current study successfully established the DLLMEmethod for extracting α-tocopherol from grains. TheDLLME method possesses several advantages over thenational standardized extraction, ASE, SFE-CO2, andconventional heat-reflux extraction methods, including easyoperation procedure, short extraction time, low organicsolvent consumption, high sensitivity and precision, andlow analysis costs, which, in turn, shows its great potentialfor micronutrients analysis in food.

The proposed method was successfully applied todetermine α-tocopherol in 42 samples of 14 purple wheatvarieties. The average α-tocopherol content ranged from13.08 to 18.97 μg/g, indicating the purple wheat is rich inα-tocopherol. The pigment content in the purple sample wasalso observed to be significantly inversely correlated withthe α-tocopherol content, which, however, needs to beconfirmed by analyzing samples being cultivated fromdifferent regions.

Acknowledgments The authors acknowledge with gratitude andappreciation financial support from Northwest A&F University.

Conflict of Interest Qilong Xie declares that he has no conflict ofinterest. Shuhui Liu declares that she has no conflict of interest.Yingying Fan declares that she has no conflict of interest. XiaokeZhang declares that he has no conflict of interest. This article doesnot contain any studies with human or animal subjects.

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