CN112345474A - A kind of method for rapid detection of tert-butyl hydroquinone in food - Google Patents

Abstract

Translated fromChinese

本发明公开了一种快速检测食品中特丁基对苯二酚的方法，该方法采用铜碘掺杂碳点作为还原剂，聚丙烯亚胺为保护剂，还原氯金酸，得到金纳米粒子，同时过量的Cu‑I/CDs与AuNPs组成混合纳米酶体系，以特丁基对苯二酚为底物，氧化TBHQ为红色氧化型醌类物质，基于TBHQ浓度与红色氧化TBHQ线性关系，建立高灵敏、选择性强TBHQ检测新方法，检出限为0.5mg/kg；将本方法应用于食品中TBHQ的检测分析，结果与GB5009.32‑2016食品安全国家标准食品中9种抗氧化剂的测定方法相符；本发明方法只能选择性氧化TBHQ，而其他抗氧化剂却无此反应，具有灵敏度高、特异性强、操作简单、快速等特点。

The invention discloses a method for rapidly detecting tert-butyl hydroquinone in food. The method adopts copper iodine doped carbon dots as a reducing agent and polypropyleneimine as a protecting agent to reduce chloroauric acid to obtain gold nanoparticles , while excess Cu-I/CDs and AuNPs form a mixed nanoenzyme system, using tert-butylhydroquinone as a substrate to oxidize TBHQ to red oxidized quinones. Based on the linear relationship between TBHQ concentration and red oxidized TBHQ, a A new method for the detection of TBHQ with high sensitivity and selectivity, with a detection limit of 0.5 mg/kg; this method was applied to the detection and analysis of TBHQ in food, and the results were consistent with the results of 9 antioxidants in GB5009.32‑2016 national food safety standard food. The determination methods are consistent; the method of the present invention can only selectively oxidize TBHQ, while other antioxidants have no such reaction, and has the characteristics of high sensitivity, strong specificity, simple operation and rapidity.

Description

Method for rapidly detecting tert-butyl hydroquinone in food

Technical Field

The invention relates to the technical field of chemical analysis and detection, in particular to a method for rapidly detecting tert-butylhydroquinone in food.

Background

Tert-butyl hydroquinone (TBHQ) is a common antioxidant in food, and has stronger antioxidant performance compared with the traditional antioxidants such as butyl hydroxy anisole, 2, 6-dibutyl hydroxy toluene and the like; in addition, TBHQ is relatively stable to heat and does not have any off-flavors when added to the grease samples. However, TBHQ has a certain toxicity to human body, and TBHQ is decomposed after long-term storage or repeated cooking, and the metabolite tert-butyl-p-benzoquinone has a large toxicity. GB2760-2014 clearly specifies that the maximum addition amount of TBHQ in the edible vegetable oil is 200 mg/kg. Common detection methods of TBHQ include a colorimetric method, a liquid chromatography, a gas chromatography-mass spectrometry method and the like, the colorimetric method is frequently adopted due to simple and rapid operation and no need of large-scale instruments and equipment, but the colorimetric method becomes a difficult problem to be solved in detection due to poor characteristics and serious detection interference.

Gold nanoparticles (AuNPs) are common nanomaterials, and because of their simple preparation method and unique optical properties, general attention has been paid, and it has been reported that gold nanoparticle solutions with good stability and high luminous intensity are synthesized by reducing chloroauric acid with tetrakis (hydroxymethyl) phosphonium chloride (THPC) at room temperature and using polyethyleneimine as a protective agent. According to the method for enhancing the luminous intensity of the gold nano-grade by the TBHQ, a novel method for rapidly determining the trace TBHQ is established and successfully used for determining the TBHQ in the food, but the problems of narrow detection range and certain interference in detection exist, because the similar synthetic phenolic antioxidants such as BHA, BHT and PG have certain interference.

