CN110763796A - Method for measuring sweetener for cigarettes by liquid chromatography-evaporative light scattering detector - Google Patents
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Abstract
The invention relates to a method for measuring a sweetening agent for cigarettes by a liquid chromatography-evaporative light scattering detector, which adopts a pretreatment method of solvent extraction in the technical scheme, establishes a detection and analysis method for simultaneously measuring 7 sweetening agents including tipping paper for cigarettes, oral smokeless tobacco products, essence for cigarettes, bead blasting for cigarettes, acesulfame potassium, aspartame, neotame, sucralose, alitame, mogroside V or stevioside in electronic cigarette liquid by HPLC-ELSD, and detects partial samples by the established method. The method is simple to operate, short in analysis time and high in sensitivity, and provides a technical means for detecting the sweetener in the tobacco material and the tobacco product.
Description
Technical Field
The invention belongs to the technical field of physical and chemical inspection of tobacco materials and food additive residues in tobacco products, and mainly relates to a method for measuring 5 sweetening agents in tobacco tipping paper, oral smokeless tobacco products, tobacco essence, tobacco bead blasting and electronic tobacco juice, in particular to a method for directly measuring by using a high performance liquid chromatography-evaporative light scattering detector by extracting the sweetening agents in a solvent extraction mode.
Background
Sweeteners are generally used in foods for the purpose of improving flavor and increasing sweetness, and can be classified into natural sweeteners and artificial sweeteners according to their source. The common natural sweetening agents comprise stevioside, momordica grosvenori glucoside V and the like, the common artificial sweetening agents mainly comprise acesulfame potassium, saccharin sodium, alitame, sodium cyclamate, sucralose and the like, and the sweetness of the sweetening agents is generally 30-700 times that of sucrose. GB2760-2014 national food safety standard food additive use standard specifies 21 food additives allowed to be added, and specifies the maximum addition amount of the food additives in different food varieties. In tipping paper for cigarettes, oral smokeless tobacco products, essence for cigarettes, bead blasting for cigarettes or electronic cigarette liquid, the type and the addition amount of a sweetening agent have great influence on the stability of the sensory quality of the tobacco products, and in order to realize the supervision and determination of the sweetening agent in the tobacco materials and the tobacco products, the development of a rapid, accurate and high-sensitivity determination method is urgently needed.
Disclosure of Invention
The invention aims to provide a method for measuring a sweetening agent for cigarettes by using a liquid chromatography-evaporative light scattering detector, which aims to solve the problems of low detection efficiency and low sensitivity of the sweetening agent for cigarettes in the prior art.
The invention is realized by the following technical scheme:
a method for measuring a sweetener for cigarettes by using a liquid chromatography-evaporative light scattering detector comprises the following steps:
1) preparation of standard working solution:
weighing each sweetener standard substance, performing constant volume by using ultrapure water, and preparing a mixed standard working solution with a concentration gradient;
2) and (3) carrying out chromatography calibration:
analyzing by using a chromatograph by using the standard working solution prepared in the step 1) to obtain corresponding retention time, and obtaining standard concentration data of the sweetener according to calibration data;
3) and (3) extracting a sample:
selecting a sample to be detected, adding a set amount of extractant, then carrying out mechanical oscillation, standing for a set time, absorbing supernatant, filtering by using a water-phase filter membrane, and carrying out chromatographic analysis on the sample to be detected as a sample solution;
4) liquid chromatography-evaporative light scattering detector analysis
Detecting the to-be-detected sample solution and the mixed standard working solution in the steps 1) and 3) by using a liquid chromatogram-evaporative light scattering detector;
wherein the parameters of the liquid chromatogram are as follows:
a chromatographic column: ZORBAX SB-C18 with specification of 4.6mm × 250mm,5 μm; column temperature: 25 ℃; sample introduction amount: 10 mu L of the solution; flow rate: 0.8 mL/min; mobile phase: a is ultrapure water, B is methanol, and 0.01% trifluoroacetic acid is added;
parameters of the evaporative light scattering detector were:
atomization temperature: 100 ℃; evaporation temperature: 100 ℃; atomizing gas: air; gas flow rate: 1.7L/min.
