CN111781286B - Propyl ester analysis method for organic acid substances in tobacco and tobacco products - Google Patents
Propyl ester analysis method for organic acid substances in tobacco and tobacco products Download PDFInfo
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Abstract
The invention provides a propyl propylate analysis method of organic acid substances in tobacco and tobacco products, which comprises the following steps: 1) Adding tobacco and tobacco products into sulfuric acid-n-propanol solution for propyl ester derivatization, and then oscillating and extracting to obtain sample solution; 2) Respectively adding a solvent into standard samples of various organic acid substances for dilution and volume fixing to obtain mixed standard solutions; 3) And respectively carrying out gas chromatography mass spectrometry determination on the sample solution and the mixed standard solution, comparing retention time for qualitative determination, determining various organic acid substances in the sample solution, and calculating the content of the various organic acid substances in the sample solution by an internal standard curve method. The propyl propylate analysis method for the organic acid substances in the tobaccos and the tobacco products, provided by the invention, can be used for quantitatively detecting volatile organic acids, non-volatile organic acids and higher fatty acids in the tobaccos and the tobacco products at the same time, and has the advantages of high precision, good repeatability and excellent recovery rate.
Description
Technical Field
The invention belongs to the technical field of tobacco chemical component analysis, relates to a propylation analysis method of organic acid substances in tobacco and tobacco products, and particularly relates to a propylation analysis method of organic acid substances in tobacco and tobacco products, including volatile organic acids, non-volatile organic acids and higher fatty acids.
Background
Organic acid substances in the tobacco are important indexes influencing the sensory evaluation of the tobacco and are important aroma components in the tobacco. The organic acid substances comprise volatile organic acid, non-volatile organic acid and higher fatty acid, wherein the volatile organic acid, such as small molecular acid, such as isobutyric acid, isovaleric acid, 2-methyl butyric acid, 3-methyl valeric acid and the like, can directly enter smoke due to low boiling point and volatility, so that the fragrance style of the cigarettes is enriched, and the smoking sensory evaluation of the tobaccos is directly influenced; in addition, nonvolatile organic acid and higher fatty acid are also important aroma components, and can play a role in adjusting acid-base balance of tobacco leaves and balancing smoke, so that the tobacco leaves have mellow taste. Therefore, the accurate quantitative detection of the organic acid substances in the tobacco has important significance for the quality evaluation of the tobacco leaves.
At present, the quantitative detection of volatile organic acids, nonvolatile organic acids and higher fatty acids is carried out separately. The detection of the volatile organic acid has the industrial standard of 'YC/T500-2014 tobacco and tobacco product volatile organic acid determination gas chromatography-mass spectrometry combined method', and the method can accurately and quantitatively detect the volatile organic acid in the tobacco and the tobacco products. The detection of non-volatile organic acids in tobacco and tobacco products has the industrial standard of' YC/T288-2009 gas chromatography for detecting polybasic acids (oxalic acid, malic acid and citric acid) of tobacco and tobacco products, the method adopts a sulfuric acid-methanol methyl esterification method to detect the free polybasic acid and the bound polybasic acid, but for the volatile organic acids, the molecular weight is small, and the boiling point of the organic acid methyl ester generated after the methyl esterification is too low, so the organic acid methyl ester flows out along with a solvent in the initial stage of temperature programming, and the analysis is difficult. The detection of the high-grade fatty acid in the tobacco and the tobacco products can adopt a high-grade fatty acid methyl esterification method commonly used in the industry, and can reduce the boiling point of non-to-be-detected substances and improve the separation degree; the fatty acid in food can also be detected by the existing industry standard GB 5009.168-2016 food safety national standard; the measurement can be carried out by a methyl esterification method of acetyl chloride-methanol.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a propylation analysis method for organic acids in tobacco and tobacco products, which solves the problem of the lack of a method for simultaneously analyzing volatile organic acids, nonvolatile organic acids and higher fatty acid substances in organic acids in the prior art.
In order to achieve the above objects and other related objects, the present invention provides a propylation analysis method for organic acids in tobacco and tobacco products, comprising the following steps:
1) Adding the tobacco or tobacco products into sulfuric acid-n-propanol solution for propyl ester derivatization and then performing oscillation extraction to obtain sample solution;
2) Respectively adding a solvent into standard samples of various organic acid substances for dilution and volume fixing to obtain mixed standard solutions;
3) Respectively carrying out gas chromatography mass spectrometry (GC-MS) determination on the sample solution obtained in the step 1) and the mixed standard solution obtained in the step 2), comparing retention time for qualitative determination, determining various organic acid substances in the sample solution, and calculating the content of the various organic acid substances in the sample solution by an internal standard curve method.
The above tobacco (scientific name: nicotiana tabacum L.) is a plant belonging to the genus Nicotiana of the family Solanaceae, and is a raw material for the tobacco industry.
The tobacco product is a tobacco product, and includes, but is not limited to, cigarettes, buccal cigarettes, electronic cigarettes, and the like.
The organic acid is selected from one or more of formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, crotonic acid, valeric acid, isovaleric acid, 2-methylbutyric acid, 3-methylpentanoic acid, caproic acid, oxalic acid, enanthic acid, lactic acid, malonic acid, benzoic acid, caprylic acid, phenylacetic acid, pelargonic acid, malic acid, capric acid, lauric acid, myristic acid, citric acid, palmitic acid, linoleic acid, oleic acid, alpha-linolenic acid and stearic acid.