The nano enzyme is a mimic enzyme which not only has the unique performance of nano materials, but also has a catalytic function. Carbon Dots (CDs) are a new type of carbon nanomaterials with size less than 10nm, formed by sp²/sp³The hybridized carbon atoms have different functional groups on the surface, have fluorescence properties depending on the components thereof, and have attracted great interest to researchers due to good photoinduced electron transfer, electron energy storage characteristics, excellent up-conversion photoluminescence behavior and two-photon absorption characteristics. Research on carbon dots as nanoenzymes has been reported, and gold nano preparation by utilizing carbon dot reducibility has also been reported, but composite nanoenzymes formed by combining the carbon dots and the nanoenzymes and catalytic oxidation by taking TBHQ as a substrate have been reported only rarely.

Disclosure of Invention

The invention aims to provide a method for rapidly detecting tert-butylhydroquinone in food, which adopts copper-iodine doped carbon dots (Cu-I/CDs) as a reducing agent and polypropyleneimine as a protective agent to reduce chloroauric acid to obtain gold nanoparticles, simultaneously excessive Cu-I/CDs and AuNPs form a mixed nano enzyme system, tert-butylhydroquinone (TBHQ) is used as a substrate, oxidized TBHQ is red oxidized quinone substances, a novel high-sensitivity and high-selectivity TBHQ detection method is established based on the linear relation between the concentration of TBHQ and red oxidized TBHQ, and the detection limit is 0.5 mg/kg; the invention has the advantages of strong specificity, and basically has no influence on interfering substances which are possibly generated and coexist in practical application, such as phenol antioxidants like synthetic phenols, amino acids, sugars and the like; the method has the characteristics of high sensitivity, specificity, simple and quick operation and the like.

The method for rapidly detecting the tert-butyl hydroquinone in the food comprises the following steps:

(1) preparing a working curve of the tert-butyl hydroquinone: adding 50-100 mu L of a standard solution of tert-butylhydroquinone (TBHQ) prepared by reduction of copper-iodine doped carbon dots (Cu-I/CDs) in a 10mL colorimetric tube with a plug, diluting the standard solution to a scale with a citric acid-disodium hydrogen phosphate buffer solution with pH of 7.4, shaking up, standing for 5-10 min, measuring absorbance A at a 492nm wavelength, and drawing a standard curve by taking the concentration of the TBHQ as a horizontal coordinate and the absorbance A as a vertical coordinate to obtain a regression equation;

(2) sample processing

Solid type samples: weighing 5g (accurate to 0.01 g) of crushed and uniform sample in a 100mL conical flask, adding 8-10 mL of absolute ethyl alcohol, carrying out vortex mixing for 1-2 min, then carrying out ultrasonic extraction for 15-20 min, standing for layering, sucking supernatant liquid in a 50mL centrifugal tube, and extracting residues with 8-10 mL of absolute ethyl alcohol for 2 times; mixing the supernatants, adding 1g of neutral alumina into a centrifuge tube, carrying out vortex oscillation for 1-2 min, centrifuging for 5-10 min at 3000r/min, transferring the supernatant into a 25mL volumetric flask, fixing the volume with absolute ethyl alcohol, and shaking up to obtain a sample solution to be detected;

oils: accurately weighing 2g of vegetable oil or grease food into a 25mL colorimetric tube, adding 6mL of ethanol solution with volume concentration of 95%, swirling for 1min, fully mixing, standing for a moment, and heating in a water bath at 90 ℃ for 10-15 s to promote layering; transferring the supernatant into a concentration bottle by using a suction pipe, repeatedly extracting for 2 times by using 6mL of 95% ethanol solution, combining the extracting solutions, and fixing the volume to 25mL to obtain a sample solution to be detected;

(3) and (3) sample determination: adding 50-100 mu L of gold nano prepared by reduction of copper-iodine doped carbon points into a 10mL colorimetric tube with a plug, adding the sample liquid to be detected prepared in the step (2), diluting the sample liquid to be detected to a scale by using a citric acid-disodium hydrogen phosphate buffer solution with the pH of 7.4, shaking up, standing for 5-10 min, measuring the absorbance A at the position of 492nm of wavelength, substituting the absorbance A into the regression equation in the step (1), and calculating the content of tert-butyl hydroquinone in the sample;

the preparation method of the gold nano-particles prepared by Cu-I/CDs reduction comprises the following steps: adding 2mL of polypropyleneimine into 15-20 mL of ultrapure water, adding 70-90 mu L of chloroauric acid under stirring, adding 180-220 mu L of copper-iodine-doped carbon dots, stirring for 30-40 min, and then enabling the solution to become reddish brown to obtain gold nanoparticles prepared by reduction of the copper-iodine-doped carbon dots; wherein the concentration of the polypropylene imine is 0.25g/mL, and the mass concentration of the chloroauric acid is 1%.

