A method for simultaneously detecting multiple sweeteners in cigarette sweet flavor tipping paper

Abstract

The application discloses a method for simultaneously detecting multiple sweeteners in cigarette sweet tipping paper, comprising the following steps: cutting a cigarette along a part between tobacco and a filter, discarding the tobacco and the filter, obtaining tipping paper, and cutting the tipping paper into pieces; adding the tipping paper pieces into a methanol aqueous solution, performing ultrasonic extraction and centrifugal treatment, collecting a supernatant extraction liquid filter membrane, and obtaining a sample filtrate; drawing a standard curve: preparing a mixed standard solution of multiple sweeteners, using an HPLC-ELSD method to measure a chromatogram, taking the concentration of the standard solution as the abscissa and the corresponding peak area as the ordinate, and drawing a standard curve of each sweetener; and using the HPLC-ELSD method to detect the sample filtrate, and calculating the content of each sweetener in the tipping paper according to the standard curve. The method for simultaneously detecting multiple sweeteners in cigarette sweet tipping paper can simultaneously detect 15 sweeteners in cigarette tipping paper, and is simple in operation, high in analysis efficiency, and accurate and reliable in detection result.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis methods, and more specifically, to a method for simultaneously detecting multiple sweeteners in cigarette sweetener tipping paper. Background Technology

[0002] Sweeteners are food additives that impart a sweet taste to food. Due to their higher sweetness and lower energy content compared to sucrose, they are gradually replacing traditional high-calorie sugars in the food industry. According to my country's newly promulgated "GB 2760-2024 Standard for the Use of Food Additives" in 2024, a total of 20 sweeteners are permitted, including 9 artificial sweeteners, 3 natural sweeteners, and 7 sugar alcohols. Some artificial sweeteners may have side effects such as exacerbating nerve inflammation, impairing normal tissue function, and causing cancer; therefore, their dosage must be strictly controlled.

[0003] Sweetness is one of the important taste characteristics of tobacco and a crucial indicator for the sensory evaluation of Chinese cigarettes. Sweetness can mitigate the bitterness, spiciness, and astringency of tobacco, effectively improving smoking quality and comfort, and significantly influencing the style characteristics of cigarettes. Tipping paper, a special material connecting the tobacco leaves, cigarette paper, and cigarette filter, is the most direct and obvious factor affecting smoke indicators among the cigarette auxiliary materials—the "three papers and one filter." In recent years, to meet consumers' demands for sensory comfort in cigarette products, cigarette manufacturers have begun to enhance the refreshing and sweet taste of cigarettes by adding artificial or natural sweeteners to the surface of the tipping paper. The type and amount of sweetener have a significant impact on the stability of cigarette sensory quality; therefore, developing a stable and efficient method for sweetener detection is of great importance.

[0004] However, previous detection methods have limitations, as they can only detect one or a few sweeteners, and methods for detecting some novel sweeteners are lacking. Therefore, there is an urgent need for a method to simultaneously detect multiple sweeteners in cigarette sweetening tipping paper. Summary of the Invention

[0005] The purpose of this invention is to provide a method for simultaneously detecting multiple sweeteners in cigarette tipping paper, thereby solving the problems in the prior art. This method can simultaneously detect 15 sweeteners in cigarette tipping paper, and is simple to operate, highly efficient in analysis, and provides accurate and reliable detection results.

[0006] This invention provides a method for simultaneously detecting multiple sweeteners in cigarette sweetener tipping paper, comprising:

[0007] Sample processing: Cut the cigarette along the part where the tobacco and the filter meet, discard the tobacco and the filter rod, and obtain the tipping paper. Cut each tipping paper into pieces.

[0008] Sample preparation: Add the tipped paper fragments to a methanol-water solution, perform ultrasonic extraction and centrifugation, collect the supernatant extract and filter it through a membrane to obtain the sample filtrate;

[0009] Plotting standard curves: Prepare mixed standard solutions of various sweeteners, and obtain chromatograms using high performance liquid chromatography-evaporative light scattering detection. Plot the standard curves for each sweetener with the concentration of the standard solution on the x-axis and the corresponding peak area on the y-axis.

