Rapid detection method for exogenous sweetener in glycyrrhizin

Abstract

The invention discloses a rapid detection method of an exogenous sweetener in glycyrrhizin, which comprises the following steps: (1) weighing a certain amount of glycyrrhizin, adding an internal standard substance and pure water, carrying out ultrasonic extraction, and filtering to obtain a test sample; and (2) determining the exogenous sweetening agent in the standard solution and the test sample solution by adopting ultra-high performance liquid chromatography-tandem mass spectrometry. According to the method disclosed by the invention, chromatographic separation conditions, solvent components and detection conditions are optimized, and the eight exogenous sweeteners in glycyrrhizin are rapidly and simultaneously analyzed and detected by improving the component separation degree and the chromatographic peak shape and improving the mass spectrum response.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analytical chemistry, and particularly relates to a method for rapidly detecting eight exogenous sweeteners, namely acesulfame potassium, sodium saccharin, cyclamate, sucralose, alitame, aspartame, neotame and stevioside, in glycyrrhizin. BACKGROUND

[0002] Glycyrrhiza uralensis is a plant with medicinal and edible properties; glycyrrhizin is an important component in Glycyrrhiza uralensis, and the content of glycyrrhizin in Glycyrrhiza uralensis is 3% to 14%. Glycyrrhizin has a special flavor-enhancing effect and is 50 to 100 times sweeter than sucrose, and has almost no calories, and is very suitable for food processing such as candies, biscuits, seasonings, beverages and weight loss products, and is a natural sweetener with broad application prospects. However, due to differences in extraction and processing technology, some production enterprises often add other synthetic sweeteners such as sodium saccharin, cyclamate, aspartame and neotame to mask the defects of the taste characteristics of glycyrrhizin products, so that glycyrrhizin with the same content will also have different taste characteristics and flavor profiles. The detection of glycyrrhizic acid and glycyrrhizinate by using methods such as GB / T22248 and GB1886.240 can intuitively understand the content of effective components in glycyrrhizin, but it is difficult to evaluate the influence of sweeteners on the taste characteristics and flavor profiles of glycyrrhizin.

[0003] Meanwhile, the use range and amount of sweeteners are required in GB 2760-2024 "National Food Safety Standard - Standard for the Use of Food Additives", and the opaque use, excessive use and shortcomings of sweeteners pose a safety risk to their application. Due to the differences in the physicochemical properties of different synthetic sweeteners, the detection methods of synthetic sweeteners are scattered in various standards such as GB5009.28, GB5009.263-2016 and GB 5009.247-2025, and the methods mainly use liquid chromatography detection. Generally, the amount of exogenous sweeteners added in glycyrrhizin is low, and the interference of natural extraction substrates is large. The sensitivity of the evaporation light scattering or ultraviolet detector method is low, and it is difficult to operate, the process is long, and the practical applicability is not strong. Therefore, the qualitative and quantitative detection of sweeteners by using mass spectrometry ion pair has small interference, low detection limit and higher accuracy, and avoids the sample enrichment pretreatment operation.

[0004] High-selectivity ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) is used for the simultaneous detection of sweeteners, which are commonly found in samples such as baijiu (Zhang Miao et al., Food Industry, 2021, 42(12)), seasonings (Tang Lichang et al., Journal of Food Safety and Quality Inspection, 2021, 12(6)), pastries (Zhang Shuquan et al., Journal of Food Safety and Quality Inspection, 2021, 12(1)), and food packaging paper (Liao Huiyun et al., Food Science, 2021, 42(12)). However, the sample processing methods and chromatographic separation conditions are not suitable for the determination of sweeteners in glycyrrhizin. Therefore, this invention is proposed. Summary of the Invention

[0005] This invention provides a rapid and simultaneous method for the detection of eight sweeteners in glycyrrhizin, namely acesulfame potassium, sodium saccharin, cyclamate, sucralose, alitane, aspartame, neotame, and steviol glycosides, using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). This method innovatively optimizes chromatographic separation conditions, solvent components, and detection conditions. By improving component resolution and peak shape, it enhances mass spectrometry response, enabling rapid and simultaneous analysis of these eight sweeteners in glycyrrhizin. This provides technical support for the flavor quality control and safety evaluation of glycyrrhizin.

[0006] The objective of this invention is achieved through the following technical solutions.

