Determination of Safranin T Pigment in Food Samples by Ionic Liquid Coated Magnetic Core/Shell Fe3O4@SiO2 Nanoparticles Coupled with Fluorescence Spectrophotometry

Three hydrophobic ionic liquids (IL) 1-octyl-3-methylimidazole hexafluorophosphate ([OMIM]PF6) (1-butyl-3methylimidazole hexafluorophosphate ([BMIM]PF6),1-hexyl-3-methyl-imidazole hexafluoro-phosphate ([HMIM]PF6), were coated by Fe3O4@SiO2 nanoparticles with core-shell structure to prepare magnetic solid phase extraction agent (Fe3O4@SiO2@IL) and establish a new method of magnetic solid phase extraction (MSPE) coupled with Fluorescence spectrophotometery for separation/analysis of safranin T (ST). The results showed that safranin T was adsorbed rapidly by Fe3O4@SiO2@[OMIM]PF6 and eluted by ethanol., pre-concentration factor of the proposed method was 20-fold. Under the optimal conditions the linear range, detection limit (DL), correlation coefficient (R) and relative standard deviation (RSD) were found to be 0.30-130.00 μg L, 0.05μg L, 0.9998 and 0.35% (n=3, c=10.00 μg L), respectively. The Fe3O4@SiO2 NPs can be used repeatedly for 10 times. This proposed method has been successfully applied to the determination of safranin T in food samples.


Introduction
Safranine T (ST) is a type of alkali industrial dye or stain (Figure 1), that could be used in different dyeing manufactories [1]. ST was also known as food additives [2], however, in many food manufactories ST was illegally used to improve the properties of food products [3], so introducing a simple and fast method for the separation of ST in dietary products is very important. The well-known techniques for ST separation were performed by using high performance liquid chromatography (HPLC), UV-visible spectrophotometry and the fluorescencemethod depend on calixarene derivatives. Among these methods, Fluorescence spectrophotometery has many advantages of easy operation and low-cost analysis which should be combined with separation and enrichment techniques to improve the selectivity and sensitivity of detection..
The definition of magnetic solid phase extraction (MSPE) was that, it is a process depend on using magnetic sorbents for separation of various analytes in different volume of the sample [4],. In MSPE procedures. The magnetic absorbent was mixed with sample and the analyte was absorbed by magnetic sorbents. The analyte-magnetic sorbents were then separated from the sample by using an external magnetic field, after being eluted by a perfect eluent [5]. However, MSPE has been used in widely in many analysis fields such as food, environmental and biological analysis [6,7] Nowadays, scientists are focusing on the use of Fe 3 [9]. Ionicliquid (IL), are a type of organic salts possess special physicochemical properties, like good stability and hydrophobic properties [10]. Many studies had described the utilization of IL through SPE [11,12]. Ionic liquids coated MNPs were used as an adsorbent in separation of aromatic hydrocarbons in water [13]. Fe 3 O 4 @IL@methyl orange NPs were used for separation of PAHs in water [14]. However, it has not been reported to separate or extract ST with Fe 3 O 4 @SiO 2 @[OMIM]PF 6 . In the present work, MSPE sorbents (Fe 3 O 4 @SiO 2 @[OMIM]PF 6 ) were prepared, these sorbents have the property of the ionic liquids and (MNPs). When compared other studies, these MSPE sorbents provides a rapid, and efficient sample preparation process, which enables the treatment of large volume samples in a short period of time [15]., this adsorbent based MSPE providing a rapid, and efficient sample preparation process, which enables the treatment of large volume samples in a short period of time. A novel MSPE method coupled with Fluorescence spectrophotometery was therefore established for separation/analysis of ST from food samples.

Equipment and Reagents
FTIR spectra were measured with a Bruker Tensor 27 spectrometer (Bruker Company, Germany). Samples were pressed into potassium bromide KBr pellets and recorded at the frequencies from 4000 to 400 cm -1 with resolution of 4 cm -1 . A Tecnai 12 TEM (Philips, Netherlands) was used to obtain micrographs of the MNPs. spectrophotometricseparation of analytes was achieved with Fluorescence spectrophotometery.
Fe 3 O 4 MNPs were prepared by conventional coprecipitation method [18,19]. First, FeCl 3 (3.30 g) was dissolved in deionized water (80 mL) followed by addition of polyethylene glycol (40 mL, 10%, w/w) and of (NH 4 ) 2 Fe(SO 4 ) 2 (4.23 g) in water solution under stirring. Then ammonium hydroxide (8 mL, 26.5%, w/w) was added rapidly under vigorous stirring. The resultant solution was stirred (3000 r min -1 ) at 80°C for 60 min. After cooling to room temperature, the obtained Fe 3 O 4 precipitate was collected by an external magnetic field, washed with deionized water five times and dried at 60°C for 12 h in vacuum.
The preparation of Fe 3 O 4 @SiO 2 was done according to literature. Fe 3 O 4 (1.0 g) were dissolved in 200 mL of the ethanol and 50 mL of deionized water by sonication for 15 min, and then 4 mL ammonium hydroxide and 6 mL TEOS were added sequentially. The mixture was reacted for 6 h at 60°C under a continuous stirring. The resultant product was collected by an external magnetic field, and rinsed with deionized water and ethanol for six times thoroughly, and then dried in vacuum to obtain Fe 3 O 4 @SiO 2 .
IL functionalized MNPs Fe 3 O 4 @SiO 2 @[OMIM]PF 6 ( Figure  2) were prepared according to process detailed in literature [20]. A 4.00 g of [OMIM]PF 6 was dissolved in 60.0 mL acetone, and then 3.00 g Fe 3 O 4 @SiO 2 nanoparticles were added. Stir the mixture inside the fume hood until the acetone completely evaporated.

