CN112394125A - Method for detecting content of rebaudioside A in tipping paper - Google Patents
Method for detecting content of rebaudioside A in tipping paper Download PDFInfo
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- CN112394125A CN112394125A CN202011357761.4A CN202011357761A CN112394125A CN 112394125 A CN112394125 A CN 112394125A CN 202011357761 A CN202011357761 A CN 202011357761A CN 112394125 A CN112394125 A CN 112394125A
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- G—PHYSICS
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Abstract
The invention provides a method for detecting the content of rebaudioside A in tipping paper, which comprises the steps of taking a mixed solution of acetonitrile and water as a mobile phase, carrying out dipping pretreatment on a tipping paper sample, carrying out ultrasonic oscillation extraction, carrying out organic membrane filtration, and measuring the content of rebaudioside A in the tipping paper sample by HPLC. The pretreatment condition provided by the invention can completely extract the rebaudioside A printed on the tipping paper, the chromatographic condition provided by the invention can rapidly and accurately measure the rebaudioside A content in the tipping paper, the repeatability and the recovery rate are good, and the method can be used for controlling and detecting the rebaudioside A content in a finished product in the process of producing the tipping paper containing rebaudioside A.
Description
Technical Field
The invention relates to a method for detecting the content of rebaudioside A in tipping paper, and belongs to the technical field of instrument analysis.
Background
Rebaudioside A is one of stevioside extracted from leaves of stevia rebaudiana Bertoni of Compositae, has sweetness about 450 times of that of sucrose, and is a novel natural sweetener with better taste. After being eaten, the rebaudioside A does not generate heat energy, so the rebaudioside A is widely applied to the production of foods, drinks and seasonings.
At present, research on rebaudioside A mainly focuses on purification and application, China lacks a relatively complete test standard for content of such substances, and only has a national standard GB8270-2014 food additive stevioside in food safety, and the method can only be applied to rebaudioside A in stevia rebaudiana extracts. Other related technologies such as "simultaneous detection of 6 sweeteners by high performance liquid chromatography" are only applicable to the measurement of the content of liquids such as beverages and wines, or easily-crushed samples such as preserves and pickles, and no research has been made on the content of rebaudioside a in paper. Tipping paper is a more important cigarette auxiliary material, is not easy to crush, has complex components, and has larger one-sided performance as a result of detection by using the technology.
Disclosure of Invention
In order to solve the problem that the content of rebaudioside A on tipping paper in the prior art cannot be accurately detected, the invention provides a method for detecting the content of rebaudioside A in the tipping paper, which achieves accurate detection of rebaudioside A on the tipping paper based on a liquid chromatography technology.
In order to solve the technical problems, the invention provides the following technical scheme:
a method of detecting the rebaudioside a content of tipping paper comprising the steps of, (1) pre-treating: cutting the tipping paper into pieces with the size of less than 0.5cm multiplied by 0.5cm, adding a mobile phase for impregnation, and obtaining a pretreatment product; the mobile phase comprises one or a mixture of acetonitrile and water, and the mass ratio of the added amount of the mobile phase to the tipping paper is 40: 1; (2) and (3) extraction: carrying out ultrasonic extraction on the pretreatment product in the step (1), and filtering to obtain filtrate; (3) and (3) detection: subjecting the filtrate to liquid chromatography with the following parameters, wherein the chromatographic column is Eclipse XDB-C18; the detection wavelength is 210 nm; the flow rate is 1.2 mL/min; the sample injection volume is 2 mu L; the column temperature was 30 ℃, the run time was 2.5min, the post run time was 7min, and the rebaudioside a retention time was 2.087 min.
Preferably, the mobile phase is a mixture of acetonitrile and water, and the volume ratio of the acetonitrile to the water is 4: 1.
preferably, the ultrasonic extraction is performed at an ultrasonic power of 500W for 10-50 min.
Preferably, the detection range of the rebaudioside A is 0.005081-0.030703 mg/mL-1。
Preferably, the rebaudioside A has an intra-day precision RSD of 0.63% and an inter-day precision RSD of 1.33%.
