CN112345474B - A kind of method for rapid detection of tert-butyl hydroquinone in food - Google Patents

Abstract

The invention discloses a method for rapidly detecting tert-butylhydroquinone in food, which adopts copper-iodine doped carbon dots as a reducing agent, adopts polypropyleneimine as a protective agent, reduces chloroauric acid to obtain gold nanoparticles, simultaneously adopts excessive Cu-I/CDs and AuNPs to form a mixed nano enzyme system, adopts tert-butylhydroquinone as a substrate and oxidizes TBHQ into red oxidized quinine substances, and establishes a novel high-sensitivity and high-selectivity TBHQ detection method based on the linear relationship between the concentration of TBHQ and the red oxidized TBHQ, wherein the detection limit is 0.5 mg/kg; the method is applied to detection and analysis of TBHQ in food, and the result accords with the determination method of 9 antioxidants in GB5009.32-2016 food safety national standard food; the method only can selectively oxidize TBHQ, but does not have the reaction of other antioxidants, and has the characteristics of high sensitivity, strong specificity, simple and quick operation and the like.

Description

Translated fromChinese

一种快速检测食品中特丁基对苯二酚的方法A kind of method for rapid detection of tert-butyl hydroquinone in food

技术领域technical field

本发明涉及化学分析检测技术领域，具体为一种快速检测食品中特丁基对苯二酚的方法。The invention relates to the technical field of chemical analysis and detection, in particular to a method for rapidly detecting tert-butyl hydroquinone in food.

背景技术Background technique

特丁基对苯二酚(Tert-butyl hydroquinone，TBHQ) 是食品中比较常见的抗氧化剂，与传统的丁基羟基茴香醚、2，6-二丁基羟基甲苯等抗氧化剂相比，其抗氧化性能更强；另外，TBHQ对热相对稳定，添加在油脂样品中无任何异味。但TBHQ对人体有一定的毒性，长期储存或反复烹饪会使TBHQ分解，其代谢物特丁基对苯醌具有较大毒性。GB2760-2014中明确规定食用植物油中TBHQ最大添加量为200mg/kg。TBHQ常见的检测方法有比色法、液相色谱法、气相色谱法、气质联用法等，比色法由于操作简单、快速、不需要大型仪器设备，而被常采用，但由于特性差、检测干扰严重，成为检测中需要解决的难题。Tert-butyl hydroquinone (TBHQ) is a common antioxidant in food. Compared with traditional antioxidants such as butylated hydroxyanisole and 2,6-dibutylhydroxytoluene, its anti-oxidant The oxidation performance is stronger; in addition, TBHQ is relatively stable to heat, and there is no odor when added to the oil sample. However, TBHQ has certain toxicity to the human body. Long-term storage or repeated cooking will decompose TBHQ, and its metabolite, tert-butyl-p-benzoquinone, is highly toxic. GB2760-2014 clearly stipulates that the maximum addition amount of TBHQ in edible vegetable oil is 200mg/kg. Common detection methods for TBHQ include colorimetry, liquid chromatography, gas chromatography, and gas chromatography-mass spectrometry. Serious interference has become a difficult problem to be solved in detection.

金纳米粒子（AuNPs）是常见的纳米材料，因其制备方法简单及独特的光学特性受到普遍关注，有报道在室温下以四羟甲基氯化磷（THPC）还原氯金酸，以聚乙烯亚胺作保护剂，合成一种稳定性好、发光强度高的金纳米溶液。根据TBHQ增强金纳米的发光强度，建立了快速测定痕量TBHQ的新方法，并成功用于食品中TBHQ的测定，但也存在检测范围窄、检测有一定干扰的问题，因为同类合成酚类抗氧化剂，如BHA、BHT、PG有一定干扰。Gold nanoparticles (AuNPs) are common nanomaterials and have attracted widespread attention due to their simple preparation method and unique optical properties. Imine was used as a protective agent to synthesize a gold nano-solution with good stability and high luminescence intensity. According to the enhanced luminescence intensity of gold nanoparticles by TBHQ, a new method for the rapid determination of trace amounts of TBHQ was established, and it was successfully used for the determination of TBHQ in food. Oxidants such as BHA, BHT, PG have certain interference.

