CN110174367B - Method for measuring content of free nicotine and application of probe compound - Google Patents
Method for measuring content of free nicotine and application of probe compound Download PDFInfo
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- CN110174367B CN110174367B CN201910552396.3A CN201910552396A CN110174367B CN 110174367 B CN110174367 B CN 110174367B CN 201910552396 A CN201910552396 A CN 201910552396A CN 110174367 B CN110174367 B CN 110174367B
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- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 title claims abstract description 96
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 title claims abstract description 92
- 229960002715 nicotine Drugs 0.000 title claims abstract description 92
- 239000000523 sample Substances 0.000 title claims abstract description 78
- 150000001875 compounds Chemical class 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 29
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 35
- 239000002904 solvent Substances 0.000 claims abstract description 33
- 241000208125 Nicotiana Species 0.000 claims abstract description 28
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims abstract description 16
- 235000019505 tobacco product Nutrition 0.000 claims abstract description 15
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000002835 absorbance Methods 0.000 claims abstract description 9
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 40
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 37
- 239000007788 liquid Substances 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 8
- 230000003287 optical effect Effects 0.000 claims description 3
- 238000004445 quantitative analysis Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims 2
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 abstract description 8
- 238000001514 detection method Methods 0.000 abstract description 6
- RTZZCYNQPHTPPL-UHFFFAOYSA-N 3-nitrophenol Chemical compound OC1=CC=CC([N+]([O-])=O)=C1 RTZZCYNQPHTPPL-UHFFFAOYSA-N 0.000 abstract description 5
- RBXVOQPAMPBADW-UHFFFAOYSA-N nitrous acid;phenol Chemical class ON=O.OC1=CC=CC=C1 RBXVOQPAMPBADW-UHFFFAOYSA-N 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 2
- 239000003571 electronic cigarette Substances 0.000 description 17
- 238000000605 extraction Methods 0.000 description 15
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 9
- 244000061176 Nicotiana tabacum Species 0.000 description 8
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 8
- 239000012452 mother liquor Substances 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 7
- 239000000284 extract Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 6
- 239000012086 standard solution Substances 0.000 description 6
- 235000019504 cigarettes Nutrition 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000005711 Benzoic acid Substances 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 235000010233 benzoic acid Nutrition 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000000391 smoking effect Effects 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 125000003158 alcohol group Chemical group 0.000 description 2
- HFVAFDPGUJEFBQ-UHFFFAOYSA-M alizarin red S Chemical compound [Na+].O=C1C2=CC=CC=C2C(=O)C2=C1C=C(S([O-])(=O)=O)C(O)=C2O HFVAFDPGUJEFBQ-UHFFFAOYSA-M 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- DUEPRVBVGDRKAG-UHFFFAOYSA-N carbofuran Chemical compound CNC(=O)OC1=CC=CC2=C1OC(C)(C)C2 DUEPRVBVGDRKAG-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000012733 comparative method Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 239000000779 smoke Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000027 toxicology Toxicity 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 1
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 1
- 244000020518 Carthamus tinctorius Species 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 208000021063 Respiratory fume inhalation disease Diseases 0.000 description 1
- 241000208292 Solanaceae Species 0.000 description 1
- 241000710959 Venezuelan equine encephalitis virus Species 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000021028 berry Nutrition 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001055 chewing effect Effects 0.000 description 1
- 235000019506 cigar Nutrition 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000003595 mist Substances 0.000 description 1
- 210000002200 mouth mucosa Anatomy 0.000 description 1
- 238000001139 pH measurement Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000002470 solid-phase micro-extraction Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to the field of detection and analysis, and particularly relates to a method for determining the content of free nicotine, which comprises the following steps: 1) reacting free nicotine in a sample with a probe compound in a solvent to produce a reaction product; wherein the probe compound is selected from nitrophenols (e.g., p-nitrophenol, m-nitrophenol) and alizarin red; 2) and detecting the reaction product by using an ultraviolet-visible spectrophotometer, and calculating the content of free nicotine in the sample according to the absorbance value of the wavelength of 380-790 nm. The invention also relates to the use of the probe compounds. The measuring method of the invention has low cost and can quickly and accurately measure the content of the free nicotine in the tobacco and/or the tobacco products.
