CN115452967B - Method for detecting content of organic acid and solvent in tobacco essence - Google Patents

Method for detecting content of organic acid and solvent in tobacco essence Download PDF

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CN115452967B
CN115452967B CN202210883621.3A CN202210883621A CN115452967B CN 115452967 B CN115452967 B CN 115452967B CN 202210883621 A CN202210883621 A CN 202210883621A CN 115452967 B CN115452967 B CN 115452967B
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acid
solvent
detection
liquid
organic acid
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CN115452967A (en
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李远根
王蕊
寇天舒
何爱民
苏国岁
沈薇
刘远上
王龙
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HEBEI CHINA TOBACCO INDUSTRY CO LTD
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HEBEI CHINA TOBACCO INDUSTRY CO LTD
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/065—Preparation using different phases to separate parts of sample

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Abstract

The invention relates to a method for detecting the content of organic acid and solvent in tobacco essence. The detection method comprises the following steps: pretreating tobacco essence to be detected to obtain liquid to be detected; separating the liquid to be detected by adopting a gas chromatography, wherein a chromatographic column adopts an inert polyethylene glycol polymer chromatographic column; separating the separated liquid flowing out through the chromatographic column to obtain a first detection liquid and a second detection liquid; introducing the first detection liquid into a mass spectrometer for analysis, and determining the content of organic acid; and (3) introducing the second detection liquid into a thermal conductivity detector for analysis, and determining the content of the solvent. The method does not need derivatization reaction, has simple sample pretreatment, high efficiency and high accuracy; the method can detect the content of the organic acid and the solvent simultaneously, is beneficial to simplifying the operation and improving the detection efficiency. The method is suitable for the situation that the detection requirements are met on the content of the organic acid and the content of the solvent, and provides technical reference for understanding the internal quality of various essences and designing the formula of the essences.

Description

Method for detecting content of organic acid and solvent in tobacco essence
Technical Field
The invention relates to the technical field of analysis of tobacco essence components, in particular to a method for detecting the content of organic acid and solvent in tobacco essence.
Background
The tobacco essence is an important tobacco additive, and the use of the tobacco essence for perfuming cigarettes is one of the important means for improving the smoking quality of cigarettes in cigarette enterprises. The tobacco essence has complex components and mainly comprises alcohols, esters, acids and the like. The organic acid is an important component, is one of main chemical components affecting the smoking quality of the tobacco, has the functions of balancing the pH value of smoke, relieving the irritation of the smoke and increasing the smoke concentration in the combustion process of the cigarette, and has an important influence on the smoking quality of the cigarette. Therefore, the method for accurately measuring the types and the contents of the organic acids in the tobacco essence has important significance for knowing the internal quality of various perfume raw materials and designing the essence formula.
At present, the common method for determining the organic acid in the tobacco is to firstly derivatize the acidic component in the tobacco into methyl ester for analysis, and the common method for esterifying uses reagents including: methanol and diazomethane sulfate, and a silicon etherification reagent, etc. The treatment method has complicated steps and long time consumption, and the condition that the acid is not completely converted into the ester and the short-chain organic acid methyl ester is lost in the extraction process possibly occurs, so that the accuracy of the detection result is affected.
In addition, the solvents in the tobacco essence are the components with the highest content, and the common tobacco essence solvents mainly comprise water, ethanol, 1, 2-propylene glycol, glycerol and the like. The stability of the solvent content is critical to the quality of the essence and the spice, and is an important index for judging whether the essence and the spice for cigarettes are qualified or not. Therefore, the detection of the solvent content in the tobacco essence has important significance in the aspects of enhancing the quality control of the essence, improving the quality stability of Chinese cigarette products and the like. At present, a common detection method for tobacco essence is gas chromatography. In the prior art, under the condition that the content of the organic acid and the content of the solvent are required to be detected respectively, the method is required to be used for detecting respectively, the operation is complicated, the time is long, and the accuracy of the detection result is required to be improved.
Disclosure of Invention
In order to solve or partially solve the problems existing in the related art, the invention provides a method for detecting the content of organic acid and solvent in tobacco essence.
