Ratio-dependent fluorescence analysis method for determining sodium dehydroacetate

Abstract

The invention discloses a ratio-type fluorescence analysis method for determining sodium dehydroacetate, which comprises the following steps: under an alkaline condition, synthesizing tetrafluoroterephthalic acid oxide, namely a fluorescent substance TFAox, by oxidizing tetrafluoroterephthalic acid with hydrogen peroxide, and forming a ratio-type fluorescence probe by the fluorescent substance and rhodamine B, a ratio-type fluorescence analysis method of sodium dehydroacetate is constructed based on the ratio-type fluorescence probe, and the method can be successfully applied to determination of sodium dehydroacetate in a water sample and a bread sample. The ratio-dependent fluorescence detection method for determining sodium dehydroacetate provided by the invention has the characteristics of wide linear range, high detection sensitivity, no interference of coexisting substances on determination, good selectivity and the like.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection technology for preservatives, specifically relating to a ratiometric fluorescence analysis method for determining sodium dehydroacetate. Background Technology

[0002] Sodium dehydroacetate (Na-DHA), readily soluble in water and heat-resistant, possesses broad-spectrum antibacterial and bacteriostatic properties, making it a novel chemical preservative widely used in food, feed, and cosmetics. However, studies have shown that sodium dehydroacetate also exhibits acute and chronic toxicity, anticoagulant effects, sensitizing effects, and cardiovascular toxicity. In recent years, relevant national departments have implemented stricter regulations on sodium dehydroacetate; for example, the National Food Safety Standard for the Use of Food Additives (GB2760-2024) prohibits its use in the production and processing of bread, pastries, and fruit and vegetable juices (pulps).

[0003] Common methods for detecting sodium dehydroacetate include liquid chromatography, gas chromatography, gas chromatography-mass spectrometry, and ultraviolet spectrophotometry. However, these methods generally suffer from drawbacks such as complex operation, long processing times, and even low sensitivity. Therefore, developing a rapid, accurate, and sensitive fluorescence analytical method for detecting sodium dehydroacetate is crucial for ensuring food safety and quality control. Summary of the Invention

[0004] The technical problem solved by this invention is to provide a ratiometric fluorescence analysis method for determining sodium dehydroacetate, wherein tetrafluoroterephthalic acid (TFA) is oxidized with hydrogen peroxide (H2O2) under alkaline conditions to synthesize tetrafluoroterephthalic acid oxide (TFA). ox A fluorescent substance was used, which, together with Rhodamine B, constituted a ratiometric fluorescent probe. Based on this ratiometric fluorescent probe, a ratiometric fluorescence analysis method for sodium dehydroacetate was constructed. This method can be successfully applied to the determination of sodium dehydroacetate in water and bread samples.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a ratiometric fluorescence analysis method for determining sodium dehydroacetate, the specific steps of which are as follows:

[0006] Step S1, fluorescent substance TFA ox Preparation: Tetrafluoroterephthalic acid oxide, i.e., the fluorescent substance TFA, is synthesized by oxidizing tetrafluoroterephthalic acid with peroxide under alkaline conditions. ox ;

[0007] Step S2, Plotting the standard curve for ratiometric fluorescence analysis: Add 0.70 mL of Britton-Robinson (BR) buffer solution (pH 7.0) and 0.50 mL of the fluorescent substance TFA to a series of centrifuge tubes respectively. ox And 150 μL concentration of 1×10 - 4 A mol / L rhodamine B solution was prepared; then, sodium dehydroacetate standard solution with increasing concentrations was added sequentially, and the volume was adjusted to 4.00 mL with purified water. The solution was shaken well and allowed to react at room temperature for 10 min. The fluorescence emission spectrum was scanned with an excitation wavelength of 310 nm, and the fluorescence intensity values ​​at emission wavelengths of 450 nm and 580 nm were recorded. 450 and I 580 Calculate (I) 450,0 -I 450 ) / I 580 The ratio, where I 450,0 The fluorescence intensity at a wavelength of 450 nm in the above mixture without the addition of sodium dehydroacetate solution is given by the linear regression equation: (I 450,0 -I 450 ) / I 580 =0.0281C - 0.09045, R 2 =0.9985, where C is the concentration of sodium dehydroacetate, the linear range is 1–150 μmol / L, and the limit of detection is 0.084 μmol / L;