[0007] A rapid and simultaneous detection method for exogenous sweeteners in glycyrrhizin includes the following steps: (1) weigh a certain amount of glycyrrhizin, add internal standard and pure water, extract by ultrasonication, and filter to obtain a test sample; (2) use ultra-high performance liquid chromatography-tandem mass spectrometry to determine the exogenous sweeteners in the standard solution and the test sample solution.

[0008] Preferably, the exogenous sweetener is one or more of acesulfame potassium, sodium saccharin, cyclamate, sucralose, alitame, aspartame, neotame, or steviol glycosides.

[0009] Preferably, in step (2), the retention time and qualitative ions of the exogenous sweetener peak in the test sample are compared with the standard substance to determine the type of exogenous sweetener in the test sample, i.e., qualitative analysis;

[0010] The ratio of the quantitative ion mass spectrometry response values ​​of the exogenous sweetener standard substance and the internal standard substance is used as the ordinate and the concentration of the standard solution is used as the abscissa to establish a standard working curve for the exogenous sweetener, and the curve fitting equation and correlation coefficient are obtained. The ratio of the quantitative ion mass spectrometry response values ​​of the exogenous sweetener and the internal standard substance in the test sample is substituted into the standard working curve to calculate the concentration of the exogenous sweetener solution, i.e., quantitative analysis.

[0011] Preferably, the concentration of the standard solution is 0.01 μg / mL-20 μg / mL, the concentration gradient is set to 5-10, and a standard working curve is established between the mass spectrometry response value of the exogenous sweetener and the corresponding concentration, and the correlation coefficient of the fitting equation is greater than 0.99.

[0012] Preferably, in step (1), the internal standard is warfarin sodium with a concentration of 5-500 μg / mL; the mass ratio of glycyrrhizin to the internal standard and pure water is 1:(2-20):(200-600), and the ultrasonic extraction is performed for 5-30 minutes; the pore size of the filter membrane used for filtration is 0.10-0.30 μm, and the filter membrane can be a nylon filter membrane. The internal standard can also be an internal standard substance with equivalent efficacy.

[0013] Preferably, in step (2), the ultra-high performance liquid chromatography conditions are as follows: column: HSS T3 column, specifications: 1.8μm, 100mm×2.1mm; column temperature: 25-30℃; injection volume: 2μL; elution mode: gradient elution; gradient elution conditions: 0min, 100% A+0% B; 3min, 60% A+40% B; 6-7.5min, 0% A+100% B; 8-9min, 100% A+0% B; flow rate: 0.25mL / min; wherein, mobile phase A is 0.2% (V / V) ammonia solution, and B is methanol.

[0014] Preferably, in step (2), the mass spectrometry conditions are: electrospray negative ion mode, multiple reaction monitoring; spray voltage: 3500V; atomizing gas pressure: 0.45MPa; auxiliary gas pressure: 0.35MPa; ion source temperature: 350℃.

[0015] Preferably, the retention time of the internal standard warfarin sodium is 4.20 minutes; the retention times of the sweeteners acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitame, sucralose, neotame, and steviol glycosides are 2.07 minutes, 2.43 minutes, 2.67 minutes, 3.68 minutes, 3.97 minutes, 4.67 minutes, 5.29 minutes, and 6.53 minutes, respectively.

[0016] Preferably, the qualitative ion pair (m / z) of the internal standard warfarin sodium is 307.1 / 250, and the qualitative ion pairs (m / z) of the sweeteners acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitane, sucralose, neotame, and steviol glycoside are 162.0 / 78.0, 181.9 / 42.0, 178.0 / 96.1, 293.1 / 261.1, 330.2 / 295.0, 397.0 / 361.1, 377.2 / 345.0, and 803.4 / 479.5, respectively.

[0017] Preferably, the quantitative ion pair (m / z) of the internal standard warfarin sodium is 307.1 / 160.9, and the quantitative ion pairs (m / z) of the sweeteners acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitane, sucralose, neotame, and steviol glycoside are 162.0 / 82.0, 181.9 / 106.0, 178.0 / 80.0, 293.1 / 200.0, 330.2 / 312.0, 395.0 / 359.1, 377.2 / 200.0, and 803.4 / 641.2, respectively.