The invention provides a method for detecting the content of organic acid and solvent in tobacco essence, which comprises the following steps:
Step a), carrying out pretreatment on tobacco essence to be detected to obtain liquid to be detected;
step b), separating the liquid to be detected by adopting a gas chromatography, wherein a chromatographic column adopts an inert polyethylene glycol polymer chromatographic column;
step c), separating the separated liquid separated by the chromatographic column to obtain a first detection liquid and a second detection liquid;
Step d), introducing the first detection liquid into a mass spectrometer for analysis, and determining the content of the organic acid; and (3) introducing the second detection liquid into a thermal conductivity detector for analysis, and determining the content of the solvent.
Further, the step a) is specifically: and adding an extractant and an internal standard substance into the tobacco essence to be detected, and filtering the tobacco essence by a microporous filter membrane to obtain the liquid to be detected.
Further, the extractant is methanol, acetone or dichloromethane.
Further, the internal standard comprises a solvent internal standard and an organic acid internal standard, wherein the solvent internal standard is n-butanol, 1, 3-butanediol and 1, 4-butanediol; the organic acid internal standard is 2-ethylbutyric acid or adipic acid.
Further, the conditions for separating the liquid to be detected by gas chromatography in the step b) are as follows:
chromatographic column: an inert treated polyethylene glycol polymer chromatographic column, 60m x 0.25mm x 0.25 μm;
Carrier gas: he; flow rate: 1ml/min;
Sample inlet temperature: 150 ℃; sample injection amount: 1 μl; split ratio: 15:1, a step of;
heating program: the initial temperature is 100 ℃, and the temperature is kept for 2min; then heating to 152 ℃ at a speed of 6 ℃/min, and keeping for 2min; heating to 173 ℃ at a speed of 6 ℃/min, and keeping for 2min; heating to 250 ℃ at 20 ℃/min, and keeping for 10min.
Further, the step c) specifically includes: and (3) splitting the separation liquid separated and flowed out by the chromatographic column through a capillary column splitter 1:1 to obtain a first detection liquid and a second detection liquid.
Further, the analysis conditions under which the first detection liquid is introduced into the mass spectrometer for analysis in the step d) are as follows:
transmission line temperature: transmission line temperature: 250 ℃;
ion source temperature: 250 ℃;
Quadrupole temperature: 150 ℃;
Ionization energy: 70eV;
Scanning mode: a SIM mode;
Solvent delay: 4.50min.
Further, the analysis conditions under which the second detection liquid is introduced into the thermal conductivity detector for analysis are as follows:
temperature: 260 ℃; reference flow rate: 20ml/min; tail blow flow: 10ml/min.
Further, the organic acid is one or more of acetic acid, formic acid, propionic acid, isovaleric acid, valeric acid, 2-methyl valeric acid, caproic acid, heptanoic acid, caprylic acid, sorbic acid and capric acid.
Further, the solvent is one or more of ethanol, water, 1, 2-propanediol and glycerol.
The method for detecting the content of the organic acid and the solvent in the tobacco essence provided by the invention can comprise the following beneficial effects:
The detection method adopts GC-MS/TCD parallel gas chromatography to directly sample, and can simultaneously detect the content of organic acid and solvent in the tobacco essence. The method does not need derivatization reaction, has simple sample pretreatment, high efficiency and high accuracy; the method can detect the content of the organic acid and the solvent simultaneously, is beneficial to simplifying the operation and improving the detection efficiency. The method is suitable for the situation that the detection requirements are met on the content of the organic acid and the content of the solvent, and provides technical reference for understanding the internal quality of various essences and designing the formula of the essences.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
Drawings
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular descriptions of exemplary embodiments of the invention as illustrated in the accompanying drawings wherein like reference numbers generally represent like parts throughout the exemplary embodiments of the invention.
FIG. 1 is a chromatogram of 11 organic acids in standard stock solutions in an example of the present invention, with compounds corresponding to each reference number in the figure being shown in Table 1 below;
FIG. 2 is a chromatogram of 4 solvent components in an example of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.