The preparation method of the Cu-I/CDs comprises the following steps: weighing 1g of CuCl₂Dissolving 3-5 g of 3-iodine-L-tyrosine in 400-600 mL of ultrapure water, uniformly mixing, adding 800-1200 mu L of ethylenediamine, carrying out ultrasonic treatment for 10-20 min, transferring to a polytetrafluoroethylene reaction kettle, heating at 180 ℃ for 8-10 h, naturally cooling, filtering with a filter membrane with the pore size of 0.22 mu m, and carrying out dialysis treatment for 24h with a dialysis bag with the molecular weight cutoff of 3000Da to obtain the water-soluble Cu-I/CDs.

The invention has the advantages that:

1. according to the invention, copper-iodine doped carbon dots (Cu-I/CDs) are used as a reducing agent, polypropylene imine is used as a protective agent, chloroauric acid is reduced, a mixed nano enzyme system consisting of gold nanoparticles and Cu-I/CDs is obtained, and due to the excellent catalytic property of the mixed nano enzyme system, tert-butylhydroquinone (TBHQ) is selectively oxidized into red oxidized quinone substances, and a novel high-sensitivity and strong-selectivity TBHQ detection method is established based on the linear relation between the TBHQ concentration and the red oxidized TBHQ;

2. the catalytic oxidation system established by the invention has specific oxidation on TBHQ, other synthetic phenolic antioxidants with similar structures and properties do not have the reaction, the reaction is quick, sensitive and stable, and the catalytic oxidation system is used for detecting TBHQ and has the characteristics of high sensitivity, good reproducibility and accuracy;

3. the copper-iodine doped carbon dots prepared by the method can be used as a reducing agent and a nano enzyme, and the reaction is rapid and sensitive due to the synergistic catalysis effect of the copper-iodine doped carbon dots and the gold nano.

Drawings

FIG. 1 is the ultraviolet-visible absorption spectrum of the composite nanoenzyme oxidized TBHQ in example 1 for detecting TBHQ;

FIG. 2 shows the effect of coexisting antioxidants on TBHQ, wherein TBHQ represents the single use thereof, and the others are a mixture of TBHQ with one of Propyl Gallate (PG), 2,4, 5-Trihydroxyphenylbutanone (THBP), nordihydroguaiaretic acid (NDGA), t-butyl p-hydroxyanisole (BHA), 2, 6-di-t-butyl-4-hydroxymethylphenol (Ionox-100), Octyl Gallate (OG), 2, 6-di-t-butyl p-methylphenol (BHT), Dodecyl Gallate (DG), glucose, maltose, sucrose, starch, and ascorbic acid;

FIG. 3 shows the effect of coexisting ions on TBHQ, where Black is TBHQ and the others are ions (Na) coexisting with TBHQ⁺、K⁺、Mg²⁺、Cu²⁺、Zn²⁺、Fe²⁺、Cl^-、SO₄^2-、NO₃^-Etc.).

Detailed Description

The technical solutions of the present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.

Example 1: determination of TBHQ in Soybean oil samples

1. Preparation of Cu-I/CDs: 0.1g of CuCl₂0.4g of 3-iodo-L-tyrosine was addedMixing and dissolving the materials in 40mL of ultrapure water, adding 100 mu L of ethylenediamine, carrying out ultrasonic treatment for 10min, transferring the materials into a polytetrafluoroethylene reaction kettle, heating the materials at 180 ℃ for 8h, naturally cooling the materials, filtering the materials by using a filter membrane with the pore diameter of 0.22 mu m, and carrying out dialysis treatment for 24h by using a dialysis bag with the molecular weight cutoff of 3000Da to obtain water-soluble Cu-I/CDs;

2. gold nanoparticles prepared by Cu-I/CDs reduction: adding 2mL of polypropylene imine with the concentration of 0.25g/mL into 20mL of ultrapure water, adding 80 mu L of chloroauric acid with the mass concentration of 1% under stirring, adding 200 mu L of Cu-I/CDs, and stirring for 30min to obtain a solution which is reddish brown, thus obtaining the gold nano-particles prepared by reduction of the Cu-I/CDs.