纳米酶是一类既有纳米材料的独特性能，又有催化功能的模拟酶。碳点（carbondots, CDs）是一种尺寸小于10nm的新型碳纳米材料，由sp²/sp³杂化的碳原子组成，表面具有不同的官能团，具有依赖于其成分的荧光性质，由于良好的光诱导电子转移、电子储能特性及优异的上转换光致发光行为和双光子吸收的特性引起研究者极大的兴趣。碳点作为纳米酶研究已有报道，利用碳点还原性进行金纳米制备也有报道，但由二者组合形成的复合纳米酶及以TBHQ为底物的催化氧化却鲜有报道。Nanozymes are a class of mimetic enzymes that have both the unique properties of nanomaterials and catalytic functions. Carbondots (CDs) are a new type of carbon nanomaterials with a size of less than 10 nm, which are composed of sp² /sp³ hybridized carbon atoms, with different functional groups on the surface, and have fluorescence properties depending on their composition. The properties of photoinduced electron transfer, electron energy storage, excellent upconversion photoluminescence behavior and two-photon absorption have attracted great interest of researchers. The research of carbon dots as nanozymes has been reported, and the preparation of gold nanoparticles by the reducibility of carbon dots has also been reported, but the composite nanozymes formed by the combination of the two and the catalytic oxidation with TBHQ as the substrate are rarely reported.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供一种快速检测食品中特丁基对苯二酚的方法，本发明采用铜碘掺杂碳点（Cu-I/CDs）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（TBHQ）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；本发明最大优点特异性强，对于可能产生及实际应用中共存的干扰物质，如同类合成酚类抗氧化剂、氨基酸、糖等基本没有影响；本方法具有灵敏度高、特异性、操作简单、快速等特点。The object of the present invention is to provide a method for rapidly detecting tert-butyl hydroquinone in food. The present invention adopts copper iodine doped carbon dots (Cu-I/CDs) as reducing agent, polypropyleneimine as protective agent, Gold nanoparticles were obtained by reducing chloroauric acid. At the same time, excess Cu-I/CDs and AuNPs formed a mixed nanoenzyme system, using tert-butyl hydroquinone (TBHQ) as a substrate, and oxidizing TBHQ to red oxidized quinones , based on the linear relationship between the concentration of TBHQ and red oxidized TBHQ, a new method for detection of TBHQ with high sensitivity and selectivity was established, with a detection limit of 0.5 mg/kg; the greatest advantage of the present invention is strong specificity, and it is possible for the interference that may occur and coexist in practical applications. Substances, such as synthetic phenolic antioxidants, amino acids, sugars, etc. have basically no effect; this method has the characteristics of high sensitivity, specificity, simple operation, and rapidity.

本发明快速检测食品中特丁基对苯二酚的方法如下：The method for rapidly detecting tert-butyl hydroquinone in food of the present invention is as follows:

（1）特丁基对苯二酚工作曲线制作：在10mL具塞比色管中加入50~100µL铜碘掺杂碳点（Cu-I/CDs）还原制得的金纳米、浓度在1~200mg/kg范围内的特丁基对苯二酚（TBHQ）标准溶液，用pH 7.4的柠檬酸-磷酸氢二钠缓冲溶液稀释至刻度，摇匀，静置5~10min，在492nm波长处，测定吸光度A，以TBHQ浓度为横坐标，吸光度A为纵坐标，绘制标准曲线，得到回归方程；(1) Preparation of working curve of tert-butyl hydroquinone: add 50~100µL of copper-iodine-doped carbon dots (Cu-I/CDs) to a 10mL colorimetric tube with a stopper to reduce the gold nanoparticles with a concentration of 1~ The standard solution of tertiary butyl hydroquinone (TBHQ) in the range of 200mg/kg, diluted to the mark with pH 7.4 citric acid-disodium hydrogen phosphate buffer solution, shake well, stand for 5~10min, at 492nm wavelength, Measure the absorbance A, take the TBHQ concentration as the abscissa and the absorbance A as the ordinate, draw a standard curve, and obtain a regression equation;

（2）样品处理(2) Sample processing

固体类样品：称取5g (精确至0. 01g ) 粉粹均匀的样品于100mL锥形瓶中，加入 8~10mL无水乙醇，旋涡混合1~2 min 后超声提取15~20min，静置分层，吸取上清液于50mL离心管中，残余物每次用 8~10mL无水乙醇提取2次；合并上清液于离心管中加入1g中性氧化铝，漩涡震荡1~2 min，3000r / min 离心5~10min，移取上清液于25mL容量瓶中，用无水乙醇定容，摇匀，得待测样品液；

