Liquid chromatography method for simultaneously detecting three sweeteners in tipping paper

Abstract

The invention discloses a liquid chromatography method for simultaneously detecting three sweeteners in tipping paper, which comprises the following steps: (1) sample extraction: weighing the cut tipping paper, adding into an acetic acid-ammonium acetate-acetonitrile aqueous solution, and carrying out ultrasonic extraction for a period of time to obtain an extracting solution; and (2) sample determination: detecting by adopting a high performance liquid chromatography to respectively obtain the contents of neotame, sucralose and glycyrrhizin in the tipping paper. According to the method disclosed by the invention, the neotame, the sucralose and the glycyrrhizin in the tipping paper can be fully extracted at one time, and the detection time is short.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and particularly relates to a liquid chromatography method for simultaneously detecting three sweeteners in tipping paper. Background Art

[0002] Sweeteners such as neotame, glycyrrhizin, and sucralose are commonly used additives in the food industry. Among them, ammonium glycyrrhizate, tripotassium glycyrrhizinate, and glycyrrhizic acid are all glycyrrhizin. Food additives are harmless to humans when used within the prescribed dosage range. However, excessive use can cause various forms of toxicity. Therefore, their usage must be strictly managed to maximize their beneficial effects and prevent their adverse effects. With the continuous optimization and adjustment of cigarette structure, a variety of products that add sweeteners to the tipping paper have appeared on the market to directly improve the taste and flavor of cigarettes. To achieve the regulation and sample measurement of these products, there is an urgent need to develop a simultaneous, accurate, and highly sensitive sample measurement method.

[0003] Currently, the literature reports on the determination of sweeteners in foods such as dairy products [Song Ge et al., Chromatography, 2011, 29(12)], liquor [Yao Mingjing et al., Brewing Technology, 2017(10)], and beverages [Ji Chao et al., Chromatography, 2009, 27(1)]. Methods for determining sweeteners include ion chromatography, liquid chromatography, liquid chromatography-tandem mass spectrometry, gas chromatography, and gas chromatography-mass spectrometry. These methods for determining sweeteners are not suitable for the determination of sweeteners in paper matrices. Using patents CN108776187B and CN118914396A can fully extract neotame from the tipping paper, but cannot fully extract glycyrrhizin, and the chromatographic peak of glycyrrhizin does not meet the detection requirements. At the same time, the efficient extraction of neotame, sucralose and glycyrrhizin from the wrapping paper and the simultaneous detection of their content in the sample will help evaluate the effect of adding neotame, sucralose and glycyrrhizin to the wrapping paper and promote the high-quality development of cigarette products.

[0004] To this end, the present invention establishes a liquid chromatography method that can simultaneously and fully extract neotame, sucralose, and glycyrrhizin and accurately detect them. Summary of the Invention

[0005] The present invention provides a liquid chromatography method for simultaneously detecting three sweeteners in tipping paper. The method of the present invention provides a sample extraction and detection method, achieving simultaneous and accurate detection of neotame, sucralose, and glycyrrhizin in tipping paper.

[0006] The technical solutions of the present invention are as follows:

[0007] A liquid chromatography method for simultaneously detecting three sweeteners in tipping paper comprises the following steps:

[0008] (1) Sample extraction: Weigh the shredded tipping paper and add it to an aqueous solution of acetic acid, ammonium acetate, and acetonitrile. Ultrasonic extraction is performed for a period of time to obtain an extract.

[0009] (2) Sample determination: High performance liquid chromatography was used to determine the contents of neotame, sucralose, and glycyrrhizin in the tipping paper.

[0010] Preferably, in step (1), the mass ratio of the tipping paper to the aqueous solution of acetic acid-ammonium acetate-acetonitrile is 1:(200-600); and the ultrasonic extraction time is 15 to 30 minutes.

[0011] Preferably, in step (1), the weight ratio of acetic acid, ammonium acetate, acetonitrile and water is 0.001:0.001:50:50.

[0012] Preferably, in step (2), the extract is diluted 5-100 times before detection by high performance liquid chromatography.