Magnetic Solid Extraction (MSPE) Procedure
The procedure was done as follows. First, Fe 3 O 4 @SiO 2 @[OMIM]PF 6 MNPs were added to a 90 mL water sample containing ST (pH=7.0), and the mixture was placed on a slow-moving platform shaker and allowed to equilibrate for 15 min at 25°C. Then, a strong magnet was applied to the bottom of the beaker, isolating the Fe 3 O 4 @SiO 2 @[OMIM]PF 6 MNPs from the suspension. After 15 min, the suspension was decanted and the residual solution of Fe 3 O 4 @SiO 2 @[OMIM]PF 6 MNPs was transferred to centrifuge tube. The Fe 3 O 4 @SiO 2 @[OMIM]PF 6 MNPs were aggregated again by positioning a magnet to the outside of the tube wall so that the residual solution could be completely removed by pipette. Finally, the isolated MNPs were mixed with 3 mL of ethanol and sonicated for 5 min to elute the pre-concentrated target analytes. After wards, a magnet was positioned on the outside of the centrifuge tube, and the supernatant solution was collected and F-7000 FL spectrophotometer (Hitachi, Japan) was used for all the fluorescence measurement.

Extraction Efficiency of Different MNPs
In this work, six types of NPs (Fe 3

Fluorescence Measurements
In a 10.0 mL test tube, 0.05 g 0.01mol/L IL-MNPs, 0.3mL of ethanol, 2.0 mL of buffer solution (pH=3.0) and adequate safranin T standard solution or sample solution were added; the solution was diluted to the mark with distilled water. Then fluorescence spectra were recorded in the range of 300-700 nm upon excitation at 570 nm.

Sample Preparation
An amounts of 20.0 g of2 different manufactured tomato sauce was accurately weighed into the beakers, dissolved in 40.0 mL of ethanol + 1.0 mL of ammonia. Then 0.4 ml of The resultant clear solution was was added to 1.5g of IL [OMIM]PF 6 and 2.0mL of NaCl 15% was added to 10.0mL test tubes from3 different types of samples.

Characterization of the MNPs
The synthesized Fe 3 O 4 @SiO 2 @ILsMNPs were characterized by FT-IR spectroscopy, thermogravimetrical analysis and magnetic characterization.
Magnetic characterization using a magnetometer at 27°C indicates that the maximal saturation magnetizations of  Figure 3.C describes the magnetic hysteresis loops of the three MNPs, and it is apparent that all of the NPs show superparamagnetic properties in the presence of magnetite particles in the core. Therefore, the Fe 3 O 4 @SiO 2 @[OMIM]PF 6 MNPs prepared here could be rapidly separated from solution with a magnet on account of their superparamagnetism and large saturation magnetization.

Optimization of Adsorption
The factors affecting the adsorption process of safranin T such as pH, temperature and solution volume were studied and the adsorption behavior of Fe 3 O 4 @SiO 2 @PIL on ST was compared with that of MNPs.

Effect of pH
As shown in Figure 4, the adsorption efficiency of ST was varied with the pH, which was between 3.0 to 12. It could be concluded that the adsorption (retention) efficiency of ST on IL MNPs was above 80.0%. It reached the highest value 83.28% when pH was7.0, Therefore, pH 7.0 was selected for the subsequent assays.

Effect of Adsorption Temperature and Time
The adsorption efficiency of STon IL MNPs and MNPs at different temperatures (5.0-50°C) were studied ( Figure 5). The adsorption efficiency of AR on Fe 3 O 4 @SiO 2 @IL was higher than that on MNPs and was always above 70% from 10°C to 15°C and was above 80% from 0.20°C to 30°C. The experiment was carried out at room temperature.  Effect of adsorption time on extraction efficiency shows the extraction process was completed within 15.0 min, and the adsorption efficiency remained almost stable (88.0%). 15.0 min as the adsorption time for ST was adopted ( Figure 6).

