A high performance liquid chromatography method for detecting rhodamine B in food

Abstract

The present invention relates to the field of food additive detection and discloses a high performance liquid chromatography (HPLC) method for detecting rhodamine B in food, comprising the following steps: (1) sample pretreatment: adding a food sample to be tested to ethyl acetate, mixing uniformly, ultrasonically extracting, and then subjecting the sample to be tested to centrifugal separation and filtration to obtain a test solution; (2) preparing a rhodamine B standard solution; (3) detecting the standard solution using a HPLC instrument to obtain a standard curve; then detecting the test solution and calculating the content of rhodamine B in the test solution by an external standard method; the chromatographic column used in the detection is a phenyl column. The present invention uses a phenyl chromatographic column to detect rhodamine B in food, which can simplify the HPLC pretreatment process and improve detection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of food additive detection, in particular to a high performance liquid chromatography detection method for rhodamine B in food. Background Art

[0002] The complex pre-treatment process makes the detection cycle of Rhodamine B in the existing technology long and the operation complicated, making it difficult to quickly and effectively detect Rhodamine B in food. Summary of the Invention

[0003] The present invention aims to overcome the problem that when determining rhodamine B in food by high performance liquid chromatography in the prior art, relatively complex pretreatment of the sample is generally required, resulting in a long detection cycle, complicated operation, and difficulty in quickly and effectively detecting rhodamine B in food. A high performance liquid chromatography method for detecting rhodamine B in food is provided. A phenyl chromatographic column is used to detect rhodamine B in food, which can simplify the pretreatment process of the high performance liquid chromatography and improve the detection efficiency.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A high performance liquid chromatography method for detecting rhodamine B in food comprises the following steps:

[0006] (1) Sample pretreatment: Add the food sample to be tested into ethyl acetate, mix well, and then perform ultrasonic extraction. Then, centrifuge and filter to obtain the test solution.

[0007] (2) Prepare rhodamine B standard solution;

[0008] (3) The standard solution is tested using a high performance liquid chromatograph to obtain a standard curve; the test solution is then tested and the content of rhodamine B in the test solution is calculated by an external standard method; the chromatographic column used in the test is a phenyl column.

[0009] In the high-performance liquid chromatography method of the present invention, a phenyl column is used to detect rhodamine B in food. The stationary phase in the phenyl column is a phenyl group bonded to a silica gel matrix via a carbon chain. Due to the presence of the carbon chain, the phenyl column has a certain hydrophobic retention capacity, and the presence of the benzene ring enables it to form a π-π interaction with aromatic compounds. Therefore, the phenyl column has a certain selectivity for rhodamine B molecules with a conjugated system. When the phenyl column is used as a chromatographic column to detect rhodamine B in food, the remaining components in the sample have little effect on the detection results. Therefore, the sample only needs to undergo a simple extraction treatment before it can be directly tested on the machine without the need for other purification treatments, thereby simplifying the detection steps and improving the detection efficiency.

[0010] Preferably, in the phenyl column used in step (3), the stationary phase adopts a phenyl periodic organic-inorganic mesoporous hybrid material, and the preparation method of the stationary phase comprises the following steps:

[0011] A) reacting epoxy bisphenol fluorene with γ-aminopropyltriethoxysilane to prepare a polyphenyl silicon source reagent;

[0012] B) adding a surfactant to the ethanol solution, mixing well, adding hydrochloric acid, then adding a polyphenyl silicon source reagent under stirring, and continuing to stir well to obtain a reaction solution;

[0013] C) reacting the reaction solution at 80-90° C. for 4-6 hours, then reacting at 140-150° C. for 12-24 hours, and washing and drying the obtained product after cooling;

[0014] D) adding the dried product in step C) to a mixed solution of hydrochloric acid and anhydrous ethanol, stirring and eluting at 60-70° C. for 12-24 hours, washing, drying, and grinding the eluted product to obtain the stationary phase.