[0013] Preferably, the liquid chromatography conditions are as follows: chromatographic column: Ecosil Carbohydrate column, specifications: 5 μm, 4.6 mm×250 mm; flow rate: 0.8 mL / min; column temperature: 25-35° C.; injection volume: 10 μL; elution mode: gradient elution; detector: evaporative light scattering detector (DAD).

[0014] Preferably, the gradient elution conditions are: time 0.00 min, 85 wt% mobile phase A + 15 wt% mobile phase B; 10.00 min, 65 wt% mobile phase A + 35 wt% mobile phase B.

[0015] Preferably, mobile phase A is acetonitrile, and mobile phase B is a mixed solution of acetic acid, ammonium acetate and water in a weight ratio of 0.001:0.001:100.

[0016] Preferably, the detector used is an evaporative light scattering detector, and the detection parameters are: atomization temperature: 40°C; evaporation temperature: 80°C; atomization gas: nitrogen; gas flow rate: 2.0 L / min.

[0017] Preferably, high performance liquid chromatography is used for detection, and the contents of neotame, sucralose and glycyrrhizin in the tipping paper are obtained according to the standard curve equations of neotame, sucralose and glycyrrhizin.

[0018] Preferably, the standard curve equation of neotame is y=6598.2x-2.0034, the standard curve equation of sucralose is y=32624x-1983.3, and the standard curve equation of glycyrrhizin is y=5322x-1.0601.

[0019] Beneficial effects of the present invention:

[0020] 1. The method of the present invention uses an aqueous solution of acetic acid-ammonium acetate-acetonitrile, which is an aqueous solution of 0.1wt% acetic acid-0.1wt% ammonium acetate-50wt% acetonitrile. When the mass ratio of the tipping paper to the aqueous solution of acetic acid-ammonium acetate-acetonitrile is 1:(200-600), ultrasonic extraction of neotame, sucralose and glycyrrhizin in the tipping paper can be fully extracted in one time.

[0021] 2. The method of the present invention utilizes an evaporative light scattering detector, which is immune to interference from impurities and can accurately and individually determine the content of neotame, sucralose, and glycyrrhizin in tipping paper. The results are accurate and the test is completed in a short time of 10 minutes.

[0022] 3. The extraction and detection methods of the present invention are simple and achieve the full extraction and simultaneous determination of neotame, sucralose, and glycyrrhizin in the tipping paper. This method is characterized by being simultaneous, accurate, and reproducible. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The liquid chromatogram of the standard solution.

[0024] Figure 2 It is the liquid chromatogram of the sample solution of Example 1.

[0025] Figure 3 It is the liquid chromatogram of the sample solution of Comparative Example 1.

[0026] Figure 4 It is the liquid chromatogram of the sample solution of Comparative Example 2. DETAILED DESCRIPTION

[0027] In order to make the present invention easier to understand, the present invention will be described in detail below with reference to specific embodiments of the present invention, but this is only for illustration and not for limitation.

[0028] Example 1: Simultaneous determination of the contents of neotame, sucralose, and glycyrrhizin in packaging paper A, the steps are as follows:

[0029] (1) Sample extraction: Accurately weigh 1.0000 g of shredded tipping paper, add 20 mL of a 0.1 wt% acetic acid-0.1 wt% ammonium acetate-50 wt% acetonitrile aqueous solution, and extract with ultrasound for 15 min;

[0030] (2) Sample determination: HPLC was used to determine the contents of neotame, sucralose, and glycyrrhizin in the tipping paper. The extract was diluted 5-fold before HPLC. The instrument conditions were as follows:

[0031] Liquid chromatography conditions were as follows: chromatographic column: Ecosil Carbohydrate column, dimensions: 5 μm, 4.6 mm × 250 mm; flow rate: 0.8 mL / min; column temperature: 25-35°C; injection volume: 10 μL; elution mode: gradient elution; mobile phase A: acetonitrile; mobile phase B: a mixture of acetic acid, ammonium acetate, and water in a weight ratio of 0.001:0.001:100; elution mode: gradient elution; detector: evaporative light scattering detector (DAD); detection parameters: nebulization temperature: 40°C; evaporation temperature: 80°C; nebulizing gas: nitrogen; gas flow rate: 2.0 L / min. Gradient elution conditions were: time 0.00 min, 85 wt% mobile phase A + 15 wt% mobile phase B; 10.00 min, 65 wt% mobile phase A + 35 wt% mobile phase B.

