Method for simultaneously determining content of acesulfame potassium, phloretin and perillartine in food essence based on UPLC

Abstract

The application discloses a method for simultaneously determining contents of advantame, phloretin and perillartine in edible essence based on UPLC, and belongs to the technical field of chemical analysis and detection, and comprises preparation of three sweetener series standard solutions, preparation of a to-be-measured sample solution, and subsequent detection and quantitative analysis by means of an ultra-high performance liquid chromatograph, and is suitable for simultaneous detection of the three sweeteners advantame, phloretin and perillartine. Compared with the prior art, different detection methods need to be adopted for detection of the three sweeteners, and the method can effectively separate and quantitatively analyze the three sweeteners with different absorption wavelengths at one time, and is rapid and convenient to operate, high in detection efficiency and easy to popularize.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection, and in particular to a method for simultaneously determining the contents of advantran, phlorizin and perilla stigma in food flavorings based on UPLC. Background Technology

[0002] Sweeteners are a class of food additives that impart sweetness. With the rapid development of the modern food industry, sweeteners have become crucial in food processing due to their ability to improve the taste and flavor of food. However, the overuse of sweeteners and other food additives has raised concerns about health issues, leading to strict restrictions on their addition. The amount of sweeteners added to flavorings is now limited to a certain range. Therefore, accurate detection of sweetener content has become an important task for food safety and quality control. Since a batch of flavorings usually contains more than one type of sweetener, accurate qualitative and quantitative detection of different sweeteners simultaneously has become particularly important for testing efficiency. In a batch of food flavorings, it is often necessary to simultaneously detect the content of three sweeteners: Advil, phlorizin, and perilla oleracea. Current detection technologies typically target single sweeteners. Advil is often detected using LC-MS, perilla oleracea using GC-MS, and phlorizin using liquid chromatography. This involves different detection methods and equipment. The disadvantages of using LC-MS and GC-MS are that the instruments are expensive, maintenance costs are high, experimental conditions are demanding, and the detection methods are relatively cumbersome and time-consuming, which not only increases the complexity and cost of detection but also affects the detection efficiency. Furthermore, the chemical properties and absorption spectra of the three substances differ, making it difficult to simultaneously detect multiple sweeteners in the same batch of samples. Therefore, there is an urgent need to develop a method that can simultaneously, rapidly, accurately, and conveniently determine their content to ensure that it is within the food safety standard range. Summary of the Invention

[0003] The purpose of this invention is to provide a method for simultaneously determining three sweeteners—Advantian, phlorizin, and perilla frutescens—using ultra-high performance liquid chromatography (UHPLC) to solve the problem that the three sweeteners cannot be detected simultaneously.

[0004] This invention is achieved through the following technical solution:

[0005] A method for simultaneously determining the contents of advansin, phlorizin, and perilla stigma in edible flavorings based on UPLC includes the following steps:

[0006] (1) Preparation of standard solutions for three sweeteners: Accurately weigh Advantest, phlorizin and perilla lepidium standards respectively, mix them, dissolve and dilute with solvent to obtain stock solutions with concentrations of 200 μg / mL, 100 μg / mL and 100 μg / mL for Advantest, phlorizin and perilla lepidium respectively; accurately measure 0.5 mL, 1.0 mL, 3.0 mL, 5.0 mL and 10.0 mL of stock solution respectively, dilute and dilute to 10 mL with solvent to obtain standard solutions for three sweeteners.

[0007] (2) Preparation of the test solution: Accurately weigh 0.5000g of food flavoring into a 25mL volumetric flask, extract it with the dissolving solution by ultrasonic extraction for 10min, cool it to room temperature, dilute it with the dissolving solution and make up to volume, centrifuge, filter and obtain the test solution;

[0008] (3) Chromatographic determination:

[0009] The three sweetener standard solutions and the test solution were filtered, and the filtrates were analyzed by ultra-high performance liquid chromatography.

[0010] The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) are as follows:

[0011] The chromatographic column was an HSS T3, with dimensions of 2.1 × 100 mm and a diameter of 1.7 μm; the column temperature was 32℃; the injection volume was 10 μL; the flow rate was 0.3 mL / min; the detector was a PDAD; and the detection wavelengths were 213 nm, 285 nm, and 234 nm.