[0010] Sample testing: The sample filtrate was tested using the high performance liquid chromatography-evaporative light scattering detection method, and the content of each sweetener in the tipping paper was calculated based on the standard curve.

[0011] The method for simultaneously detecting multiple sweeteners in cigarette sweetener tipping paper as described above, wherein preferably, the multiple sweeteners include acesulfame potassium, sodium saccharin, cyclamate, sucralose, aspartame, alitane, naringin dihydrochalcone, neohesperidin dihydrochalcone, advansat, neotame, mogroside, rebaudioside D, stevioside, rebaudioside A, and ammonium glycyrrhizate.

[0012] The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper, as described above, preferably includes the following sample processing: cutting the cigarette along the boundary between the tobacco and the filter, discarding the tobacco and the filter rod to obtain the tipping paper, and cutting each sheet of tipping paper into fragments, including:

[0013] Use tweezers to remove the cigarette from the box, cut it along the part where the tobacco and the filter meet, discard the tobacco and the filter rod to get the tipping paper, cut each tipping paper into pieces, and for tipping papers with printed patterns, separate the printed side from the non-printed side and cut them into pieces respectively.

[0014] The tipped paper fragments should be placed in a dry centrifuge tube and left for 25-30 minutes at a temperature of 20℃-25℃ and a humidity of 40%-60% to balance the moisture content of the sample.

[0015] The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper, as described above, preferably includes the following sample preparation: adding tipping paper fragments to a methanol-water solution, performing ultrasonic extraction and centrifugation, collecting the supernatant extract and filtering it through a membrane to obtain a sample filtrate, comprising:

[0016] Place the tipped paper fragments into 10 mL centrifuge tubes, weigh 0.200±0.020 g into each tube, add 4.0 mL of 50% methanol-water mixture, and sonicate in an ice-water bath for 35 min-45 min. Then centrifuge at 8000 r / min-10000 r / min for 10 min-15 min, collect the supernatant extract and filter it through a 0.22 μm organic filter membrane to obtain the sample filtrate.

[0017] The method for simultaneously detecting multiple sweeteners in cigarette sweetener tipping paper as described above, wherein preferably, the extraction temperature during the ultrasonic extraction process is 10℃-15℃; the filtered filtrate is sealed and stored at 4℃ for no more than 24 hours.

[0018] The method for simultaneously detecting multiple sweeteners in cigarette sweetener tipping paper, as described above, preferably includes the following step: preparing a mixed standard solution of multiple sweeteners, obtaining the chromatogram using high-performance liquid chromatography-evaporative light-dissipative detection, and plotting a standard curve for each sweetener with the concentration of the standard solution on the x-axis and the corresponding peak area on the y-axis, including:

[0019] Mixed standard stock solution: Weigh 10.0 mg of each of 15 sweetener standards using a high-precision electronic balance and place them in a 25 mL volumetric flask. Dilute to volume with 50% methanol aqueous solution filtered through a 0.22 μm filter membrane, shake well, and prepare a mixed standard stock solution with a concentration of 400 μg / mL for later use.

[0020] Prepare a series of mixed standard solutions; dilute the mixed standard stock solution with 50% methanol aqueous solution to prepare a series of mixed standard solutions with a concentration range of 40 μg / mL-400 μg / mL, and set at least 5 concentration gradients;

[0021] Chromatograms of each concentration gradient were obtained using high performance liquid chromatography-evaporative light heat dissipation detection. Each concentration gradient was measured in triplicate, and the average peak area was taken. Standard curves for each sweetener were plotted with the concentration of the standard solution on the x-axis and the average peak area on the y-axis.

[0022] The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper, as described above, preferably involves preparing a series of mixed standard solutions; diluting the mixed standard stock solution with a concentration range of 40 μg / mL to 400 μg / mL using a 50% methanol aqueous solution, and setting at least 5 concentration gradients, including:

[0023] Transfer 100 μL, 200 μL, 400 μL, 600 μL, 800 μL, and 1000 μL of the mixed standard stock solution into six 1.5 mL centrifuge tubes, respectively, and dilute to 1000 μL with 50% methanol-water solution. Shake well to obtain a series of mixed standard solutions with mass concentrations of 40 μg / mL, 80 μg / mL, 160 μg / mL, 240 μg / mL, 320 μg / mL, and 400 μg / mL, respectively.