[0015] To ensure good selectivity and retention of rhodamine B on a phenyl column, the present invention uses a polyphenyl silicon source reagent, prepared by reacting epoxybisphenol fluorene with γ-aminopropyltriethoxysilane. Under the structure-guiding action of a surfactant, the polyphenyl silicon source is hydrolyzed and condensed, and then the template is washed away to form a phenyl periodic organic-inorganic mesoporous hybrid material as the stationary phase. Research by the present invention has found that using the periodic organic-inorganic mesoporous hybrid material with a polyphenyl ring structure in its skeleton as the stationary phase can achieve highly selective separation of rhodamine B in foods, significantly improving the separation of rhodamine B from other structurally similar aromatic compounds in foods. This simplifies the pretreatment process while ensuring the accuracy and reproducibility of the measurement results.

[0016] Preferably, the molar ratio of epoxybisphenol fluorene to γ-aminopropyltriethoxysilane in step A) is 1:2 to 2.5; the reaction conditions are: epoxybisphenol fluorene and γ-aminopropyltriethoxysilane are added to DMF, deoxygenated with nitrogen, and then heated at 70 to 90° C. for 2 to 3 hours to obtain the polyphenyl silicon source reagent.

[0017] Preferably, in the ethanol solution described in step B), the volume ratio of anhydrous ethanol to water is 1 to 2:5; the surfactant comprises F127 and cetyltrimethylammonium bromide in a molar ratio of 1:10 to 15; the mass volume ratio of the added F127 to the ethanol solution is 1 g:35 to 45 mL; the mass concentration of the hydrochloric acid is 36 to 38%, and the volume ratio of the added hydrochloric acid to the ethanol solution is 1:35 to 45; the mass ratio of the added polyphenyl silicon source reagent to F127 is 2 to 3:1.

[0018] Preferably, the mass concentration of the hydrochloric acid in step D) is 36-38%, the volume ratio of hydrochloric acid to anhydrous ethanol in the mixed solution is 4-5:150; and the mass volume ratio of the dried product to the mixed solution is 1 g:150-200 mL.

[0019] Preferably, the phenyl column used in step (3) has an inner diameter of 2.1 to 4.6 mm and a length of 100 to 150 mm.

[0020] Preferably, in step (1), the mass volume ratio of the food sample to ethyl acetate is 2 g:10-15 mL, and the ultrasonic extraction time is 15-20 min; the rotation speed during centrifugation is 3000-4000 r / min, the centrifugation time is 5-10 min, and a 0.22 μm microporous filter membrane is used for filtration.

[0021] Preferably, the concentration range of the Rhodamine B standard solution in step (2) is 5 to 50 ng / mL.

[0022] Preferably, the mobile phase for detection in step (3) is methanol and water in a volume ratio of 75 to 85:25 to 15. The present invention selects the type and ratio of the mobile phase to ensure that rhodamine B has an appropriate retention time, thereby improving the sensitivity and repeatability of the detection.

[0023] Preferably, the detection conditions in step (3) are: column temperature 25-35°C; flow rate 0.8-1.0 mL / min; injection volume 10.0-10.2 μL; excitation wavelength 550 nm, emission wavelength 580 nm.

[0024] Therefore, the present invention has the following beneficial effects:

[0025] (1) Phenyl columns are used to detect rhodamine B in food. The other components in the sample have little effect on the test results. The sample can be directly tested on the machine after a simple extraction process without the need for other purification processes, which simplifies the test steps and improves the test efficiency.

[0026] (2) The use of periodic organic-inorganic mesoporous hybrid materials with multiple benzene ring structures in the skeleton as the stationary phase in the phenyl column can achieve highly selective separation of rhodamine B in food, and can significantly improve the separation effect of rhodamine B and other aromatic compounds with similar structures in food, thereby simplifying the pretreatment process while ensuring the accuracy and reproducibility of the measurement results. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a chromatogram of the standard working solution of each concentration gradient in Example 1;

[0028] Figure 2is the standard curve after fitting in Example 1;

[0029] Figure 3 is the spiked chromatogram of the test solution in Example 1;

[0030] Figure 4 is the standard curve after fitting in Example 2;