[0008] Step S3, determination of sodium dehydroacetate by ratiometric fluorescence analysis: Add 0.70 mL of Britton-Robinson buffer solution (pH 7.0) and 0.50 mL of the fluorescent substance TFA to a centrifuge tube. ox And 150 μL concentration of 1×10 -4 Add mol / L Rhodamine B solution; then add sodium dehydroacetate test solution, and then dilute to 4.00 mL with purified water. Shake well and let it react at room temperature for 10 min. Scan its fluorescence emission spectrum with 310 nm as the excitation wavelength, and record the fluorescence intensity values ​​at emission wavelengths of 450 nm and 580 nm respectively. 450 and I 580 Calculate (I) 450,0 -I 450 ) / I 580 The ratio, where I 450,0 The fluorescence intensity at a wavelength of 450 nm in the above mixed system without the addition of sodium dehydroacetate as the test solution is calculated based on (I 450,0 -I 450 ) / I 580 The concentration of sodium dehydroacetate in the test solution was obtained by combining the ratio with the linear regression equation.

[0009] Further specifying, the fluorescent material TFA ox The specific preparation process is as follows: 13.10 mL of purified water, 0.10 mL of 1 mol / L sodium hydroxide solution, and 5.00 mL of 10 mmol / L tetrafluoroterephthalic acid are added sequentially to a 100 mL reaction vessel. The mixture is stirred for 5 min, and then 1.80 mL of 3 wt% H2O2 solution is added dropwise to the mixed solution. The mixture is stirred at 90 °C for 5 h, and then cooled to room temperature to obtain a colorless and transparent solution, i.e., the fluorescent substance TFA. ox The fluorescent substance TFA ox It possesses water solubility, light stability, long-term storage stability, pH adaptability, and high ionic strength stability.

[0010] Further specifying, the fluorescent material TFA ox A ratiometric fluorescent probe constructed with rhodamine B can selectively detect sodium dehydroacetate in an interference system, wherein the interference system is K. + Zn 2+ Hg 2+ Pb 2+ Mg 2+ Mn 2+ Cu 2+ Na + Ca 2+ Cr 6+ Fe 3+ Ba 2+ Cl - SO4 2- SO3 2- NO3 - NO2 - CO3 2- HCO3 - AC - It contains one or more of glucose, sucrose, citric acid, and benzoic acid.

[0011] Compared with the prior art, the present invention has the following advantages and beneficial effects: the ratiometric fluorescence detection method for determining sodium dehydroacetate provided by the present invention has a wide linear range and high detection sensitivity, and coexisting substances do not interfere with the determination, and the method has good selectivity. Attached Figure Description

[0012] Figure 1 To synthesize the fluorescent substance TFA ox Feasibility analysis.

[0013] Figure 2 TFA is a fluorescent substance ox The excitation and emission spectra.

[0014] Figure 3 TFA fluorescent material under different excitation wavelengths ox The fluorescence spectrum.

[0015] Figure 4 pH effect on fluorescent substance TFA ox The impact on stability.

[0016] Figure 5 For storage time of fluorescent material TFA ox The impact on stability.

[0017] Figure 6 The effect of illumination time on the fluorescent substance TFA ox The impact on stability.

[0018] Figure 7 The ionic strength of the fluorescent substance TFA ox The impact on stability.

[0019] Figure 8 The fluorescence intensity variation curves of sodium dehydroacetate at different concentrations and the standard curve are shown.