The invention has the beneficial effects that:
the pretreatment method provided by the invention can completely extract the rebaudioside A in the tipping paper, and the provided chromatographic conditions can ensure that the chromatographic peak and other impurity peaks in the tipping paper generate good separation effects, so that the content of the rebaudioside A in a sample can be accurately measured, and the method can be used for quality control in the production process of the tipping paper containing the rebaudioside A and content detection of the rebaudioside A in a finished product. The high performance liquid chromatography adopted by the invention has the advantages of stable method and short analysis time, and can realize continuous sample injection detection.
Drawings
FIG. 1 is a rebaudioside A standard curve;
FIG. 2 is a chromatogram of rebaudioside A in standard solution;
FIG. 3 is a chromatogram of rebaudioside A in a J-R01 sample solution.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
Instruments and reagents, materials: agilent 1260 infinity liquid chromatograph (Agilent Technologies, usa); agilent Eclipse XDB-C18 column (250 mm. times.4.6 mm, 5 μm) (Agilent Technologies, USA); BP211D electronic analytical balance (sydoris scientific instruments ltd, germany); 0.45 μm microfiltration membrane, Elma ultrasonic oscillator (Elma corporation, germany); rebaudioside a standard (biotechnology limited, yuhimu, shanghai).
The sample is cut into pieces of 0.5cm x 0.5cm in a shredding mode recommended by the tobacco industry.
Examples
Measuring the content of rebaudioside A in tipping paper for cigarettes according to the following steps
First, tipping paper sample preparation containing rebaudioside A
(1) Blending natural sweetener gloss oil: adding the aqueous gloss oil, the deionized water and the rebaudioside A into a stirrer one by one according to the weight ratio of 63 parts of the aqueous gloss oil, 17 parts of the deionized water and 2 parts of the rebaudioside A, and stirring for 30 min;
(2) preparation of sweet tipping paper: directly printing the prepared natural sweetener gloss oil on the surface of the tipping paper which is subjected to color printing and is not cut, wherein the printing amount is 1-1.5g/m2。
(3) The sweet tipping paper is used for rolling and manufacturing cigarettes.
Second, examination of sample pretreatment method
1. Selection of extraction solvent
Taking a tipping paper sample, accurately cutting and weighing 2 parts of tipping paper sample, 1dm each2After crushing, respectively putting the crushed materials into 40mL headspace bottles, respectively adding 20mL mobile phase and water, sealing the bottles, respectively carrying out ultrasonic oscillation (45kHz, 500W) extraction treatment for 30min, cooling the bottles, shaking the bottles evenly, continuously filtering the bottles for 6 times by using a 0.45-micrometer microporous membrane, taking filtrate to obtain 6 solutions of different extraction solvents, measuring peak area values by adopting HPLC (the detection conditions are the same as those in the step 3), and representing the content of the rebaudioside A by using the peak areas.
The working parameters of the liquid chromatogram are as follows: the chromatographic column is Eclipse XDB-C18; the detection wavelength is 210 nm; the flow rate is 1.2 mL/min; the sample injection volume is 2 mu L; the column temperature was 30 ℃, run time 2.5min, post run time 7min, rebaudioside a retention time 2.087min, mobile phase 4: 1 acetonitrile and water.
TABLE 1 examination of different extraction solvents
As shown in table 1, the mobile phase was selected as the solvent because the mobile phase was used as the solvent, the extraction was relatively uniform, the relative deviation of the detection results was small, and the solution was not uniform due to the foaming of the solution and the non-uniformity of the extract solution.
2. Selection of extraction mode
Taking a tipping paper sample, accurately cutting and weighing 2 parts of tipping paper sample, 1dm each2After crushing, respectively putting into 40mL headspace bottles, respectively adding 20mL mobile phase, sealing, respectively performing shaking extraction (100r/min, 100W) and ultrasonic shaking extraction (45kHz, 500W) by using a shaking table for 30min, shaking uniformly, filtering by using a 0.45-micrometer microporous filter membrane, taking subsequent filtrate to obtain extraction solutions with different extraction modes, measuring each peak area value by using HPLC (detection conditions are the same as those in step 4), and representing the content of rebaudioside A by the peak area.
TABLE 2 examination results of different extraction modes
As shown in table 2, the extraction rate of rebaudioside a in the tipping paper by ultrasonic extraction was high, so the ultrasonic method was selected as the optimal extraction method for preparing the sample extraction solution.