[0018] The beneficial effects of this invention are:

[0019] 1. The rapid and simultaneous detection method for exogenous sweeteners in glycyrrhizin of the present invention uses ammonia as a buffer for the aqueous mobile phase in liquid chromatography. Compared with one or more buffers such as formic acid, acetic acid, ammonium formate, and ammonium acetate added in commonly used methods, it can obtain better component separation and chromatographic peak shape, and improve the mass spectrometry response of each component by more than 25%. The liquid chromatography used an HSS T3 column as the separation column and warfarin sodium as the internal standard, which effectively reduced the interference of complex natural components in glycyrrhizin on the analysis of the target analytes, thus improving the accuracy and sensitivity of the method of this invention. Compared with the national standard method, the limits of quantification for sodium saccharin (10.0 μg / g), neotame (0.2 μg / g), aspartame (3.0 μg / g), alitame (3.0 μg / g), and sucralose (3.0 μg / g) were improved to 0.6 μg / g, 0.15 μg / g, 1.0 μg / g, 0.25 μg / g, and 0.6 μg / g, respectively, enabling the quantitative analysis of exogenous sweeteners in glycyrrhizin without sample enrichment.

[0020] 2. The method of this invention optimizes chromatographic separation conditions, solvent components, and detection conditions. By improving component separation and chromatographic peak shape, it enhances mass spectrometry response, enabling a rapid and simultaneous analysis of eight exogenous sweeteners in glycyrrhizin. The analysis of the target analytes can be completed within seven minutes, effectively shortening the detection process and steps. The method is simple, fast, sensitive, and efficient, accurately reflecting the content of common exogenous sweeteners in glycyrrhizin and providing scientific data support for the evaluation of the taste characteristics and flavor profile of glycyrrhizin. Attached Figure Description

[0021] Figure 1 The chemical structural formulas of acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitame, sucralose, neotame, and steviol glycosides are given.

[0022] Figure 2 The liquid chromatograms are those of standard solutions of acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitame, sucralose, neotame, steviol glycosides, and warfarin sodium (internal standard).

[0023] Figure 3The liquid chromatogram of glycyrrhizin A sample in Case 1 is shown. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the scope of the invention.

[0025] Example 1: Rapid and simultaneous detection of exogenous sweeteners in commercially available glycyrrhizin A, the steps are as follows:

[0026] (1) Sample preparation: Accurately weigh 50 mg glycyrrhizin A, add 0.1 mL of warfarin sodium internal standard solution with a concentration of 100 μg / mL and 20 mL of pure water, extract by ultrasonication for 30 min, and filter with a 0.22 μm microporous membrane to obtain the sample to be tested.

[0027] (2) Determination: Ultra-high performance liquid chromatography-tandem mass spectrometry was used for analysis. The instrument conditions are as follows:

[0028] The liquid chromatography conditions were as follows: column: HSS T3 column, specifications: 1.8μm, 100mm×2.1mm; column temperature: 25-30℃; injection volume: 2μL; elution mode: gradient elution; gradient elution conditions: 0 min, 100% A + 0% B; 3 min, 60% A + 40% B; 6-7.5 min, 0% A + 100% B; 8-9 min, 100% A + 0% B; flow rate: 0.25 mL / min; wherein, mobile phase A was 0.2% (V / V) ammonia solution, and B was methanol.

[0029] The mass spectrometry conditions were: electrospray negative ion mode, multiple reaction monitoring; spray voltage: 3500V; nebulizer gas pressure: 0.45MPa; auxiliary gas pressure: 0.35MPa; ion source temperature: 350℃. The qualitative ion pair (m / z) of the internal standard, warfarin sodium, was 307.1 / 250. The qualitative ion pairs (m / z) of acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitane, sucralose, neotame, and steviol glycosides were 162.0 / 78.0, 181.9 / 42.0, 178.0 / 96.1, 293.1 / 261.1, 330.2 / 295.0, 397.0 / 361.1, 377.2 / 345.0, and 803.4 / 479.5, respectively. The quantitative ion pair (m / z) of warfarin sodium is 307.1 / 160.9. The quantitative ion pairs (m / z) of sweeteners acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitane, sucralose, neotame, and steviol glycoside are 162.0 / 82.0, 181.9 / 106.0, 178.0 / 80.0, 293.1 / 200.0, 330.2 / 312.0, 395.0 / 359.1, 377.2 / 200.0, and 803.4 / 641.2, respectively.