3. Preparing a working curve of the tert-butyl hydroquinone: adding 50 mu L of TBHQ standard solution with the concentration being 1-200 mg/kg, which is prepared by reduction of Cu-I/CDs, into a 10mL colorimetric tube with a plug, diluting the gold nanoparticles with citric acid-disodium hydrogen phosphate buffer solution with the pH of 7.4 to a scale, shaking up, standing for 10min, measuring the absorbance A at the position of 492nm wavelength, and drawing a standard curve by taking the concentration of TBHQ as a horizontal coordinate and A as a vertical coordinate to obtain a regression equation; see fig. 1, the regression equation, correlation coefficient, relative standard deviation, linear range, etc. are obtained as shown in table 1;

TABLE 1 Linear equation, correlation coefficient, relative standard deviation, Linear Range

4. Method specificity investigation: TBHQ was mixed with other antioxidants and ions as a mixed antioxidant, and the effect of the coexisting antioxidant and coexisting ions on TBHQ in the above-mentioned detection system was examined, wherein the concentration of TBHQ was 10mg/kg, and FIG. 2 shows the effect of the coexisting antioxidant (propyl gallate (PG), 2,4, 5-trihydroxy phenylbutanone (THBP), nordihydroguaiaretic acid (NDGA), t-butyl p-hydroxyanisole (BHA), 2, 6-di-t-butyl-4-hydroxymethylphenol (Ionox-100), Octyl Gallate (OG), 2, 6-di-t-butyl p-methylphenol (BHT), Dodecyl Gallate (DG)) and glucose, maltose, sucrose, starch and ascorbic acid on TBHQ, and FIG. 3 shows the effect of the coexisting ions on TBHQSeed (Na)⁺、K⁺、Mg²⁺、Cu²⁺、Zn²⁺、Fe²⁺、Cl^-、SO₄^2-、NO₃^-Etc.) on TBHQ, the concentration of the above interfering substances is 50mg/kg, and as can be seen from the figure, the nano enzyme system has better selection specificity for oxidizing TBHQ, only TBHQ has obvious oxidation reaction, other substances are almost not present, and the method has good selection specificity;

5. determination of TBHQ in soybean blend oil sample

(1) Sample treatment: accurately weighing 2g of soybean blend oil into a 25mL colorimetric tube, adding 6mL of ethanol solution with volume concentration of 95%, swirling for 1min, fully mixing, standing for a moment, and heating in a 90 ℃ water bath for 10-15 s to promote layering; transferring the supernatant into a concentration bottle by using a suction pipe, repeatedly extracting for 2 times by using 6mL of 95% ethanol solution, combining the extracting solutions, and fixing the volume to 25mL to obtain a sample solution to be detected;

(2) and (3) sample determination: adding 50 mu L of gold nano prepared by Cu-I/CDs reduction into a 10mL colorimetric tube with a plug, adding 4mL of sample liquid to be detected prepared in the step (1), diluting the sample liquid to be detected to a scale by using citric acid-disodium hydrogen phosphate buffer solution with pH of 7.4, shaking up, standing for 10min, measuring absorbance A at a 492nm wavelength, substituting the absorbance A into the regression equation in the step (1), and calculating the TBHQ content of the sample to be 54.90 mg/kg;

(3) recovery and precision experiments: respectively adding 2 TBHQ standard solutions with different concentrations into a soybean blend oil sample; the concentration is parallelly measured for 3 times, the standard adding recovery rate is calculated, the relative standard deviation RSD is calculated, the result is shown in table 2, the standard adding recovery rate of TBHQ is measured to be 98.0% -101.9%, the RSD is measured to be 0.20% -1.3%, and the method has good accuracy and precision.