Description
Technical Field
The invention belongs to the field of detection and analysis, and particularly relates to a method for determining the content of free nicotine, and application of a probe compound in detection of free nicotine.
Background
Nicotine is a peculiar alkaloid in tobacco and is also a main index for evaluating tobacco products. Nicotine exists in tobacco products mainly in free form and in proton form, wherein the proton form is the proton nicotine, and the non-protonized nicotine is the free nicotine (called free nicotine). In 1970, Armitage et al (Nature, 1970,226(5252): 1231-1232) found that free nicotine is more lipophilic than protic nicotine and more readily penetrates the oral mucosa and is rapidly absorbed by the body. Tobacco chemists have also carried out a great deal of work on the delivery of free nicotine [ Tobacco Control,2006,15, 189-. The research shows that: the content of free nicotine has obvious linear relation with the smoking quality of the cigarettes, and has strong addiction. In 2013, PAX Labs uses the concept of nicotine salt in electronic cigarettes, and uses the nicotine salt in Juul which is a product of the company so as to provide 2-3 times of physiological strength and good mouthfeel, and then the use of the nicotine salt is gradually increased. The use of nicotine salts greatly reduces the irritation of the smoke inhalation process by reducing the free nicotine content of the tobacco product.
A large amount of development work on free nicotine detection methods has been carried out, mainly including: (1) pH measurement method, in which the content of free nicotine is obtained by measuring the pH value of the cigarette smoke and indirectly calculating by using the relationship between the pH value and the nicotine acid-base equilibrium constant [ Tobacco Science,1972 (16): 56]The method is simple and convenient but is limited by the sensitivity of a pH meter, and the content data of free nicotine cannot be directly obtained; (2) gas chromatography: using the solubility difference between the aqueous phase and the organic phase of the protonic nicotine and the free nicotine, gas chromatography was used to determine the free nicotine more inclined to the organic phase [ tobacco science, 2003, 6-10; chemical Research in biology, 2015,28,1532-1537]Or the fiber head is extracted by solid phase micro extraction to enrich the free nicotine by utilizing the obvious difference of the volatility of the protonic nicotine and the free nicotine, so as to achieve the aim of respectively measuring [ Analytica Chimica Acta,663(2010), 49-54; journal of Hunan university (Nature science edition), 38(11),70-75](ii) a (3) Liquid chromatography: determination of absolute nicotine and ion in samples using anion exchange/cation exchange/reverse chromatography triple liquid chromatography stationary phase and phosphate buffer mobile phaseThe content of [ LcGc North America,29(2):52-52]However, this method does not reflect the presence of nicotine in the sample; (4) a nuclear magnetic method: the method uses a nuclear magnetic spectrometer to determine the content of nicotine in a sample1H-NMR chemical shifts with nicotine in free and single molecular form1The proportion of free nicotine in the sample is known by comparison with H-NMR. This method most truly reflects the proportion of free nicotine present in the sample.
In summary, although many methods have been developed for measuring free nicotine and all reflect the relative size of free nicotine in a sample to some extent, they require the use of large amounts of solvents or long extraction and separation processes;1H-NMR can reduce the real existing form of free nicotine in a sample, but is limited by expensive instruments and reagents in nuclear magnetic experiments, so that the content information of the free nicotine is difficult to obtain quickly in the production process.
Therefore, a low-cost method for quickly and accurately measuring the content of free nicotine in tobacco or tobacco products is established, the content of the free nicotine in the tobacco products is objectively and truly reflected, and the method has guiding significance on aspects such as cigarette formula design, novel product development and quality control.