[0030] Preparation of standard solutions: Accurately prepare eight sweeteners with a concentration of 100 μg / mL, such as... Figure 1 The mixed standard stock solutions of acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitane, sucralose, neotame, and steviol glycosides were prepared sequentially into six gradient standard solutions of 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL. Standard working curves for the above eight sweeteners were established with the ratio of the quantitative ion mass spectrometry response values ​​of the standard substance to the internal standard substance as the ordinate and the concentration of the standard solution as the abscissa. The curve fitting equations and correlation coefficients are shown in Table 1.

[0031] The glycyrrhizin A sample solution was analyzed, and consistency was determined by comparing the retention time and qualitative ion pairs with those of the standard substance. This confirmed the presence of three sweeteners: sodium saccharin, cyclamate, and neotame. Acesulfame potassium, aspartame, alitame, sucralose, and steviol glycosides were not detected. Figure 3 As shown.

[0032] The ratios of the quantitative ion mass spectrometry response values ​​of sodium saccharin, cyclamate, neotame, and the internal standard were substituted into the standard working curve fitting equation, and the contents of sodium saccharin, cyclamate, and neotame were calculated to be 2.75 μg / g, 3.68 μg / g, and 3.50 μg / g, respectively.

[0033] Table 1

[0034]

[0035] Example 2: Rapid and simultaneous detection of exogenous sweeteners in commercially available glycyrrhizin B and glycyrrhizin C, with the following steps:

[0036] (1) Sample preparation: Accurately weigh 50 mg of glycyrrhizin B and glycyrrhizin C respectively, add 0.1 mL of internal standard solution with a concentration of 100 μg / mL and 20 mL of pure water to each, extract by ultrasonication for 30 min, and filter with a 0.22 μm microporous membrane to obtain the sample to be tested.

[0037] (2) Determination: Ultra-high performance liquid chromatography-tandem mass spectrometry was used for analysis.

[0038] The instrument conditions are the same as in Example 1.

[0039] The standard solution was prepared in the same manner as in Example 1.

[0040] Glycyrrhizin B and glycyrrhizin C sample solutions were measured separately. By comparing the retention time and qualitative ion pairs with those of the standard substances, it was determined that the samples contained only neotame; other substances such as acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitame, sucralose, and steviol glycosides were not detected.

[0041] The ratio of the quantitative ion mass spectrometry response values ​​of neotame to the internal standard was substituted into the standard working curve fitting equation, and the neotame content in glycyrrhizin B and glycyrrhizin C samples was calculated to be 12.37 μg / g and 12.72 μg / g, respectively.

[0042] Example 3: The following steps were taken to detect the sweeteners acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitame, sucralose, neotame, and steviol glycosides in glycyrrhizin D with known amounts of standard substances added:

[0043] (1) Sample preparation: Accurately weigh 50 mg glycyrrhizin D, add 0.1 mL of internal standard solution with a concentration of 100 μg / mL and 20 mL of pure water, extract by ultrasonication for 30 min, and filter with a 0.22 μm microporous membrane to obtain the sample to be tested.

[0044] (1) Determination: Ultra-high performance liquid chromatography-tandem mass spectrometry was used for analysis.

[0045] The instrument conditions are the same as in Example 1.

[0046] The standard solution was prepared in the same manner as in Example 1.

[0047] The glycyrrhizin D sample solution was tested, and the presence of acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitame, sucralose, neotame, and steviol glycosides was determined by comparing the retention time and qualitative ion pairs with those of the standard substance.

[0048] The ratios of the quantitative ion mass spectrometry response values ​​of the sweetener and the internal standard were substituted into the standard working curve fitting equation to calculate the contents of acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitane, sucralose, neotame, and steviol glycosides in the sample as 1.12 μg / g, 1.11 μg / g, 1.13 μg / g, 1.87 μg / g, 0.48 μg / g, 1.09 μg / g, 1.10 μg / g, and 0.47 μg / g, respectively. The recoveries between the detected contents and the added amounts were 93.2%, 92.4%, 94.3%, 93.6%, 96.4%, 90.8%, 91.7%, and 93.4%, respectively.