[0024] The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper, as described above, preferably employs the following chromatographic conditions for the high-performance liquid chromatography-evaporative light scattering (HPLC-ELSD) method in the standard curve plotting and sample detection: A ZORBAX 300SB-C18 column is used, rinsed with a 1:1 methanol-water solution for 30 minutes before use; mobile phase A is a 0.1% formic acid aqueous solution, adjusted to pH 4.0 ± 0.1 with ammonia; mobile phase B is methanol; the gradient elution program is as follows: 0 min-15 min, A is 85%; 15 min-75 min, A decreases from 85% to 20%; 75 min-80 min, A increases from 20% to 85%; flow rate is 1.0 mL / min; injection volume is 20 μL; column temperature is 35℃.

[0025] The method for simultaneously detecting multiple sweeteners in cigarette sweetener tipping paper as described above, wherein, preferably, the ELSD conditions for the high-performance liquid chromatography-evaporative light scattering detection method in the plotting of the standard curve and the sample detection are as follows: instrument preheating for 30 minutes before detection; gain: 10; atomization power: 67%, 40°C; evaporation temperature: 60°C; atomizing gas: air; gas pressure: 20 psi.

[0026] The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper as described above, wherein preferably, the regression equation of the standard curve has a correlation coefficient R² ≥ 0.998; the detection limit for 15 sweeteners is 7 μg / mL-10 μg / mL, the quantitation limit is 20-30 μg / mL; the spiked recovery rate is 87.8%-113.3%, and the relative standard deviation RSD < 3.04%.

[0027] This invention provides a method for simultaneously detecting multiple sweeteners in cigarette sweetened tipping paper. It has a wide detection range, enabling the simultaneous detection of 15 sweeteners in cigarette sweetened tipping paper, including acesulfame potassium, sodium saccharin, cyclamate, sucralose, aspartame, alitane, naringin dihydrochalcone, neohesperidin dihydrochalcone, advansat, neotame, mogroside, riboflavin D, steviol glycoside, riboflavin A, and ammonium glycyrrhizate. This method overcomes the limitations of previous detection methods that could only detect one or a few sweeteners, and supplements the detection methods for some novel sweeteners. The sample processing and preparation steps are simple, requiring no complex pretreatment processes, thus reducing operational difficulty and labor intensity. The method is simple to operate and highly efficient in analysis; the detection results are accurate and reliable. By optimizing experimental conditions, increasing the number of parallel determinations, and conducting method verification experiments, the accuracy and precision of the detection results are improved. The method of this invention has a low limit of detection, a reasonable limit of quantitation, a high spiked recovery rate, and a small relative standard deviation, which can meet the requirements for the detection of sweeteners in cigarette tipping paper. It provides a convenient and efficient analytical method for detecting sweeteners in cigarette tipping paper, and can be used for safety monitoring and process quality control of sweetened tipping paper. It provides technical support for the production and quality supervision of cigarette enterprises. Attached Figure Description

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to the accompanying drawings, wherein:

[0029] Figure 1 A flowchart illustrating an embodiment of the method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper provided by the present invention;

[0030] Figure 2 Standard chromatograms of 15 sweeteners;

[0031] Figure 3 The effect of the volume of the extraction solvent on the content of the extract;

[0032] Figure 4 The effect of extraction method on extract content;

[0033] Figure 5 The effect of extraction time on the content of extract. Detailed Implementation

[0034] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings. The descriptions of the exemplary embodiments are merely illustrative and are in no way intended to limit the present disclosure or its application or use. The present disclosure may be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided so that the present disclosure will be thorough and complete, and will fully express the scope of the disclosure to those skilled in the art. It should be noted that, unless specifically stated otherwise, the relative arrangement of components and steps, the composition of materials, numerical expressions, and values ​​set forth in these embodiments should be interpreted as exemplary only and not as limiting.