Effect of the Sample Volume
The adsorption efficiency of ST varied with the increase of sample volume. The amount of ST was fixed at 50.0 µg and the volume of the sample solution increased from 10.0 mL to 90.0 mL. The adsorption efficiency of ST was above 89% from 10.0 to 80.0 mL and remain steady. and 77.85% when sample volume was 90.0 mL. So the largest sample volume allowed was 80.0 mL (Figure 7).

Adsorption Capacity
The adsorption capacity is defined as the maximum amountof ST adsorbed per gram of IL MNPs. The adsorption capacity of ST on IL MNPs was studied (Figure 8). When the concentration of ST was 50.0 µg/mL (volume: 50 mL ), the adsorption of ST for 0.0225g IL MNPs reached the maximum. The adsorption capacity for IL MNPs was calculated as 105.80 mg/g.

Selection of Eluent
In this work, different eluents were investigated. The order of elution efficiency was ethanol >methanol> cetyltrimethyl ammonium bromide (CTAB)> sodium dodecyl sulfonate (SDS) > NaOH (0.1 M) > HCl (0.1 M). So ethanol was adopted as the eluent. Figure 9. The effect of ethanol volume was detailedly evaluated on the elution of ST. The results described that quantitative elution (above 95%) was observed as the volume of ethanol was higher than 3.0 mL. The pre-concentration factor is 25fold. The optimum volume of ethanol was chosen at 3.0 mL.
The elution process was completed within 10.0 min, and the elution efficiency did not change with a stable elution efficiency of 95.% thereafter. The elution time of 10.0min for ST was adopted.
Effect of elution temperature The elution efficiency of ST at different temperatures (5-60°C) was studied. The elution efficiencywas increased progressively with an increase of elutiontemperature from below 40°C. The elution efficiency of ST was greater than 85% and decreased at the elution temperature range from 50 to 60°C. Accordingly, the elution was performed at room temperature.

The Reusability of Fe 3 O 4 @SiO 2 @IL
In order to investigate there cycling of the Fe 3 O 4 @SiO 2 NPs, they were washed with 2mL ethanol for twice after each MSPE run, and subsequently assembled with IL. Each re-prepared adsorbent was used for MSPE. The experimental results are shown in Figure 10. It was clear that no obvious loss of the sorption capacity occurred after ten times of recycling.

Evaluation of Interferents
With a relative error of less than ±5%, the influence of some interferents that food samples contain on the determination of ST in the presence of foreign substances was investigated. the tolerance limit for various foreign substances is in Table 1. The results indicated that the majority of these substances in samples had no remarkable interference on the ST determination.

Analytical Performance of the Method
Under the optimum conditions, the linear calibration curve was obtained in the concentration range of 0.30-130.00 µg L -1 . The equations of calibration graph is I (peak area) = 11.97c+12.11 (µg L -1 ), with a correlation coefficient of 0.9998. The limit of detection (DL) was 0.05µg L -1 . The relative standard deviation was 0.35% (n=3, c=10.00 µg L -1 ). The preconcentration factor, defined as the quotient of volume before absorption and after elution, is 25-fold.

Sample Analysis
The proposed method was applied to determine ST in Tomato sauce A, Tomato sauce B. ST is 0.29 µg kg -1 in Tomato sauce A, with a recovery rate of ST of 97.4% to 103 %. and it was 0.37 µg kg -1 in Tomato sauce B with a recovery rate of ST of 110.0% to 120.9 %..

Comparison of the Proposed Method with Relevant Literature
The comparison of current work with some other methods on the determination of ST (Table 3) reveals that this method is either comparable or has rather pronounced advantages over them. Moreover, it is obvious that the present work has high sensitivity, wide linear range, and easy operation. Table 3 listed the linear range and the limit of detection for the analysis of ST in real samples obtained by the reported methods, compared with other reported methods, the method adopted in the present work obviously had a satisfactory linear range and limit of detection.

Conclusion
In this work, IL MNPs was synthesized as magnetic solid phase extraction adsorbent to pre-concentrate/separate ST from real samples. In summary, we have prepared Fe 3 O 4 @SiO 2 @IL as adsorbent, for MSPE of. The magnetic nano-adsorbent is prepared by a self-assembly technique, which is very simple and mild. Compared with traditional SPE, this MSPE based on the Fe 3 O 4 @SiO 2 @IL MNPs as an SPE adsorbent is fast, and the adsorbent can be easily separated from the sample solution. Moreover, this adsorbent has a high extraction capacity and high enrichment factors and is able to treat large-volume samples in a short period of time. The hydrocarbon chains of ILs on the surface of the adsorbent can provide adsorption sites for ST other organic pollutants through π-π and hydrophobic interactions. The proposed method for the analysis of ST is satisfactory. Thus, this adsorbent may also find potential application in the extraction and analysis of other analytes.