The sweetener content in the sample was calculated according to the following formula:
obtaining a linear range curve formula of the sweetener according to the standard concentration data of the sweetener in the step 2):
and Ci is Kx + a, wherein K is a coefficient, the types of the sweeteners are different, K is different, a is a constant, and i is the type of the sweetener.
The sweetener is one or more of acesulfame potassium, aspartame, neotame, sucralose, alitame, mogroside V or stevioside.
When the sweetener is acesulfame K, the value of K is 5 × 10-5A is-14.085;
when the sweetener is sucralose, the value of K is 2X 10-5A is 20.852;
when the sweetener is aspartame, the value of K is 2X 10-5A is 35.957;
when the sweetener is alitame, the value of K is 2 x 10-5A is 23.246;
when the sweetener is mogroside V, K has a value of 2 × 10-5A is 21.932;
when the sweetener is neotame, the value of K is 1X 10-5A is 28.422;
when the sweetener is stevioside, K has a value of 2 × 10-5And a is 23.115.
The extractant is ultrapure water.
The water phase filter membrane is a 0.22 mu m water phase filter membrane.
The sweetener in the sample is calculated according to the following formula:
xi ═ (Ci × V) ÷ M, where Xi is the amount of sweetener i in the sample, in μ g/g; ci is the concentration of the sweetener i in the sample obtained from the standard working curve, and the unit is mu g/mL; v is the volume of the extractant, and the unit is mL; m is the sample mass, and the unit g, i is the sweetener type.
The invention has the beneficial effects that:
the technical scheme adopts a pretreatment method of solvent extraction, establishes a detection and analysis method for simultaneously measuring 7 sweetening agents, namely tipping paper for cigarettes, oral smokeless tobacco products, essence for cigarettes, bead blasting for cigarettes, acesulfame, aspartame, neotame, sucralose, alitame, mogroside V or stevioside in electronic cigarette liquid by HPLC-ELSD, and detects part of samples by using the established method. The method is simple to operate, short in analysis time and high in sensitivity, and provides a technical means for detecting the sweetener in the tobacco material and the tobacco product.
Drawings
FIG. 1 is a flow chart of the assay method of the present invention;
FIG. 2 is an HPLC-DAD chromatogram of a standard solution of 7 sweeteners.
1 acesulfame potassium, 2 sucralose, 3 aspartame, 4 alitame, 5 mogroside V, 6 neotame, 7 stevioside.
Detailed Description
The technical solutions of the present invention are described in detail below by examples, and the following examples are only exemplary and can be used only for explaining and explaining the technical solutions of the present invention, but not construed as limiting the technical solutions of the present invention.
As shown in fig. 1 and fig. 2, in the technical scheme, a method for detecting 7 sweeteners in tipping paper for cigarettes, oral smokeless tobacco products, essence for cigarettes, bead blasting for cigarettes and electronic cigarette liquid is mainly adopted, wherein the 7 sweeteners are acesulfame potassium 1, aspartame 3, neotame 6, sucralose 2, alitame 4, mogroside V5 or stevioside 7.
1) Preparation of standard working solutions
Preparing a mixed standard working solution, weighing each sweetener standard substance, fixing the volume by using ultrapure water, preparing the mixed standard working solution with a concentration gradient, mixing 5 sweeteners according to a set standard, preparing the mixed standard working solution with different concentrations, and preparing different concentrations by using a single sweetener and mapping by using a chromatogram to determine the retention time of a certain sweetener.
The method specifically comprises the following steps:
weighing 7 sweetener standard substances 0.050g (accurate to 0.1mg) respectively, placing in a 50mL volumetric flask, fixing the volume to scale with ultrapure water, shaking up to obtain mixed standard solution with concentration of each target compound of 1mg/mL, and using the mixed standard solution as standard stock solution for later use; transferring certain volumes of 0.10mL, 0.75mL, 1.00mL, 1.50mL, 2.00mL, 3.00mL and 4.00mL into 10mL volumetric flasks, adding ultrapure water to the scale, shaking up to obtain 1-7 grade standard working solutions with concentrations of 10. mu.g/mL, 75. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL, 200. mu.g/mL, 300. mu.g/mL and 400. mu.g/mL.