[0035] The terms “first,” “second,” and similar terms used in this disclosure do not indicate any order, quantity, or importance, but are merely used to distinguish different parts. Terms such as “including” or “contains” mean that the element preceding the term encompasses the element listed after it, and do not exclude the possibility of encompassing other elements as well. Terms such as “above” and “below” are used only to indicate relative positional relationships; when the absolute position of the described object changes, this relative positional relationship may also change accordingly.

[0036] In this disclosure, when a specific component is described as being located between a first component and a second component, an intermediary component may or may not be present between the specific component and the first or second component. When a specific component is described as connecting to other components, the specific component may be directly connected to the other components without having an intermediary component, or it may not be directly connected to the other components but may have an intermediary component.

[0037] All terms used in this disclosure (including technical or scientific terms) have the same meaning as understood by one of ordinary skill in the art to which this disclosure pertains, unless otherwise specifically defined. It should also be understood that terms defined in a general dictionary, such as a dictionary, should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and not as having an idealized or highly formalized meaning, unless expressly defined herein.

[0038] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, they should be considered part of the specification.

[0039] like Figure 1 As shown, the method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper provided in this embodiment includes the following steps in actual implementation:

[0040] Step S1, Sample processing: Cut the cigarette along the boundary between the tobacco and the filter, discard the tobacco and the filter rod, and obtain the splice paper. Cut each splice paper into pieces.

[0041] In one embodiment of the method for simultaneously detecting multiple sweeteners in cigarette sweetener tipping paper according to the present invention, step S1 may specifically include:

[0042] Step S11: Use tweezers to remove the cigarette from the box, cut it along the part where the tobacco and the filter meet, discard the tobacco and the filter rod to obtain the tipping paper, cut each tipping paper into pieces, and for tipping papers with printed patterns, separate the printed side from the non-printed side and cut them into pieces respectively.

[0043] In one embodiment of the present invention, each sheet of splicing paper is cut into 8 small pieces, approximately 5mm × 10mm.

[0044] Step S12: The spliced ​​paper fragments should be placed in a dry centrifuge tube and kept in an environment with a temperature of 20℃-25℃ and a humidity of 40%-60% for 25-30 minutes to balance the moisture content of the sample.

[0045] Step S2, Sample preparation: Add the spliced ​​paper fragments to a methanol-water solution, and after ultrasonic extraction and centrifugation, collect the supernatant extract and filter it through a membrane to obtain the sample filtrate.

[0046] Specifically, the collected paper fragments were placed in 10mL centrifuge tubes, with 0.200±0.020g weighed per tube. 4.0mL of 50% methanol-water solution was added, and the mixture was ultrasonically extracted in an ice-water bath for 35-45 minutes. Then, it was centrifuged at 8000-10000 rpm for 10-15 minutes. The supernatant extract was collected and filtered through a 0.22μm organic filter membrane to obtain the sample filtrate. The extraction temperature during the ultrasonic extraction process was 10℃-15℃. The filtered filtrate was sealed and stored at 4℃ for no more than 24 hours.

[0047] In one embodiment of the present invention, the extraction conditions are selected, specifically the extraction solvent volume, extraction method, and extraction time.

[0048] Specifically, regarding the extraction solvent volume: using 50% methanol-water as the extraction solvent, 0.200 g of the tipped paper sample was accurately weighed, and the effect of extraction solvent volumes of 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL on the sweetener extraction concentration was investigated. The results are shown in […]. Figure 3 The results showed that an experimental volume of 4 mL yielded the best extraction effect.

[0049] Extraction methods: The experiment investigated the effects of two different extraction methods, ultrasonic extraction and shaking, on the content of the extract. The results are as follows: Figure 4 As shown. Figure 4The results showed that the extract mass fractions under shaking and ultrasonic oscillation conditions were 2604.19 μg / g and 3155.30 μg / g, respectively. Ultrasonic extraction was significantly more effective than shaking extraction. Therefore, ultrasonic extraction was chosen for this experiment.