[0031] Figure 5 is the spiked chromatogram of the test solution in Example 2;

[0032] Figure 6 is the standard curve after fitting in Example 3;

[0033] Figure 7 is the spiked chromatogram of the test solution in Example 3;

[0034] Figure 8 is the chromatogram of the standard working solution of each concentration gradient in Comparative Example 1;

[0035] Figure 9 is the standard curve after fitting in Comparative Example 1;

[0036] Figure 10 This is the spiked chromatogram of the test solution in Comparative Example 1;

[0037] Figure 11 is the standard curve after fitting in Comparative Example 2;

[0038] Figure 12 This is the spiked chromatogram of the test solution in Comparative Example 2. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0040] In the present invention, unless otherwise specified, all raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0041] Example 1:

[0042] A high performance liquid chromatography method for detecting rhodamine B in food, comprising the following steps:

[0043] (1) Sample pretreatment: Weigh 2 g of chili powder sample, add 10 mL of ethyl acetate, mix in a vortex mixer for 2 min, and ultrasonically extract for 15 min; then centrifuge at 3500 rpm for 5 min, filter through a 0.22 μm microporous membrane, and obtain the test solution;

[0044] (2) Preparation of Rhodamine B standard solution: Weigh 0.10000 g of Rhodamine B standard using a 100,000th balance, dilute to volume with methanol into a 100 mL volumetric flask to obtain a 1.00 mg / mL stock solution; dilute the stock solution 100 times to obtain a 10.0 μg / mL application solution 1, and then dilute the application solution 1 100 times to obtain a 100 ng / mL application solution 2; take 0.10 mL, 0.20 mL, 0.40 mL, 1.00 mL, 1.50 mL, and 2.00 mL of application solution 2 and dilute to 10 mL, respectively, to obtain standard working solutions with concentrations of 1.00 ng / mL, 2.00 ng / mL, 4.00 ng / mL, 10.00 ng / mL, 15.00 ng / mL, and 20.00 ng / mL;

[0045] The standard working solution was measured by high performance liquid chromatography (Waters2695 nano) to make a standard curve. The results are as follows Figure 1 and Figure 2 As shown in;

[0046] The test conditions are:

[0047] Chromatographic column: inner diameter 4.6 mm, length 150 mm, stationary phase: phenyl periodic organic-inorganic mesoporous hybrid material;

[0048] Mobile phase: methanol and water in a volume ratio of 80:20;

[0049] Column temperature: 30°C;

[0050] Flow rate: 1 mL / min;

[0051] Injection volume: 10 μL;

[0052] Excitation wavelength: 550 nm;

[0053] Emission wavelength: 580nm;

[0054] Under the same detection conditions, the test solution was tested by high performance liquid chromatography for spike recovery test, the content of rhodamine B in the test solution was calculated by external standard method, and the accuracy of the method was evaluated by spike recovery rate (GB / T 27404-2008). The results are as follows Figure 3 and as shown in Table 1;

[0055] Among them, the preparation method of the stationary phase phenyl periodic organic-inorganic mesoporous hybrid material is:

[0056] A) adding epoxybisphenol fluorene and γ-aminopropyltriethoxysilane in a molar ratio of 1:2.3 to DMF, purging with nitrogen for 10 minutes to remove oxygen, and then heating at 80° C. for 3 hours to obtain a polyphenyl silicon source reagent;

[0057] B) adding a surfactant to an ethanol solution, wherein the volume ratio of anhydrous ethanol to water in the ethanol solution is 1.5:5; the surfactant comprises F127 and cetyltrimethylammonium bromide in a molar ratio of 1:12; the mass volume ratio of the added F127 to the ethanol solution is 1 g:40 mL; after mixing, 36 wt % hydrochloric acid is added, and the volume ratio of the added hydrochloric acid to the ethanol solution is 1:40; then, a polyphenyl silicon source reagent is added under stirring, and the mass ratio of the added polyphenyl silicon source reagent to the F127 is 2.5:1, and stirring is continued to obtain a reaction solution;