[0012] The mobile phase conditions were: Phase A was methanol:0.1% formic acid = 5:95, and Phase B was a 0.1% formic acid methanol solution;

[0013] The gradient elution conditions are shown in the table below:

[0014]

[0015] The blank experimental group used the solution as a blank control. The solution was filtered, and the filtrate was analyzed by ultra-high performance liquid chromatography.

[0016] All of the above solutions are 70% methanol aqueous solutions;

[0017] In step (2) above, after centrifugation at 8000 r / min for 10 minutes, the solution is filtered through a 0.22 μm microporous membrane to obtain the filtrate to be tested.

[0018] In step (2) above, the volumetric flask is a brown volumetric flask;

[0019] In step (3) above, a 0.22μm filter membrane is used for filtration.

[0020] The beneficial effects of this invention are:

[0021] Compared to existing technologies that require specific detection methods for each sweetener, ultra-high performance liquid chromatography (UPLC) can achieve simultaneous detection of three analytes in a single experiment. The detection time is significantly shorter than that of liquid chromatography, GC-MS, and LC-MS, with an analysis time of only 20 minutes per injection, greatly improving detection efficiency, simplifying experimental procedures, and saving on consumable costs. The core of this invention—a method for simultaneously determining multiple sweeteners using UPLC—has significant practical application value. Through technological innovation, it significantly improves detection efficiency and simplifies the operation process while ensuring detection accuracy. This method has a positive impact on food safety monitoring and the development of the food industry. Furthermore, the simultaneous detection of different sweeteners may provide scientific basis and technical support for the formulation of relevant regulations and standards. Attached Figure Description

[0022] Figure 1 This is a liquid chromatogram of the standard of the present invention;

[0023] Figure 2 This is the liquid chromatogram of sample A of the present invention;

[0024] Figure 3 This is the liquid chromatogram of sample B of the present invention;

[0025] Figure 4 This is the liquid chromatogram of sample C of the present invention;

[0026] Figure 5 This is the liquid chromatogram of sample D of the present invention;

[0027] Figure 6 This is the liquid chromatogram of sample E of the present invention;

[0028] Figure 7 This is the liquid chromatogram of sample F of the present invention;

[0029] Among them, Adv (Advansine); Phl (Phlorizin); Per (Perilla frutescens). Detailed Implementation

[0030] To more clearly illustrate the present invention, the invention will be further described below with reference to the accompanying drawings.

[0031] In the following description, detailed examples are provided to provide a more in-depth understanding of the invention. It is obvious that the described embodiments are merely a part of the embodiments of the invention, and not all of them. It should be understood that the specific embodiments described are for illustrative purposes only and are not intended to limit the invention.

[0032] It should be understood that when the terms “comprising” and / or “including” are used in this specification, they indicate the presence of the said feature, integral, step, operation, element, or component, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, or combinations thereof.

[0033] This invention discloses a method for the simultaneous determination of Advantest, Phloretin, and Perilla Leaf content in food flavorings using UPLC, falling under the technical category of chemical analysis and detection. The invention includes the precise preparation of standard solutions for the three sweeteners, the preparation of the sample solutions to be tested, and subsequent detection and quantitative analysis using UPLC. This method is particularly suitable for the simultaneous detection of these three sweeteners. In current technologies, due to the differences in the physicochemical properties and absorption spectra of these three sweeteners, different analytical methods and equipment are usually required. In contrast, the method of this invention can achieve the simultaneous separation and quantification of these three sweeteners with different absorption wavelengths in a single analytical process, significantly improving detection efficiency and offering advantages such as ease of operation, speed, and applicability in related fields.

[0034] The specific implementation method includes the following steps:

[0035] (1) Preparation of standard solutions for three sweeteners: Accurately weigh Advantest, phlorizin and perilla lepidium standards respectively, mix them, dissolve and dilute with solvent to obtain stock solutions with concentrations of 200 μg / mL, 100 μg / mL and 100 μg / mL for Advantest, phlorizin and perilla lepidium respectively; accurately measure 0.5 mL, 1.0 mL, 3.0 mL, 5.0 mL and 10.0 mL of stock solution respectively, dilute and dilute to 10 mL with solvent to obtain standard solutions for three sweeteners.

[0036] (2) Preparation of the test solution: Accurately weigh 0.5000g of food flavoring into a 25mL volumetric flask, extract it with the dissolving solution by ultrasonic extraction for 10min, cool it to room temperature, dilute it with the dissolving solution and make up to volume, centrifuge, filter and obtain the test solution;

[0037] (3) Chromatographic determination:

[0038] The three sweetener standard solutions and the test solution were filtered, and the filtrates were analyzed by ultra-high performance liquid chromatography.