[0032] Preparation of standard solutions: Accurately weigh 5.0 mg of neotame, 10.0 mg of sucralose, and 10.0 mg of glycyrrhizin standard into a 10 mL volumetric flask. Dissolve the stock solutions in an aqueous solution of acetic acid, ammonium acetate, and acetonitrile (0.1 wt% acetic acid, 0.1 wt% ammonium acetate, 50 wt% acetonitrile). Prepare six standard gradients ranging from 1.0 μg / mL to 500 μg / mL based on the concentrations of the stock solutions. Based on the concentrations and peak areas of the neotame, sucralose, and glycyrrhizin standard solutions, the calibration curve equations for this experiment were: y = 6598.2x - 2.0034, with a correlation coefficient R² = 0.9979; y = 32624x - 1983.3, with a correlation coefficient R² = 0.9952; and y = 5322x - 1.0601, with a correlation coefficient R² = 0.9957.

[0033] Figure 1 The liquid chromatogram of the standard solution. Figure 2 It is the liquid chromatogram of the sample solution of this embodiment.

[0034] The contents of neotame, sucralose and glycyrrhizin in tipping paper A were measured to be 1.77 mg / g, 7.59 mg / g and 34.61 mg / g, respectively.

[0035] Example 2: Simultaneous determination of the contents of neotame, sucralose, and glycyrrhizin in packaging paper B, the steps are as follows:

[0036] (1) Sample extraction: Accurately weigh 1.0000 g of shredded tipping paper, add 20 mL of a 0.1 wt% acetic acid-0.1 wt% ammonium acetate-50 wt% acetonitrile aqueous solution, and extract with ultrasound for 15 min;

[0037] (2) Sample determination: HPLC was used to determine the contents of neotame, sucralose, and glycyrrhizin in the tipping paper. Before HPLC, the extract was diluted 10 times.

[0038] The instrument conditions are as follows:

[0039] Liquid chromatography conditions were as follows: chromatographic column: Ecosil Carbohydrate column, dimensions: 5 μm, 4.6 mm × 250 mm; flow rate: 0.8 mL / min; column temperature: 25-35°C; injection volume: 10 μL; elution mode: gradient elution; mobile phase A: acetonitrile; mobile phase B: a mixture of acetic acid, ammonium acetate, and water in a weight ratio of 0.001:0.001:100; elution mode: gradient elution; detector: evaporative light scattering detector (DAD); detection parameters: nebulization temperature: 40°C; evaporation temperature: 80°C; nebulizing gas: nitrogen; gas flow rate: 2.0 L / min. Gradient elution conditions were: time 0.00 min, 85 wt% mobile phase A + 15 wt% mobile phase B; 10.00 min, 65 wt% mobile phase A + 35 wt% mobile phase B.

[0040] Preparation of standard solutions: Accurately weigh 5.0 mg of neotame, 10.0 mg of sucralose, and 10.0 mg of glycyrrhizin standard into a 10 mL volumetric flask. Dissolve the stock solutions in an aqueous solution of acetic acid, ammonium acetate, and acetonitrile (0.1 wt% acetic acid, 0.1 wt% ammonium acetate, 50 wt% acetonitrile). Prepare six standard gradients ranging from 1.0 μg / mL to 500 μg / mL based on the concentrations of the stock solutions. Based on the concentrations and peak areas of the neotame, sucralose, and glycyrrhizin standard solutions, the calibration curve equations for this experiment were: y = 6598.2x - 2.0034, with a correlation coefficient R² = 0.9979; y = 32624x - 1983.3, with a correlation coefficient R² = 0.9952; and y = 5322x - 1.0601, with a correlation coefficient R² = 0.9957.

[0041] The contents of neotame, sucralose and glycyrrhizin in tipping paper B were measured to be 1.59 mg / g, 6.06 mg / g and 32.72 mg / g, respectively.