[0058] C) reacting the reaction solution at 85° C. for 5 h, then at 145° C. for 18 h, and after cooling, washing the obtained product with water and anhydrous ethanol, respectively, and then drying at 55° C. for 5 h;

[0059] D) adding the dried product from step C) to a mixed solution of 36 wt % hydrochloric acid and anhydrous ethanol, wherein the volume ratio of hydrochloric acid to anhydrous ethanol in the mixed solution is 4.5:150; the mass volume ratio of the dried product to the mixed solution is 1 g:160 mL, stirring and eluting at 65° C. for 18 hours, washing the eluted product with anhydrous ethanol, drying it at 55° C. for 5 hours, and grinding it to obtain the stationary phase; and loading the stationary phase into a stainless steel chromatographic column (4.6 mm*150 mm) using a homogenization method to obtain a phenyl column.

[0060] Example 2:

[0061] A high performance liquid chromatography method for detecting rhodamine B in food, comprising the following steps:

[0062] (1) Sample pretreatment: Weigh 2 g of chili oil sample, add 10 mL of ethyl acetate, mix in a vortex mixer for 2 min, and ultrasonically extract for 20 min; then centrifuge at 3000 rpm for 10 min, filter through a 0.22 μm microporous membrane, and obtain the test solution;

[0063] (2) Preparation of Rhodamine B standard working solution: the preparation method is the same as that in Example 1;

[0064] The standard working solution was measured by high performance liquid chromatography (Waters2695 nano) to make a standard curve. The results are as follows Figure 4 As shown; the detection conditions are the same as in Example 1;

[0065] The preparation method of the phenyl periodic organic-inorganic mesoporous hybrid material as the stationary phase of the chromatographic column is as follows:

[0066] A) adding epoxybisphenol fluorene and γ-aminopropyltriethoxysilane in a molar ratio of 1:2 to DMF, purging with nitrogen for 10 minutes to remove oxygen, and then heating at 70° C. for 3 hours to obtain a polyphenyl silicon source reagent;

[0067] B) adding a surfactant to an ethanol solution, wherein the volume ratio of anhydrous ethanol to water in the ethanol solution is 1:5; the surfactant comprises F127 and cetyltrimethylammonium bromide in a molar ratio of 1:10; the mass volume ratio of the added F127 to the ethanol solution is 1 g:35 mL; after mixing, 36 wt % hydrochloric acid is added, and the volume ratio of the added hydrochloric acid to the ethanol solution is 1:35; then, a polyphenyl silicon source reagent is added under stirring, and the mass ratio of the added polyphenyl silicon source reagent to the F127 is 2:1, and stirring is continued to obtain a reaction solution;

[0068] C) reacting the reaction solution at 80° C. for 6 h, then at 150° C. for 12 h, and after cooling, washing the obtained product with water and anhydrous ethanol, respectively, and then drying at 50° C. for 6 h;

[0069] D) The dried product in step C) was added to a mixed solution of 36 wt% hydrochloric acid and anhydrous ethanol, wherein the volume ratio of hydrochloric acid to anhydrous ethanol in the mixed solution was 4:150; the mass volume ratio of the dried product to the mixed solution was 1 g:150 mL, and the mixture was stirred and eluted at 60° C. for 24 h. The eluted product was washed with anhydrous ethanol and dried at 50° C. for 6 h, and then ground to obtain the stationary phase; (3) Under the same detection conditions, a high performance liquid chromatograph was used to perform a spike recovery test on the test solution, and the content of rhodamine B in the test solution was calculated by the external standard method. The accuracy of the method was evaluated by the spike recovery rate. The results are as follows: Figure 5 and as shown in Table 1.