Disclosure of Invention
The invention provides a method for measuring the content of free nicotine, which has low cost and can quickly and accurately measure the content of the free nicotine in tobacco and/or tobacco products (such as electronic cigarette oil). In addition, the invention also provides application of probe compounds nitrophenol (such as p-nitrophenol and m-nitrophenol) and/or alizarin red in detection of free nicotine.
One aspect of the invention relates to a method for determining the content of free nicotine, comprising the steps of:
1) reacting free nicotine in a sample with a probe compound in a solvent to generate a reaction product; wherein the probe compound is selected from nitrophenols (e.g., p-nitrophenol, m-nitrophenol) and alizarin red;
2) detecting the reaction product by using an ultraviolet-visible spectrophotometer, and calculating the content of the free nicotine in the sample according to the absorbance values of 380-790 nm (such as 390nm, 400nm, 419nm, 430nm, 450nm, 480nm, 500nm, 520nm, 540nm, 560nm, 580nm, 600nm, 630nm, 650nm, 670nm, 700nm, 720nm, 740nm, 760nm and 780 nm).
In some embodiments of the first aspect of the present invention, in step 1), the solvent is selected from alcohol and water, preferably a mixture of alcohol and water; more preferably, the alcohol is selected from the group consisting of ethanol, methanol, isopropanol, and n-propanol.
In some embodiments of the first aspect of the present invention, in step 1), the volume ratio of the alcohol to the water in the solvent is 1 (1 to 13), such as 1:2, 1:3, 1:4, 1:6, 1:8, 1:10, 1: 12.
In some embodiments of the first aspect of the present invention, the method comprises the following steps (1) to (2) and optionally (1-1):
(1-1) mixing a sample with a solvent to obtain a first mixture;
(1) mixing the sample or the first mixture with a probe compound or a solution thereof to obtain a second mixture; wherein the probe compound is selected from nitrophenols (e.g., p-nitrophenol, m-nitrophenol) and alizarin red;
(2) detecting the second mixture with an ultraviolet-visible spectrophotometer, and calculating the free nicotine content in the sample based on absorbance values at wavelengths of 400-480 nm (e.g., 410nm, 419nm, 430nm, 440nm, 450nm, 460nm, 470 nm) or 500-600 nm (e.g., 510nm, 519nm, 530nm, 540nm, 550nm, 570nm, 580nm, 590 nm).
In some embodiments of the first aspect of the present invention, in step (1-1), the solvent is selected from alcohol and water, preferably a mixture of alcohol and water; more preferably, the alcohol is selected from the group consisting of ethanol, methanol, isopropanol, and n-propanol.
In some embodiments of the first aspect of the present invention, in the step (1-1), the volume ratio of the alcohol to the water in the solvent is 1 (1-13), such as 1:2, 1:3, 1:4, 1:6, 1:8, 1:10, 1: 12.
In certain embodiments of the first aspect of the present invention, in step 1) or step (1), the reaction temperature or mixing temperature is from 15 ℃ to 35 ℃, e.g., 18 ℃,20 ℃, 23 ℃, 25 ℃, 26 ℃,28 ℃, 30 ℃, 32 ℃, 34 ℃.
In some embodiments of the first aspect of the present invention, in step 1) or step (1), the reaction time or mixing time is 0.5-10 min, such as 1min, 2min, 3min, 4min, 6min, 7min, 8min, 9 min.
In some embodiments of the first aspect of the invention, the molar ratio of probe compound to nicotine in the sample in step 1) or step (1) is ≧ 1, e.g., 1.1, 1.2, 1.5, 1.8, 2, 3,4, 5, 6, 7, 8, 10.
In some embodiments of the first aspect of the present invention, in step (1), the molar ratio of probe compound to nicotine in the first mixture is ≧ 1, e.g., 1.1, 1.2, 1.5, 1.8, 2, 3,4, 5, 6, 7, 8, 10.