Wherein the volatile organic acid is selected from one or more of formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, crotonic acid, valeric acid, isovaleric acid, 2-methylbutyric acid, 3-methylvaleric acid, caproic acid, enanthic acid, benzoic acid, caprylic acid, phenylacetic acid, pelargonic acid and capric acid.
The non-volatile organic acid is selected from one or more of oxalic acid, lactic acid, malonic acid and malic acid.
The higher fatty acid is selected from one or more of lauric acid, myristic acid, citric acid, palmitic acid, linoleic acid, oleic acid, alpha-linolenic acid, and stearic acid.
Preferably, in the step 1), the propyl ester derivatization IS to add the first internal standard working solution (IS-1) and the second internal standard working solution (IS-2) into the tobacco or the tobacco product, respectively, add the sulfuric acid-n-propanol solution, perform water bath heating after vortex oscillation, take out, stand and cool to room temperature.
Preferably, the first internal standard working solution (IS-1) IS an n-propanol solution of trans-3-hexenoic acid at a concentration of 0.3-0.8 mg/mL. More preferably, the first internal standard working solution (IS-1) IS an n-propanol solution of trans-3-hexenoic acid at a concentration of 0.5 mg/mL.
Preferably, the second internal standard working solution (IS-2) IS an n-propanol solution of adipic acid at a concentration of 9-11 mg/mL. More preferably, the second internal standard working solution (IS-2) IS an n-propanol solution of adipic acid at a concentration of 10 mg/mL.
And placing the first internal standard working solution and the second internal standard working solution in a refrigerator at 4 ℃ for dark storage, wherein the storage life of the first internal standard working solution and the second internal standard working solution is 6 months.
Preferably, the ratio of the mass added to the tobacco or tobacco product to the volume added to the first internal standard working solution and the volume added to the second internal standard working solution is from 0.095 to 0.105. More preferably, the ratio of the mass added to the tobacco or tobacco product to the volume added to the first internal standard working solution and the volume added to the second internal standard working solution is 0.1.
Preferably, the ratio of the mass added to the tobacco or tobacco product to the volume added to the sulfuric acid-n-propanol solution is from 0.1 to 0.5 to 1.5,g/mL. More preferably, the ratio of the mass added to the tobacco or tobacco product to the volume added of the sulfuric acid-n-propanol solution is 0.1.
Preferably, the sulfuric acid-n-propanol solution is a mixed aqueous solution of 9-11% by volume of sulfuric acid and n-propanol, and the volume ratio of the sulfuric acid to the n-propanol is 1. More preferably, the sulfuric acid-n-propanol solution is a 10% by volume mixed aqueous solution of sulfuric acid and n-propanol, and the volume ratio of the sulfuric acid to the n-propanol is 1.
Preferably, the time of the vortex oscillation is 1-3min. More preferably, the time of the vortex oscillation is 2min.
Preferably, the vortex oscillation has an oscillation frequency of 2000-3000rpm.
Preferably, the time of the water bath heating is 2.5-3.5h, and the temperature of the water bath heating is 65-75 ℃. More preferably, the time of the water bath heating is 3h, and the temperature of the water bath heating is 70 ℃.
Preferably, the room temperature is 20-30 ℃.
Preferably, in the step 1), the oscillating extraction is to add water and n-hexane into the tobacco or tobacco product subjected to propyl ester derivatization treatment, perform vortex oscillation, take out, stand for layering, and take out supernatant to obtain the required sample solution.
Preferably, the ratio of the added mass of the tobacco or tobacco products to the added volume of the water and the added volume of the n-hexane is 0.1-4-6. More preferably, the ratio of the mass added to the volume added to the water and the volume added to the n-hexane is 0.1.
Preferably, the time of the vortex oscillation is 0.5-1.5min. More preferably, the time of the vortex oscillation is 1min.
Preferably, the vortex oscillation has an oscillation frequency of 2000-3000rpm.
Preferably, in step 2), the solvent is n-propanol.
Preferably, in the step 2), the concentration range of the plurality of organic acids in the mixed standard solution is 0.122 μ g/g-11.25mg/g.
Preferably, in the step 2), a first internal standard working solution (IS-1) and a second internal standard working solution (IS-2) are also added into the mixed standard solution, wherein the added first internal standard working solution (IS-1) in the mixed standard solution IS 25-75 μ L, and the added second internal standard working solution (IS-2) IS 75-125 μ L. Preferably, the mixed standard solution IS added with 50. Mu.L of the first internal standard working solution (IS-1) and 100. Mu.L of the second internal standard working solution (IS-2).
Preferably, the first internal standard working solution (IS-1) IS an n-propanol solution of trans-3-hexenoic acid at a concentration of 0.3-0.8 mg/mL. More preferably, the first internal standard working solution (IS-1) IS an n-propanol solution of trans-3-hexenoic acid at a concentration of 0.5 mg/mL.
Preferably, the second internal standard working solution (IS-2) IS an n-propanol solution of adipic acid at a concentration of 9-11 mg/mL. More preferably, the second internal standard working solution (IS-2) IS an n-propanol solution of adipic acid at a concentration of 10 mg/mL.
Preferably, in step 3), in the gas chromatography-mass spectrometry, the chromatographic column used for gas chromatography is a DB-fatcax capillary chromatographic column [30m (length) × 0.25mm (inner diameter) × 0.25 μm (film thickness) ].
Preferably, in step 3), in the gas chromatography-mass spectrometry, the temperature rise procedure of the gas chromatography is as follows: the initial temperature was maintained at 40 ℃ for 2min and increased to 220 ℃ at a rate of 15 ℃/min for 15min.
