Chinese Chemical Letters  2014, Vol.25 Issue (03):465-468   PDF    
Determination of spices in food samples by ionic liquid aqueous solution extraction and ion chromatography
Hai-Bao Zhua , Yun-Chang Fanb, Ya-Ling Qiana, Hong-Fang Tanga, Zheng Ruana, Dan-Hua Liua, Han Wanga    
Corresponding authors at:a Zhejiang Academy of Medical Sciences, Hangzhou 310013, China;
b College of Physics and Chemistry, Henan Polytechnic University, Jiaozuo 454003, China
Abstract: In the present work, a novel method to extract three kinds of spices, namely vanillin, ethyl vanillin and ethyl maltol from food products such as biscuit, chocolate and milk powder was developed. 1-Octyl-3-methylimidazolium chloride ([Omim]Cl) aqueous solution was selected as the extracting medium. A 0.5 g powder of food product was extracted by 5.0 mL of [Omim]Cl aqueous solution (0.3 mol/L, pH 6.0) under ultrasonication at 50 ℃, and then the extract was centrifuged for 10 min at 4000 rpm. The extract was filtered through a syringe filter and injected into ion chromatography system for analysis. The separation of the three spices was carried out on an anion exchange column. The detection wavelength was set at 280 nm. Compared with traditional extraction solvents, [Omim]Cl aqueous solution displayed particular advantages. The applicability of the proposed method to real sample was confirmed. Under the optimal conditions, good reproducibility of extraction performance was obtained, with the relative standard deviation (RSD) values ranging from 1.9% to 6.3%. The recoveries of spiked samples were between 79.8% and 95.8%. The detection limits (LOD, S/N = 3) of vanillin, ethyl vanillin and ethyl maltol were in the range of 20-45 μg/kg. The use of ionic liquid aqueous solution as extraction solvent was operationally easy and environmental-friendly.
Key words: Spices     Ionic liquid     Food     Ion chromatography    
1. Introduction

Vanillin,ethyl vanillin and ethyl maltol are extensively used as spices in food industry to improve the taste of the products of interest. However,excessive usage of spices might be harmful. So, it is of great importance to monitor the level of the three spices in food samples. High performance liquid chromatography (HPLC) [1],capillary electrophoresis [2] and spectrometric methods [3] were developed for this purpose. However,few methods were reported using ion chromatography (IC). The three spices investigated in the present work are weak organic acids. Therefore, they could be separated by anion-exchange chromatography. However,suitable sample pretreatment procedure is a prerequisite for the determination of the three spices in food samples by ion chromatography.

Extraction methods for spices in different food samples predominantly consist of solid phase extraction (SPE),solvent extraction and matrix solid phase expression (MSPE) [4, 5]. Organic solvents were usually used in these conventional methods. They pose a threat to the environment and human health. Thus developing a relatively simple and environmentally benign extraction method for the determination of spices in food samples is urgent. Recently,ionic liquids (ILs) have attracted extensive interest owing to their potential use as environmentally benign solvents to replace traditional volatile organic solvents in many applications,including some extraction processes like liquid-phase microextraction [6, 7],solid-phase microextraction [8, 9] and microwave-assisted extraction [10, 11].

It has been reported that a series of ILs based on the 1-alkyl-3- methylimdazolium salts act as short chain cationic surfactants in aqueous solution and form aggregations above critical aggregation concentration [12, 13]. Therefore,IL-based aqueous solution is also a good medium for the extraction of polycyclic aromatic hydrocarbons from sediments.

The main purpose of the present work was to evaluate the possibility of using an IL aqueous solution,1-octyl-3-methylimidazolium chloride ([Omim]Cl),to replace traditional organic solvents to extract vanillin,ethyl vanillin and ethyl maltol from biscuit,chocolate and milk powder. After investigating the parameters affecting the extraction efficiency,the optimal extraction conditions were established for the IC-UV/vis determination of vanillin,ethyl vanillin and ethyl maltol. Because of the nonvolatility of the IL,the proposed method was environmentally friendly with good reproducibility and spiked recoveries. 2. Experimental 2.1. Reagents and samples

Vanillin,ethyl vanillin and ethyl maltol standard agents were obtained from Dingfu Chem. Co. (Shanghai,China). HPLC-grade acetonitrile was purchased from Tedia Co. (USA). 1-Chlorooctane (99.5%) was purchased from Bangcheng Chem. Co. (Shanghai, China). 1-Methylimidazole (99.0%) was obtained from Kaile Chem. Co. (Zhejiang,China). Analytically pure sodium hydroxide was purchased from Sinopharm Chem. Reagent Co.,Ltd. (Beijing,China). Deionized (18.2 MΩ/cm) water generated by a Millipore Milli-Q Plus system (Millopore,Milford,USA) was used throughout.