[0050] Extraction time: The experiment investigated the effect of extraction times of 30, 40, 50, 60, and 70 min on the content of the extract. The results are shown in […]. Figure 4 .Depend on Figure 4 It can be seen that the extract content reaches its maximum when the extraction time is 40 minutes. Further extending the extraction time leads to decomposition and volatilization of the extract due to the increased temperature under ultrasonic treatment, resulting not only in a decrease in the average extract content but also a gradual increase in error until it becomes unusable. Therefore, the experiment selected an extraction time of 40 minutes.

[0051] Therefore, the tipped paper fragments were placed in 10 mL centrifuge tubes, with 0.200 ± 0.020 g weighed per tube. 4.0 mL of 50% methanol-water was added, and the mixture was ultrasonically extracted for 40 min followed by centrifugation. An appropriate amount of the supernatant extract was filtered through a 0.22 μm organic filter membrane to obtain the filtrate.

[0052] Step S3: Plotting standard curves: Prepare mixed standard solutions of various sweeteners, and obtain chromatograms using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD). Plot the standard curves for each sweetener with the concentration of the standard solution on the x-axis and the corresponding peak area on the y-axis.

[0053] The various sweeteners mentioned include acesulfame potassium, sodium saccharin, cyclamate, sucralose, aspartame, alitame, naringin dihydrochalcone, neohesperidin dihydrochalcone, advansat, neotame, mogroside, rebaudioside D, stevioside, rebaudioside A, and ammonium glycyrrhizate. In step S3, the chromatographic conditions for the high-performance liquid chromatography-evaporative light scattering detection method are as follows: a ZORBAX 300SB-C18 column (4.6 mm × 250 mm, 5 μm) is used, which is rinsed with a 1:1 methanol-water solution for 30 minutes before use; the mobile phase A is a 0.1% formic acid aqueous solution, with the pH adjusted to 4.0 ± 0.1 by ammonia, and the mobile phase B is methanol; the gradient elution program is as follows: 0 min - 15 min, A is 85%; 15 min - 75 min, A decreases from 85% to 20%; 75 min - 80 min, A increases from 20% to 85%; the flow rate is 1.0 mL / min; the injection volume is 20 μL; and the column temperature is 35℃. Furthermore, the ELSD conditions for the high performance liquid chromatography-evaporative light scattering detection method are as follows: preheat the instrument for 30 minutes before detection; gain: 10; nebulization power: 67%, 40°C; evaporation temperature: 60°C; nebulizing gas: air; gas pressure: 20 psi.

[0054] In one embodiment of the present invention, the chromatographic conditions were optimized, specifically the mobile phase and instrument conditions.

[0055] Specifically, mobile phase optimization: The effects of different mobile phase systems on the separation of 15 sweeteners were compared. Five different mobile phase systems are shown in Table 1. The results showed that using 0.1% formic acid solution as phase A and ammonia solution as a buffer to adjust the acidity could maintain a stable pH value of the mobile phase. When the pH was 4.0, the separation of the 15 sweeteners was relatively good.

[0056] Table 1 Investigation of mobile phase

[0057]

[0058] Instrument condition optimization: The effects of column temperature, flow rate, nebulization power (temperature), evaporation temperature, and gas pressure on peak shape were investigated, and the results are shown in Table 2. The results indicate that the best detection performance was achieved at a column temperature of 35℃, a flow rate of 1.0 mL / min, a nebulization power of 67% (temperature 40℃), an evaporation temperature of 60℃, and a gas pressure of 20 psi.

[0059] Table 2 Instrument Condition Assessment

[0060]

[0061] In one embodiment of the method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to the present invention, step S3 may specifically include:

[0062] Step S31: Mixed standard stock solution: Weigh 10.0 mg of each of the 15 sweetener standards using a high-precision electronic balance and place them in a 25 mL volumetric flask. Dilute to volume with 50% methanol aqueous solution filtered through a 0.22 μm filter membrane, shake well, and prepare a mixed standard stock solution with a concentration of 400 μg / mL for later use.

[0063] Step S32: Prepare a series of mixed standard solutions; dilute the mixed standard stock solution with 50% methanol aqueous solution to prepare a series of mixed standard solutions with a concentration range of 40 μg / mL-400 μg / mL, and set at least 5 concentration gradients.