In some embodiments of the first aspect of the present invention, in step (1), the solvent used for the probe compound solution is selected from the group consisting of alcohol and water, preferably a mixture of alcohol and water;
preferably, the alcohol is selected from the group consisting of ethanol, methanol, isopropanol, and n-propanol.
In some embodiments of the first aspect of the present invention, in step (1), the probe compound solution is used in a solvent in which the volume ratio of alcohol to water is 1 (1 to 13), for example, 1:2, 1:3, 1:4, 1:6, 1:8, 1:10, or 1: 12.
In some embodiments of the first aspect of the present invention, the sample is selected from tobacco and tobacco products, preferably electronic cigarette oil.
In some embodiments of the first aspect of the present invention, one or more of the following a to F:
A. in the step (1), the concentration of the probe compound solution is 10 to 500. mu. mol/L, such as 20. mu. mol/L, 40. mu. mol/L, 60. mu. mol/L, 80. mu. mol/L, 100. mu. mol/L, 120. mu. mol/L, 150. mu. mol/L, 180. mu. mol/L, 200. mu. mol/L, 230. mu. mol/L, 260. mu. mol/L, 280. mu. mol/L, 300. mu. mol/L, 320. mu. mol/L, 350. mu. mol/L, 370. mu. mol/L, 400. mu. mol/L, 420. mu. mol/L, 450. mu. mol/L, 480. mol/L, 500. mu. mol/L;
B. in the step 2) or the step (2), the optical distance detected by the ultraviolet-visible spectrophotometer is 1-100 mm, such as 5mm, 8mm, 10mm, 13mm, 16mm, 20mm, 24mm, 25mm, 28mm, 30mm, 35mm, 40mm, 45mm, 50mm, 55mm, 60mm, 65mm, 70mm, 75mm, 80mm, 85mm, 90 mm;
C. in the step 2) or the step 2, the scanning step of the ultraviolet-visible spectrophotometer is 0.1-0.5 nm, such as 0.2nm, 0.3nm and 0.4 nm;
D. in the step 2) or the step (2), the scanning speed of the ultraviolet-visible spectrophotometer is 180-300 nm/min, such as 200nm/min, 220nm/min, 250nm/min, 270nm/min, 290nm/min and 300 nm/min;
E. in the step 2) or the step (2), calculating by adopting an external standard quantitative method;
F. in the step (1-1), the volume ratio of the sample to the solvent is 1 (2-20), such as 1:3, 1:5, 1:7, 1:10, 1:12, 1:14, 1:16, 1: 18.
A second aspect of the invention relates to the use of a probe compound for the detection of free nicotine; wherein the probe compound is selected from nitrophenols (e.g., p-nitrophenol, m-nitrophenol) and alizarin red.
In some embodiments of the second aspect of the invention, the use is the use of determining the free nicotine content in a sample.
In some embodiments of the second aspect of the present invention, the sample is tobacco and/or a tobacco product, preferably e-liquid.
In the present invention, the E-liquid is an E-liquid conventionally used in the art, for example, an E-liquid commercially available from commercial sources.
In some embodiments of the invention, one or more of the following e-liquid is used:
1) electronic cigarette oil with the brand number Juul Cr (r) brubee produced by Juul Labs;
2) e-liquid Tobacco available from British American Tobacco under the brand name Vype Wild Berries;
3) e-liquid Tobacco manufactured by Phillip Morris corporation under the trademark VEEV Mellow Tobacco;
4) chinese style flue-cured tobacco electronic tobacco tar produced by tobacco industry Limited liability company in Fujian province.
In one embodiment of the invention, the conventional Chinese style flue-cured tobacco electronic tobacco tar comprises nicotine, acid, tobacco extract, glycerol and propylene glycol, and is preferably prepared by mixing the components according to a conventional ratio.