TABLE 2 sample recovery with addition of standard and RSD (n = 3)

。

Example 2: determination of TBHQ in sunflower seed oil samples

1. Preparation of Cu-I/CDs：1 g CuCl₂Dissolving 3g of 3-iodine-L-tyrosine in 500mL of purified water, mixing and dissolving, adding 900 mu L of ethylenediamine, carrying out ultrasonic treatment for 15min, transferring to a polytetrafluoroethylene reaction kettle, heating at 180 ℃ for 10h, naturally cooling, filtering with a filter membrane with the pore diameter of 0.22 mu m, and carrying out dialysis treatment for 24h with a dialysis bag with the molecular weight cutoff of 3000D to obtain water-soluble Cu-I/CDs;

2. preparing gold nano-particles prepared by reduction of Cu-I/CDs: 2mL of 0.25g/mL polypropylene imine, 18mL of ultrapure water, 90 mu L of 1% chloroauric acid under stirring, 190 mu L of Cu-I/CDs under stirring, and after stirring for 35min, the solution turns into reddish brown, so that the Cu-I/CDs are reduced to obtain gold nano-particles;

3. the working curve of tert-butylhydroquinone was prepared as in example 1;

4. determination of TBHQ in sunflower seed oil samples

(1) Sample treatment: accurately weighing 2g of sunflower seed oil into a 25mL colorimetric tube, adding 6mL of ethanol solution with volume concentration of 95%, vortexing for 1min, mixing uniformly, standing for a moment, and heating in a 90 ℃ water bath for 15s to promote layering; transferring the supernatant into a concentration bottle by using a suction pipe, repeatedly extracting for 2 times by using 6mL of 95% ethanol solution, combining the extracting solutions, and fixing the volume to 25mL to obtain a sample solution to be detected;

(2) and (3) sample determination: the TBHQ content of the sunflower seed oil sample was 31.33 mg/kg, as in example 1.

Example 3: determination of TBHQ in cookies

1. Cu-I/CDs were prepared as in example 1;

2. the preparation of gold nanoparticles by reduction of Cu-I/CDs was the same as in example 1;

3. the working curve of tert-butylhydroquinone was prepared as in example 1;

4. determination of TBHQ in cookies

(1) Sample treatment: weighing 5.00g of crushed and uniformly-ground biscuit sample in a 100mL conical flask, adding 8mL of absolute ethyl alcohol, carrying out vortex mixing for 1min, then carrying out ultrasonic extraction for 15min, standing for layering, sucking supernatant into a 50mL centrifuge tube, and extracting residues with 8mL of absolute ethyl alcohol for 2 times each time; mixing the supernatants, adding 1g of neutral alumina into a 50mL centrifuge tube, carrying out vortex oscillation for 1min, centrifuging at 3000r/min for 8min, transferring the supernatant into a 25mL volumetric flask, carrying out constant volume with absolute ethyl alcohol, and shaking up to obtain a sample solution to be detected;

(2) and (3) sample determination: as in example 1, the TBHQ content of the cookie was 5.38 mg/kg.

Example 4: determination of TBHQ in instant noodles

1. Cu-I/CDs were prepared as in example 1;

2. the preparation of gold nanoparticles by reduction of Cu-I/CDs was the same as in example 1;

3. the working curve of tert-butylhydroquinone was prepared as in example 1;

4. determination of TBHQ in instant noodles:

(1) sample treatment: weighing 5.00g of crushed and uniformly-ground instant noodle sample into a 100mL conical flask, adding 9mL of absolute ethyl alcohol, carrying out vortex mixing for 1min, then carrying out ultrasonic extraction for 20min, standing for layering, absorbing supernatant into a 50mL centrifuge tube, and extracting residues with 9mL of absolute ethyl alcohol for 2 times each time; mixing the supernatants, adding 1g of neutral alumina into a 50mL centrifuge tube, carrying out vortex oscillation for 1min, centrifuging for 10min at 3000r/min, transferring the supernatant into a 25mL volumetric flask, carrying out constant volume with absolute ethyl alcohol, and shaking up to obtain a sample solution to be detected;

(2) and (3) sample determination: in the same manner as in example 1, the TBHQ content of the instant noodles was 1.25 mg/kg;

comparing the method of the invention with the method for measuring 9 antioxidants in national food safety standard GB5009.32-2016 in examples 1-4, the results are shown in Table 3; as can be seen from the results, the two methods were consistent in result;

TABLE 3 recovery with spiking and relative standard deviation (mg/kg)

The TBHQ measuring method established by the invention has the advantages of few processing steps, no need of GB5009.32-2016 sample purification treatment, short time, low processing cost, simple and convenient operation, no need of large instruments and equipment, and strong advantage in actual detection.