[0064] Specifically, 100 μL, 200 μL, 400 μL, 600 μL, 800 μL, and 1000 μL of the mixed standard stock solution were transferred into six 1.5 mL centrifuge tubes, respectively, and diluted to 1000 μL with 50% methanol-water solution. The solutions were then shaken well to obtain a series of mixed standard solutions with mass concentrations of 40 μg / mL, 80 μg / mL, 160 μg / mL, 240 μg / mL, 320 μg / mL, and 400 μg / mL, respectively.

[0065] Mixed standard stock solutions and series of mixed standard solutions must be prepared and used immediately. If storage is required, they must be placed in an environment of -20°C and the storage period shall not exceed 7 days.

[0066] Step S33: The chromatograms of each concentration gradient were determined using high-performance liquid chromatography-evaporative light-dissipative detection (HPLC-ELSD), as shown below. Figure 2 As shown in Table 3, each concentration gradient was measured in triplicate, and the average peak area was taken. The standard curves for each sweetener were plotted with the concentration of the standard solution on the x-axis and the average peak area on the y-axis. The results are shown in Table 3. The mass concentrations of the 15 sweeteners showed a Log-Log linear relationship with the peak area in the range of 40-400 μg / mL, with correlation coefficients r ≥ 0.9980. The limits of detection were 7 μg / mL-10 μg / mL, and the limits of quantitation were 20 μg / mL-30 μg / mL.

[0067] Table 3 Standard curves, limits of detection, and limits of quantitation for 15 sweeteners

[0068]

[0069] Furthermore, in one embodiment of the method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to the present invention, step S3 further includes:

[0070] Step S34, Precision and Recovery Experiment: Weigh three blank sweetened tipping paper samples and add mixed standard solutions of 15 sweeteners at three different content levels to each sample, and process them according to the experimental method. After three parallel determinations, calculate the recovery rate and relative standard deviation (RSD) of each sweetener. The results are shown in Table 4. The recovery rates of each sweetener ranged from 87.8% to 113.3%, and the highest RSD was 3.04%, proving that the detection method has high precision and recovery rate, which can meet the needs of sample detection.

[0071] Table 4 Precision and Recovery Rate

[0072]

[0073]

[0074] Step S4, Sample detection: The sample filtrate is detected using the high performance liquid chromatography-evaporative light scattering detection method (HPLC-ELSD), and the content of each sweetener in the tipping paper is calculated according to the standard curve.

[0075] The chromatographic and ELSD conditions in step S4 are the same as those in step S3, and will not be repeated here. In one embodiment of the present invention, tipping paper A, tipping paper B, and tipping paper C are processed according to steps S1-S4 respectively, and the detection results of each tipping paper sample are shown in Table 5. The sweetener content in tipping paper A is 1674.58 μg / g of steviol glycoside, 1725.47 μg / g of ribobandibosin A, and the content of other sweeteners is 0; the sweetener content in tipping paper B is 3155.73 μg / g of sodium saccharin, and the content of other sweeteners is 0; the sweetener content in tipping paper C is 1043.44 μg / g of acesulfame potassium, 281.59 μg / g of cyclamate, and the content of other sweeteners is 0.

[0076] Table 5. Detection results of each sample

[0077]

[0078] In one embodiment of the present invention, the method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper further includes:

[0079] Method validation: Stability experiments, inter-laboratory validation, and matrix effect experiments were conducted.

[0080] The stability test involves placing the same filtrate at 4°C for 0, 6, 12, and 24 hours, and then measuring the change rate of each sweetener content. The change rate must be ≤5%.

[0081] Interlaboratory validation involves sending the same standard solution and sample to three different laboratories for testing, calculating the relative deviation of the test results from each laboratory, and ensuring that the relative deviation is ≤5%.

[0082] The matrix effect experiment involves determining the recovery rate of the standard added to the blank tipping paper matrix and the recovery rate of the pure standard solution, respectively, and calculating the matrix effect factor. The matrix effect factor should be between 0.9 and 1.1.

