CN110763797A - Method for measuring sweetening agent for cigarettes by high performance liquid chromatography - Google Patents
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Abstract
The invention relates to a method for measuring a sweetening agent for cigarettes by high performance liquid chromatography, which adopts a pretreatment method of solvent extraction in the technical scheme, establishes a detection and analysis method for simultaneously measuring 5 sweetening agents such as acesulfame, aspartame, neotame, alitame, saccharin sodium and the like in tipping paper for cigarettes, oral smokeless tobacco products, essence for cigarettes, blasting beads for cigarettes and electronic cigarette liquid by HPLC-DAD, and detects partial samples by utilizing 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 physicochemical inspection of residual food additives in tobacco materials and tobacco products, and mainly relates to a method for measuring 5 sweetening agents in tobacco tipping paper, oral smokeless tobacco products, tobacco essence, tobacco blasting beads and electronic tobacco juice, in particular to a method for directly measuring by adopting a high performance liquid chromatography-diode array method 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 agent comprises stevioside, momordica grosvenori glucoside V and the like, the common artificial sweetening agent mainly comprises acesulfame potassium, saccharin sodium, alitame, sodium cyclamate, sucralose and the like, and the sweetness of the sweetening agent 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 and 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 high performance liquid chromatography, 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 sweetening agent for cigarettes by high performance liquid chromatography comprises the following steps:
1) preparation of standard working solution:
weighing a standard substance of the sweetener to be detected, using ultrapure water to perform constant volume, 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 a linear range curve formula of the standard concentration of the sweetener according to calibration data:
ci is Kx + a, wherein K is a coefficient, the types of the sweeteners are different, K values are different, a is a constant, and i is the type of the sweetener;
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) the sweetener content 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 sweetener includes but is not limited to one or more of acesulfame potassium, aspartame, neotame, alitame or saccharin sodium.
In the step 2), the step (c) is carried out,
when the sweetener is acesulfame K, the value of K is 5 × 10-5A is-14.085;
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 10-5，aIs 23.246;
when the sweetener is neotame, the value of K is 1X 10-5A is 28.422;
when the sweetener is saccharin sodium, 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 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 5 sweetening agents such as acesulfame, aspartame, neotame, alitame, saccharin sodium and the like in cigarette tipping paper, oral smokeless tobacco products, essence for cigarettes, bead blasting for cigarettes and electronic cigarette liquid by using HPLC-DAD, and detects partial 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.
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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 5 sweeteners.
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.
The technical scheme mainly comprises a method for detecting 5 sweetening agents in tipping paper for cigarettes, oral smokeless tobacco products, essence for cigarettes, bead blasting for cigarettes and electronic cigarette liquid through a high performance liquid chromatography-diode array detector, wherein the 5 sweetening agents are acesulfame potassium 1, aspartame 2, neotame 3, alitame 4 and saccharin sodium 5 respectively.
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:
respectively weighing 0.050g (accurate to 0.1mg) of each of 5 sweetener standard substances, placing in a 50mL volumetric flask, performing constant volume to scale with ultrapure water, shaking up to obtain a mixed standard solution with the concentration of each target compound being 1mg/mL, and taking the mixed standard solution as a 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 15 sweetener Standard Linear equation, correlation coefficient, 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-DAD 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 cigarette tipping paper, the mouth-use smokeless tobacco products, the cigarette essence, the cigarette blasting beads 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-DAD conditions:
a chromatographic column: poroshell 120EC-C18(3 mm. times.150 mm,2.7 μm); column temperature: 30 ℃; sample introduction amount: 5 mu L of the solution; flow rate: 0.3 mL/min; mobile phase: a is 0.01% acetic acid water solution, B is methanol; diode array detector 210 nm; mobile phase gradient elution conditions are shown in table 2:
TABLE 2 mobile phase gradient elution conditions
The recovery and precision of the 5 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.
Example 1
1. Instruments and reagents
5 kinds of sweetening agents and methanol are chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.
High performance liquid chromatography (Agilent 1260Infinity LC, Agilent inc.); diode array detector (Agilent 1260Infinity DAD, Agilent Inc. USA); hukang centrifuge (TG16-WS, Hunan Hukang centrifuge Limited); MS303S electronic balance (sensory 0.0001g, Mettler Toledo, Switzerland); full temperature oscillator (HZQ-F160, Experimental facilities in Large warehouse).
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 carrying out HPLC-DAD analysis.
3. Preparing a standard working solution; the preparation method is shown in the standard working solution preparation part and is not repeated here.
4. HPLC-DAD 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 337.23. mu.g/g.
Example 2
1. Instruments and reagents
5 kinds of sweetening agents and methanol are chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.
High performance liquid chromatography (Agilent 1260Infinity LC, Agilent inc.); diode array detector (Agilent 1260Infinity DAD, Agilent Inc. USA); ultrasonic extractor (SB-3200DT, Ningbo Xinzhi Biotechnology Co., Ltd.).
2. 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-DAD analysis on the extract liquid serving as a sample to be detected.
3. Preparation of Standard working solutions
4. HPLC-DAD 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 measured, wherein only acesulfame K was detected in the sample at 168.86. mu.g/g.
Example 3
1. Instruments and reagents
5 kinds of sweetening agents and methanol are chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.
High performance liquid chromatography (Agilent 1260Infinity LC, Agilent inc.); diode array detector (Agilent 1260Infinity DAD, Agilent Inc. USA); ultrasonic extractor (SB-3200DT, Ningbo Xinzhi Biotechnology Co., Ltd.).
2. 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-DAD analysis as a sample to-be-detected liquid.