[0049] The foregoing description and presentation of the main features and advantages of the present invention are not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A rapid detection method for exogenous sweeteners in glycyrrhizin, characterized in that, Includes the following steps: (1) Weigh a certain amount of glycyrrhizin, add internal standard and pure water, extract by ultrasonication, and filter to obtain the test sample; (2) Use ultra-high performance liquid chromatography-tandem mass spectrometry to determine the exogenous sweetener in the standard solution and the test sample solution.

2. The method according to claim 1, characterized in that, The exogenous sweetener is one or more of acesulfame potassium, sodium saccharin, cyclamate, sucralose, alitame, aspartame, neotame, or steviol glycosides.

3. The method according to claim 1, characterized in that, In step (2), the retention time and qualitative ions of the exogenous sweetener peak in the test sample are compared with the standard substance to determine the type of exogenous sweetener in the test sample; The ratio of the quantitative ion mass spectrometry response values ​​of the exogenous sweetener standard to the internal standard was used as the ordinate and the concentration of the standard solution as the abscissa to establish a standard working curve for the exogenous sweetener, and the curve fitting equation and correlation coefficient were obtained. The ratio of the quantitative ion mass spectrometry response values ​​of the exogenous sweetener to the internal standard in the test sample was substituted into the standard working curve to calculate the concentration of the exogenous sweetener solution.

4. The method according to claim 3, characterized in that, The standard solution concentrations ranged from 0.01 μg / mL to 20 μg / mL, with concentration gradients set to 5-10. Standard working curves were established to compare the mass spectrometry response values ​​of exogenous sweeteners with their corresponding concentrations.

5. The method according to claim 1, characterized in that, In step (1), the internal standard is warfarin sodium with a concentration of 5-500 μg / mL; the mass ratio of glycyrrhizin to internal standard and pure water is 1:(2-20):(200-600); ultrasonic extraction is performed for 5-30 minutes; and the pore size of the filter membrane used for filtration is 0.10-0.30 μm.

6. The method according to claim 1, characterized in that, In step (2), the ultra-high performance liquid chromatography (UHPLC) conditions are as follows: column: HSS T3 column, specifications: 1.8μm, 100mm×2.1mm; column temperature: 25-30℃; injection volume: 2μL; elution mode: gradient elution; gradient elution conditions: 0min, 100%A+0%B; 3min, 60%A+40%B; 6-7.5min, 0%A+100%B; 8-9min, 100%A+0%B; flow rate: 0.25mL / min; wherein, mobile phase A is 0.2% (V / V) ammonia solution, and B is methanol.

7. The method according to claim 1, characterized in that, In step (2), the mass spectrometry conditions are: electrospray negative ion mode, multiple reaction monitoring; spray voltage: 3500V; nebulizer gas pressure: 0.45MPa; auxiliary gas pressure: 0.35MPa; ion source temperature: 350℃.

8. The method according to claim 3, characterized in that, The retention time of the internal standard warfarin sodium was 4.20 minutes; the retention times of the sweeteners acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitane, sucralose, neotame, and steviol glycosides were 2.07 minutes, 2.43 minutes, 2.67 minutes, 3.68 minutes, 3.97 minutes, 4.67 minutes, 5.29 minutes, and 6.53 minutes, respectively.

9. The method according to claim 3, characterized in that, The qualitative ion pair (m / z) of the internal standard warfarin sodium was 307.1 / 250. The qualitative ion pairs (m / z) of the sweeteners acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitane, sucralose, neotame, and steviol glycosides were 162.0 / 78.0, 181.9 / 42.0, 178.0 / 96.1, 293.1 / 261.1, 330.2 / 295.0, 397.0 / 361.1, 377.2 / 345.0, and 803.4 / 479.5, respectively.

10. The method according to claim 3, characterized in that, The quantitative ion pair (m / z) of the internal standard warfarin sodium was 307.1 / 160.

9. The quantitative ion pairs (m / z) of the sweeteners acesulfame potassium, sodium saccharin, cyclamate, aspartame, alitane, sucralose, neotame, and steviol glycosides were 162.0 / 82.0, 181.9 / 106.0, 178.0 / 80.0, 293.1 / 200.0, 330.2 / 312.0, 395.0 / 359.1, 377.2 / 200.0, and 803.4 / 641.2, respectively.

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