[0070] Example 3:

[0071] A high performance liquid chromatography method for detecting rhodamine B in food, comprising the following steps:

[0072] (1) Sample pretreatment: Weigh 2 g of bacon sample, add 10 mL of ethyl acetate, mix in a vortex mixer for 2 min, and ultrasonically extract for 15 min; then centrifuge at 4000 rpm for 5 min, filter through a 0.22 μm microporous membrane, and obtain the test solution;

[0073] (2) Preparation of Rhodamine B standard working solution: the preparation method is the same as that in Example 1;

[0074] The standard working solution was measured by high performance liquid chromatography (Waters2695 nano) to make a standard curve. The results are as follows Figure 6 As shown; the detection conditions are the same as in Example 1;

[0075] The preparation method of the phenyl periodic organic-inorganic mesoporous hybrid material as the stationary phase of the chromatographic column is as follows:

[0076] A) epoxybisphenol fluorene and γ-aminopropyltriethoxysilane in a molar ratio of 1:2.5 were added to DMF, nitrogen was passed through for 10 minutes to remove oxygen, and then heated at 90° C. for 2 hours to obtain a polyphenyl silicon source reagent;

[0077] B) adding a surfactant to an ethanol solution, wherein the volume ratio of anhydrous ethanol to water in the ethanol solution is 2:5; the surfactant comprises F127 and cetyltrimethylammonium bromide in a molar ratio of 1:15; the mass volume ratio of the added F127 to the ethanol solution is 1 g:45 mL; after mixing, 36 wt % hydrochloric acid is added, and the volume ratio of the added hydrochloric acid to the ethanol solution is 1:45; then, a polyphenyl silicon source reagent is added under stirring, and the mass ratio of the added polyphenyl silicon source reagent to the F127 is 3:1, and stirring is continued to obtain a reaction solution;

[0078] C) reacting the reaction solution at 90° C. for 4 h, then at 140° C. for 24 h, and after cooling, washing the obtained product with water and anhydrous ethanol, respectively, and then drying at 60° C. for 5 h;

[0079] D) The dried product in step C) was added to a mixed solution of 36 wt% hydrochloric acid and anhydrous ethanol, wherein the volume ratio of hydrochloric acid to anhydrous ethanol in the mixed solution was 5:150; the mass volume ratio of the dried product to the mixed solution was 1 g:200 mL, and the mixture was stirred and eluted at 70° C. for 12 h. The eluted product was washed with anhydrous ethanol and dried at 60° C. for 5 h, and then ground to obtain the stationary phase; (3) Under the same detection conditions, a high performance liquid chromatograph was used to perform a spike recovery test on the test solution, and the content of rhodamine B in the test solution was calculated by the external standard method. The accuracy of the method was evaluated by the spike recovery rate. The results are as follows: Figure 7 and as shown in Table 1.

[0080] Comparative Example 1 (using C18 column):

[0081] The chromatographic column used in the detection in Comparative Example 1 was a commercially available C18 column, and the rest was the same as in Example 1;

[0082] The standard curve in Comparative Example 1 is as follows Figure 8 and Figure 9 As shown in the figure, the spiked chromatogram of the test solution is as follows Figure 10 shown.

[0083] Comparative Example 2 (changing the mobile phase ratio):

[0084] The mobile phase for detection in Comparative Example 2 was methanol and water in a volume ratio of 50:50; the rest was the same as in Example 1;

[0085] The standard curve and the spiked chromatogram of the test solution in Comparative Example 2 are as follows: Figure 11 and Figure 12 shown.

[0086] Table 1: Spike recovery test results.

[0087]

[0088] From Table 1 and Figures 1 to 11 It can be seen that the method of the present invention in Examples 1 to 3 can effectively detect Rhodamine B in food, with fast peak emission, high detection efficiency, and little interference from other impurities ( Figure 3 、 Figure 5 and Figure 7 ); and the response value of the standard curve is high ( Figure 1 ), the sample spike recovery rate meets the standard requirements and the detection accuracy is high.

[0089] When the C18 column was used for determination in Comparative Example 1, the response value of the standard curve was low ( Figure 8 ), when only simple pretreatment is performed on the sample, the sample recovery rate is low and the accuracy of the method is not high. In comparative example 2, changing the mobile phase ratio will cause the peak time to be delayed and the detection efficiency to be reduced ( Figure 12 ).