[0039] The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) are as follows:

[0040] The chromatographic column was an HSS T3, with dimensions of 2.1 × 100 mm and a diameter of 1.7 μm; the column temperature was 32℃; the injection volume was 10 μL; the flow rate was 0.3 mL / min; the detector was a PDAD; and the detection wavelengths were 213 nm, 285 nm, and 234 nm.

[0041] The mobile phase conditions were: Phase A was methanol:0.1% formic acid = 5:95, and Phase B was a 0.1% formic acid methanol solution;

[0042] The gradient elution conditions are shown in the table below:

[0043]

[0044] The blank experimental group used the solution as a blank control. The solution was filtered, and the filtrate was analyzed by ultra-high performance liquid chromatography.

[0045] Based on this, the method described in this invention specifically covers the preparation of standard solutions, ensuring accurate detection of various sweeteners. The sample solution is then fed into a UPLC system for accurate and reliable detection and quantitative analysis. A PDAD detector is selected, and detection is performed at three wavelengths: 213 nm, 285 nm, and 234 nm, achieving the maximum detection limit. Simultaneously, this method employs an ultra-high performance liquid chromatograph (UHPLC), which, compared to commonly used HPLC systems, offers higher operating pressure and is equipped with a smaller particle size HSS T3 column, facilitating the separation of analytes and reducing interference from impurities. It boasts advantages such as ultra-high resolution and ultra-high separation efficiency. This novel method offers numerous advantages, including, but not limited to, simplified experimental procedures, reduced detection time, enhanced operational convenience, and broader application prospects. Compared to existing multiplex detection methods, this one-stop solution significantly improves the efficiency and practicality of the analytical process, playing a vital role in food safety testing, quality control, and related scientific research.

[0046] Example

[0047] Simultaneous detection of advandan, phlorizin, and perilla lepidium in a batch of food flavorings was performed using the following specific methods:

[0048] (1) Preparation of standard solutions for three sweetener series:

[0049] Accurately weigh appropriate amounts of Advil, phlorizin, and perilla lepidium standards, mix them, dissolve them in a dissolving solution (70% methanol aqueous solution, the same below) and dilute to volume, so that the concentrations of Advil, phlorizin, and perilla lepidium in the solution are 200 μg / mL, 100 μg / mL, and 100 μg / mL, respectively. Use this solution as a stock solution. Accurately measure 0.5 mL, 1.0 mL, 3.0 mL, 5.0 mL, and 10.0 mL of the stock solution, dilute them with the dissolving solution and dilute to volume, respectively, to obtain a series of standard solutions for the three sweeteners.

[0050] (2) Preparation of the test solution

[0051] The six food flavoring samples to be tested were labeled A, B, C, D, E, and F, respectively. The samples were prepared into test solutions according to the following steps.

[0052] Accurately weigh 0.5000 g of edible flavor sample into a 25 mL brown volumetric flask, dissolve and ultrasonically extract with the dissolving solution for 10 min, cool to room temperature, then dilute and bring to volume with the dissolving solution; centrifuge at 8000 r / min for 10 min, and filter through a 0.22 μm microporous membrane to obtain the test solution;

[0053] (3) Chromatographic analysis:

[0054] The standard solutions of the three sweetener series and the test solution were filtered through a 0.22 μm microporous membrane, and the filtrate was analyzed by ultra-high performance liquid chromatography.

[0055] The chromatographic conditions were as follows: flow rate: 0.3 mL / min; column temperature: 32℃; detector: PDAD; detection wavelength: 213 nm, 285 nm, 234 nm; injection volume: 10 μL; column: HSS T3 (2.1 × 100 mm, 1.7 μm); mobile phase A: methanol: 0.1% formic acid = 5:95; mobile phase B: 0.1% formic acid and methanol.

[0056] The gradient elution conditions are shown in the table below:

[0057]

[0058] The blank experimental group used the solution as a blank control. The solution was filtered through a 0.22 μm microporous membrane, and the filtrate was analyzed by ultra-high performance liquid chromatography.