Working solutions of vanillin,ethyl vanillin and ethyl maltol were obtained by appropriate dilution of the corresponding standard solutions (1000 mg/L) with deionized water. Ionic liquids [Omim]Cl was prepared according to the reported literature [14, 15]. Biscuit,chocolate and milk powder were randomly purchased from local supermarket. 2.2. Equipment

IC analysis was performed on a Dionex 500 ion chromatographic system (Dionex,USA) consisting of a quaternary pump,a manual injector with a 15 mL loop and an ultraviolet detector setting at 280 nm. An AS18 column equipped with an AG18 guard column (Dionex,USA) was used for the separation of the three spices extracted in the IL phase. Chromeleon 6.5 software was used to acquire data and control the instrumentation.

TDL-60B ultrasonic machine from Anting Instrument Co. (Shanghai,China) was used to facilitate the extraction of the three spices from solid matrices. TG16-W centrifugal machine from Weierkang Xiangying Centrifuge Co.,Ltd. (Changsha,China) was used to clarify and simultaneous separate the liquid phase. 2.3. Chromatographic conditions

A mixture of 25 mmol/L sodium hydroxide–acetonitrile (85:15, v/v) was used as mobile phase by pneumatic means,i.e. by putting the high-density polyethylene (HDPE) bottles in a chamber pressurized at 250 psi with nitrogen. The flow rate of the eluent was 0.25 mL/min. 2.4. Extraction procedure

Typically,0.5 g of sample,which was crushed into powders,was weighed into a 10-mL plastic conical bottom tube,and then 5.0 mL IL aqueous solution (0.3 mol/L) was added. After extraction for 20 min under ultrasonic irradiation,the mixture was centrifuged for 10 min at 4000 rpm. The extract was filtered through a syringe filter (pore size 0.45 mm) and injected into the IC system for analysis. 3. Results and discussion 3.1. Influence of IL concentration

To evaluate the IL concentration effect on the extraction efficiency,additional experiments were performed using different concentrations of [Omim]Cl. Biscuit sample spiked with 80 mg/kg of vanillin,ethyl vanillin and ethyl maltol was used as a representative in the experiment. The result in Fig. 1 shows that the extraction recoveries of vanillin,ethyl vanillin and ethyl maltol increased with the IL concentration up to 0.3 mol/L and plateaued above this level.

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Fig. 1.Effect of IL concentration on extraction efficiency. Ethyl vanillin; ethyl maltol; vanillin. Condition: 0.5 g biscuit sample spiked with 80 mg/kg of vanillin,ethyl vanillin and ethyl maltol was extracted with 5.0 mL extractant for 20 min under ultrasonication.
3.2. Effect of volume of IL

In order to investigate the effect of IL volume on extraction efficiency,0.5 g of biscuit sample spiked with 80 mg/kg of vanillin, ethyl vanillin and ethyl maltol was extracted with different volumes of extractant (2–7 mL) for 20 min. The results shown in Fig. 2 demonstrate that the recoveries of the three spices are almost constant with the volume of [Omim]Cl ranging from 5.0 to 7.0 mL. Since unnecessary IL aqueous solution was not used to avoid an excessive dilution of the sample,5.0 mL was then selected as the extractant volume.

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Fig. 2.Effect of extractant volume on extract efficincy of the three spices. Ethyl vanillin; ethyl maltol; vanillin. Condition: 0.5 g biscuit sample spiked with 80 mg/kg of vanillin,ethyl vanillin and ethyl maltol was extracted with 0.3 mol/L IL for 20 min under ultrasonication.
3.3. Effect of pH value

The effect of pH value on the efficiency of extraction was studied using 2 mol/L hydrochloric acid or 1 mol/L sodium hydroxide regulating the pH values of extraction phase. Results showed that,in the pH 3–8 range,the extraction efficiency of the IL aqueous solution on the three spices did not vary with the change of pH values. However,the extraction efficiency decreased significantly when the pH value was above 8. A reasonable explanation for this phenomenon is that the hydroxyl groups (– OH) of the three spices exist in neutral forms (–OH) and thus can form hydrogen bonds with the IL [6]. Thus,the extraction efficiency of the [Omim]Cl was higher under acidic conditions than in alkaline environments. Considering the fact that the pH value of deionized water is very close to 6,pH 6 was then selected. Additionally,the pKa values of three spices were in the following order: ethyl vanillin > ethyl maltol > vanillin,therefore,the percentages of the neutral form for the three spices were also in the same order,which explained why the extraction efficiency decreased in the following sequence: ethyl vanillin > ethyl maltol > vanillin. 3.4. Effect of temperature

In this study,the effect of extraction temperature on extraction efficiency was examined in the range of 40–70°C. The results showed that,the extraction efficiency at 50°C is higher than the extraction efficiency at 40°C; however,above this level,no obvious increase in the extraction efficiency was observed while the concentrations of co-existences clearly increased. So,50°C was selected as the best extraction temperature. 3.5. Effect of extraction time

Since extraction time is another factor that would influence the extraction efficiency in most extraction processes. Therefore,in the present work,the range of extraction time from 5 to 30 min for spiked blank biscuit sample (ultrasonication time) was investigated. Results shown in Fig. 3 indicate that the extraction recoveries of the three spices increased with the extraction time up to 20 min, and remained constant after that. Based on this result,the extraction time of 20 min was adopted in the following experiments.