[0020] Figure 9 To determine the selectivity and anti-interference performance of sodium dehydroacetate using a ratiometric fluorescent probe. Detailed Implementation

[0021] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0022] Example

[0023] Synthetic fluorescent substance TFA ox The optimal condition is:

[0024] 13.10 mL of purified water, 0.10 mL of 1 mol / L sodium hydroxide solution, and 5.00 mL of 10 mmol / L tetrafluoroterephthalic acid (TFA) were added sequentially to a 100 mL round-bottom flask. The mixture was stirred at room temperature for 5 min. Then, 1.80 mL of H₂O₂ (3 wt%) solution was added dropwise to the mixture. The mixture was stirred at 90 °C for 5 h. After cooling to room temperature, a colorless and transparent solution was obtained, which was designated as the fluorescent substance TFA. ox To verify the effects of experimental conditions such as tetrafluoroterephthalic acid (TFA), sodium hydroxide (NaOH), and hydrogen peroxide (H2O2) on the synthesis of TFA. oxAll three conditions are indispensable. Under optimal experimental conditions, such as keeping the reaction temperature, reaction time, and total reaction volume (20.00 mL) constant, the fluorescence properties of different reaction combinations, including TFA+H2O, TFA+H2O2, TFA+NaOH, and TFA+NaOH+H2O2, were further investigated. Figure 1 (As shown). By Figure 1 It is known that the fluorescent substance TFA can only be synthesized efficiently under experimental conditions where the three reactants tetrafluoroterephthalic acid (TFA), sodium hydroxide (NaOH), and hydrogen peroxide (H2O2) coexist. ox .

[0025] Figure 2 TFA is a fluorescent substance ox The excitation and emission spectra. (From...) Figure 2 It can be seen that the fluorescent substance TFA ox The maximum excitation wavelength is 310 nm, and the maximum emission wavelength is 452 nm. Figure 3 TFA fluorescent material under different excitation wavelengths ox The fluorescence spectrum. (From) Figure 3 It can be seen that when the excitation wavelength is increased from 280nm to 360nm, the fluorescent substance TFA... ox The position of the maximum emission peak remained almost unchanged. This indicates that the fluorescent substance TFA... ox Fluorescence spectroscopy is not dependent on the excitation wavelength. Figure 4 pH effect on fluorescent substance TFA ox The impact on stability. Figure 4 It can be seen that within the pH range of 5.0-8.0, the fluorescent substance TFA... ox The fluorescence intensity of the fluorescent substance TFA is almost unaffected by pH. Further increasing or decreasing the pH will affect its fluorescence intensity. ox The fluorescence intensity will decrease significantly. Figure 5 For storage time of fluorescent material TFA ox The impact on stability. Figure 5 It can be seen that the fluorescent substance TFA ox After 45 days of continuous storage, the fluorescent substance TFA... ox The fluorescence intensity remained stable and even showed an increasing trend. This indicates that the fluorescent substance TFA ox It is durable and can be stored for a long time. Figure 6 The effect of illumination time on the fluorescent substance TFA ox Effects on stability. Fluorescent substance TFA ox The solution was irradiated at a wavelength of 365 nm, and its fluorescence intensity was measured every 10 minutes. Figure 6 As shown, the fluorescent substance TFA ox The fluorescence properties remained stable after 120 minutes of illumination. Figure 7The ionic strength of the fluorescent substance TFA ox The impact on stability. For example... Figure 7 As shown, when the sodium chloride concentration is increased to 500 mM, the fluorescent substance TFA... ox The fluorescence properties remain stable.

[0026] Plotting the standard curve:

[0027] First, add 0.70 mL of Britton-Robinson buffer solution (pH 7.0) and 0.50 mL of the fluorescent substance TFA to a series of centrifuge tubes. ox And 150 μL concentration of 1×10 -4 Prepare a mol / L rhodamine B solution; then add sodium dehydroacetate standard solution in increasing concentrations, and finally dilute to 4.00 mL with purified water and shake well. Let the reaction proceed at room temperature for 10 min. Scan its fluorescence emission spectrum with an excitation wavelength of 310 nm. Figure 8 (A) shows the fluorescence spectrum as the concentration of sodium dehydroacetate increases from 0 μmol / L to 250 μmol / L. The fluorescence intensity values ​​I at emission wavelengths of 450 nm and 580 nm were recorded respectively. 450 and I 580 Calculate (I) 450,0 -I 450 ) / I 580 The ratio, where I 450,0 The value represents the fluorescence intensity at a wavelength of 450 nm when no sodium dehydroacetate solution was added to the above mixed solution. Figure 8 (B) is (I) 450,0 -I 450 ) / I 580 The linear relationship between the ratio and the concentration of sodium dehydroacetate. The linear regression equation is: (I 450,0 -I 450 ) / I 580 =0.0281C - 0.09045, R 2 =0.9985, where C is the concentration of sodium dehydroacetate; the linear range is 1–150 μmol / L, and the limit of detection is 0.084 μmol / L.