Preferably, in step 3), in the gas chromatography-mass spectrometry, the sample introduction conditions of the gas chromatography are as follows: sample inlet temperature: 250 ℃; sample introduction amount: 2 mu L of the solution; shunting mode: shunting and sampling; carrier gas: high-purity helium (He), wherein the purity of carrier gas is more than or equal to 99.999 percent; carrier gas flow rate: 2.0mL/min; and (4) a constant current mode.
Preferably, in step 3), in the gas chromatography-mass spectrometry, the determination conditions of the mass spectrum are as follows:
an ionization mode: an Electron Impact (EI) ion source; ionization energy: 70eV; ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; auxiliary interface temperature: 220 ℃; solvent retardation: 2min; the scanning mode comprises the following steps: full Scan (Scan) and Selective Ion Monitoring (SIM); scanning range of the full scanning mode: 29-300amu.
Preferably, the quantitative ions selected in the mass spectrometric assay are respectively: formic acid 42, acetic acid 73, propionic acid 75, butyric acid 89, isobutyric acid 89, crotonic acid 69, valeric acid 103, isovaleric acid 103, 2-methylbutyric acid 85, 3-methylvaleric acid 117, caproic acid 117, oxalic acid 133, heptanoic acid 113, lactic acid 75, malonic acid 105, benzoic acid 123, caprylic acid 145, phenylacetic acid 178, pelargonic acid 159, malic acid 117, capric acid 173, lauric acid 183, myristic acid 211, citric acid 261, palmitic acid 239, linoleic acid 263, oleic acid 265, alpha-linolenic acid 320, stearic acid 285.
Preferably, in step 3), the internal standard curve method comprises the following steps:
a) Preparing a series of mixed standard solutions with different concentrations in the step 2), respectively carrying out GC-MS detection to obtain a linear relation between the chromatographic peak area ratio of the multiple organic acid substances/internal standards and the concentration ratio of the corresponding organic acid substances/internal standards, drawing corresponding standard working curves, and calculating to obtain a regression equation of the standard working curves of the multiple organic acid substances;
b) Carrying out GC-MS detection on the sample solution in the step 1), substituting the obtained chromatographic peak area ratio of the multiple organic acid substances to the internal standard into a regression equation of the standard working curve of the multiple organic acid substances in the step A), and calculating the concentration of the multiple organic acid substances in the sample solution according to the known concentration of the internal standard.
Preferably, in the standard working curve, the area ratio of the chromatographic peaks of the plurality of organic acids to the internal standard is taken as an ordinate (Y axis), and the concentration ratio of the corresponding organic acids to the internal standard is taken as an abscissa (X axis).
Preferably, in step 3), in the internal standard curve method, the corresponding internal standards of the organic acid substances for quantification are respectively: formic acid IS-1, acetic acid IS-1, propionic acid IS-1, butyric acid IS-1, isobutyric acid IS-1, crotonic acid IS-1, valeric acid IS-1, isovaleric acid IS-1, 2-methylbutyric acid IS-1, 3-methylpentanic acid IS-1, caproic acid IS-1, oxalic acid IS-2, enanthic acid IS-1, lactic acid IS-2, malonic acid IS-2, benzoic acid IS-1, caprylic acid IS-1, phenylacetic acid IS-1, pelargonic acid IS-1, malic acid IS-2, capric acid IS-1, lauric acid IS-2, myristic acid IS-2, citric acid IS-2, palmitic acid IS-2, linoleic acid IS-2, oleic acid IS-2, alpha-IS-linolenic acid IS-2, stearic acid IS-2.
As mentioned above, the propyl propylate analysis method for organic acid substances in tobacco and tobacco products provided by the invention has the following beneficial effects:
(1) The propyl ester analysis method for the organic acid substances in the tobaccos and the tobacco products, provided by the invention, can be used for simultaneously and quantitatively detecting the volatile organic acids, the nonvolatile organic acids and the higher fatty acids in the tobaccos and the tobacco products aiming at the current situation that the conventional volatile organic acids, the nonvolatile organic acids and the higher fatty acids are independently and separately measured, so that the three original detection methods are reduced into one, the pretreatment process is saved, the pretreatment step is shortened, and the method is more efficient and convenient.
(2) According to the propyl esterification analysis method of the organic acid substances in the tobaccos and the tobacco products, the propyl esterification method of sulfuric acid-n-propanol is developed on the basis of a sulfuric acid-methanol methyl esterification method, volatile organic acids, non-volatile organic acids and higher fatty acids in the tobaccos and the tobacco products are derived into propyl esters of the organic acids, and then gas chromatography-mass spectrometry quantitative detection is carried out, so that the pretreatment process is saved, the pretreatment step is shortened, and the method is more efficient and convenient.
(3) The propyl ester analysis method of the organic acid substances in the tobacco and the tobacco products, provided by the invention, aims at various organic acid substances, and has the advantages of high precision, good repeatability and excellent recovery rate.
Drawings
FIG. 1 shows a total ion flow diagram of a propylated propyl ester analysis method of organic acids in tobacco and tobacco products, wherein FIG. 1a is the total ion flow diagram with retention time of 1-10 minutes, and FIG. 1b is the total ion flow diagram with retention time of 10-22 minutes.