3. Selection of extraction time
Taking a tipping paper sample, accurately cutting and weighing 2 parts of tipping paper sample, 1dm each2Dividing the sample into 60mL headspace bottles, adding 40mL mobile phase, sealing the bottles, performing ultrasonic oscillation treatment for 10min, 20min, 30min, 40min and 50min respectively, cooling, shaking uniformly, filtering with a 0.45-micrometer microporous membrane, collecting the filtrate to obtain sample solutions with different extraction times, measuring each peak area value by adopting HPLC (the detection condition is the same as that in step 4), and representing the content of rebaudioside A by the peak area.
TABLE 3 investigation results at different extraction times
As a result, as shown in table 3, the ultrasonic oscillation extraction time 40min was finally selected as the optimum extraction time to extract rebaudioside a in the tipping paper more completely and to reduce the cost.
4 volume of extraction solvent
Taking a tipping paper sample, accurately cutting and weighing 5 parts of tipping paper sample, 1dm each2Placing into a 60mL headspace bottle, adding mobile phases of 10mL, 20mL, 30mL, 40mL and 50mL respectively, sealing, treating with ultrasonic oscillation (45kHz and 500W) for 40min, cooling, shaking, filtering with 0.45 μm microporous membrane, collecting filtrate to obtain solutions with different extraction solvent volumes, measuring each peak area by HPLC (detection conditions same as step 4), and converting into 10mL area, wherein the peak area represents rebaudioside A content.
TABLE 4 examination of different extraction solvents
As a result, as shown in table 4, 40mL of mobile phase can extract rebaudioside a in the tipping paper more completely, and the optimal extraction volume of mobile phase 40mL is finally selected in order to save cost.
Thirdly, establishment of method for measuring rebaudioside A content in tipping paper
1. Preparation of a rebaudioside A Standard Curve
Precisely weighing 40.mg of rebaudioside A, placing into a 20mL headspace bottle, adding 19.9984g of mobile phase for dissolving, sealing, shaking to obtain 2.0002mg/g of rebaudioside A standard stock solution, sequentially diluting with mobile phase to obtain 0.005081 mg/mL-1、0.009980mg·mL-1、0.014955mg·mL-1、0.019769mg·mL-1、0.024551mg·mL-1Standard working solution of (2). The peak area values (results are shown in Table 5) were measured by HPLC (detection conditions are the same as in step 4) as the solution concentrationThe standard curve is plotted with the abscissa and the peak area the ordinate (see FIG. 1). Obtaining the regression equation of the rebaudioside A as Y being 1159.6372X-0.3166 and r being 0.9997, which shows that the rebaudioside A is 0.005081-0.030703 mg/mL-1Has a good linear relationship in the range of (1); the liquid chromatogram of rebaudioside A is shown in FIG. 2.
TABLE 5 Standard Curve assay data for rebaudioside A
2. Precision experiment
The above concentration was sucked at 0.014955 mg/mL-1The sample injection is repeated 6 times in the same day under the chromatographic conditions of step 4, and the peak area of the rebaudioside a is recorded as the precision in the day (see table 6). Rebaudioside a has an intra-day precision RSD of 0.63%; the sample solutions were continuously injected for 3 days, 6 needles were injected for each sample, and the peak area was recorded as the daytime precision (see table 7). The RSDs of rebaudioside a with daytime precision were 1.33%, and were each less than 3.0%, indicating good precision.
TABLE 6 results of measurement of precision within day
TABLE 7 results of day precision measurement
3. Repeatability test
Taking a tipping paper sample, accurately cutting and weighing 6 parts of tipping paper sample, 1dm each2According to the steps of the present application, the analysis of the sample was carried out to obtain the area of the peak of rebaudioside ASee table 10. The relative standard deviation RSD value of the peak area of rebaudioside A was 1.87%.
TABLE 10 results of the repeatability tests
4. Sample application recovery rate test
Taking 2 rebaudioside A containing tipping paper samples, 3 portions each, making 1dm2After an appropriate amount of rebaudioside a standard was added, the samples were analyzed according to the procedure of the present application, and the measurement results are shown in table 11. The average recovery and RSD of rebaudioside A were 97.91% and 1.13%, respectively
TABLE 11 measurement of rebaudioside A sample recovery
Fourthly, detection of rebaudioside A in tipping paper
Taking 4 samples of different batches respectively, carrying out measurement according to the experimental steps provided by the application, taking the average value of the detection as the final detection result of the rebaudioside A content in the tipping paper, and the result is shown in Table 13, and the liquid chromatogram of the sample is shown in figure 3.