2) Chromatography calibration
Using the prepared mixed standard working solution, analyzing by using a chromatograph to obtain corresponding retention time, and obtaining a linear range curve formula of the standard concentration of the sweetener according to calibration data, as shown in table 1:
and Ci is Kx + a, wherein K is a coefficient, the types of the sweeteners are different, K is different, a is a constant, and i is the type of the sweetener.
Table 17 sweetener standard linear equations, correlation coefficients, detection limits and quantitation limits
3) Sample extraction
Selecting a sample to be detected, adding ultrapure water with a set amount, then carrying out mechanical oscillation, standing for a set time, sucking supernatant, filtering by using a 0.22 mu m water-phase filter membrane, and carrying out chromatographic analysis on the sample to be detected.
And (3) carrying out HPLC-ELSD detection on the sample solution to be detected in the steps (1) and (3) and the prepared mixed standard working solution with different concentrations, and carrying out qualitative and quantitative analysis on the types and the contents of the sweetening agents in the tipping paper for cigarettes, the oral smokeless tobacco products, the essence for cigarettes, the bead blasting for cigarettes and the electronic cigarette solution samples through a map.
During detection and analysis, the specific reference settings of the instrument parameters are as follows:
HPLC detection parameters:
a chromatographic column: ZORBAX SB-C18(4.6 mm. times.250 mm,5 μm); column temperature: 25 ℃; sample introduction amount: 10 mu L of the solution; flow rate: 0.8 mL/min; mobile phase: a is ultrapure water, B is methanol (with 0.01% trifluoroacetic acid added);
ELSD detection parameters:
atomization temperature: 100 ℃; evaporation temperature: 100 ℃; atomizing gas: air; gas flow rate: 1.7L/min.
TABLE 2 mobile phase gradient elution conditions
The standard recovery and precision of the 7 sweeteners are shown in Table 3.
In the following examples of the present application, the preparation of the standard working solution is the same as the preparation of the standard working solution described above, and a description thereof will not be repeated.
For HPLC-DAD measurement analysis, the above-mentioned chromatographic calibration and specific reference setup for instrument parameters are included, and will not be described repeatedly below.
In the following examples of the present application, the 7 sweeteners used in the preparation of the standard working solution, methanol, were all chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.
The apparatus used in the following examples of the present application:
high performance liquid chromatography (Agilent 1260Infinity LC, Agilent inc.); an evaporative light scattering detector (Alltech ELSD 2000ES, Onta technologies, Inc. USA); hukang centrifuge (TG16-WS, Hunan Hukang centrifuge Limited); MS303S electronic balance (sensory 0.0001g, Mettler Toledo, Switzerland); a full temperature oscillator (HZQ-F160, Experimental facilities in Large warehouse); ultrasonic extractor (SB-3200DT, Ningbo Xinzhi Biotechnology Co., Ltd.).
Example 1
1. Instruments and reagents
7 kinds of sweetening agents and methanol are chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.
2. Extraction of samples
Weighing a proper amount of tipping paper sample, adding a proper amount of ultrapure water after pretreatment, mechanically oscillating and extracting, standing, taking a proper amount of extract liquid, passing through a 0.22 mu m organic filter membrane, and performing HPLC-ELSD analysis.
3. Preparation of Standard working solutions
4. HPLC-ELSD assay.
5. The amount of each sweetener in the sample was calculated according to formula (1):
Xi＝(Ci×V)÷M (1)
in the formula: xi is the content of sweetener i in the sample (μ g/g); ci is the concentration of sweetener i in the sample (μ g/mL) from the standard working curve; v is the volume of extract (mL); m is the sample mass (g).
The extracted sample was assayed, in which neotame was only detected in the sample at 329.96. mu.g/g.