It should be understood that although the terms "first," "second," "third," etc. may be used in this disclosure to describe various information, these information should not be limited to these terms. These terms are only used to distinguish one type of information from another. For example, first information may also be referred to as second information, and similarly, second information may also be referred to as first information, without departing from the scope of the invention. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present invention, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
The embodiment of the invention provides a method for detecting the content of organic acid and solvent in tobacco essence, which comprises the following steps:
Step a), carrying out pretreatment on tobacco essence to be detected to obtain liquid to be detected;
step b), separating the liquid to be detected by adopting a gas chromatography, wherein an inert chromatographic column is adopted as a chromatographic column;
Step c), separating the separated liquid which flows out through the chromatographic column to obtain a first detection liquid and a second detection liquid;
Step d), introducing the first detection liquid into a mass spectrometer for analysis, and determining the content of the organic acid; and (3) introducing the second detection liquid into a thermal conductivity detector for analysis, and determining the content of the solvent.
The step a) is to pre-treat the tobacco flavor to be detected so as to be suitable for gas chromatographic separation. The method specifically comprises the following steps: and adding an extractant and an internal standard substance into the tobacco essence to be detected, and filtering the tobacco essence by a microporous filter membrane to obtain the liquid to be detected which can be detected on machine. The purpose of the extractant is to extract the organic acid in the tobacco essence. The extractant is preferably methanol, acetone or dichloromethane. The inventor finds that the three extracting agents have similar extraction effects on organic acids in the tobacco essence; however, the other two extractants except methanol have different degrees of interference between the solvent peak and the sample peak, so that the methanol is more preferably selected as the organic acid extractant in the tobacco essence according to the consideration of the factors.
The function of adding the internal standard in the step is that: the influence of the fluctuation of the operation condition on the analysis result is calibrated and eliminated to improve the accuracy of the analysis result. The detection method provided by the invention can be used for simultaneously detecting the solvent and the organic acid in the tobacco essence, so that the internal standard preferably comprises the solvent internal standard and the organic acid internal standard. As for the internal standard of the solvent, n-butanol, 1, 3-butanediol or 1, 4-butanediol is preferably used, the peak of the internal standard of the solvent can be well separated from the target peak, the retention time of each internal standard is in the middle section of each component peak, the internal standard is not overlapped with other components in the sample, the peak shape is symmetrical, and n-butanol is more preferably used. As the internal standard of the organic acid, 2-ethylbutyric acid or adipic acid is preferably used, and 2-ethylbutyric acid is more preferably used. In summary, as a most preferred embodiment of this embodiment, the internal standard comprises n-butanol and 2-ethylbutyric acid.
In the step, preferably, a vibration extraction mode is adopted, namely, the vibration extraction is carried out after the extractant and the internal standard substance are added into the tobacco essence to be detected, the vibration extraction can be adopted to achieve high extraction efficiency, and the vibration extraction time is preferably 20-50 min. Further, the present inventors have found that the extraction effect is improved with the prolonged shaking extraction time, but the change of the extraction effect is not obvious after 30min. Therefore, as a more preferable scheme, the time of the shaking extraction is 30min.
The step b) is a step of separating the liquid to be detected obtained by pretreatment by adopting a gas chromatography method, so as to separate the target detection object. Gas chromatography is a good separation means, and can separate each component in the compound mixture, and quantitatively analyze the organic acid by using gas chromatography, and a common method is to carry out derivatization reaction on the organic acid first and then detect the organic acid by using a capillary gas chromatography column. Because the strong polar compound in the underivatized organic acid has stronger interaction with the active site on the chromatographic column, tailing or peak separation is poor, the quantitative analysis difficulty under low concentration is increased, and the method is not suitable for the quantitative analysis of the organic acid. Further, since the present example also requires measurement of the tobacco flavor solvent, the above problems are certainly aggravated by the presence of a large amount of strongly polar compounds such as water in the separated liquid. Therefore, the inventor considers to overcome the problems by improving the chromatographic column, and further, according to the physicochemical properties of the organic acid and the solvent of the essence for cigarettes, the inventor adopts the polyethylene glycol polymer chromatographic column subjected to inert treatment, on one hand, the polyethylene glycol polymer chromatographic column has stronger polarity, is favorable for adsorbing the organic acid, and further, ensures better separation effect on the organic acid; on the other hand, the interaction between strong polar compounds such as organic acid, water and the like and the chromatographic column can be reduced by inert treatment, and the column loss is weakened. Therefore, the polyethylene glycol polymer chromatographic column subjected to inert treatment can realize simultaneous detection of the organic acid and the solvent, and the organic acid is directly injected without derivatization treatment, so that the detection step is shortened, and the detection accuracy is improved. More preferably, the DB-FATWAX ultra-high inert chromatographic column of Agilent company is adopted, the chromatographic column has good separation effect on all organic acids in the tobacco essence, and the peak shape is sharp and symmetrical, and the best peak shape.