TABLE 13 content of rebaudioside A (mg. dm) in tipping paper of different lots2)
The invention discloses a method for detecting the content of rebaudioside A in tipping paper by an HPLC (high performance liquid chromatography) method. The pretreatment condition provided by the invention can completely extract the rebaudioside A printed on the tipping paper, the chromatographic condition provided by the invention can rapidly and accurately measure the rebaudioside A content in the tipping paper, the repeatability and the recovery rate are good, and the method can be used for controlling and detecting the rebaudioside A content in a finished product in the process of producing the tipping paper containing rebaudioside A.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (5)
1. A method for detecting the content of rebaudioside A in tipping paper, characterized by: comprises the following steps of (a) carrying out,
(1) pretreatment: cutting tipping paper into fragments of less than 0.5cm multiplied by 0.5cm, adding a mobile phase, wherein the mobile phase comprises one or more of acetonitrile and water, and the mass ratio of the addition amount of the mobile phase to the tipping paper is 40: 1;
(2) and (3) extraction: carrying out ultrasonic oscillation extraction on the pretreatment product in the step (1), and filtering to obtain filtrate;
(3) and (3) detection: the filtrate was subjected to liquid chromatography with the following parameters,
the chromatographic column is Eclipse XDB-C18, and the detection wavelength is 210 nm; the flow rate is 1.2 mL/min; the sample injection volume is 2 mu L; the column temperature was 30 ℃, the run time was 2.5min, the post run time was 7min, and the rebaudioside a retention time was 2.087 min.
2. The method of detecting the rebaudioside a content in the tipping paper according to claim 1, characterized in that: the volume ratio of acetonitrile to water in the mobile phase is 4: 1.
3. the method of detecting the rebaudioside a content in the tipping paper according to claim 1, characterized in that: the ultrasonic oscillation extraction has ultrasonic instrument parameters of 45kHz frequency, 500W power and 10-50min time.
4. The method of detecting the rebaudioside a content in the tipping paper according to claim 1, characterized in that: the detection range of the rebaudioside A is 0.005081-0.030703 mg/mL-1。
5. The method of detecting the rebaudioside a content in the tipping paper according to claim 1, characterized in that: the RSD of the rebaudioside A is 0.63 percent in the day precision and 1.33 percent in the daytime precision.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120269954A1 (en) * | 2011-01-28 | 2012-10-25 | Tate & Lyle Ingredients Americas Llc | Stevia Blends Containing Rebaudioside B |
| CN102766177A (en) * | 2012-06-06 | 2012-11-07 | 天津北洋百川生物技术有限公司 | Crystallization process for increasing rebaudioside A content in stevioside |
| US20170211113A1 (en) * | 2014-01-28 | 2017-07-27 | Pepsico, Inc. | Method for preparing rebaudioside m by using enzyme method |
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2020
- 2020-11-27 CN CN202011357761.4A patent/CN112394125B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120269954A1 (en) * | 2011-01-28 | 2012-10-25 | Tate & Lyle Ingredients Americas Llc | Stevia Blends Containing Rebaudioside B |
| CN102766177A (en) * | 2012-06-06 | 2012-11-07 | 天津北洋百川生物技术有限公司 | Crystallization process for increasing rebaudioside A content in stevioside |
| US20170211113A1 (en) * | 2014-01-28 | 2017-07-27 | Pepsico, Inc. | Method for preparing rebaudioside m by using enzyme method |
Non-Patent Citations (4)
| Title |
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| 彭应枝等: "反相高效液相法测定甜叶菊中莱鲍迪苷A和甜菊糖苷的含量", 《中南药学》 * |
| 邓佐等: "简易HPLC法测定甜菊叶中甜菊糖苷", 《农产品加工(学刊)》 * |
| 闫莉等: "高效液相色谱法测定新疆地区甜叶菊中甜菊苷和莱鲍迪苷A的含量", 《新疆医科大学学报》 * |
| 阿依提拉·吾布力卡斯穆等: "基于HPLC法的不同产地甜叶菊叶中莱鲍迪苷A含量测定", 《塔里木大学学报》 * |
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