3. Preparation of Standard working solutions
4. HPLC-DAD 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 measured, wherein only acesulfame K was detected in the sample at 201.06. mu.g/g.
Example 4
1. Instruments and reagents
5 kinds of sweetening agents and methanol are chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.
High performance liquid chromatography (Agilent 1260Infinity LC, Agilent inc.); diode array detector (Agilent 1260Infinity DAD, Agilent Inc. USA); ultrasonic extractor (SB-3200DT, Ningbo Xinzhi Biotechnology Co., Ltd.).
2. 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 by using a 0.22 mu m water-phase filter membrane, and carrying out HPLC-DAD analysis on the sample liquid to be detected.
3. Preparation of Standard working solutions
4. HPLC-DAD 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 only aspartame was detected at a content of 215.03. mu.g/g.
Example 5
1. Instruments and reagents
5 kinds of sweetening agents and methanol are chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.
High performance liquid chromatography (Agilent 1260Infinity LC, Agilent inc.); diode array detector (Agilent 1260Infinity DAD, Agilent Inc. USA); ultrasonic extractor (SB-3200DT, Ningbo Xinzhi Biotechnology Co., Ltd.).
2. Extraction of samples
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-DAD analysis on the extract liquid serving as a sample to be detected.
3. Preparation of Standard working solutions
4. HPLC-DAD 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).
And (3) determining the extracted sample, wherein the content of the acesulfame potassium, the aspartame and the neotame in the sample is 184.75 mu g/g, 201.05 mu g/g and 102.57 mu g/g respectively.
Example 6
1. Instruments and reagents
5 kinds of sweetening agents and methanol are chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.
High performance liquid chromatography (Agilent 1260Infinity LC, Agilent inc.); diode array detector (Agilent 1260Infinity DAD, Agilent Inc. USA); hukang centrifuge (TG16-WS, Hunan Hukang centrifuge Limited); MS303S electronic balance (sensory 0.0001g, Mettler Toledo, Switzerland); full temperature oscillator (HZQ-F160, Experimental facilities in Large warehouse).
2. Extraction of samples
Weighing a proper amount of tobacco essence, pre-treating, filtering supernatant liquid by 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.
3. Preparation of Standard working solutions
4. HPLC-DAD 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).
And (3) determining the extracted sample, wherein the content of the acesulfame potassium, the saccharin sodium, the alitame and the neotame in the sample is 212.36 mu g/g, 196.86 mu g/g, 234.69 mu g/g and 186.07 mu g/g respectively.
Example 7
1. Instruments and reagents
5 kinds of sweetening agents and methanol are chromatographic grade reagents; meets the requirement of first-grade water in GB/T6682.
High performance liquid chromatography (Agilent 1260Infinity LC, Agilent inc.); diode array detector (Agilent 1260Infinity DAD, Agilent Inc. USA); hukang centrifuge (TG16-WS, Hunan Hukang centrifuge Limited); MS303S electronic balance (sensory 0.0001g, Mettler Toledo, Switzerland).
2. Extraction of samples
Weighing a proper amount of sample, pre-treating, sucking supernatant, filtering by a 0.22 mu m water-phase filter membrane into a chromatographic bottle, and performing HPLC-DAD analysis as a sample to-be-detected solution;
3. preparation of Standard working solutions
4. HPLC-DAD assay analysis
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 measured, wherein only acesulfame K was detected in the sample at 196.36. mu.g/g.
Example 8
Another e-liquid sample was selected as described in example 7, and acesulfame-K and neotame were detected in the sample at 156.42 μ g/mL and 182.48 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 (5)
1. A method for measuring a sweetening agent for cigarettes by high performance liquid chromatography is characterized by comprising the following steps:
1) preparation of standard working solution:
weighing a standard substance of the sweetener to be detected, using ultrapure water to perform constant volume, 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 a linear range curve formula of the standard concentration of the sweetener according to calibration data:
ci is Kx + a, wherein K is a coefficient, the types of the sweeteners are different, K values are different, a is a constant, and i is the type of the sweetener;
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) the sweetener content 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.
2. The method for measuring the sweetening agent for the cigarettes by using the high performance liquid chromatography as claimed in claim 1, wherein the sweetening agent comprises but is not limited to one or more of acesulfame potassium, aspartame, neotame, alitame or saccharin sodium.
3. The method for measuring the sweetening agent for the cigarettes by using the high performance liquid chromatography as claimed in claim 1, wherein in the step 2):
when the sweetener is acesulfame K, the value of K is 5 × 10-5A is-14.085;
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 neotame, the value of K is 1X 10-5A is 28.422;
when the sweetener is saccharin sodium, K has a value of 2 × 10-5And a is 23.115.
4. The method for measuring the sweetening agent for the cigarettes by using the high performance liquid chromatography as claimed in claim 1, wherein the extracting agent is ultrapure water.
5. The method for measuring the sweetening agent for the cigarettes by using the high performance liquid chromatography as claimed in claim 1, wherein the water phase filter membrane is a 0.22 μm water phase filter membrane.
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| CN113281288A (en) * | 2021-05-18 | 2021-08-20 | 福建中烟工业有限责任公司 | Pretreatment method of tipping paper for cigarettes and method for measuring saccharin sodium in tipping paper for cigarettes |
| CN113281447A (en) * | 2021-05-18 | 2021-08-20 | 福建中烟工业有限责任公司 | Extraction device, determination method and system for saccharin sodium in paper surface layer and application |
| CN113281287A (en) * | 2021-05-18 | 2021-08-20 | 福建中烟工业有限责任公司 | Paper surface layer pretreatment method and method for measuring content of saccharin sodium in paper surface layer |
| 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 |
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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 |
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