Solid samples: Weigh 5g (accurate to 0.01g) of the powdered and homogeneous sample into a 100mL conical flask, add 8~10mL of absolute ethanol, vortex for 1~2 minutes, and then ultrasonically extract for 15~20 minutes. layer, suck the supernatant into a 50mL centrifuge tube, and extract the residue twice with 8~10mL absolute ethanol each time; combine the supernatant and add 1g of neutral alumina to the centrifuge tube, vortex for 1~2 min, 3000r /min Centrifuge for 5~10min, transfer the supernatant to a 25mL volumetric flask, dilute to volume with absolute ethanol, and shake well to obtain the sample solution to be tested;

油类：准确称取植物油或油脂食品2g于25mL比色管中，加入6mL体积浓度95％的乙醇溶液，涡旋1min充分混匀，静置片刻，于90℃水浴中加热10~15s促使其分层；将上层清液用吸管转移到浓缩瓶中，再用6mL 95％乙醇溶液重复提取2次，合并提取液并定容到25mL，得待测样品液；

Oils: Accurately weigh 2g of vegetable oil or oily food into a 25mL colorimetric tube, add 6mL of 95% ethanol solution by volume, vortex for 1min to fully mix, let stand for a while, and heat it in a 90°C water bath for 10~15s to promote it. Layering; transfer the supernatant to a concentrated bottle with a pipette, repeat the extraction twice with 6 mL of 95% ethanol solution, combine the extracts and set the volume to 25 mL to obtain the sample solution to be tested;

（3）样品测定：在10mL具塞比色管中加入50~100µL铜碘掺杂碳点还原制得的金纳米，加入步骤（2）制备待测样品液，用pH7.4的柠檬酸-磷酸氢二钠缓冲溶液稀释至刻度，摇匀，静置5~10min，在492nm波长处，测定吸光度A，代入步骤（1）回归方程，计算样品中特丁基对苯二酚的含量；(3) Sample determination: add 50~100µL of gold nanoparticles obtained by reduction of copper iodine doped carbon dots into a 10mL colorimetric tube with a stopper, add step (2) to prepare the sample solution to be tested, and use pH 7.4 citric acid- Dilute the sodium hydrogen phosphate buffer solution to the mark, shake well, let stand for 5~10min, measure the absorbance A at the wavelength of 492nm, and substitute it into the regression equation of step (1) to calculate the content of tert-butyl hydroquinone in the sample;

所述的Cu-I/CDs还原制得的金纳米的制备方法如下：铜碘掺杂碳点还原制得的金纳米是将2mL聚丙烯亚胺加入15~20mL超纯水中，在搅拌下加入70~90μL氯金酸，再加入180~220μL的铜碘掺杂碳点，搅拌30~40min后，溶液变为红褐色，即得铜碘掺杂碳点还原制得的金纳米；其中聚丙烯亚胺浓度为0.25g/mL，氯金酸质量浓度为1%。The preparation method of the gold nanometers prepared by the reduction of Cu-I/CDs is as follows: the gold nanometers prepared by the reduction of copper iodine doped carbon dots are: add 2 mL of polypropyleneimide to 15-20 mL of ultrapure water, and stir under stirring. Add 70-90 μL of chloroauric acid, and then add 180-220 μL of copper-iodine-doped carbon dots, and after stirring for 30-40 min, the solution turns reddish-brown to obtain gold nanoparticles prepared by reduction of copper-iodine-doped carbon dots; The concentration of propylene imine is 0.25g/mL, and the mass concentration of chloroauric acid is 1%.