In the drawings, the reference numerals are: 1: formic acid, 2: acetic acid, 3: propionic acid, 4: isobutyric acid, 5: butyric acid, 6: isovaleric acid, 7: 2-methylbutyric acid, 8: valeric acid, 9: crotonic acid, 10: 3-methylvaleric acid, 11: caproic acid, 12: trans-3-hexenoic acid (IS 1), 13: heptanoic acid, 14: lactic acid, 15: octanoic acid, 16: oxalic acid, 17: pelargonic acid, 18: decanoic acid, 19: malonic acid, 20: benzoic acid, 21: phenylacetic acid, 22: lauric acid, 23: adipic acid (IS 2), 24: myristic acid, 25: malic acid, 26: palmitic acid, 27: stearic acid, 28: oleic acid, 29: linoleic acid, 30: α -linolenic acid, 31: and (4) citric acid.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
The following embodiments of the present invention are provided by way of specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The materials, reagents and equipment used in the following examples are as follows:
1. materials and reagents
Formic acid (98%, J & K), acetic acid (99.5%, J & K), propionic acid (99.0%, DR Ehrenstontorfer Gmbh), isobutyric acid (99.0%, DR Ehrentorfer Gmbh), butyric acid (99.0%, TCI), crotonic acid (98%, J & K), isovaleric acid (99.0%, DR Ehrentorfer Gmbh), valeric acid (98.0%, TCI), 2-methylbutyric acid (95%, xideng perfume), 3-methylvaleric acid (99.0%, CNW), hexanoic acid (98.0%, TCI), lactic acid (99.0%, CNW), oxalic acid (98.8%, DR Ehrentorfer Gmbh), heptanoic acid (98.0%, TCI), malonic acid (97.0%, CNW), octanoic acid (99.2%, DR Ehrenstorfer Gmbh), benzoic acid (99.5%, sigma-aldrich), phenylacetic acid (95%, heliotropin essence), nonanoic acid (97.0%, DR Ehrenstorfer Gmbh), malic acid (99.0%, CNW), decanoic acid (98.7%, DR Ehrenstorfer Gmbh), lauric acid (98.0%, CNW), myristic acid (99.0%, CNW), citric acid (98.0%, CNW), palmitic acid (97.0%, CNW), linoleic acid (99.0%, CNW), oleic acid (98.0%, CNW), α -linolenic acid (98.5%, CNW), stearic acid (95.0%, CNW);
trans-3-hexenoic acid (95.0%, CNW); adipic acid (99.0%, CNW); n-propanol (greater than or equal to 99.0%, shanghai Lingfeng Chemicals Co., ltd.); sulfuric acid (with the concentration more than or equal to 98wt%, chinese medicine reagent); n-hexane (95%, J & K);
deionized water (made by MILLIPORE water purifier).
2. Instrument for measuring the position of a moving object
7890B gas chromatography-5977A mass spectrometer (Agilent); DB-FATWAX capillary column (Agilent); vortex shaker model Vortex-Genie 2 (Scientific Industries); HH-S11-25 Water bath (Shanghai Hede laboratory instruments, ltd.).
The analysis process of the propyl propylate analysis method of the organic acid substances in the tobacco and the tobacco products comprises the following steps
1. Sample pretreatment
Accurately weighing a sample of tobacco or a tobacco product, respectively adding a first internal standard working solution (IS-1) and a second internal standard working solution (IS-2), then adding a sulfuric acid-n-propanol solution for propyl ester derivatization treatment, namely quickly sealing, performing vortex oscillation for 1-3min at the frequency of 2000-3000rpm, heating in a water bath kettle at the temperature of 65-75 ℃ for 2.5-3.5h, taking out, standing and cooling to room temperature. Wherein the first internal standard working solution (IS-1) IS an n-propanol solution of trans-3-hexenoic acid with the concentration of 0.3-0.8 mg/mL; the second internal standard working solution (IS-2) IS an n-propanol solution of adipic acid with the concentration of 9-11 mg/mL; the sulfuric acid-n-propanol solution is a mixed aqueous solution of 9-11% by volume of sulfuric acid and n-propanol, and the volume ratio of the sulfuric acid to the n-propanol is 1. The ratio of the mass added to the tobacco or tobacco product to the volume added to the first internal standard working solution and the volume added to the second internal standard working solution is 0.095-0.105. The ratio of the mass added to the tobacco or tobacco product to the volume added to the sulfuric acid-n-propanol solution is 0.1.
And then, performing oscillation extraction on the tobacco or the tobacco product subjected to propyl ester derivatization treatment, namely adding water and n-hexane for vortex oscillation for 0.5-1.5min at the frequency of 2000-3000rpm, taking out, standing for layering, and taking supernatant to obtain the required sample solution. Wherein the ratio of the added mass of the tobacco or the tobacco products to the added volume of the water and the added volume of the n-hexane is 0.1-6.
2. Preparing standard solution
Adding normal propyl alcohol into a standard sample of various organic acid substances for diluting to a constant volume to obtain a mixed standard solution. In the mixed standard solution, 29 organic acid substances are contained, including formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, crotonic acid, valeric acid, isovaleric acid, 2-methylbutyric acid, 3-methylvaleric acid, caproic acid, oxalic acid, heptanoic acid, lactic acid, malonic acid, benzoic acid, caprylic acid, phenylacetic acid, pelargonic acid, malic acid, capric acid, lauric acid, myristic acid, citric acid, palmitic acid, linoleic acid, oleic acid, alpha-linolenic acid and stearic acid. Wherein the volatile organic acid comprises formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, crotonic acid, valeric acid, isovaleric acid, 2-methylbutyric acid, 3-methylvaleric acid, caproic acid, enanthic acid, benzoic acid, caprylic acid, phenylacetic acid, pelargonic acid, and capric acid. The non-volatile organic acid includes oxalic acid, lactic acid, malonic acid, and malic acid. The higher fatty acid includes lauric acid, myristic acid, citric acid, palmitic acid, linoleic acid, oleic acid, alpha-linolenic acid, and stearic acid.