[0083] In this invention, all indicators of the method verification for splicing paper A, splicing paper B, and splicing paper C meet the requirements.

[0084] The present invention provides a method for simultaneously detecting multiple sweeteners in cigarette sweetened tipping paper. This method has a wide detection range, enabling the simultaneous detection of 15 sweeteners in cigarette sweetened tipping paper, including acesulfame potassium, sodium saccharin, cyclamate, sucralose, aspartame, alitane, naringin dihydrochalcone, neohesperidin dihydrochalcone, advansat, neotame, mogroside, riboflavin D, steviol glycoside, riboflavin A, and ammonium glycyrrhizate. This overcomes the limitations of previous methods that could only detect single or a few sweeteners, and supplements the detection methods for some novel sweeteners. The sample processing and preparation steps are simple, requiring no complex pretreatment, thus reducing operational difficulty and labor intensity. The method is simple to operate and highly efficient; the detection results are accurate and reliable. By optimizing experimental conditions, increasing the number of parallel determinations, and conducting method verification experiments, the accuracy and precision of the detection results are improved. The method of this invention has a low limit of detection, a reasonable limit of quantitation, a high spiked recovery rate, and a small relative standard deviation, which can meet the requirements for the detection of sweeteners in cigarette tipping paper. It provides a convenient and efficient analytical method for the detection of sweeteners in cigarette tipping paper, and can be used for safety monitoring and process quality control of sweetened tipping paper. It provides technical support for the production and quality supervision of cigarette enterprises.

[0085] The embodiments of this disclosure have now been described in detail. To avoid obscuring the concept of this disclosure, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0086] While specific embodiments of this disclosure have been described in detail by way of examples, those skilled in the art should understand that the examples are for illustrative purposes only and not intended to limit the scope of this disclosure. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this disclosure. The scope of this disclosure is defined by the appended claims.

Claims

1. A method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper, characterized in that, include: Sample processing: Cut the cigarette along the part where the tobacco and the filter meet, discard the tobacco and the filter rod, and obtain the tipping paper. Cut each tipping paper into pieces. Sample preparation: Add the tipped paper fragments to a methanol-water solution, perform ultrasonic extraction and centrifugation, collect the supernatant extract and filter it through a membrane to obtain the sample filtrate; Plotting standard curves: Prepare mixed standard solutions of various sweeteners, and obtain chromatograms using high performance liquid chromatography-evaporative light scattering detection. Plot the standard curves for each sweetener with the concentration of the standard solution on the x-axis and the corresponding peak area on the y-axis. Sample testing: The sample filtrate was tested using the high performance liquid chromatography-evaporative light scattering detection method, and the content of each sweetener in the tipping paper was calculated based on the standard curve.

2. The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to claim 1, characterized in that, The various sweeteners include acesulfame potassium, sodium saccharin, cyclamate, sucralose, aspartame, alitame, naringin dihydrochalcone, neohesperidin dihydrochalcone, advans, neotame, mogroside, rebaudioside D, stevioside, rebaudioside A, and ammonium glycyrrhizate.

3. The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to claim 1, characterized in that, The sample processing involves cutting the cigarette along the boundary between the tobacco and the filter, discarding the tobacco and filter rod to obtain the tipping paper, and then cutting each tipping paper into fragments, including: Use tweezers to remove the cigarette from the box, cut it along the part where the tobacco and the filter meet, discard the tobacco and the filter rod to get the tipping paper, cut each tipping paper into pieces, and for tipping papers with printed patterns, separate the printed side from the non-printed side and cut them into pieces respectively. The tipped paper fragments should be placed in a dry centrifuge tube and left for 25-30 minutes at a temperature of 20℃-25℃ and a humidity of 40%-60% to balance the moisture content of the sample.