所述Cu-I/CDs制备方法如下：称取1g CuCl₂、3~5g 3-碘-L-酪氨酸溶于400~600mL超纯水中，混合均匀后，加入800~1200μL乙二胺，超声10~20min，并转移至聚四氟乙烯反应釜中在180℃下加热8~10h，自然冷却后，先用孔径为0.22μm滤膜过滤，后用截留分子量为3000Da 的透析袋进行透析处理24h，得到水溶性Cu-I/CDs。The preparation method of the Cu-I/CDs is as follows: Weigh 1g CuCl₂ and 3~5g 3-iodo-L-tyrosine and dissolve them in 400~600mL ultrapure water, after mixing evenly, add 800~1200μL ethylenediamine , ultrasonic for 10~20min, and transferred to a polytetrafluoroethylene reactor, heated at 180℃ for 8~10h, after natural cooling, first filtered with a 0.22μm filter membrane, and then dialyzed with a dialysis bag with a molecular weight cut-off of 3000Da. After treatment for 24 h, water-soluble Cu-I/CDs were obtained.

本发明的优点在于：The advantages of the present invention are:

1、本发明利用铜碘掺杂碳点（Cu-I/CDs）作为还原剂，聚丙烯亚胺为保护剂，还原氯金酸，得到金纳米粒子与Cu-I/CDs组成混合纳米酶体系，由于其优越的催化特性，选择性氧化特丁基对苯二酚（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；1. The present invention uses copper iodine doped carbon dots (Cu-I/CDs) as a reducing agent, and polypropyleneimide as a protective agent to reduce chloroauric acid to obtain a mixed nanoenzyme system composed of gold nanoparticles and Cu-I/CDs. , Due to its superior catalytic properties, the selective oxidation of tert-butyl hydroquinone (TBHQ) is a red oxidized quinone. Based on the linear relationship between the concentration of TBHQ and red oxidized TBHQ, a new method for the detection of TBHQ with high sensitivity and selectivity was established. ;

2、本发明建立的催化氧化体系对TBHQ有特异性氧化，其他类似结构及性质的合成酚类抗氧化剂无此反应，反应快速、灵敏、稳定，用于TBHQ检测，具有灵敏度高、重现性好、准确的特点；2. The catalytic oxidation system established in the present invention has specific oxidation on TBHQ, and other synthetic phenolic antioxidants with similar structures and properties have no such reaction. The reaction is fast, sensitive and stable. It is used for TBHQ detection with high sensitivity and reproducibility. good and accurate characteristics;

3、本发明制备的铜碘掺杂碳点既作为还原剂，又作为纳米酶，由于与金纳米的协同催化作用，使反应快速、灵敏。3. The copper-iodine-doped carbon dots prepared by the present invention serve as both a reducing agent and a nano-enzyme, and the reaction is fast and sensitive due to the synergistic catalysis with gold nanoparticles.

附图说明Description of drawings

图1为实施例1中复合纳米酶氧化TBHQ紫外-可见吸收光谱，用于TBHQ检测；Fig. 1 is the ultraviolet-visible absorption spectrum of composite nanozyme oxidizing TBHQ in Example 1, which is used for TBHQ detection;

图2为共存抗氧化剂对TBHQ的影响结果，图中TBHQ代表单独使用，其他为TBHQ与没食子酸丙酯 ( PG )、2 , 4 , 5-三羟基苯丁酮 ( THBP )、去甲二氢愈创木酸( NDGA )、叔丁基对羟基茴香醚( BHA )、2 , 6-二叔丁基-4-羟甲基苯酚(Ionox-100 )、没食子酸辛酯( OG )、 2 , 6- 二叔丁基对甲基苯酚( BHT )、没食子酸十二酯( DG )、葡萄糖、麦芽糖、蔗糖、淀粉、抗坏血酸中的一个混合；Figure 2 shows the effect of coexisting antioxidants on TBHQ. In the figure, TBHQ stands for single use. guaiaretic acid (NDGA), tert-butyl-p-hydroxyanisole (BHA), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), octyl gallate (OG), 2,6 - A mixture of di-tert-butyl-p-cresol (BHT), dodecyl gallate (DG), glucose, maltose, sucrose, starch, ascorbic acid;

图3为共存离子对TBHQ的影响结果，图中Black为TBHQ，其他为与TBHQ共存的离子（Na⁺、K⁺、Mg²⁺、Cu²⁺、Zn²⁺、Fe²⁺、Cl^-、SO₄^2-、NO₃^-等）。Figure 3 shows the effect of coexisting ions on TBHQ, in which Black is TBHQ, and the others are ions that coexist with TBHQ (Na⁺ , K⁺ , Mg²⁺ , Cu²⁺ , Zn²⁺ , Fe²⁺ , Cl^- , SO₄^2- , NO₃^- , etc.).