The concentration range of various organic acid substances in the mixed standard solution is 0.122 mu g/g-11.25mg/g.
The mixed standard solution IS also added with a first internal standard working solution (IS-1) and a second internal standard working solution (IS-2), wherein the added first internal standard working solution (IS-1) in the mixed standard solution IS 25-75 mu L, and the added second internal standard working solution (IS-2) IS 75-125 mu L. . The first internal standard working solution (IS-1) IS an n-propanol solution of trans-3-hexenoic acid at a concentration of 0.3-0.8 mg/mL. The second internal standard working solution (IS-2) was an n-propanol solution of adipic acid at a concentration of 9-11 mg/mL.
3. Measurement of
And respectively carrying out gas chromatography mass spectrometry (GC-MS) determination on the sample solution and the mixed standard solution, comparing retention time for qualitative determination, determining various organic acid substances in the sample solution, and calculating the content of the various organic acid substances in the sample solution by an internal standard curve method.
Specifically, a series of mixed standard solutions with different concentrations are prepared, GC-MS detection is carried out respectively, the linear relation between the chromatographic peak area ratio of the organic acid substances/internal standards and the concentration ratio of the corresponding organic acid substances/internal standards is obtained, corresponding standard working curves are drawn, and the regression equation of the standard working curves of the organic acid substances is obtained through calculation. And carrying out GC-MS detection on the sample solution, substituting the obtained chromatographic peak area ratio of the multiple organic acid substances to the internal standard into a regression equation of a standard working curve of the multiple organic acid substances, and calculating the concentration of the multiple organic acid substances in the sample solution according to the known concentration of the internal standard.
Wherein, the measuring conditions of the gas chromatography are as follows: a chromatographic column: is DB-FATWAX capillary chromatography column [30m (length) × 0.25mm (inner diameter) × 0.25 μm (film thickness) ]; sample inlet temperature: 250 ℃; sample introduction amount: 2 mu L of the solution; a shunting mode: split-flow sample injection; carrier gas: high-purity helium (He), wherein the purity of carrier gas is more than or equal to 99.999 percent; carrier gas flow: 2.0ml/min; a constant current mode; the temperature rising procedure is as follows: the initial temperature was maintained at 40 ℃ for 2min, and the temperature was increased to 220 ℃ at a rate of 15 ℃/min for 15min.
The mass spectrum measurement conditions were: an Electron Impact (EI) ion source; ionization energy: 70eV; ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; auxiliary interface temperature: 220 ℃; solvent retardation: 2min; the scanning mode is as follows: full Scan (Scan) and Selective Ion Monitoring (SIM); scanning range of full scan mode: 29-300amu.
The quantitative ions selected in the mass spectrometry were: formic acid 42, acetic acid 73, propionic acid 75, butyric acid 89, isobutyric acid 89, crotonic acid 69, valeric acid 103, isovaleric acid 103, 2-methylbutyric acid 85, 3-methylvaleric acid 117, caproic acid 117, oxalic acid 133, heptanoic acid 113, lactic acid 75, malonic acid 105, benzoic acid 123, caprylic acid 145, phenylacetic acid 178, pelargonic acid 159, malic acid 117, capric acid 173, lauric acid 183, myristic acid 211, citric acid 261, palmitic acid 239, linoleic acid 263, oleic acid 265, alpha-linolenic acid 320, stearic acid 285.
The corresponding internal standards for quantification of organic acids were: formic acid IS-1, acetic acid IS-1, propionic acid IS-1, butyric acid IS-1, isobutyric acid IS-1, crotonic acid IS-1, valeric acid IS-1, isovaleric acid IS-1, 2-methylbutyric acid IS-1, 3-methylpentanic acid IS-1, caproic acid IS-1, oxalic acid IS-2, enanthic acid IS-1, lactic acid IS-2, malonic acid IS-2, benzoic acid IS-1, caprylic acid IS-1, phenylacetic acid IS-1, pelargonic acid IS-1, malic acid IS-2, capric acid IS-1, lauric acid IS-2, myristic acid IS-2, citric acid IS-2, palmitic acid IS-2, linoleic acid IS-2, oleic acid IS-2, alpha-IS-linolenic acid IS-2, stearic acid IS-2.
Example 1
1. Sample pretreatment
Accurately weighing 0.1g (accurate to 0.1 mg) of tobacco or tobacco product sample into a 15mL thick-walled centrifuge tube, adding 50 μ L of a first internal standard working solution (IS-1) with a concentration of 0.5 mg/mL: n-propanol solution of trans-3-hexenoic acid and 100. Mu.L of a second internal standard working solution (IS-2) at a concentration of 10 mg/mL: adding 1mL of 10% sulfuric acid-n-propanol solution into n-propanol solution of adipic acid, performing propyl ester derivatization treatment, namely rapidly sealing the bottle mouth, performing vortex oscillation at the frequency of 2500rpm for 2min, heating in a 70 ℃ water bath kettle for 3h, taking out, standing and cooling to room temperature. Wherein, in the sulfuric acid-n-propanol solution, the volume ratio of sulfuric acid to n-propanol is 1. The sulfuric acid-n-propanol solution is prepared by slowly draining concentrated sulfuric acid through a glass rod and adding the concentrated sulfuric acid into the n-propanol solution under the condition of stirring in an ice bath.