[0028] Analysis of the selectivity and anti-interference performance of ratiometric fluorescent probes for the determination of sodium dehydroacetate:

[0029] First, add 0.70 mL of Britton-Robinson buffer solution (pH 7.0) and 0.50 mL of the fluorescent substance TFA to a series of centrifuge tubes. ox And 150 μL concentration of 1×10 -4A solution of 50 μmol / L Rhodamine B was prepared; then a certain amount of sodium dehydroacetate and / or interfering substance solution was added, and the volume was adjusted to 4.00 mL with purified water. The solution was shaken well. The concentration of sodium dehydroacetate was 50 μmol / L, and the concentration of the interfering substance was 100 μmol / L. The reaction was allowed to proceed at room temperature for 10 min. The fluorescence emission spectrum was scanned at an excitation wavelength of 310 nm, and the fluorescence intensity values ​​at emission wavelengths of 450 nm and 580 nm were recorded. 450 and I 580 Calculate I 450 / I 580 ratio.

[0030] Depend on Figure 9 As can be seen from (A), the method for determining sodium dehydroacetate provided by the present invention is applicable to cation K at a concentration of 100 μmol / L. + Zn 2+ Hg 2+ Pb 2+ Mg 2+ Mn 2+ Cu 2+ Na + Ca 2+ Cr 6+ Fe 3+ Ba 2+ anion Cl - SO4 2- SO3 2- NO3 - NO2 - CO3 2- HCO3 - AC - Compounds such as glucose, sucrose, citric acid, and benzoic acid showed almost no response. Furthermore, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 9 As shown in section (B), these common anions, cations, and compounds have almost no effect on the determination of sodium dehydroacetate when coexisting with a 50 μmol / L sodium dehydroacetate solution. This indicates that the sodium dehydroacetate fluorescence determination method provided by this invention has strong selectivity and high anti-interference performance. Applications of the determination method:

[0031] Different water samples, including tap water, lake water, and river water, were filtered and used for later use. Bread samples were purchased from a local supermarket. 0.50g of minced bread was weighed, added to 6.00mL of purified water, mixed thoroughly, and left to stand for 10 minutes. Then, 1.00mL of 0.50mol / L NaOH solution and 1.00mL of 0.40mol / L MgSO4 solution were added, followed by 2.00mL of purified water. After mixing, the mixture was centrifuged at 4000r / min for 10 minutes. The supernatant was then adjusted to neutral pH with 1mol / L hydrochloric acid solution and used for later use.

[0032] Take 0.70 mL of Britton-Robinson buffer solution (pH 7.0) and 0.50 mL of the fluorescent substance TFA. ox And 150 μL concentration of 1×10 -4 A certain volume of the sample solution and mol / L Rhodamine B solution were added and diluted to 4.00 mL with purified water, and the mixture was shaken well. The reaction was allowed to proceed at room temperature for 10 min. The fluorescence emission spectrum was scanned using 310 nm as the excitation wavelength, and the fluorescence intensity values ​​at emission wavelengths of 450 nm and 580 nm were recorded. 450 and I 580 Calculate (I) 450,0 -I 450 ) / I 580 The ratio, where I 450,0 The fluorescence intensity at 450 nm is the emission wavelength of the above mixed solution without the addition of the test sample solution. Sodium dehydroacetate was not detected in any of the four water samples and two bread samples. Subsequent spiked recovery tests were performed with spikes of 20 μmol / L, 50 μmol / L, and 100 μmol / L. The results are shown in Table 1. The recoveries ranged from 93.26% to 100.30%, and the RSDs ranged from 0.06% to 1.70%.

[0033] Table 1. Analytical results of sodium dehydroacetate in water and bread samples.