The conditions for separating the liquid to be detected by the above gas chromatography are preferably as follows:
chromatographic column: an inert treated polyethylene glycol polymer chromatographic column, 60m x 0.25mm x 0.25 μm;
Carrier gas: he; flow rate: 1ml/min;
Sample inlet temperature: 150 ℃; sample injection amount: 1 μl; split ratio: 15:1, a step of;
heating program: the initial temperature is 100 ℃, and the temperature is kept for 2min; then heating to 152 ℃ at a speed of 6 ℃/min, and keeping for 2min; heating to 173 ℃ at a speed of 6 ℃/min, and keeping for 2min; heating to 250 ℃ at 20 ℃/min, and keeping for 10min.
Among the above analysis conditions:
the inventors have found that the sample injection temperature has an effect on the determination of organic acids and solvents:
Preparing common 11 organic acids (acetic acid, formic acid, propionic acid, isovaleric acid, valeric acid, 2-methyl valeric acid, caproic acid, heptanoic acid, caprylic acid, sorbic acid and capric acid) and common 4 solvents (ethanol, water, 1, 2-propylene glycol and glycerol) in tobacco essence into a standard stock solution serving as a sample;
And (3) using a standard stock solution as a sample, and comparing different sample inlet temperatures to study the influence of the organic acid and the solvent to be detected at the sample inlet temperature. Comparing the temperatures of sample inlets at 100 ℃, 150 ℃,200 ℃, 250 ℃ and 300 ℃, and finding that when the temperature is 100 ℃, the solvent peak is too large, which can interfere the detection and quantification of the object to be detected afterwards, and the temperature is too low, the sample gasification speed is slow, and the expansion of the solvent peak is related; when the temperature reaches 250 ℃ and above, the variety of the finally detected analyte is significantly reduced, and the sample may be decomposed due to an excessively high temperature. Therefore, the temperature of the sample inlet is finally selected to be 150 ℃.
The inventors have found that the split ratio of the sample injection has an effect on the determination of the organic acid and the solvent:
The standard stock solutions were used as samples, and were not split, 1:10,1:50,1:100,1:200 shunt effect on detection of organic acid and solvent to be detected. As a result, it was found that when the split ratio was less than 1: at 50, a portion of the chromatographic peak of the analyte appears as a distinct deformation tail, which may be associated with a decrease in separation efficiency due to excessive sample entry into the chromatographic column. When the split ratio is 1: at 200, some of the organic acid components are not detected, which may be associated with too low a concentration of the sample after fractionation, or may be associated with an increase in the branching discrimination effect with an increase in the branching ratio. Although the effect of the split-flow discrimination can be reduced by increasing the gasification temperature, too high a temperature may cause decomposition of the organic acid in the sample, so the invention uses a lower split-flow 1:50 split ratio.
The organic acid and the solvent are separated through the chromatographic column, and due to the difference of physical properties of the organic acid and the solvent, the method does not consider that one method is used for simultaneously detecting the two types of compounds, but the method which is more suitable for the two types of substances is selected for respectively and simultaneously detecting the two types of compounds. In order to realize simultaneous detection of the same separating liquid by adopting two methods, in the embodiment, the separating liquid is split first and is divided into two parts, namely, the step c) is performed, so as to obtain a first detecting liquid and a second detecting liquid; therefore, the two detection liquids can be respectively and simultaneously guided into different analysis instruments for detection, and a precondition is provided for subsequent MS/TCD detection. Further, in order to ensure that the first detection liquid and the second detection liquid flow out in equal amounts, the present step is preferably as follows: and (3) splitting the separation liquid separated and flowed out by the chromatographic column through a capillary column splitter 1:1 to obtain a first detection liquid and a second detection liquid. After being split by a capillary column splitter 1:1, the separation liquid enters a damping column in the splitter respectively and reaches MS and TCD simultaneously to be detected, so that sample injection can be performed once, and simultaneous analysis and detection of organic acid and solvent can be realized.