4. The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to claim 1, characterized in that, Sample preparation: Fragments of tipped paper are added to a methanol-water solution, followed by ultrasonic extraction and centrifugation. The supernatant extract is collected and filtered through a membrane to obtain the sample filtrate, which includes: Place the tipped paper fragments into 10 mL centrifuge tubes, weigh 0.200±0.020 g into each tube, add 4.0 mL of 50% methanol-water mixture, and sonicate in an ice-water bath for 35 min-45 min. Then centrifuge at 8000 r / min-10000 r / min for 10 min-15 min, collect the supernatant extract and filter it through a 0.22 μm organic filter membrane to obtain the sample filtrate.

5. The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to claim 4, characterized in that, The extraction temperature during the ultrasonic extraction process is 10℃-15℃; the filtered filtrate is sealed and stored at 4℃ for no more than 24 hours.

6. The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to claim 1, characterized in that, The standard curve is plotted as follows: A mixed standard solution of various sweeteners is prepared, and the chromatogram is obtained using high-performance liquid chromatography-evaporative light-dissipative detection. The concentration of the standard solution is plotted on the x-axis, and the corresponding peak area is plotted on the y-axis to create a standard curve for each sweetener, including: Mixed standard stock solution: Weigh 10.0 mg of each of 15 sweetener standards using a high-precision electronic balance and place them in a 25 mL volumetric flask. Dilute to volume with 50% methanol aqueous solution filtered through a 0.22 μm filter membrane, shake well, and prepare a mixed standard stock solution with a concentration of 400 μg / mL for later use. Prepare a series of mixed standard solutions; dilute the mixed standard stock solution with 50% methanol aqueous solution to prepare a series of mixed standard solutions with a concentration range of 40 μg / mL-400 μg / mL, and set at least 5 concentration gradients; Chromatograms of each concentration gradient were obtained using high performance liquid chromatography-evaporative light heat dissipation detection. Each concentration gradient was measured in triplicate, and the average peak area was taken. Standard curves for each sweetener were plotted with the concentration of the standard solution on the x-axis and the average peak area on the y-axis.

7. The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to claim 6, characterized in that, The preparation of the series of mixed standard solutions; the mixed standard stock solution is diluted with 50% methanol aqueous solution to form a series of mixed standard solutions with a concentration range of 40 μg / mL-400 μg / mL, with at least 5 concentration gradients, including: Transfer 100 μL, 200 μL, 400 μL, 600 μL, 800 μL, and 1000 μL of the mixed standard stock solution into six 1.5 mL centrifuge tubes, respectively, and dilute to 1000 μL with 50% methanol-water solution. Shake well to obtain a series of mixed standard solutions with mass concentrations of 40 μg / mL, 80 μg / mL, 160 μg / mL, 240 μg / mL, 320 μg / mL, and 400 μg / mL, respectively.

8. The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to claim 1, characterized in that, In the plotting of the standard curve and the detection of the samples, the chromatographic working conditions of the high-performance liquid chromatography-evaporative light scattering detection method are as follows: a ZORBAX 300SB-C18 column is used, which is rinsed with a methanol-water solution with a volume ratio of 1:1 for 30 minutes before use; the mobile phase A is a 0.1% formic acid aqueous solution, and the pH value is adjusted to 4.0±0.1 with ammonia; the mobile phase B is methanol; the gradient elution program is as follows: 0 min-15 min, A is 85%; 15 min-75 min, A decreases from 85% to 20%; 75 min-80 min, A increases from 20% to 85%; the flow rate is 1.0 mL / min; the injection volume is 20 μL; and the column temperature is 35℃.

9. The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to claim 1, characterized in that, In the plotting of the standard curve and the sample detection, the ELSD conditions of the high-performance liquid chromatography-evaporative light scattering detection method are as follows: preheat the instrument for 30 minutes before detection; gain: 10; nebulization power: 67%, 40℃; evaporation temperature: 60℃; nebulizing gas: air; gas pressure: 20 psi.

10. The method for simultaneously detecting multiple sweeteners in cigarette sweetening tipping paper according to claim 1, characterized in that, The regression equation of the standard curve had a correlation coefficient R² ≥ 0.998; the detection limits for 15 sweeteners were 7 μg / mL-10 μg / mL, and the quantitation limits were 20-30 μg / mL; the spiked recoveries were 87.8%-113.3%, and the relative standard deviations (RSD) were < 3.04%.

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