CN112394125B - 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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- CN112394125B CN112394125B CN202011357761.4A CN202011357761A CN112394125B CN 112394125 B CN112394125 B CN 112394125B CN 202011357761 A CN202011357761 A CN 202011357761A CN 112394125 B CN112394125 B CN 112394125B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
Abstract
The invention provides a method for detecting the content of rebaudioside A in tipping paper, which takes a mixed solution of acetonitrile and water as a mobile phase, performs dipping pretreatment on a tipping paper sample, performs organic membrane filtration after ultrasonic oscillation extraction, and measures 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 that of sucrose, and is a novel natural sweetener with good taste. The rebaudioside A does not generate heat energy after being eaten, 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 mobile phase to the tipping paper is 40; (2) extraction: performing ultrasonic extraction on the pretreatment product in the step (1), and filtering to obtain a filtrate; and (3) detection: performing liquid chromatography analysis on the filtrate, wherein the parameters are as follows, and the chromatographic column is Eclipse XDB-C18; the detection wavelength is 210nm; the flow rate is 1.2mL/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 2.087min.
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 10-50min.
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 chromatography column (250 mm. Times.4.6 mm,5 μm) (Agilent Technologies, inc., USA); BP211D electronic analytical balance (sydows scientific instruments ltd, germany); 0.45 μm microfiltration membrane, elma ultrasonic oscillator (Elma corporation, germany); rebaudioside a standard (biotechnology limited, yuhima, 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
1. Preparation of tipping paper sample 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 30min;
(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/m 2 。
(3) The sweet tipping paper is used for rolling and manufacturing cigarettes.
2. 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, each 1dm 2 After crushing, respectively putting into 40mL headspace bottles, respectively adding 20mL mobile phase and water, sealing, respectively carrying out ultrasonic oscillation (45kHz, 500W) extraction treatment for 30min, cooling, shaking up, continuously filtering for 6 times by using a 0.45-micrometer microporous membrane, taking filtrate to obtain 6 solutions of different extraction solvents, measuring each peak area value by adopting HPLC (the detection condition is the same as that in step 3), and representing the content of rebaudioside A by the peak area.
The working parameters of the liquid chromatogram are as follows: the chromatographic column is Eclipse XDB-C18; the detection wavelength is 210nm; the flow rate is 1.2mL/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 extract solution was relatively uniform and the relative variation of the detection results was small, and the ultra-pure water was used as the solvent.
2. Selection of extraction mode
Taking a tipping paper sample, accurately cutting and weighing 2 parts of tipping paper sample, 1dm each 2 After crushing, respectively putting into 40mL headspace bottles, respectively adding 20mL mobile phase, sealing, respectively shaking by shaking table extraction (100 r/min, 100W), ultrasonic shaking extraction (45kHz, 500W) for 30min, shaking uniformly, filtering with 0.45 μm microporous filter membrane, taking the subsequent filtrate to obtain the extract solution with different extraction modes, measuring each peak area value by HPLC (detection conditions same as step 4), and representing the content of rebaudioside A by the peak area.
TABLE 2 examination results of different extraction methods
As a result, as shown in table 2, the extraction rate of rebaudioside a in the tipping paper was high by ultrasonic extraction, so that 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 each 2 Dividing 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 in order to extract rebaudioside a in the tipping paper more completely and save cost.
4. Volume of extraction solvent
Taking a tipping paper sample, accurately cutting and weighing 5 parts of tipping paper sample, 1dm each 2 Placing into 60mL headspace bottles, adding mobile phase 10mL, 20mL, 30mL, 40mL, 50mL respectively, sealing, treating with ultrasonic oscillation (45kHz, 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 the mobile phase extracted rebaudioside a more completely from the tipping paper, and in order to save costs, the optimal extraction volume of the mobile phase of 40mL was finally selected.