The step d) is a step of simultaneously measuring the content of the organic acid and the solvent by adopting different methods. Specifically, for the content measurement of organic acid, a mass spectrometer is adopted, a mass spectrometer technology is adopted, a vaporized molecular sample is converted into charged particles in a high-vacuum ion source, the charged particles enter a mass analyzer after ionization, extraction and focusing, mass-to-charge ratio separation is carried out according to time front and back or space positions under the action of a magnetic field or an electric field, the left back is detected by a particle detector, MS fragments obtained under certain conditions and corresponding intensities are easy to identify just like a fingerprint image, and the method has the advantage of high sensitivity. For the content measurement of the solvent, the thermal conductivity detector is adopted, and has response to inorganic matters and organic matters, does not damage the sample, and has the advantages of simple structure, good stability, wide linear range and low influence on the detection result due to high solvent content in the detection liquid although the sensitivity is relatively low. And the reason for adopting the two methods is that: because the mass spectrometer scans in a selected ion detection (SIM) mode, the mode only detects selected ions, and other ions are not recorded, the presence of the solvent does not affect the detection of the organic acid by the MS; since the organic acid and solvent have been effectively separated by the column, the presence of the organic acid does not affect the detection of the solvent by TCD.
The analysis conditions under which the first detection liquid is introduced into the mass spectrometer for analysis are preferably as follows:
transmission line temperature: transmission line temperature: 250 ℃;
ion source temperature: 250 ℃;
Quadrupole temperature: 150 ℃;
Ionization energy: 70eV;
Scanning mode: a SIM mode;
Solvent delay: 4.50min.
The analysis conditions under which the second detection liquid is introduced into the thermal conductivity detector for analysis are preferably as follows:
temperature: 260 ℃; reference flow rate: 20ml/min; tail blow flow: 10ml/min.
In this step, for the determination of the organic acid content and the solvent content, an external standard method may be employed.
The organic acid detection object suitable for the present invention is preferably: one or more of acetic acid, formic acid, propionic acid, isovaleric acid, valeric acid, 2-methyl valeric acid, caproic acid, heptanoic acid, caprylic acid, sorbic acid and capric acid;
the solvent detection object suitable for the present invention is preferably: the solvent is preferably one or more of ethanol, water, 1, 2-propanediol and glycerol.
The technical scheme of the invention is further described below with reference to specific examples:
examples
[ Object to be detected ]
Organic acid (11): acetic acid, formic acid, propionic acid, isovaleric acid, valeric acid, 2-methyl valeric acid, caproic acid, heptanoic acid, caprylic acid, sorbic acid and capric acid
Solvent (4): ethanol, water, 1, 2-propanediol and glycerol
Materials, reagents and instruments
Essence for cigarettes: is provided by the research of essence and spice of Hebei Zhongyan technology center.
Acetic acid, formic acid, propionic acid, isovaleric acid, valeric acid, 2-ethylbutyric acid (internal standard), 2-methylpentanoic acid, caproic acid, heptanoic acid, caprylic acid, sorbic acid, capric acid, ethanol, 1, 2-propanediol, glycerol: the purity is more than or equal to 98 percent, and the products are purchased from the carbofuran company.
Gas chromatography mass spectrometer: model 7890B-5977A, available from Agilent corporation, U.S.A.;
Thermal Conductivity Detector (TCD): purchased from Agilent corporation of united states;
chromatographic column: purchased from Agilent corporation, usa, DB-FATWAX ultra high inert (UI) column;
1:1 capillary column flow divider: the inner diameter of the damping column is 0.22mm, and is purchased from Agilent company of America;
EL204 type electronic balance: purchased from mertrer-tolidor instruments (Shanghai); ;
organic phase filtration membrane: 0.22 μm, purchased from Shanghai Annotation;
Mili-Q ultra-pure water instrument: purchased from Millipore corporation;
GFL3017 shaker: purchased from Burgwedel, germany.
[ Detection System ]
The outlet of the chromatographic column of the gas chromatography-mass spectrometer is connected with a 1:1 capillary column diverter, one interface (namely the first detection liquid flow outlet) of the diverter is connected with the sample injection end of the mass spectrometer, and the other interface (namely the second detection liquid flow outlet) is connected with the sample injection end of the thermal conductivity detector.