Example 2
Another sample of tipping paper for cigarettes was selected as described in example 1, and 3 sweeteners acesulfame K, sucralose and neotame were detected in the sample at 167.35 μ g/g, 219.12 μ g/g and 193.34 μ g/g, respectively.
In the following examples, the steps of the detection method are the same, the calculation of the content of each sweetener in the sample is the same, and the difference is only that the sample is different, so that the following examples are not repeated and only the difference is listed.
Example 3
Extraction of gum-based tobacco samples
Weighing a proper amount of gum-based tobacco sample, adding a proper amount of extract liquid after pretreatment, carrying out ultrasonic oscillation extraction, sucking a proper amount of extract liquid, filtering the extract liquid through a 0.22 mu m water-phase filter membrane into a chromatographic bottle, and carrying out HPLC-ELSD analysis on the extract liquid serving as a sample to be detected.
The extracted sample was assayed, wherein only acesulfame K was detected in the sample at a content of 172.05. mu.g/g.
Example 4
Extraction of bagged buccal cigarette sample
Weighing a proper amount of buccal tobacco sample, adding a proper amount of extract liquid after pretreatment, carrying out ultrasonic oscillation, sucking a proper amount of extract liquid, filtering the extract liquid through a 0.22 mu m aqueous phase filter membrane to a chromatographic bottle, and carrying out HPLC-ELSD analysis as a sample to-be-detected liquid.
The extracted sample was measured, wherein only acesulfame K was detected in the sample at 203.15. mu.g/g.
Example 5
Extraction of smoke-containing samples
Weighing a proper amount of tobacco-containing sample, pretreating, adding a proper amount of extract liquid, carrying out ultrasonic oscillation, sucking a proper amount of extract liquid, filtering the extract liquid through a 0.22 mu m water-phase filter membrane, and carrying out HPLC-ELSD analysis on the extract liquid serving as a sample to be detected.
The extracted sample was assayed in which only aspartame was detected at a content of 209.85. mu.g/g.
Example 6
Extraction of bead blasting sample for cigarette
Taking a proper amount of bead blasting samples for cigarettes, adding a proper amount of extract liquid after pretreatment, carrying out ultrasonic oscillation, sucking a proper amount of extract liquid, filtering the extract liquid through a 0.22 mu m water-phase filter membrane to a chromatographic bottle, and carrying out HPLC-ELSD analysis on the extract liquid serving as a sample to be detected.
And (3) determining the extracted sample, wherein the content of the acesulfame potassium, the aspartame and the neotame in the sample is 180.04 mu g/g, 212.01 mu g/g and 111.02 mu g/g respectively.
Example 7
Extraction of tobacco flavor sample
Weighing a proper amount of essence for cigarettes, pre-treating, filtering supernatant liquid by a 0.22 mu m water-phase filter membrane into a chromatographic bottle, and performing HPLC-ELSD analysis as a sample to-be-detected liquid.
And (3) determining the extracted sample, wherein the content of the acesulfame-K, the alitame and the neotame in the sample is 211.42 mu g/g, 219.20 mu g/g and 188.72 mu g/g respectively.
Example 8
Another tobacco flavor sample was selected as described in example 6, and the amounts of sucralose, mogroside V, and aspartame 3 sweeteners were detected at 256.02 μ g/mL, 176.43 μ g/mL, and 183.15 μ g/mL, respectively.
Example 9
Extraction of electronic cigarette liquid sample
Weighing a proper amount of electronic cigarette liquid sample, pre-treating, filtering supernatant liquid through a 0.22 mu m water-phase filter membrane into a chromatographic bottle, and performing HPLC-DAD analysis as a sample to-be-detected liquid.
The extracted sample was measured, wherein only acesulfame K was detected in the sample at 181.35. mu.g/g.
