Materials
Five different brands of pickle samples and canned mix congee were purchased respectively at a supermarket in Beijing; Disodium Ethylenediaminetetraacetate acid (>99.0%, C10H16N2O8, J&K Technology Co., Ltd., Beijing); Zirconium oxychloride octahydrate (≥98%, ZrOCl2·8H2O, J&K Technology Co., Ltd., Beijing); Quercetin (≥99%, C15H14O9, Yi Xiu Bogut Biological Technology Co., Ltd., Beijing); Hexadecyl trimethyl ammonium bromide (≥99%, C19H42BrN, blue-Yi Chemical Products Co., Ltd., Beijing); Ferric chloride (FeCl3); ascorbic acid (C6H8O6); tetrabutylammonium hydroxide (C16H39NO2); Acetic acid (CH3COOH); anhydrous ethanol (C2H6O); hydrochloric acid (HCL); chloroform (CHCl3); n-butanol (CH3(CH2)3OH); ammonia water (NH3·H2O) used in this work were of analytical grade (Beijing chemical Works); Acetonitrile CH3CN and Formic acid HCOOH (Chromatographic grade, J & K Technology Co., Ltd., Beijing).
Instruments and Equipment
A 1260 high performance liquid chromatograph with a diode array detector (Agilent, USA); ODS-C18 liquid chromatography column (250 × 4.60 mm, 5 µm, Agilent, USA); F-7000 fluorescence spectrophotometer (Hitachi, Japan); QL861 vortex mixer (Kylin-bell Instrument Co., Ltd., Jiangsu, China); Feige TDL-5-A desktop centrifuge (Anke Scientific Instruments corp., Shanghai, China); Milli-Q ultrapure water machine (Millipore, USA); FA1104B electronic analytical balance (sensitivity: 1/10000, Vietnam Scientific Instruments Ltd., Shanghai, China); KQ-500DE CNC heated ultrasonic cleaning instrument (Kunshan Ultrasonic Instrument Co., Ltd., Jiangsu, China); RE-52AA rotary evaporator (Shanghai Yarong Biochemistry Instrument Factory, Shanghai, China)
HPLC-DAD method
Preparation of calibration standard solutions
Disodium ethylene diamine tetra acetate standard 100 mg (accurate to 0.0001g) was dispersed in deionized water with a concentration of 1000 µg/mL. The 10.0, 20.0, 50.0, 100.0, 200.0, 400.0 and 600.0 µL solutions were added in 10mL volumetric flask respectively and mixed with 0.1 mol/L FeCl3 (1 mL) and 0.23 mol/L Ascorbic acid solution (100 µL). All solutions were stored at room temperature for one night. Quantification was performed using an external calibration method. Calibration curves were prepared at concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, 40.0, 60.0 µg/mL.
Pretreatment and Derivatization of Samples
The homogenized canned mix congee, pickled vegetables (5.0g, respectively) were added to a 50 mL Teflon screw centrifugal tube and 20 mL of deionized water was added. The samples were then extracted by ultrasonication (80W) for 30 min. Next, the solutions were centrifuged for 10 min at 3500 r/min. Then the residues were re-extracted twice by using the same methods. The filtered solution extractions were transferred into the volumetric flask (100 mL) and complemented with deionized water. The obtained filtrate (500 µL) was diluted with 0.1 mol/L FeCl3 solution (100 µL) and 0.23 mol/L ascorbic acid (10 µL). Next, reaction mixture was made up to 1 mL with ultrapure water, vortexed for 10 s, and kept for overnight.
HPLC condition
The elution was isocratic with a flow rate of 1.0 mL/min using a mixture of 0.13% tetrabutylammonium hydroxide aqueous solution adjusted pH to 4 by formic acid (80%, V) and acetonitrile (20%, V) as the eluent. The target compound was determined at a wavelength of 254 nm.
Three-dimensional fluorescence method
Optimization of Derivative Conditions
In order to obtain the optimal reaction conditions for the derivatization of the ternary mixed complexation, the different volumes of NH3·H2O (0.2, 0.4, 0.6, 0.8 and 1.0 mL) and absolute ethanol (0.5, 1.0, 1.5, 2.0, 2.25, 2.5, 2.75, 3.0 and 3.25 mL) were mixed with 2×10-3 mol/L Zr (IV) (1 mL), 1.0×10-2 mol/L quercetin (1 mL) and 1000.0 µg/mL EDTA-2Na solution (0.4 mL). Furthermore, hexadecyl trimethyl ammonium bromide (CTMAB) was also used to study the effect on the fluorescence intensity of the system. EDTA-2Na (10.0, 20.0, 50.0, 100.0, 200.0 and 400.0 µL), 2×10-3 mol/L Zr (IV) (1 mL), 1.0×10-2 mol/L quercetin (1 mL) were mixed with 1.0×10-2 mol/L CTMAB (3.5 mL) or without the solution. The all solutions were made up to 10.0 mL with ultrapure water in a volumetric flask. After vortexing for 10 s, fluorescence analysis was performed within 3 min.