And then, oscillating and extracting the sample of the tobacco or the tobacco product subjected to propyl ester derivatization at the frequency of 2500rpm, namely adding 5mL of deionized water and 2mL of normal hexane, carrying out vortex oscillation for 1.0min, taking out, standing for layering, and taking supernatant to obtain the required sample solution 1#.
2. Preparing standard solution
Adding n-propanol into a standard sample of various organic acid substances to dilute to a constant volume, and obtaining a mixed standard solution. In the mixed standard solution, 29 kinds of organic acid substances comprise formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, crotonic acid, valeric acid, isovaleric acid, 2-methylbutyric acid, 3-methylvaleric acid, caproic acid, oxalic acid, heptanoic acid, lactic acid, malonic acid, benzoic acid, caprylic acid, phenylacetic acid, pelargonic acid, malic acid, capric acid, lauric acid, myristic acid, citric acid, palmitic acid, linoleic acid, oleic acid, alpha-linolenic acid and stearic acid. Wherein the volatile organic acid comprises formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, crotonic acid, valeric acid, isovaleric acid, 2-methylbutyric acid, 3-methylvaleric acid, caproic acid, enanthic acid, benzoic acid, caprylic acid, phenylacetic acid, pelargonic acid, and capric acid. The non-volatile organic acid includes oxalic acid, lactic acid, malonic acid, and malic acid. The higher fatty acid includes lauric acid, myristic acid, citric acid, palmitic acid, linoleic acid, oleic acid, alpha-linolenic acid, and stearic acid.
The concentration range of the 29 organic acid substances in the mixed standard solution is 0.122 mu g/g-11.25mg/g. .
The mixed standard solution was also added with 50. Mu.L of the first internal standard working solution (IS-1) and 100. Mu.L of the second internal standard working solution (IS-2). The first internal standard working solution (IS-1) was an n-propanol solution of trans-3-hexenoic acid at a concentration of 0.5 mg/mL. The second internal standard working solution (IS-2) was an n-propanol solution of adipic acid at a concentration of 10 mg/mL.
3. Measurement of
And respectively carrying out gas chromatography mass spectrometry (GC-MS) determination on the sample solution 1# and the mixed standard solution, comparing retention time for qualitative determination, determining 29 organic acid substances in the sample solution 1# and calculating the content of the 29 organic acid substances in the sample solution 1# by an internal standard curve method.
Specifically, a series of mixed standard solutions with different concentrations are prepared, GC-MS detection is performed respectively, a linear relation between the chromatographic peak area ratio of the 29 organic acid substances/internal standards and the concentration ratio of the corresponding organic acid substances/internal standards is obtained, corresponding standard working curves are drawn, and a regression equation of the standard working curves of the 29 organic acid substances is obtained through calculation. And then carrying out GC-MS detection on the sample solution 1#, substituting the obtained chromatographic peak area ratios of the 29 organic acid substances and the internal standard into a regression equation of a standard working curve of the 29 organic acid substances, and calculating the concentrations of the 29 organic acid substances in the sample solution 1#, according to the known concentrations of the internal standard substances.
Wherein, the measuring conditions of the gas chromatography are as follows: a chromatographic column: is DB-FATWAX capillary chromatography column [30m (length) × 0.25mm (inner diameter) × 0.25 μm (membrane thickness) ]; sample inlet temperature: 250 ℃; sample introduction amount: 2 mu L of the solution; a shunting mode: split-flow sample injection; carrier gas: high-purity helium (He), wherein the purity of carrier gas is more than or equal to 99.999%; carrier gas flow: 2.0ml/min; a constant current mode; the temperature-raising program is: the initial temperature was maintained at 40 ℃ for 2min and increased to 220 ℃ at a rate of 15 ℃/min for 15min.
The mass spectrum measurement conditions were: an Electron Impact (EI) ion source; ionization energy: 70eV; ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; auxiliary interface temperature: 220 ℃; solvent retardation: 2min; the scanning mode is as follows: full Scan (Scan) and Selective Ion Monitoring (SIM); scanning range of the full scanning mode: 29-300amu.
The retention times, the quantitative selection ions and the internal standard selection of 29 organic acids in sample solution 1# are shown in table 1. The chromatograms of 29 organic acids and internal standard in sample solution 1# are shown in fig. 1a and 1b.