3. Establishment of method for measuring rebaudioside A content in tipping paper
1. Preparation of a rebaudioside A Standard Curve
Precisely weighing 40mg of rebaudioside A, placing into a 20mL headspace bottle, adding 19.9984g of mobile phase for dissolving, capping, shaking to obtain standard stock solution of rebaudioside A2.0002 mg/g, 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 -1 Standard working solution of (2). The peak area values were measured by HPLC (the detection conditions were the same as in step 4) (the results are shown in Table 5), and a standard curve was drawn with the solution concentration as the abscissa and the peak area as the ordinate (see FIG. 1). The regression equation for rebaudioside A was Y =1159.6372X-0.3166, r =0.9997, indicating that rebaudioside A was in the range 0.005081-0.030703 mg.mL -1 Has a good linear relationship in the range of (1); the liquid chromatogram of rebaudioside A is shown in FIG. 2.
TABLE 5 Standard Curve data for rebaudioside A
2. Precision experiment
The above-mentioned concentration was 0.014955 mg/mL -1 The 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 measurements
3. Repeatability test
Taking a tipping paper sample, accurately cutting and weighing 6 parts of tipping paper sample which are 1dm respectively 2 According to the steps of the present application, the analysis of the sample was performed, and the peak area of rebaudioside a was found in table 8. The relative standard deviation RSD value of the peak area of rebaudioside A was 1.87%.
TABLE 8 results of the measurement of the reproducibility test
4. Sample application recovery rate experiment
Taking 3 parts of 2 tipping paper samples containing rebaudioside A, and preparing each part into 1dm 2 Adding an appropriate amount of rebaudioside A standard, and performing the sample according to the procedure of the present applicationThe product analysis and the measurement results are shown in Table 9. The mean recovery of rebaudioside a and RSD were 97.91% and 1.13%, respectively.
TABLE 9 measurement of rebaudioside A sample recovery
4. Detection of rebaudioside a in tipping paper
Taking 4 samples of different batches respectively, measuring according to the experimental steps provided by the application, taking the average value of the detection as the final detection result of the content of rebaudioside A in the tipping paper, wherein the result is shown in Table 10, and the liquid chromatogram of the sample is shown in FIG. 3.
TABLE 10 content of rebaudioside A in various lots of tipping paper (mg. Dm) 2 )
The invention discloses a method for detecting the content of rebaudioside A in tipping paper by an HPLC 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 (3)
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 pieces of less than 0.5cm multiplied by 0.5cm, adding a mobile phase, wherein the mobile phase is a mixed solution of acetonitrile and water, and the mass ratio of the added amount of the mobile phase to the tipping paper is 40; the volume ratio of acetonitrile to water in the mobile phase is 4;
(2) Extraction: performing ultrasonic oscillation extraction on the pretreatment product in the step (1), and filtering to obtain filtrate;
the ultrasonic oscillation extraction is carried out, wherein the ultrasonic instrument parameters are 45kHz in frequency, 500W in power and 10-50min in time;
(3) And (3) detection: the filtrate was subjected to liquid chromatography with the following parameters,
the chromatographic column is Eclipse XDB-C18; the detection wavelength is 210nm; the flow rate is 1.2mL/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 2.087min.
2. 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 。
3. 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% in the intra-day precision and 1.33% in the inter-day precision.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9402411B2 (en) * | 2011-01-28 | 2016-08-02 | Tate & Lyle Ingredients Americas Llc | Stevia blends containing rebaudioside B |
| WO2015113231A1 (en) * | 2014-01-28 | 2015-08-06 | 苏州汉酶生物技术有限公司 | Rebaudioside m enzymatic preparation method |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (4)
| Title |
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| 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;彭应枝等;《中南药学》;20130930;第11卷(第09期);700-702 * |
| 基于HPLC法的不同产地甜叶菊叶中莱鲍迪苷A含量测定;阿依提拉·吾布力卡斯穆等;《塔里木大学学报》;20150930;第27卷(第03期);19-24 * |
| 简易HPLC法测定甜菊叶中甜菊糖苷;邓佐等;《农产品加工(学刊)》;20131031(第20期);14-15,18 * |
| 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;闫莉等;《新疆医科大学学报》;20161130;第39卷(第11期);1444-1446,1451 * |
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