[0042] Example 3: Simultaneous determination of the contents of neotame, sucralose, and glycyrrhizin in packaging paper C, the steps are as follows:

[0043] (1) Sample extraction: Accurately weigh 1.0000 g of shredded tipping paper, add 20 mL of a 0.1 wt% acetic acid-0.1 wt% ammonium acetate-50 wt% acetonitrile aqueous solution, and extract with ultrasound for 15 min;

[0044] (2) Sample determination: HPLC was used to determine the contents of neotame, sucralose, and glycyrrhizin in the tipping paper. Before HPLC, the extract was diluted 20 times.

[0045] The instrument conditions are as follows:

[0046] Liquid chromatography conditions were as follows: chromatographic column: Ecosil Carbohydrate column, dimensions: 5 μm, 4.6 mm × 250 mm; flow rate: 0.8 mL / min; column temperature: 25-35°C; injection volume: 10 μL; elution mode: gradient elution; mobile phase A: acetonitrile; mobile phase B: a mixture of acetic acid, ammonium acetate, and water in a weight ratio of 0.001:0.001:100; elution mode: gradient elution; detector: evaporative light scattering detector (DAD); detection parameters: nebulization temperature: 40°C; evaporation temperature: 80°C; nebulizing gas: nitrogen; gas flow rate: 2.0 L / min. Gradient elution conditions were: time 0.00 min, 85 wt% mobile phase A + 15 wt% mobile phase B; 10.00 min, 65 wt% mobile phase A + 35 wt% mobile phase B.

[0047] Preparation of standard solutions: Accurately weigh 5.0 mg of neotame, 10.0 mg of sucralose, and 10.0 mg of glycyrrhizin standard into a 10 mL volumetric flask. Dissolve the stock solutions in an aqueous solution of acetic acid, ammonium acetate, and acetonitrile (0.1 wt% acetic acid, 0.1 wt% ammonium acetate, 50 wt% acetonitrile). Prepare six standard gradients ranging from 1.0 μg / mL to 500 μg / mL based on the concentrations of the stock solutions. Based on the concentrations and peak areas of the neotame, sucralose, and glycyrrhizin standard solutions, the calibration curve equations for this experiment were: y = 6598.2x - 2.0034, with a correlation coefficient R² = 0.9979; y = 32624x - 1983.3, with a correlation coefficient R² = 0.9952; and y = 5322x - 1.0601, with a correlation coefficient R² = 0.9957.

[0048] The contents of neotame, sucralose and glycyrrhizin in tipping paper C were measured to be 1.57 mg / g, 6.15 mg / g and 32.73 mg / g, respectively.

[0049] Comparative Example 1: Simultaneous determination of the contents of neotame, sucralose, and glycyrrhizin in packaging paper C, the steps are as follows:

[0050] (1) Sample extraction: 1.0000 g of shredded tipping paper was accurately weighed, and 20 mL of a 0.1 wt% acetic acid-0.1 wt%-50 wt% acetonitrile aqueous solution was added, and ultrasonic extraction was performed for 15 min.

[0051] (2) Sample determination: HPLC was used for detection. Before HPLC detection, the extract was diluted 20 times. The instrument conditions were as follows:

[0052] Liquid chromatography conditions were as follows: chromatographic column: Ecosil Carbohydrate column, dimensions: 5 μm, 4.6 mm × 250 mm; flow rate: 0.8 mL / min; column temperature: 25-35°C; injection volume: 10 μL; elution mode: gradient elution; mobile phase A: acetonitrile; mobile phase B: a mixture of acetic acid and water in a weight ratio of 0.001:100; elution mode: gradient elution; detector: evaporative light scattering detector (DAD); detection parameters: nebulization temperature: 40°C; evaporation temperature: 80°C; nebulizing gas: nitrogen; gas flow rate: 2.0 L / min. Gradient elution conditions were: time 0.00 min, 85 wt% mobile phase A + 15 wt% mobile phase B; 10.00 min, 65 wt% mobile phase A + 35 wt% mobile phase B.

[0053] Test results: Figure 3 As shown in the figure, the baseline drift is serious, and no chromatographic peaks appear for neotame and glycyrrhizin, making it impossible to achieve simultaneous qualitative and quantitative detection of neotame, sucralose, and glycyrrhizin in the tipping paper.