Example 10
Another e-liquid sample was selected as described in example 9, and acesulfame-K and neotame were detected in the sample at 146.32 μ g/mL and 179.59 μ g/mL, respectively.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A method for measuring a sweetening agent for cigarettes by using a liquid chromatography-evaporative light scattering detector is characterized by comprising the following steps of:
1) preparation of standard working solution:
weighing each sweetener standard substance, performing constant volume by using ultrapure water, and preparing a mixed standard working solution with a concentration gradient;
2) and (3) carrying out chromatography calibration:
analyzing the mixed standard working solution prepared in the step 1) by using a chromatograph to obtain corresponding retention time, and obtaining standard concentration data of the sweetener according to calibration data;
3) and (3) extracting a sample:
selecting a sample to be detected, adding a set amount of extractant, then carrying out mechanical oscillation, standing for a set time, absorbing supernatant, filtering by using a water-phase filter membrane, and carrying out chromatographic analysis on the sample to be detected as a sample solution;
4) liquid chromatography-evaporative light scattering detector analysis
Detecting the to-be-detected sample solution and the mixed standard working solution in the steps 1) and 3) by using a liquid chromatogram-evaporative light scattering detector;
wherein the parameters of the liquid chromatogram are as follows:
a chromatographic column: ZORBAX SB-C18 with specification of 4.6mm × 250mm,5 μm; column temperature: 25 ℃; sample introduction amount: 10 mu L of the solution; flow rate: 0.8 mL/min; mobile phase: a is ultrapure water, B is methanol, and 0.01% trifluoroacetic acid is added;
parameters of the evaporative light scattering detector were:
atomization temperature: 100 ℃; evaporation temperature: 100 ℃; atomizing gas: air; gas flow rate: 1.7L/min.
2. The method for measuring the sweetener for cigarettes by using the liquid chromatography-evaporative light scattering detector as claimed in claim 1, wherein the content of the sweetener in the sample is calculated according to the following formula:
using the standard concentration data of the sweetener in the step 2) to obtain a linear range curve formula of the sweetener:
and Ci is Kx + a, wherein K is a coefficient, the types of the sweeteners are different, K is different, a is a constant, and i is the type of the sweetener.
3. The method for detecting the sweetener for cigarettes by using the liquid chromatography-evaporative light scattering detector as claimed in claim 2, wherein the sweetener is one or more of acesulfame potassium, aspartame, neotame, sucralose, alitame, mogroside V and stevioside.
4. The method for measuring a sweetener for cigarette according to claim 3, wherein when the sweetener is acesulfame K, K is 5X 10-5A is-14.085;
when the sweetener is sucralose, the value of K is 2X 10-5A is 20.852;
when the sweetener is aspartame,k has a value of 2X 10-5A is 35.957;
when the sweetener is alitame, the value of K is 2 x 10-5A is 23.246;
when the sweetener is mogroside V, K has a value of 2 × 10-5A is 21.932;
when the sweetener is neotame, the value of K is 1X 10-5A is 28.422;
when the sweetener is stevioside, K has a value of 2 × 10-5And a is 23.115.
5. The method for measuring the sweetener for cigarettes by using the liquid chromatography-evaporative light scattering detector as claimed in claim 1, wherein the extractant is ultrapure water.
6. The method for detecting the sweetener for cigarettes by using the liquid chromatography-evaporative light scattering detector as claimed in claim 1, wherein the water-phase filter membrane is a 0.22 μm water-phase filter membrane.
7. The method for detecting the sweetener for cigarettes by using the liquid chromatography-evaporative light scattering detector as claimed in claim 4, wherein the sweetener in the sample is calculated according to the following formula:
xi ═ (Ci × V) ÷ M, where Xi is the amount of sweetener i in the sample, in μ g/g; ci is the concentration of the sweetener i in the sample obtained from the standard working curve, and the unit is mu g/mL; v is the volume of the extractant, and the unit is mL; m is the sample mass, and the unit g, i is the sweetener type.
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| CN115015453A (en) * | 2022-06-15 | 2022-09-06 | 河南中烟工业有限责任公司 | Pretreatment method for detecting sweetener in oral smokeless tobacco product |
| CN116165304A (en) * | 2023-03-20 | 2023-05-26 | 红塔辽宁烟草有限责任公司 | Method for detecting 8 sweeteners in tipping paper for cigarettes by using ultra-high performance liquid chromatography-tandem mass spectrometry |
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