In the invention, the CAS number of alizarin red is 130-22-3, and the molecular formula is C14H7NaO7S, the chemical name is 3, 4-dihydroxy-9, 10-dioxo-2-anthracene sulfonic acid sodium salt, and the structural formula is
In the present invention, tobacco belongs to the genus Nicotiana (Nicotiana Tabacum L.) of the family Solanaceae, and is a perennial or perennial herb, favorite crop. There are more than 60 species of this genus, most of which are wild species. The main cultivated species are safflower tobacco and yellow flower tobacco.
In the present invention, the tobacco products refer to the hobby consumption products made of tobacco as raw material, and are classified into smoking tobacco products (including cigarettes, cigars, tobacco shreds for smoking, etc.) and non-smoking tobacco products (including electronic cigarettes, snuff, chewing tobacco, etc.).
In the invention, the electronic cigarette oil is also called electronic cigarette liquid, and is electronic atomized liquid used in cooperation with an electronic cigarette. By heating with an electronic smoke atomizer, a mist like a cigarette can be produced.
The invention has the following beneficial effects:
the method of the invention has low cost and can rapidly and accurately measure the content of the free nicotine in the tobacco and/or tobacco products (such as electronic cigarette oil).
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a UV-Vis spectrum of MNP probe solutions containing different nicotine concentrations in example 1 of the present invention;
FIG. 2 is a standard operating curve for example 1 of the present invention;
FIG. 3 is an appearance diagram of the mixture of solutions with different pH ratios and MNP probe solution in example 1 of the present invention;
FIG. 4 is a chart of UV-Vis spectra of ARS probe solutions containing different nicotine concentrations in example 2 of the present invention;
FIG. 5 is a standard operating curve for example 2 of the present invention;
FIG. 6 is an appearance diagram of the mixture of different pH ratio solutions and ARS probe solutions in example 2 of the present invention.
Detailed Description
Embodiments of the present invention will now be described more fully hereinafter with reference to the accompanying examples, in which some, but not all embodiments of the invention are shown. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following examples and comparative examples used the following materials:
nicotine: the purity is more than or equal to 99 percent, and the product is purchased from the national tobacco quality supervision and inspection center;
benzoic acid: purity 99.5%, purchased from carbofuran;
p-nitrophenol: the purity is more than or equal to 99 percent and the product is purchased from Sigma-Aldrich company;
alizarin red: purity not less than 90%, purchased from Sigma-Aldrich company;
ethanol: pure chromatography, purchased from carbofuran;
water: meets the requirement of first-grade water in GB/T6682-2008;
the following examples and comparative examples employ the following instruments and conditions:
lambda 35 ultraviolet-visible spectrophotometer of Perkin Elmer company, USA, with wavelength range of 200-700 nm, scanning step of 0.2nm, scanning speed of 240nm/min, and quartz cuvette optical path of 10 mm;
ML204 electronic balance, Toledo, miller, switzerland;
elmasonic S300 ultrasonic extractor from Elma, Germany.
Example 1
1. Preparation of an extraction solvent: 200mL of ethanol is transferred, water is added to the mixture to be constant volume of 1000mL, and the ethanol-water (20/80v/v) extraction solvent is obtained.
2. Preparing a probe solution: accurately weighing 0.0174g of p-nitrophenol (MNP), dissolving by using an extraction solvent and fixing the volume to 25mL to obtain MNP mother liquor with the concentration of 5.0 mmol/L. 4.0mL of MNP mother liquor is transferred and is subjected to constant volume to 100mL by using an extraction solvent, so as to obtain the MNP probe solution with the concentration of 200 mu mol/L.
3. Preparation of nicotine standard solution: accurately weighing 0.324g of nicotine, and diluting to 10.0mL by using an extraction solvent to obtain 0.2mol/L nicotine mother liquor. Taking 1.0mL of nicotine mother liquor, and diluting to 10mL by using an extraction solvent to obtain a nicotine standard solution with the concentration of 0.02 mol/L.