具体实施方式Detailed ways

下面将结合具体的实施例对本发明的技术方案作进一步详细地描述说明，但本发明的保护范围并不仅限于此。The technical solutions of the present invention will be described in further detail below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

实施例1：大豆油样品中TBHQ的测定Example 1: Determination of TBHQ in soybean oil samples

1、Cu-I/CDs的制备：0.1g CuCl₂、0.4g 3-碘-L-酪氨酸加入到40mL超纯水中混合溶解，然后加入100μL乙二胺，超声10min，并转移至聚四氟乙烯反应釜中，于180℃加热8h，自然冷却后，先用孔径为0.22μm滤膜过滤，后用截留分子量为3000Da 的透析袋进行透析处理24h，得到水溶性Cu-I/CDs；1. Preparation of Cu-I/CDs: 0.1 g of CuCl₂ and 0.4 g of 3-iodo-L-tyrosine were added to 40 mL of ultrapure water, mixed and dissolved, then 100 μL of ethylenediamine was added, sonicated for 10 min, and transferred to a polymer. In a tetrafluoroethylene reaction kettle, heated at 180 °C for 8 h, and after natural cooling, firstly filtered with a 0.22 μm pore size filter membrane, and then dialyzed with a dialysis bag with a molecular weight cut-off of 3000 Da for 24 h to obtain water-soluble Cu-I/CDs;

2、Cu-I/CDs还原制得的金纳米：将2mL浓度0.25g/mL的聚丙烯亚胺加入20mL超纯水，在搅拌下加入 质量浓度1% 的氯金酸80μL，再加入200 μL的Cu-I/CDs，搅拌30min后，溶液变为红褐色，即得Cu-I/CDs还原制得的金纳米。2. Gold nanoparticles prepared by reduction of Cu-I/CDs: Add 2 mL of polypropyleneimine with a concentration of 0.25 g/mL to 20 mL of ultrapure water, add 80 μL of 1% chloroauric acid by mass under stirring, and then add 200 μL After stirring for 30 min, the solution turned reddish-brown, that is, gold nanoparticles prepared by reduction of Cu-I/CDs were obtained.

3、特丁基对苯二酚工作曲线制作：在10mL具塞比色管中加入50µL Cu-I/CDs还原制得的金纳米、浓度在1~200mg/kg范围内的TBHQ标准溶液，用pH 7.4的柠檬酸-磷酸氢二钠缓冲溶液稀释至刻度，摇匀，静置10min，在492nm波长处，测定吸光度A，以TBHQ浓度为横坐标，A为纵坐标，绘制标准曲线，得到回归方程；见图1，得到回归方程、相关系数、相对标准偏差、线性范围等见表1；3. Preparation of the working curve of tert-butyl hydroquinone: add 50 µL of Cu-I/CDs reduction to the TBHQ standard solution with a concentration in the range of 1~200 mg/kg in a 10 mL colorimetric tube with a stopper, and use The citric acid-disodium hydrogen phosphate buffer solution of pH 7.4 was diluted to the mark, shaken well, and allowed to stand for 10 min. At the wavelength of 492 nm, the absorbance A was measured, with the concentration of TBHQ as the abscissa and A as the ordinate, and the standard curve was drawn to obtain the regression Equation; see Figure 1, the regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1;

表1线性方程、相关系数、相对标准偏差、线性范围Table 1 Linear equation, correlation coefficient, relative standard deviation, linear range