TABLE 1
| Name of substance | Retention time (min) | Quantitative ion | Internal standard | |
| 1 | Formic acid | 2.974 | 42 | IS-1 |
| 2 | Acetic acid | 3.573 | 73 | IS-1 |
| 3 | Propionic acid | 4.425 | 75 | IS-1 |
| 4 | Isobutyric acid | 4.510 | 89 | IS-1 |
| 5 | Butyric acid | 5.323 | 89 | IS-1 |
| 6 | Isovaleric acid | 5.516 | 103 | IS-1 |
| 7 | 2-methyl butyric acid | 5.692 | 85 | IS-1 |
| 8 | Valeric acid | 6.415 | 103 | IS-1 |
| 9 | Crotonic acid | 6.752 | 87 | IS-1 |
| 10 | 3-Methylpentanoic acid | 6.872 | 117 | IS-1 |
| 11 | Hexanoic acid | 7.425 | 117 | IS-1 |
| 12 | Trans-3-hexenoic acid (IS-1) | 8.095 | 114 | |
| 13 | Heptanoic acid | 8.376 | 113 | IS-1 |
| 14 | Lactic acid | 8.445 | 75 | IS-2 |
| 15 | Octanoic acid | 9.277 | 145 | IS-1 |
| 16 | Oxalic acid | 10.058 | 133 | IS-2 |
| 17 | Pelargonic acid | 10.128 | 159 | IS-1 |
| 18 | Capric acid | 10.933 | 173 | IS-1 |
| 19 | Malonic acid | 11.028 | 105 | IS-2 |
| 20 | Benzoic acid | 11.283 | 123 | IS-1 |
| 21 | Phenylacetic acid | 12.065 | 178 | IS-1 |
| 22 | Lauric acid | 12.436 | 183 | IS-2 |
| 23 | Adipic acid (IS-2) | 13.447 | 171 | |
| 24 | Myristic acid | 13.807 | 211 | IS-2 |
| 25 | Malic acid | 14.263 | 117 | IS-2 |
| 26 | Palmitic acid | 15.243 | 239 | IS-2 |
| 27 | Stearic acid | 17.307 | 285 | IS-2 |
| 28 | Oleic acid | 17.613 | 265 | IS-2 |
| 29 | Linoleic acid | 18.288 | 263 | IS-2 |
| 30 | Alpha-linolenic acid | 18.733 | 320 | IS-2 |
| 31 | Citric acid | 19.407 | 261 | IS-2 |
Example 2
Respectively taking 29 organic acid substance standards of 0.01mL, 0.02mL, 0.05mL, 0.1mL, 0.2mL, 0.5mL, 0.8mL, 1.0mL and 1.5mL, adding 50 mu L of first internal standard working solution (IS-1) and 100 mu L of second internal standard working solution (IS-2), adding n-propanol for dilution and volume fixing, and preparing 9-grade mixed standard solutions of 29 organic acid substances with different concentrations. Meanwhile, a solution prepared from 29 organic acid substances with the concentration of 0 mug/L represents a solvent blank.
In the mixed standard solution, the concentration ranges of 29 organic acid substances are respectively as follows:
formic acid: 70.88-10631.4. Mu.g/g, acetic acid: 63.76-9564.0 μ g/g, propionic acid: 0.794 to 63.520. Mu.g/g, isobutyric acid: 0.381 to 57.168. Mu.g/g, butyric acid: 0.973-29.196 mu g/g, isovaleric acid: 1.182-177.252 μ g/g, 2-methylbutyric acid: 10.616-1592.4 mu g/g, valeric acid: 0.193-28.92 mug/g, crotonic acid: 0.786-117.88. Mu.g/g, 3-methylpentanoic acid: 0.400-59.940 mu g/g, caproic acid: 0.144-21.528 mug/g, heptanoic acid: 0.122-18.270 mug/g, caprylic acid: 0.143-14.328 μ g/g, pelargonic acid: 0.138-20.640 mug/g, decanoic acid: 0.151-15.144 μ g/g, lactic acid: 0.062-4.620 mg/g, benzoic acid: 0.766-114.906 mug/g, phenylacetic acid: 1.038-103.796 μ g/g, oxalic acid: 0.131-19.695 mg/g, malonic acid: 0.050 to 4.999mg/g, lauric acid: 0.052-7.854 mg/g, myristic acid: 0.047-7.095 mg/g, malic acid: 0.750-112.5 mg/g, palmitic acid: 0.131-19.590 mg/g, stearic acid: 0.047-4.655 mg/g, oleic acid: 0.048-7.125 mg/g, linoleic acid: 0.041-6.180 mg/g, alpha-linolenic acid: 0.050-3.750 mg/g, citric acid: 0.541-40.605 mg/g.
The prepared mixed standard solutions of the 9 grades with different concentrations are respectively subjected to gas chromatography-mass spectrometry analysis as shown in the above example 1 in 3, and regression analysis is performed by taking the chromatographic peak area ratio of the 29 organic acid substances to the internal standard as the ordinate (Y axis) and the concentration ratio of the corresponding organic acid substances to the internal standard as the abscissa (X axis) to obtain a regression equation and a correlation coefficient thereof. The specific results are shown in Table 2. As can be seen from Table 2, the regression equation has good linearity and the correlation coefficient R 2 >0.995。
Meanwhile, for target object response signals in the mixed standard solution, the lowest concentration standard sample is repeatedly injected for 10 times, the standard deviation of 3 times of the concentration measurement value is taken as the detection limit, and the standard deviation of 10 times of the concentration measurement value is taken as the quantification limit. Specific results are shown in Table 2.
As is clear from Table 2, the detection limits of the 29 organic acids were 0.0142 to 20.4. Mu.g/g, respectively, and the quantitation limits were 0.0426 to 68.0. Mu.g/g, respectively, and this method had high sensitivity.
TABLE 2
y: peak area; x: concentration of
Example 3
Within the same day, a parallel experiment was performed 5 times on samples of known concentrations of tobacco or tobacco products, and the within-day reproducibility of the method was examined. Meanwhile, the recovery rates of the samples were measured at three concentration levels, i.e., low, medium, and high, and the results are shown in Table 3. As can be seen from Table 3, the relative standard deviation (RSD%) is less than 10% in the concentration range of 0.122. Mu.g/g-112.5 mg/g, and the method repeatability and recovery rate are both good.
TABLE 3 Experimental methodological data
Example 4
The method established by the invention is used for detecting different flue-cured tobacco samples, and the specific results are shown in the following table 4. As can be seen from Table 4, the method can accurately and quantitatively determine the contents of 29 organic acid substances in the flue-cured tobacco sample.