4. Drawing a standard working curve: adding nicotine standard solution into 2.0mL MNP probe solution to make the nicotine concentration in the probe solution be 0-200 mu mol/L, respectively drawing a spectrogram (figure 1) by using an ultraviolet-visible spectrophotometer, drawing a standard working curve (figure 2) by taking the absorbance value with the wavelength of 418nm to the free nicotine concentration, wherein the standard working curve is that y is 0.0549+0.0214 xx-3.1828 x10-5×x2，r2＝0.9985。
5. Appearance of mixing of solutions with different pH ratios with Probe solutions:
preparing different ethanol solutions from nicotine and benzoic acid according to a ratio from large to small, transferring 10 μ L of the prepared solutions respectively, and mixing in 2mL of MNP probe solution at 25 deg.C for 1min to obtain the appearance of each solution as shown in figure 3 (the nicotine and the benzoic acid are arranged according to a ratio from large to small).
6. Testing of E-liquid 1-4
Mixing 100 μ L of electronic cigarette oil 1-4 (manufacturer, brand name shown in Table 1) with 900 μ L of extraction solvent to obtain electronic cigarette oil extract 1-4. Transferring 10 mu L of the electronic tobacco tar extract 1-4 into 2mL of MNP probe solution, mixing for 1 minute at 25 ℃, then drawing a spectrogram by using an ultraviolet-visible spectrophotometer, calculating the content of free nicotine by adopting an external standard method according to the absorbance value with the wavelength of 418nm, wherein the content of the free nicotine in the electronic tobacco tar 1-4 is shown in Table 1.
TABLE 1 free nicotine content in E-liquid (MNP probe solution)
Note:acomparative methods reference the literature (Chemical Research in Toxicology 2015,28(8),1532-,bthe laboratory preparation of electronic cigarette oil is obtained by mixing nicotine, acid, tobacco extract, glycerin and propylene glycol according to conventional proportions.
As can be seen from Table 1, the method of the invention can rapidly and simply measure the content of free alkali in the electronic cigarette liquid, and the measurement result is accurate.
Example 2
1. Preparation of an extraction solvent: 100mL of ethanol is removed, water is added to the mixture to reach the constant volume of 1000mL, and the ethanol-water (10/90v/v) extraction solvent is obtained.
2. Preparing a probe solution: accurately weighing 0.0428g of Alizarin Red (ARS), dissolving by using an extraction solvent and fixing the volume to 25mL to obtain an ARS mother liquor with the concentration of 5.0 mmol/L. 4.0mL of ARS mother liquor is transferred and the volume is increased to 100mL by using the extraction solvent, thus obtaining the ARS probe solution with the concentration of 200 mu mol/L.
3. Preparation of nicotine standard solution: accurately weighing 0.324g of nicotine, and using an extraction solvent to fix the volume to 10.0mL to obtain 0.2mol/L nicotine mother liquor. Taking 1.0mL of nicotine mother liquor, and diluting to 10mL by using an extraction solvent to obtain a nicotine standard solution with the concentration of 0.02 mol/L.
4. Drawing a standard working curve: adding nicotine standard solution into 2.0mL ARS probe solution to make the nicotine concentration in the probe solution be 0-200 μmol/L, respectively drawing a spectrogram (figure 4) by using an ultraviolet-visible spectrophotometer, and drawing a standard working curve (figure 5) by taking the absorbance value with the wavelength of 519nm to the free nicotine concentration, wherein the standard working curve is that y is 0.0962-0.0012 xx +7.5869 x10-5×x2-2.4611×10-7×x3，r2＝0.9994。
5. Appearance of mixing of solutions with different pH ratios with Probe solutions:
different ethanol solutions were prepared from nicotine and benzoic acid at a decreasing ratio, and 10. mu.L of each prepared solution was transferred to 2mL of ARS probe solution and mixed at 25 ℃ for 1 minute to obtain solutions having the appearance shown in FIG. 6 (the solutions were arranged at decreasing ratios of nicotine and acid).