[ Detection method ]
1. Standard working curve drawing
1.1 Standard working solution configuration
Preparation of standard stock solution: 160mg of acetic acid, 100mg of formic acid, 160mg of propionic acid, 10mg of isovaleric acid, 5mg of valeric acid, 10mg of 2-methyl valeric acid, 20mg of caproic acid, 40mg of heptanoic acid, 10mg of caprylic acid, 80mg of sorbic acid, 20mg of capric acid, 200mg of ethanol, 100mg of water, 200mg of 1, 2-propanediol and 200mg of glycerol are respectively weighed accurately in a 50ml volumetric flask, and methanol is used for volume metering to prepare a mixed standard stock solution.
Preparation of internal standard stock solution: 50mg of 2-ethylbutyric acid and 50mg of n-butanol are weighed into a10 ml volumetric flask, the accuracy is 0.1mg, and methanol is added to fix the volume, so as to obtain an internal standard stock solution.
Series standard working curve configuration: accurately transferring 0.05, 0.10, 0.50, 1.00, 2.00, 3.00, 4.00, 5.00, 6.00 and 7.00mL of mixed standard stock solution into a 10mL volumetric flask respectively; 100 μl of internal standard stock solution is added respectively, and methanol is used for volume fixation to scale, so as to obtain ten-stage series standard working solutions with different concentrations.
1.2 On-machine GC-MS/TCD analysis
And respectively carrying out GC-MS/TCD analysis on the detection systems on ten-stage series standard working solutions with different concentrations.
Chromatographic condition analysis conditions:
chromatographic column: an inert treated polyethylene glycol polymer chromatographic column, 60m x 0.25mm x 0.25 μm;
Carrier gas: he; flow rate: 1ml/min;
and (3) sample injection: sample inlet temperature: 150 ℃; sample injection amount: 1 μl; split ratio: 15:1, a step of;
heating program: the initial temperature is 100 ℃, and the temperature is kept for 2min; then heating to 152 ℃ at a speed of 6 ℃/min, and keeping for 2min; heating to 173 ℃ at a speed of 6 ℃/min, and keeping for 2min; heating to 250 ℃ at 20 ℃/min, and keeping for 10min.
MS detector: transmission line temperature: 250 ℃; ion source temperature: 250 ℃; quadrupole temperature: 150 ℃; ionization energy: 70eV; scanning mode: a SIM mode; solvent delay for 4.50min;
TCD detector: temperature 260 ℃, reference flow rate: 20ml/min, tail blow flow: 10ml/min.
1.3 Qualitative and quantitative analysis
And (3) carrying out GC-MS/TCD analysis on the mixed standard sample by adopting a selected ion scanning mode (SIM), and carrying out qualitative analysis by adopting a standard sample adding method and retention time comparison and combining with a GC-MS identification result under the same analysis condition. And (3) determining the retention time and characteristic ions of standard organic acid samples of all to-be-detected substances by combining NIST98 spectrum library search, wherein the results are shown in table 1, the chromatograms of 11 organic acid standard samples are shown in figure 1, and the compounds corresponding to the reference numerals in the figure are shown in table 1. The chromatograms of the 4 solvent components in the standard stock solutions are shown in fig. 2.
TABLE 1 retention time of 11 organic acids and characteristic ions
1.4 Standard Curve, detection Limit and quantitative Limit
Regression analysis is carried out by the ratio of the quantitative ion peak area of the target compound and the internal standard peak to the ratio of the concentration of each target object and the internal standard concentration, and the linear regression equation and the correlation coefficient of 15 objects to be detected are obtained. The limit of detection (LOD) was calculated at a 3-fold signal to noise ratio (S/n=3) and the limit of quantification (LOQ) was calculated at S/n=10, and the results are shown in table 2. As can be seen from Table 2, each compound exhibited good linearity in peak area in the corresponding concentration range, and the correlation coefficient (R2) was 0.999 or more. The detection limit and the quantitative limit are respectively between 0.01-0.75 mug/ml and 0.02-2.49 mug/ml, which can meet the quantitative analysis requirement of each target.
TABLE 2 Linear parameters, detection limits and quantitative limits for 15 analytes in tobacco flavor
2. Detection of organic acid and solvent of tobacco essence
15 Essence samples for cigarettes are taken, and the serial numbers are sequentially 1-15; respectively accurately weighing 1.0g of 15 tobacco essence samples, respectively adding the samples into 25ml of triangular flasks, adding 10ml of methanol and 100 mu l of internal standard stock solution, oscillating for 30min on an oscillating table, and filtering by a 0.22 mu m microporous filter membrane to obtain a liquid to be detected;
and (3) detecting: the process and analysis conditions are the same as in step 1.