4、方法特异性考察：将TBHQ和其他抗氧化剂、离子混合作为混合抗氧化剂，检测共存抗氧化剂和共存离子在上述检测体系中的TBHQ的影响，TBHQ浓度为10mg/kg，图2是共存抗氧化剂（没食子酸丙酯 ( PG )、2 , 4 , 5-三羟基苯丁酮 ( THBP )、去甲二氢愈创木酸( NDGA )、叔丁基对羟基茴香醚( BHA )、2 , 6-二叔丁基-4-羟甲基苯酚(Ionox-100 )、没食子酸辛酯( OG )、2 , 6- 二叔丁基对甲基苯酚( BHT )、没食子酸十二酯( DG ))及葡萄糖、麦芽糖、蔗糖、淀粉和抗坏血酸对TBHQ的影响结果，图3为共存离子（Na⁺、K⁺、Mg²⁺、Cu²⁺、Zn²⁺、Fe²⁺、Cl^-、SO₄^2-、NO₃^-等）对TBHQ的影响结果，以上干扰物质浓度为50mg/kg，从图中可以看出，纳米酶体系氧化TBHQ有较好的选择特异性，仅有TBHQ有明显的氧化反应，其它物质几乎没有，方法具有好的选择特异性；4. Method specificity investigation: TBHQ was mixed with other antioxidants and ions as mixed antioxidants, and the influence of coexisting antioxidants and coexisting ions on TBHQ in the above detection system was detected. The concentration of TBHQ was 10 mg/kg. Oxidizing agents (propyl gallate (PG), 2,4,5-trihydroxybutanone (THBP), nordihydroguaiaretic acid (NDGA), tert-butyl-p-hydroxyanisole (BHA), 2, 6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), octyl gallate (OG), 2,6-di-tert-butyl-p-cresol (BHT), dodecyl gallate (DG) ) and the effects of glucose, maltose, sucrose, starch and ascorbic acid on TBHQ, Figure 3 shows the coexisting ions (Na⁺ , K⁺ , Mg²⁺ , Cu²⁺ , Zn²⁺ , Fe²⁺ , Cl^- , SO_{4 )}^2- , NO₃^- , etc.) on TBHQ. The concentration of the above interfering substances is 50mg/kg. It can be seen from the figure that the nano-enzyme system oxidizes TBHQ with good selection specificity, and only TBHQ has obvious oxidation. Reaction, almost no other substances, the method has good selection specificity;

5、大豆调和油样品中TBHQ的测定5. Determination of TBHQ in soybean blended oil samples

（1）样品处理：准确称取大豆调和油2g于25mL比色管中，加入6mL体积浓度95％的乙醇溶液，涡旋1min充分混匀，静置片刻，于90℃水浴中加热10～15s促使其分层；将上层清液用吸管转移到浓缩瓶中，再用6mL 95％乙醇溶液重复提取2次，合并提取液并定容到25mL，得待测样品液；(1) Sample processing: Accurately weigh 2g of soybean blend oil into a 25mL colorimetric tube, add 6mL of 95% ethanol solution by volume, vortex for 1min to fully mix, let stand for a while, and heat in a 90°C water bath for 10-15s To promote its stratification; transfer the supernatant liquid to a concentrated bottle with a pipette, repeat the extraction twice with 6 mL of 95% ethanol solution, combine the extracts and dilute to 25 mL to obtain the sample solution to be tested;

（2）样品测定：在10mL具塞比色管中加入50µL Cu-I/CDs还原制得的金纳米，加入步骤（1）制备待测样品液4mL，用pH 7.4的柠檬酸-磷酸氢二钠缓冲溶液稀释至刻度，摇匀，静置10min，在492 nm波长处，测定吸光度A，代入步骤（1）回归方程，计算样品TBHQ含量为54.90 mg/kg；(2) Sample determination: add 50µL of gold nanoparticles prepared by reduction of Cu-I/CDs to a 10mL colorimetric tube with a stopper, add 4mL of the sample solution to be tested in step (1), and use citric acid-hydrogen phosphate dibasic with pH 7.4. Dilute the sodium buffer solution to the mark, shake well, let stand for 10 minutes, measure the absorbance A at a wavelength of 492 nm, and substitute it into the regression equation of step (1) to calculate the TBHQ content of the sample to be 54.90 mg/kg;

（3）回收率与精密度实验：在大豆调和油样品中分别添加2个不同浓度的TBHQ标准溶液；每个浓度平行测定3次，计算加标回收率，并计算出相对标准偏差RSD，结果见表2，测得TBHQ的加标回收率在98.0％～101.9％，RSD在0.20％～1.3％，本方法有好的准确性和精密度。(3) Recovery and precision experiment: Add 2 different concentrations of TBHQ standard solutions to the soybean blend oil samples respectively; each concentration was measured 3 times in parallel, calculate the standard addition recovery rate, and calculate the relative standard deviation RSD, the results See Table 2, the measured recovery of standard addition of TBHQ is 98.0%-101.9%, and the RSD is 0.20%-1.3%. This method has good accuracy and precision.