TABLE 4
In conclusion, the propyl ester analysis method for the organic acid substances in the tobaccos and the tobacco products, provided by the invention, can be used for simultaneously and quantitatively detecting volatile organic acids, non-volatile organic acids and higher fatty acids in the tobaccos and the tobacco products, and has the advantages of high precision, good repeatability and excellent recovery rate. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims (6)
1. A propyl propylation analysis method for organic acid substances in tobaccos and tobacco products comprises the following steps:
1) Adding tobacco or tobacco products into sulfuric acid-n-propanol solution for propyl ester derivatization, and then oscillating and extracting to obtain sample solution;
2) Respectively adding a solvent into standard samples of various organic acid substances for dilution and volume fixing to obtain mixed standard solutions;
3) Respectively carrying out gas chromatography mass spectrometry determination on the sample solution obtained in the step 1) and the mixed standard solution obtained in the step 2), comparing retention time for qualitative determination, determining various organic acid substances in the sample solution, and calculating the content of the various organic acid substances in the sample solution by an internal standard curve method;
the organic acid substances comprise formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, crotonic acid, valeric acid, isovaleric acid, 2-methylbutyric acid, 3-methylpentanoic acid, caproic acid, oxalic acid, enanthic acid, lactic acid, malonic acid, benzoic acid, caprylic acid, phenylacetic acid, pelargonic acid, malic acid, capric acid, lauric acid, myristic acid, citric acid, palmitic acid, linoleic acid, oleic acid, alpha-linolenic acid and stearic acid;
in the step 1), propyl ester derivatization is to add a first internal standard working solution and a second internal standard working solution into tobacco or tobacco products respectively, then add a sulfuric acid-n-propanol solution, perform water bath heating after vortex oscillation, take out, stand and cool to room temperature;
in the step 1), the oscillating extraction is to add water and normal hexane into the tobacco or tobacco products subjected to propyl ester derivatization treatment, then carry out vortex oscillation, take out, stand for layering, and take supernatant to obtain a required sample solution;
the first internal standard working solution is an n-propanol solution of trans-3-hexenoic acid with the concentration of 0.3-0.8 mg/mL; the second internal standard working solution is an n-propanol solution of adipic acid with the concentration of 9-11 mg/mL;
in the step 3), in the gas chromatography-mass spectrometry, a chromatographic column selected by the gas chromatography is a DB-FATWAX capillary chromatographic column, and the chromatographic column is 30m multiplied by 0.25mm multiplied by 0.25 mu m; the temperature rising procedure of the gas chromatography is as follows: the initial temperature was maintained at 40 ℃ for 2min and increased to 220 ℃ at a rate of 15 ℃/min for 15min.
2. The method for propyl propylate analysis of organic acids in tobacco and tobacco products according to claim 1, wherein said propyl ester derivatization comprises any one or more of the following conditions:
a1 The ratio of the added mass of the tobacco or tobacco products to the added volume of the first internal standard working solution and the added volume of the second internal standard working solution is 0.095-0.105;
a2 The ratio of the added mass of the tobacco or tobacco product to the added volume of the sulfuric acid-n-propanol solution is 0.1 to 0.5 to 1.5 g/mL;
a3 The sulfuric acid-n-propanol solution is a mixed aqueous solution of 9-11% by volume of sulfuric acid and n-propanol, and the volume ratio of the sulfuric acid to the n-propanol is 1;
a4 Time of the vortex oscillation is 1-3min;
a5 The oscillation frequency of the vortex oscillation is 2000-3000rpm;
a6 The water bath heating time is 2.5 to 3.5 hours, and the water bath heating temperature is 65 to 75 ℃.
3. The method for propylylation analysis of organic acids in tobacco and tobacco products according to claim 1, wherein said shaking extraction comprises any one or more of the following conditions:
b1 The ratio of the added mass of the tobacco or the tobacco products to the added volume of the water and the added volume of the normal hexane is 0.1-4-6;
b2 Time of the vortex oscillation is 0.5-1.5min;
b3 The oscillation frequency of the vortex oscillation is 2000 to 3000rpm.
4. The method for propyl propylate analysis of organic acids in tobacco and tobacco products according to claim 1, wherein in step 2), the solvent is n-propanol; the method comprises the steps that a first internal standard working solution and a second internal standard working solution are further added into the mixed standard solution, the first internal standard working solution added into the mixed standard solution is 25-75 mu L, and the second internal standard working solution added into the mixed standard solution is 75-125 mu L.
5. The method for propyl propylate analysis of organic acids in tobacco and tobacco products according to claim 1, wherein in step 3), the sample introduction conditions of the gas chromatography mass spectrometry are as follows: sample inlet temperature: 250 ℃; sample introduction amount: 2 mu L of the solution; shunting mode: shunting and sampling; carrier gas: high-purity helium, wherein the purity of carrier gas is more than or equal to 99.999 percent; carrier gas flow rate: 2.0ml/min; and (4) a constant current mode.
6. The method for propyl propylate analysis of organic acids in tobacco and tobacco products according to claim 1, wherein in step 3), the conditions for mass spectrometry are as follows:
an ionization mode: electron bombardment of an EI ion source; ionization energy: 70eV; ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; auxiliary interface temperature: 220 ℃; solvent retardation: 2min; the scanning mode is as follows: full Scan and selective ion monitoring SIM; scanning range of full scan mode: 29-300amu.
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