6. Testing of E-liquid 1-4
Mixing 100 μ L of electronic cigarette oil 1-4 (manufacturer, brand name shown in Table 2) with 900 μ L of extraction solvent to obtain electronic cigarette oil extract 1-4. Transferring 10 μ L of extract 1-4 respectively, mixing in 2mL ARS probe solution at 25 deg.C for 1min, drawing spectrogram with ultraviolet-visible spectrophotometer, calculating free nicotine content according to absorbance value with wavelength 519nm by external standard method, wherein free nicotine content in E-liquid 1-4 is shown in Table 2.
TABLE 2 free nicotine content in E-liquid (ARS probe solution)
Note:acomparative methods reference literature (Chemical Research in Toxicology 2015,28(8),1532-1537.) for determination of E-liquid free nicotine by GC-MS after liquid-liquid extraction;bthe laboratory preparation of electronic cigarette oil is obtained by mixing nicotine, acid, tobacco extract, glycerin and propylene glycol according to conventional proportions.
As can be seen from Table 2, the method of the invention can rapidly and simply measure the content of free alkali in the electronic cigarette liquid, and the measurement result is accurate.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications derived therefrom are intended to be within the scope of the invention.
Claims (10)
1. A method of determining free nicotine content comprising the steps of:
1) the sample is selected from tobacco and tobacco products, free nicotine in the sample reacts with the probe compound in a solvent, the reaction temperature is 15-35 ℃, and the reaction time is 0.5-10 min, so that a reaction product is generated; the probe compound is p-nitrophenol, and the solvent is a mixed solution of alcohol and water in a volume ratio of 1 (1-13);
2) and detecting the reaction product by adopting an ultraviolet-visible spectrophotometer, and calculating the content of free nicotine in the sample according to the absorbance value with the wavelength of 400-430 nm.
2. The method according to claim 1, which comprises the following steps (1-1), (1) and (2):
(1-1) mixing a sample with a solvent to obtain a first mixture; wherein the solvent is a mixed solution of alcohol and water in a volume ratio of 1 (1-13);
(1) mixing the first mixture with a probe compound or a solution thereof at the temperature of 15-35 ℃ for 0.5-10 min to obtain a second mixture; wherein the probe compound is p-nitrophenol;
(2) and detecting the second mixture by using an ultraviolet-visible spectrophotometer, and calculating the content of free nicotine in the sample according to the absorbance value with the wavelength of 400-430 nm.
3. The method according to claim 1 or 2, wherein in step 1) or step (1), the molar ratio of probe compound to nicotine in the sample is at least 1.
4. The method according to claim 1 or 2, wherein in the step (1), the solvent used for the probe compound solution is a mixture of alcohol and water.
5. The method according to claim 4, wherein the probe compound solution uses an alcohol in a solvent selected from the group consisting of ethanol, methanol, isopropanol and n-propanol.
6. The method according to claim 4, wherein the probe compound solution is prepared using a solvent in which the alcohol and water are present in a volume ratio of 1 (1-13).
7. The method of claim 1 or 2, wherein the sample is e-liquid.
8. The method according to claim 1 or 2, characterized by one or more of the following a to F:
A. in the step (1), the concentration of the probe compound solution is 10-500 mu mol/L;
B. in the step 2) or the step (2), the optical distance detected by the ultraviolet-visible spectrophotometer is 1-100 mm;
C. in the step 2) or the step 2, the scanning step of the ultraviolet-visible spectrophotometer is 0.1-0.5 nm;
D. in the step 2) or the step (2), the scanning speed of the ultraviolet-visible spectrophotometer is 180-300 nm/min;
E. in the step 2) or the step (2), calculating by adopting an external standard quantitative method;
F. in the step (1-1), the volume ratio of the sample to the solvent is 1 (2-20).
9. Use of a probe compound for determining the free nicotine content in a sample; wherein the probe compound is p-nitrophenol, and the sample is tobacco and/or tobacco products.
10. The use of claim 9, wherein the sample is e-liquid.
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