表2 样品加标回收率及RSD（n = 3）Table 2 Sample spike recovery and RSD (n = 3)

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实施例2：葵花籽油样品中TBHQ的测定Example 2: Determination of TBHQ in Sunflower Oil Samples

1、Cu-I/CDs的制备：1 g CuCl₂和3g 3-碘-L-酪氨酸溶于500mL纯化水，混合溶解，加入900μL乙二胺，超声15min，并转移至聚四氟乙烯反应釜，于180℃加热10h，自然冷却后，先用孔径为0.22μm滤膜过滤，后用截留分子量为3000D 的透析袋进行透析处理24h，得到水溶性Cu-I/CDs；1. Preparation of Cu-I/CDs: 1 g of CuCl₂ and 3 g of 3-iodo-L-tyrosine were dissolved in 500 mL of purified water, mixed and dissolved, added 900 μL of ethylenediamine, sonicated for 15 min, and transferred to polytetrafluoroethylene The reaction kettle was heated at 180 °C for 10 h, and after natural cooling, it was filtered with a 0.22 μm pore size filter membrane, and then dialyzed with a dialysis bag with a molecular weight cut-off of 3000 D for 24 h to obtain water-soluble Cu-I/CDs;

2、Cu-I/CDs还原制得的金纳米的制备：2mL的0.25g/mL聚丙烯亚胺，加入18mL超纯水，在搅拌下加入1%氯金酸90μL，再加入190μL Cu-I/CDs，搅拌35min之后，溶液变为红褐色，既得Cu-I/CDs还原制得金纳米；2. Preparation of gold nanoparticles by reduction of Cu-I/CDs: 2 mL of 0.25 g/mL polypropyleneimine, 18 mL of ultrapure water, 90 μL of 1% chloroauric acid under stirring, and then 190 μL of Cu-I /CDs, after stirring for 35min, the solution turned reddish-brown, and the obtained Cu-I/CDs was reduced to obtain gold nanoparticles;

3、特丁基对苯二酚工作曲线制作同实施例1；3. The production of tert-butyl hydroquinone working curve is the same as that in Example 1;

4、葵花籽油样品中TBHQ的测定4. Determination of TBHQ in sunflower oil samples

（1）样品处理：准确称取葵花籽油2g于25mL比色管中，加入6mL体积浓度95％的乙醇溶液，涡旋1min充分混匀，静置片刻，于90℃水浴中加热15s促使其分层；将上层清液用吸管转移到浓缩瓶中，再用6mL 95％乙醇溶液重复提取2次，合并提取液并定容到25mL，得待测样品液；(1) Sample processing: Accurately weigh 2g of sunflower oil into a 25mL colorimetric tube, add 6mL of 95% ethanol solution by volume, vortex for 1min to fully mix, let stand for a while, and heat it in a 90°C water bath for 15s to promote it. Layering; transfer the supernatant to a concentrated bottle with a pipette, repeat the extraction twice with 6 mL of 95% ethanol solution, combine the extracts and set the volume to 25 mL to obtain the sample solution to be tested;

（2）样品测定：同实施例1，葵花籽油样品中TBHQ的含量为31.33 mg/kg。(2) Sample determination: Same as Example 1, the content of TBHQ in the sunflower oil sample was 31.33 mg/kg.

实施例3：曲奇饼干中TBHQ的测定Example 3: Determination of TBHQ in Cookies

1、Cu-I/CDs的制备同实施例1；1. The preparation of Cu-I/CDs is the same as in Example 1;

2、Cu-I/CDs还原制得的金纳米的制备同实施例1；2. The preparation of gold nanoparticles obtained by reduction of Cu-I/CDs is the same as in Example 1;

3、特丁基对苯二酚工作曲线制作同实施例1；3. The production of tert-butyl hydroquinone working curve is the same as that in Example 1;