Preparation of calibration standards
2×10-3 mol/L Zr (IV) (1 mL), 1.0×10-2 mol/L quercetin (1 mL), different volume of EDTA-2Na solution, 2.75 mL of absolute ethanol and 0.6 mL of 0.5 mol/L NH3·H2O solution were mixed and diluted to 10.0 mL with ultrapure water in volumetric flask. After vortexing for 10 s, fluorescence analysis was carried out within 3 min. Quantification was performed using an external calibration method. Calibration curves were prepared at EDTA-2Na concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, 30.0, 40.0 µg/mL.
Pretreatment and Derivatization of Samples
The extraction method was in the same way as mentioned in the section about quantification of EDTA-2Na by HPLC-DAD. For extremely complex canned mix congee samples, it is also necessary to remove sugar and protein. A 20.0 mL of the extract was placed into a 50.0 mL screw-top PTFE centrifuge tube while anhydrous ethanol (30.0 mL) was added. The tube was shaken rapidly, refrigerated at 4 °C for 1h, and then centrifuged at 5000 r/min for 5 min. The filtered mixture was evaporated by rotary evaporation under reduced pressure at 55 °C for 6 min. After removing ethanol, the volume was adjusted to 20.0 mL with ultrapure water. A 10.0 mL of the above solution was taken into a 15.0 mL screw-top PTFE centrifuge tube. Afterwards, 2 ml of Sevag reagent (chloroform and n-butanol at a ratio of 4:1/V: V) was used to deposit protein under centrifugation for 5 min at 3000 r/min. The supernatant was collected for derivatization. The above treatment was not required for pickles. Then 4.65 mL of the supernatant was derivatized in the same method as the calibration standard solution.
Selection of Scanning Scope, PMT Negative Voltage and Slit Width
In order to obtain the appropriate scanning scope, negative voltage of the photomultiplier tube and slit width, 40.0 µg/mL EDTA-2Na ternary mixed complex solution was used as the experimental mode. In the condition of excitation wavelengths of 350-500 nm and 5-nm intervals, the emission wavelengths of 450-600 nm and 5-nm intervals were scanned to determine optimal excitation and emission wavelengths to use for analysis. Moreover, the photomultiplier tube voltage and slit width were also optimized considering the resolution and intensity by comparing the results at 250 V, 400 V, and 700 V with the slit width (5 nm). Similarly, by controlling voltage, the suitable slit width can be obtained.
Method validation
According to the ICH Harmonized Tripartite Guidelines on Validation of Analytical Procedures: Methodology (ICH 2005), linearity, fortified recoveries, RSDs, and sensitivity were evaluated for the optimized method.
Linear range and sensitivity
Calibration curves were respectively diluted in solution at concentrations of 40, 30, 20, 10, 5, and 1 µg/mL, where y was the fluorescence response and x was the concentration. Three-dimensional fluorescence limits of detection (LOD) and quantification (LOQ) (µg/g) were calculated based on the standard deviation (σ, counts) of 11 readings of the standard solution blank, per Eq. 1:
where slope was the sensitivity (counts mL/µg) and k was the dilution factor (mL/g)
Recovery and Repeatability
To evaluate the accuracy of the method, the samples of spiked pickle and canned mix congee were taken to analyze the recovery. The spiked concentrations were at three level of 40.0, 100.0, 160.0 µg/g, respectively. Each level was analyzed in triplicate. And the repeatability was evaluated via the relative standard deviation of the five replicates using canned mix congee and pickle samples.
Accuracy via comparison by the HPLC and fluorescence method
In order to further verify the accuracy of the results for the determination of EDTA-2Na in food by fluorescence method, the EDTA-2Na contents in the 10 samples were analyzed by HPLC-DAD and fluorophotometry simultaneously, and each level was determined in triplicate. Moreover, the accuracy was calculated as the relative error (RE %) of the two methods.
where A was the content by Fluorescence method (µg/g) and B was the content by HPLC-DAD method (µg/g).