[0059] It is worth noting that when selecting an ultra-high performance liquid chromatograph (UHPLC), the detector selection must be made first. Experiments have shown that perilla leaves do not produce peaks in ELSD. Since the chemical properties and absorption spectra of advandy, phlorizin, and perilla leaves, which need to be detected simultaneously, are different, it is difficult to achieve the maximum detection limit of the three analytes using a single wavelength. Therefore, the detector used in this method is a PDAD, which can detect the three analytes advandy, phlorizin, and perilla leaves using three wavelengths: 213 nm, 285 nm, and 234 nm, and can perform efficient and sensitive detection.

[0060] The six batches of edible flavoring samples were tested according to the above method, and the results are shown in Table 1. Table 1 shows the contents of advantran, phlorizin, and perilla stigma in the six batches of edible flavoring samples:

[0061] Table 1

[0062]

[0063] Note: "-" indicates not detected.

[0064] The series of standard solutions in the above detection step (1) are determined according to the procedure to obtain... Figure 1 The reference chromatogram showed that the peak elution order of the three sweeteners was Advan, Phlorizin, and Perilla frutescens. Six batches of edible flavoring samples A, B, C, D, E, and F were analyzed, and the results were as follows: Figure 2-7 The liquid chromatogram; based on the peak time of the reference standard's liquid chromatogram, combined with... Figure 2-7 The liquid chromatograms and Table 1 show that the elution times of each substance do not overlap, and the main peaks achieve good baseline separation. This allows for relatively clear separation and qualitative analysis of the three substances, and quantitative analysis based on their concentration and peak area.

[0065] The beneficial effects of this invention are that ultra-high performance liquid chromatography (UHPLC) can simultaneously detect three analytes—Advansine, phlorizin, and perilla lepidium—in a single experiment. The analysis is fast, with high separation and detection efficiency, and the detection cost is low and the operation is simple. Compared with commonly used detection methods, this method greatly simplifies the detection process and reduces detection costs, playing a positive role in food safety monitoring and the development of the food industry. The high efficiency and practicality of this method have broad application prospects and are easy to promote.

[0066] The above-disclosed embodiments are merely a few specific examples of the present invention, but the present invention is not limited thereto. Any variations that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A method for simultaneously determining the contents of advantran, phlorizin, and perilla stigma in edible flavorings based on UPLC, characterized in that, Includes the following steps: (1) Preparation of standard solutions of three sweeteners: Accurately weigh Advantest, phlorizin and perilla lepidium standards respectively, mix them, dissolve and dilute with solvent to obtain stock solutions with concentrations of 200µg / mL, 100µg / mL and 100µg / mL of Advantest, phlorizin and perilla lepidium respectively; accurately measure 0.5mL, 1.0mL, 3.0mL, 5.0mL and 10.0mL of stock solution respectively, dilute and dilute with solvent to 10mL respectively to obtain standard solutions of three sweeteners. (2) Preparation of the test solution: Accurately weigh 0.5000g of food flavoring into a 25mL volumetric flask, extract with the dissolving solution by ultrasonic extraction for 10min, cool to room temperature, dilute with the dissolving solution and make up to volume, centrifuge, filter, and obtain the test solution; (3) Chromatographic determination: The three sweetener standard solutions and the test solution were filtered, and the filtrates were analyzed by ultra-high performance liquid chromatography. The chromatographic conditions for the ultra-high performance liquid chromatograph are as follows: The chromatographic column was an HSS T3, with dimensions of 2.1 × 100 mm and a diameter of 1.7 µm; the column temperature was 32 °C; the injection volume was 10 µL; the flow rate was 0.3 mL / min; the detector was a PDAD; and the detection wavelengths were 213 nm, 285 nm, and 234 nm. The mobile phase conditions are: Phase A is methanol: 0.1% formic acid = 5:95, and Phase B is a 0.1% formic acid methanol solution; The gradient elution conditions are shown in the table below: ， The blank experimental group used the solution as a blank control. The solution was filtered, and the filtrate was analyzed by ultra-high performance liquid chromatography. In steps (1), (2), and (3), the solution is a 70% methanol aqueous solution. In step (2), after centrifugation at 8000 r / min for 10 min, the solution is filtered through a 0.22 µm microporous membrane to obtain the test solution.

2. The method for simultaneous determination of advansin, phlorizin, and perilla leaf content in edible flavorings based on UPLC according to claim 1, characterized in that, In step (2), the volumetric flask is a brown volumetric flask.

3. The method for simultaneous determination of advansin, phlorizin, and perilla leaf content in edible flavorings based on UPLC according to claim 1, characterized in that, In step (3), 0.22µm filter membranes were used for filtration.

Images