[0054] Comparative Example 2: Simultaneous determination of the contents of neotame, sucralose, and glycyrrhizin in packaging paper C, the steps are as follows:

[0055] (1) Sample extraction: Accurately weigh 1.0000 g of shredded tipping paper, add 20 mL of a 0.2 wt% sodium dihydrogen phosphate-50 wt% acetonitrile aqueous solution, and extract with ultrasound for 15 min.

[0056] (2) Sample determination: HPLC was used for detection. Before HPLC detection, the extract was diluted 20 times. The instrument conditions were as follows:

[0057] Liquid chromatography conditions were as follows: chromatographic column: Ecosil Carbohydrate column, dimensions: 5 μm, 4.6 mm × 250 mm; flow rate: 0.8 mL / min; column temperature: 25-35°C; injection volume: 10 μL; elution mode: gradient elution; mobile phase A: acetonitrile; mobile phase B: a mixture of sodium dihydrogen phosphate and water in a weight ratio of 0.002:100; elution mode: gradient elution; detector: evaporative light scattering detector (DAD); detection parameters: nebulization temperature: 40°C; evaporation temperature: 80°C; nebulizing gas: nitrogen; gas flow rate: 2.0 L / min. Gradient elution conditions were: time 0.00 min, 85 wt% mobile phase A + 15 wt% mobile phase B; 10.00 min, 65 wt% mobile phase A + 35 wt% mobile phase B.

[0058] Test results: Figure 4 As shown in the figure, the baseline drift is serious and no chromatographic peak appears for glycyrrhizin, making it impossible to achieve simultaneous qualitative and quantitative detection of neotame, sucralose and glycyrrhizin in the tipping paper.

[0059] The main features and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely preferred examples of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A liquid chromatography method for simultaneously detecting three sweeteners in tipping paper, characterized in that: The steps include: (1) Sample extraction: Weigh the shredded tipping paper and add it to an aqueous solution of acetic acid, ammonium acetate, and acetonitrile. Ultrasonic extraction is performed for a period of time to obtain an extract. (2) Sample determination: High performance liquid chromatography was used to determine the contents of neotame, sucralose, and glycyrrhizin in the tipping paper.

2. The method according to claim 1, characterized in that In step (1), the mass ratio of the tipping paper to the aqueous solution of acetic acid-ammonium acetate-acetonitrile is 1:(200-600); and the ultrasonic extraction time is 15 to 30 minutes.

3. The method according to claim 1, characterized in that In step (1), the weight ratio of acetic acid, ammonium acetate, acetonitrile and water is 0.001:0.001:50:

50.

4. The method according to claim 1, wherein In step (2), the extract is diluted 5-100 times before detection by high performance liquid chromatography.

5. The method according to claim 1, wherein The liquid chromatography conditions are as follows: chromatographic column: EcosilCarbohydrate column, specifications: 5 μm, 4.6 mm×250 mm; flow rate: 0.8 mL / min; column temperature: 25-35°C; injection volume: 10 μL; elution method: gradient elution; detector: evaporative light scattering detector.

6. The method according to claim 5, characterized in that The gradient elution conditions were: time 0.00 min, 85 wt % mobile phase A + 15 wt % mobile phase B; 10.00 min, 65 wt % mobile phase A + 35 wt % mobile phase B.

7. The method according to claim 6, characterized in that Mobile phase A was acetonitrile, and mobile phase B was a mixed solution of acetic acid, ammonium acetate, and water in a weight ratio of 0.001:0.001:

100.

8. The method according to claim 5, characterized in that The detector used was an evaporative light scattering detector, and the detection parameters were: atomization temperature: 40°C; evaporation temperature: 80°C; atomization gas: nitrogen; gas flow rate: 2.0 L / min.

9. The method according to claim 1, characterized in that High performance liquid chromatography was used for detection, and the contents of neotame, sucralose and glycyrrhizin in the tipping paper were obtained according to the standard curve equations of neotame, sucralose and glycyrrhizin.

10. The method according to claim 9, characterized in that The standard curve equation for neotame is y=6598.2x-2.0034, the standard curve equation for sucralose is y=32624x-1983.3, and the standard curve equation for glycyrrhizin is y=5322x-1.0601.

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