[0034]

[0035] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A ratiometric fluorescence analytical method for determining sodium dehydroacetate, characterized in that... The specific steps are as follows: Step S1, fluorescent substance TFA ox Preparation: Tetrafluoroterephthalic acid oxide, i.e., the fluorescent substance TFA, is synthesized by oxidizing tetrafluoroterephthalic acid with peroxide under alkaline conditions. ox ; Step S2, Plotting the standard curve for ratiometric fluorescence analysis: Add 0.70 mL of Britton-Robinson buffer solution (pH 7.0) and 0.50 mL of the fluorescent substance TFA to a series of centrifuge tubes. ox And 150 μL concentration of 1×10 -4 A mol / L rhodamine B solution was prepared; then, sodium dehydroacetate standard solution with increasing concentrations was added sequentially, and the volume was adjusted to 4.00 mL with purified water. The solution was shaken well and allowed to react at room temperature for 10 min. The fluorescence emission spectrum was scanned with an excitation wavelength of 310 nm, and the fluorescence intensity values ​​at emission wavelengths of 450 nm and 580 nm were recorded. 450 and I 580 Calculate (I) 450,0 -I 450 ) / I 580 The ratio, where I 450,0 The fluorescence intensity at a wavelength of 450 nm in the above mixture without the addition of sodium dehydroacetate solution is given by the linear regression equation: (I 450,0 -I 450 ) / I 580 =0.0281C-0.09045, R 2 =0.9985, where C is the concentration of sodium dehydroacetate, the linear range is 1~150 μmol / L, and the limit of detection is 0.084 μmol / L; Step S3, determination of sodium dehydroacetate by ratiometric fluorescence analysis: Add 0.70 mL of Britton-Robinson buffer solution (pH 7.0) and 0.50 mL of the fluorescent substance TFA to a centrifuge tube. ox And 150 μL concentration of 1×10 -4 Add mol / L Rhodamine B solution; then add sodium dehydroacetate test solution, and then dilute to 4.00 mL with purified water. Shake well and let it react at room temperature for 10 min. Scan its fluorescence emission spectrum with 310 nm as the excitation wavelength, and record the fluorescence intensity values ​​at emission wavelengths of 450 nm and 580 nm respectively. 450 and I 580 Calculate (I) 450,0 -I 450 ) / I 580 The ratio, where I 450,0 The fluorescence intensity at a wavelength of 450 nm in the above mixed system without the addition of sodium dehydroacetate as the test solution is calculated based on (I 450,0 -I 450 ) / I 580 The concentration of sodium dehydroacetate in the test solution was obtained by combining the ratio with the linear regression equation.

2. The ratiometric fluorescence analysis method for determining sodium dehydroacetate according to claim 1, characterized in that... The fluorescent material TFA ox The specific preparation process is as follows: 13.10 mL of purified water, 0.10 mL of 1 mol / L sodium hydroxide solution, and 5.00 mL of 10 mmol / L tetrafluoroterephthalic acid are added sequentially to a 100 mL reaction vessel. The mixture is stirred for 5 min, and then 1.80 mL of 3 wt% H2O2 solution is added dropwise to the mixed solution. The mixture is stirred at 90 °C for 5 h, and then cooled to room temperature to obtain a colorless and transparent solution, i.e., the fluorescent substance TFA. ox The fluorescent substance TFA ox It possesses water solubility, light stability, long-term storage stability, pH adaptability, and high ionic strength stability.

3. The ratiometric fluorescence analysis method for determining sodium dehydroacetate according to claim 1, characterized in that: The fluorescent material TFA ox A ratiometric fluorescent probe constructed with rhodamine B can selectively detect sodium dehydroacetate in an interference system, wherein the interference system is K. + Zn 2+ Hg 2+ Pb 2+ Mg 2+ Mn 2+ Cu 2+ Na + Ca 2+ Cr 6+ Fe 3+ Ba 2+ Cl - SO4 2- SO3 2- NO3 - NO2 - CO3 2- HCO3 - AC - It contains one or more of glucose, sucrose, citric acid, and benzoic acid.

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