Claims

1. A high performance liquid chromatography method for detecting rhodamine B in food, characterized in that the steps include: (1) Add the food sample to be tested into ethyl acetate, mix well, perform ultrasonic extraction, and obtain the test solution by centrifugation and filtration; (2) Prepare rhodamine B standard solution; (3) The standard solution was tested by HPLC to obtain a standard curve; the test solution was then tested and the content of rhodamine B was calculated by the external standard method; the FLD detector was used for the detection, and the mobile phase was methanol and water in a volume ratio of 75-85:25-15; The chromatographic column is a phenyl column, and the stationary phase adopts a phenyl periodic organic-inorganic mesoporous hybrid material. The preparation method includes: A) reacting epoxybisphenol fluorene with γ-aminopropyltriethoxysilane to prepare a polyphenyl silicon source reagent; B) adding a surfactant to the ethanol solution, mixing uniformly, adding hydrochloric acid, then adding a polyphenyl silicon source reagent while stirring, and continuing to stir uniformly to obtain a reaction solution; C) reacting the reaction solution at 80-90°C for 4-6 hours, then at 140-150°C for 12-24 hours, and then washing and drying the product after cooling; D) Add the product of step C) to a mixed solution of hydrochloric acid and anhydrous ethanol, stir and elute at 60-70°C for 12-24 hours, wash, dry, and grind to obtain a stationary phase.

2. The high performance liquid chromatography method for detecting rhodamine B in food according to claim 1, wherein: In step A), the molar ratio of epoxybisphenol fluorene to γ-aminopropyltriethoxysilane is 1:2-2.

5. The reaction conditions are as follows: epoxybisphenol fluorene and γ-aminopropyltriethoxysilane are added to DMF, deoxygenated by nitrogen, and then heated at 70-90° C. for 2-3 hours to obtain the polyphenyl silicon source reagent.

3. The high performance liquid chromatography method for detecting rhodamine B in food according to claim 1, wherein: In the ethanol solution described in step B), the volume ratio of anhydrous ethanol to water is 1-2:5; the surfactant includes F127 and cetyltrimethylammonium bromide in a molar ratio of 1:10-15; the mass volume ratio of the added F127 to the ethanol solution is 1 g:35-45 mL; the mass concentration of the hydrochloric acid is 36-38%, and the volume ratio of the added hydrochloric acid to the ethanol solution is 1:35-45; the mass ratio of the added polyphenyl silicon source reagent to F127 is 2-3:

1.

4. The high performance liquid chromatography method for detecting rhodamine B in food according to claim 1, wherein: The mass concentration of the hydrochloric acid in step D) is 36-38%, the volume ratio of hydrochloric acid to anhydrous ethanol in the mixed solution is 4-5:150; the mass volume ratio of the dried product to the mixed solution is 1 g:150-200 mL.

5. The high performance liquid chromatography method for detecting rhodamine B in food according to claim 1, wherein: The phenyl column used in step (3) has an inner diameter of 2.1-4.6 mm and a length of 100-150 mm.

6. The high performance liquid chromatography method for detecting rhodamine B in food according to claim 1, wherein: In step (1), the mass volume ratio of the food sample to ethyl acetate is 2 g:10-15 mL, and the ultrasonic extraction time is 15-20 min. The rotation speed during centrifugal separation is 3000-4000 r / min, the centrifugation time is 5-10 min, and a 0.22 μm microporous filter membrane is used for filtration.

7. The high performance liquid chromatography method for detecting rhodamine B in food according to claim 1, wherein: The concentration range of the Rhodamine B standard solution in step (2) is 5-50 ng / mL.

8. The high performance liquid chromatography method for detecting rhodamine B in food according to claim 1, wherein: The mobile phase for detection in step (3) was methanol and water in a volume ratio of 80:

20.

9. The high performance liquid chromatography method for detecting rhodamine B in food according to claim 8, wherein: The detection conditions in step (3) are: column temperature 25~35°C; flow rate 0.8~1.0 mL / min; injection volume 10.0~10.2 μL; excitation wavelength 550 nm, emission wavelength 580 nm.

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