Claims (3)

1. A method for rapidly detecting tert-butylhydroquinone in food is characterized by comprising the following steps:

(1) preparation of working curve of tert-butylhydroquinone

Adding 50-100 mu L of standard solution of gold nanoparticles prepared by reduction of copper-iodine doped carbon points and tert-butyl hydroquinone with the concentration being within the range of 1-200 mg/kg into a 10mL colorimetric tube with a plug, diluting the standard solution to a scale with a citric acid-disodium hydrogen phosphate buffer solution with the pH value of 7.4, shaking up, standing for 5-10 min, measuring the absorbance A at the position of 492nm wavelength, drawing a standard curve by taking the concentration of the tert-butyl hydroquinone as a horizontal coordinate and the absorbance A as a vertical coordinate, and obtaining a regression equation;

(2) sample processing

Solid type samples: weighing 5.00g of crushed and uniformly distributed sample in a conical flask, adding 8-10 mL of absolute ethyl alcohol, carrying out vortex mixing for 1-2 min, then carrying out ultrasonic extraction for 15-20 min, standing for layering, absorbing supernatant into a 50mL centrifuge tube, and extracting residues for 2 times by using 8-10 mL of absolute ethyl alcohol each time; mixing the supernatants, adding 1g of neutral alumina into a centrifuge tube, carrying out vortex oscillation for 1-2 min, centrifuging for 5-10 min at 3000r/min, transferring the supernatant into a 25mL volumetric flask, fixing the volume with absolute ethyl alcohol, and shaking up to obtain a sample solution to be detected;

oils: accurately weighing 2g of vegetable oil or grease food into a 25mL colorimetric tube, adding 6mL of ethanol solution with volume concentration of 95%, swirling for 1min, fully mixing, standing for a moment, and heating in a water bath at 90 ℃ for 10-15 s to promote layering; transferring the supernatant into a concentration bottle by using a suction pipe, repeatedly extracting for 2 times by using 6mL of 95% ethanol solution, combining the extracting solutions, and fixing the volume to 25mL to obtain a sample solution to be detected;

(3) and (3) sample determination: adding 50-100 mu L of gold nano prepared by copper-iodine doped carbon point reduction into a 10mL colorimetric tube with a plug, adding the gold nano prepared by the step (2) to prepare a sample solution to be detected, diluting the sample solution to a scale by using a citric acid-disodium hydrogen phosphate buffer solution with the pH of 7.4, shaking up, standing for 5-10 min, measuring the absorbance A at the position of 492nm of wavelength, substituting the absorbance A into the regression equation in the step (1), and calculating the content of the tert-butyl hydroquinone in the sample.

2. The method for rapidly detecting tert-butylhydroquinone in food according to claim 1, wherein: adding 2mL of polypropyleneimine into 15-20 mL of ultrapure water, adding 70-90 mu L of chloroauric acid under stirring, adding 180-220 mu L of copper-iodine-doped carbon dots, stirring for 30-40 min, and then enabling the solution to become reddish brown to obtain gold nanoparticles prepared by reduction of the copper-iodine-doped carbon dots; wherein the concentration of the polypropylene imine is 0.25g/mL, and the mass concentration of the chloroauric acid is 1%.

3. The method for rapidly detecting tert-butylhydroquinone in food as claimed in claim 2, wherein the preparation method of the copper-iodine doped carbon dots is as follows: weighing 1g of CuCl₂Dissolving 3-5 g of 3-iodine-L-tyrosine in 400-600 mL of ultrapure water, uniformly mixing, adding 800-1200 mu L of ethylenediamine, carrying out ultrasonic treatment for 10-20 min, transferring to a polytetrafluoroethylene reaction kettle, heating at 180 ℃ for 8-10 h, naturally cooling, filtering with a filter membrane with the pore size of 0.22 mu m, and carrying out dialysis treatment for 24h with a dialysis bag with the molecular weight cutoff of 3000Da to obtain the water-soluble Cu-I/CDs.

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