The analysis results of 15 samples of tobacco flavor were obtained according to the detection results and the standard working curve, and are shown in table 3.
TABLE 3 analysis results (mg/g) of 15 analytes in essence samples
As can be seen from table 3: no glycerol is detected in all samples, the types and the contents of organic acids contained in different tobacco essences are obviously different, and the use amount is small, but acetic acid, formic acid, heptanoic acid, caproic acid and the like are widely used.
3. Recovery and precision of the method
Taking a No. 9 tobacco essence sample as an example, adding 15 substances to be detected with low, medium and high concentration of 3 substances to be detected with different concentrations for carrying out a labeling recovery experiment. Each concentration level was repeated 6 times, and recovery rates and RSD of 15 test substances in the samples were calculated, and the results are shown in table 4. As can be seen from table 4: the average standard adding recovery rate of 15 to-be-detected substances under different concentration levels is 91.14-113.08%, and the RSD is 0.42-6.46%. The method has high recovery rate and good precision.
Table 4 recovery and precision of 11 fragrances and 4 solvents for cigarettes (n=6)
Note that: no glycerol test data was present since no glycerol was detected in the samples.
From the above, the method for detecting the content of the organic acid and the solvent in the tobacco essence provided by the invention has the following advantages:
The detection method adopts GC-MS/TCD parallel gas chromatography to directly sample, and can simultaneously detect the content of organic acid and solvent in the tobacco essence. The method does not need derivatization reaction, has simple sample pretreatment, high efficiency and high accuracy; the method can detect the content of the organic acid and the solvent simultaneously, is beneficial to simplifying the operation and improving the detection efficiency. The method is suitable for the situation that the detection requirements are met on the content of the organic acid and the content of the solvent, and provides technical reference for understanding the internal quality of various essences and designing the formula of the essences.
The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the improvement of technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Claims (3)

1. The method for detecting the content of organic acid and solvent in the tobacco essence is characterized by comprising the following steps of:
Step a), adding an extractant and an internal standard substance into the tobacco essence to be detected, and then filtering the tobacco essence by a microporous filter membrane to obtain a liquid to be detected; the extractant is methanol, acetone or dichloromethane; the internal standard comprises n-butanol and 2-ethylbutyric acid;
and b), separating the liquid to be detected by adopting a gas chromatography method under the following conditions:
chromatographic column: DB-FATWAX ultra-high inert chromatographic column of Agilent company, 60m×0.25mm×0.25 μm;
Carrier gas: he; flow rate: 1ml/min;
Sample inlet temperature: 150 ℃; sample injection amount: 1 μl; split ratio: 15:1, a step of;
heating program: the initial temperature is 100 ℃, and the temperature is kept for 2min; then heating to 152 ℃ at a speed of 6 ℃/min, and keeping for 2min; heating to 173 ℃ at a speed of 6 ℃/min, and keeping for 2min; heating to 250deg.C at 20deg.C/min, and maintaining for 10min;
Step c), separating the separation liquid separated and flowed out by the chromatographic column through a capillary column splitter 1:1 to obtain a first detection liquid and a second detection liquid;
Step d), the first detection liquid is guided into a mass spectrometer for analysis, the scanning mode is a SIM mode, and the content of the organic acid is measured; introducing the second detection liquid into a thermal conductivity detector for analysis, and determining the content of the solvent;
the organic acid is acetic acid, formic acid, propionic acid, isovaleric acid, valeric acid, 2-methyl valeric acid, caproic acid, heptanoic acid, caprylic acid, sorbic acid and capric acid; the solvent is ethanol, water, 1, 2-propylene glycol and glycerol.
2. The method according to claim 1, wherein the analysis conditions for introducing the first detection liquid into the mass spectrometer for analysis in the step d) are as follows:
Transmission line temperature: 250 ℃;
ion source temperature: 250 ℃;
Quadrupole temperature: 150 ℃;
Ionization energy: 70eV;
Solvent delay: 4.50min.
3. The method of claim 1, wherein the second detection liquid is introduced into the thermal conductivity detector under the following analysis conditions:
temperature: 260 ℃; reference flow rate: 20ml/min; tail blow flow: 10ml/min.
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