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Fig. 3.Effect of extraction time on extraction recovery. Ethyl vanillin; ethyl maltol; vanillin. Condition: 0.5 g biscuit sample spiked with 80 mg/kg of vanillin,ethyl vanillin and ethyl maltol was extracted with 5.0 mL 0.3 mol/L IL under ultrasonication.
3.6. Comparison with the conventional solvent

In order to compare the extraction efficiency of [Omim]Cl with that of conventional solvents,acetonitrile,which is usually used for extraction of spices,was used to extract the three spices from food samples under the above mentioned conditions. The results shown in Fig. 4 indicate under the optimal conditions,the extraction efficiencies obtained with [Omim]Cl are better than the ones obtained with acetonitrile. As mentioned in Section 3.3,under acidic conditions,spice molecules and IL can form hydrogen bonds. So,the solubilities of the three spices are greater in ILs than in acetonitrile.

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Fig. 4.Effect of different solvents on the extraction recovery.

Overall,[Omim]Cl exhibits much higher extraction efficiency. Additionally,acetonitrile is volatile,flammable and harmful to the environment and lab workers. Therefore,[Omim]Cl aqueous solution is potentially an alternative solvent to replace conventional solvents for the extraction of spices. 3.7. Optimization of chromatographic conditions

In order to rapidly separate the three analytes and eliminate IL from the separation column,a small amount of acetonitrile was added to the eluent (15%,v/v).

Common conventional anions such as fluoride,chloride,sulfate and phosphate have no signals under the ultraviolet detector setting at 280 nm. Although the nitrate,nitrite and other coextracted compounds have signals,they do not interfere the determination of the three analytes. 3.8. Analytical performance

Under the optimal conditions,some parameters such as linearity,precision (RSD,n = 6),limits of detection (LOD) and limits of quantification (LOQ) of the proposed method were investigated. Calibration curves were obtained by preparing a series of standard solutions with the concentration range of 0.2– 25 mg/L. Each analyte exhibited good linearity with a correlation coefficient R ≥ 0.9993 in the studied range. The reproducibility of the peak area was studied by six repetitive determinations of 2 mg/L standard and the relative standard deviations (RSD) were not more than 3.8%. The limits of detection (LOD),estimated based on signal-to-noise ratio of 3 (S/N = 3),were in the range of 20–45 mg/kg. The limits of quantification (S/N = 10) were in the range of 0.07–0.15 mg/kg. Table 1 summarizes these analytical data.

Table 1
Analytical performance of the proposed method.
3.9. Analysis of real samples

To evaluate the feasibility of the proposed method in determining spices in real food samples,the biscuit,chocolate and milk powder samples purchased from supermarket were tested under the optimal experimental conditions. In order to test the matrix effects,biscuit,chocolate and milk powder samples were spiked with the standards of the three spices at different concentration levels. The experiments demonstrate that the recoveries of spiked samples were between 79.8% and 95.8%.

These results are listed in Table 2. The typical chromatograms are shown in Fig. 5. It should be noted that although the recovery of spiked vanillin was just 79.8%,by employing accelerated solvent extraction (ASE) instead of ultroasonication,improved recovery could be attained and further investigation is undergoing.

Table 2
Results of determination of the three spices in food samples (mean±standard deviation,n = 3).

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Fig. 5.A typical chromatogram of chocolate sample. Peaks: 1 = [Omim]Cl; 2 = ethyl maltol; 3 = vanillin; 4 = ethyl vanillin. Sample volume: 15 mL; IC column: IonPac AG18 (50 mm × 2 mm),AS18 (250 mm × 2 mm); detection: ultraviolet detector setting at 280 nm; mobile phase: 25 mmol/L sodium hydroxide–acetonitrile (85:15,v/v); flow-rate: 0.25 mL/min.
4. Conclusions

In the present work,the environmentally benign method termed ionic liquid extraction technique coupled with IC-UV/vis has been successfully applied in the determination of spices in food samples. Compared with the conventional methods,the proposed extraction method only requires a small amount of sample and a small volume of [Omim]Cl to extract sample. Moreover,no complicated solvent evaporation and reconstitution steps are involved. These facts lead to the conclusion that the IL-based extraction technique is a simple,effective and environmentally benign extraction method and can be used for the extraction and determination of spices in food samples. Acknowledgments

This research was financially supported by the National Natural Science Foundation of China (No. 21307028),Zhejiang Provincial Medical Key Discipline Group–Public Health Detection (No. XKQ- 009-003),and Zhejiang Provincial Key Technology and Laboratory of Occupational Hazards Detection (No. C71139D),and Laboratory Construction to Promote the Occupation Health Monitoring Technology (No. C71318D).

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