4、曲奇饼干中TBHQ的测定4. Determination of TBHQ in cookies

（1）样品处理：称取5.00g粉粹均匀的饼干样品于100mL锥形瓶中，加入8mL无水乙醇，旋涡混合1min 后超声提取15min，静置分层，吸取上清液于50mL 离心管中，残余物每次用8mL无水乙醇提取2次；合并上清液于50mL离心管中加入1g中性氧化铝，漩涡震荡1min，3000r/min 离心8min，移取上清液于25 mL容量瓶中，用无水乙醇定容，摇匀，得待测样品液；(1) Sample processing: Weigh 5.00g of powdered and homogeneous biscuit samples into a 100mL conical flask, add 8mL of absolute ethanol, vortex for 1min, ultrasonically extract for 15min, let stand for stratification, and draw the supernatant into a 50mL centrifuge tube The residue was extracted twice with 8 mL of absolute ethanol each time; the combined supernatant was added to 1 g of neutral alumina in a 50 mL centrifuge tube, vortexed for 1 min, centrifuged at 3000 r/min for 8 min, and the supernatant was transferred to a 25 mL volume In the bottle, dilute to volume with absolute ethanol, shake well, and obtain the sample solution to be tested;

（2）样品测定：同实施例1，曲奇饼干中TBHQ的含量为5.38 mg/kg。(2) Sample determination: Same as Example 1, the content of TBHQ in the cookie was 5.38 mg/kg.

实施例4：方便面中TBHQ的测定Example 4: Determination of TBHQ in instant noodles

1、Cu-I/CDs的制备同实施例1；1. The preparation of Cu-I/CDs is the same as in Example 1;

2、Cu-I/CDs还原制得的金纳米的制备同实施例1；2. The preparation of gold nanoparticles obtained by reduction of Cu-I/CDs is the same as in Example 1;

3、特丁基对苯二酚工作曲线制作同实施例1；3. The production of tert-butyl hydroquinone working curve is the same as that in Example 1;

4、方便面中TBHQ的测定：4. Determination of TBHQ in instant noodles:

（1）样品处理：称取5.00g粉粹均匀的方便面样品于100mL锥形瓶中，加入9mL 无水乙醇，旋涡混合1min 后超声提取20min，静置分层，吸取上清液于50mL 离心管中，残余物每次用 9mL无水乙醇提取2次；合并上清液于50mL离心管中加入1g中性氧化铝，漩涡震荡1min，3000r/min 离心10min，移取上清液于25mL容量瓶中，用无水乙醇定容，摇匀，得待测样品液；(1) Sample processing: Weigh 5.00g of powdered and uniform instant noodle sample into a 100mL conical flask, add 9mL of absolute ethanol, vortex for 1min, then ultrasonically extract for 20min, let stand for stratification, and suck the supernatant into a 50mL centrifuge tube The residue was extracted twice with 9 mL of absolute ethanol each time; the combined supernatant was added to 1 g of neutral alumina in a 50 mL centrifuge tube, vortexed for 1 min, centrifuged at 3000 r/min for 10 min, and the supernatant was transferred to a 25 mL volumetric flask , dilute to volume with absolute ethanol, shake well, and obtain the sample solution to be tested;

（2）样品测定：同实施例1，方便面中TBHQ的含量为1.25 mg/kg；(2) Sample determination: same as Example 1, the content of TBHQ in instant noodles is 1.25 mg/kg;

将实施例1-4用本发明方法与国家标准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进行比对，结果见表3；从结果可以看出，两种方法结果一致；Examples 1-4 were compared with the method of the present invention and the national standard GB5009.32-2016 national food safety standard for the determination of 9 antioxidants in food, and the results are shown in Table 3; it can be seen from the results that the results of the two methods consistent;

表3加标回收率及相对标准偏差（mg/kg）Table 3 Spike recovery and relative standard deviation (mg/kg)

本发明建立的TBHQ测定法具有处理步骤少，不需要GB5009.32-2016 样品净化处理，所用时间短，处理成本低，操作简便，不需要大型仪器设备，在实际检测中具有较强优势。The TBHQ assay method established in the present invention has few processing steps, does not require GB5009.32-2016 sample purification treatment, takes a short time, has low processing cost, is easy to operate, does not require large-scale instruments and equipment, and has strong advantages in actual detection.

Claims (2)

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 muL of gold nano particles prepared by reduction of copper-iodine doped carbon points into a 10mL colorimetric tube with a plug, adding the gold nano particles prepared by the step (2) to prepare a sample liquid to be detected, diluting the sample liquid 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 absorbance A at the position of 492nm 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;

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 allowing the solution to turn into 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%.

2. The method for rapidly detecting tert-butylhydroquinone in food according to claim 1, 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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