CN111579652B - Method for measuring free state and protonated nicotine content by ultra-high performance combined phase chromatography - Google Patents
Method for measuring free state and protonated nicotine content by ultra-high performance combined phase chromatography Download PDFInfo
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Abstract
The invention discloses a method for measuring the content of free nicotine and protonated nicotine by ultra-high performance phase-locked chromatography, which belongs to the technical field of chemical analysis detection and comprises the preparation of a nicotine standard solution and the preparation of a sample solution to be measured, wherein the preparation comprises the steps of preparing the free nicotine and the protonated nicotine, and then directly detecting the free nicotine and the protonated nicotine by the ultra-high performance phase-locked chromatography for quantitative analysis, so that the method can be suitable for simultaneously measuring the free nicotine and the protonated nicotine. The method has the advantages of high sensitivity, good stability, simple and quick operation, low cost, environmental friendliness and easy popularization and application.
Description
Technical Field
The invention relates to the technical field of chemical analysis and detection, in particular to a method for determining free-state and protonated nicotine content by ultra-high performance combined chromatography.
Background
Nicotine, commonly known as nicotine, belongs to pyridine alkaloids, is a main addictive component of tobacco, and has strong physiological effect on human. The electronic cigarette can meet the requirement of people on nicotine, does not produce tar and carbon monoxide, has various tastes, and can meet the individual requirements of different people. With the development of the global tobacco ban policy, the demand of the electronic cigarette is increasing continuously, and the market of the electronic cigarette in China shows a rapid growth situation in recent years. With the development of the electronic cigarette industry, people find that the satisfaction brought by traditional cigarettes is difficult to obtain by smoking the electronic cigarette. This is because the electronic cigarette device is not ideal in the delivery of nicotine from the blood, and only a small part of high-concentration nicotine enters the blood and the majority stays in the throat, causing excessive stimulation, resulting in poor user experience.
Nicotine salts are products of nicotine combined with organic acids and are more stable in nature. Meanwhile, according to related researches, the nicotine salt has stronger penetrating power than nicotine, can enter the blood of a human body more easily, can meet the requirement of people on the nicotine in a short time, has mellow mouthfeel, does not excessively stimulate the throat, and can provide satisfaction close to real smoke. Therefore, the addition of nicotine salt into the electronic cigarette oil becomes a new trend and selling point, the ratio of free nicotine and protonated nicotine in the cigarette oil has a great influence on the taste and strength of the electronic cigarette, and the analysis of the ratio of free nicotine and protonated nicotine is of great interest to people.
Ultra performance compatible chromatography (UPC) 2 ) The technique stems from supercritical fluid chromatography techniques, since it is performed with supercritical CO 2 The main mobile phase is separated and analyzed by depending on the solvating capacity of the mobile phase, so that samples which are not suitable for gas chromatography and have high boiling points, low volatility and unstable heat can be analyzed, and the analysis speed and column efficiency of high performance liquid chromatography can be improved; the combination of 2 μm packing technology makes it possible to obtain higher separation capacity and higher analysis efficiency, and can obtain required system resolution by precisely regulating strength, pressure and temperature of mobile phaseThe efficiency and the selectivity are effectively controlled for the retention and the separation of the object to be tested, and compared with the high performance liquid chromatography, the ultra-high performance combined chromatography has the advantages of higher diffusion efficiency, better mass transfer effect, low operation temperature, high sensitivity, high separation efficiency, good repeatability, high analysis speed, environmental protection, capability of effectively reducing the damage of toxic solvents to the body health of testers and the like.
In tobacco and tobacco products, free nicotine and protonic nicotine are in dynamic balance, the content of the free nicotine is easily interfered by factors such as moisture, temperature, solvent, ph and the like, so that accurate determination of the free nicotine in the tobacco products is still a technical problem in the field, based on the technical problem, a liquid phase detection method capable of testing the free nicotine and the protonated nicotine and having less environmental interference and more difficulty for analysis and detection personnel is established, at present, most reports about nicotine content analysis are total nicotine, only a small amount of documents report the liquid phase detection method of the free nicotine and the protonated nicotine in cigarettes, and the existing test methods have different degrees of damage to the environment of the free nicotine and the protonated nicotine, so that the method for the ultra-high efficiency synthetic phase chromatography test capable of accurately determining the content of the free nicotine and the protonated nicotine in the tobacco and tobacco products is established It is necessary that the related reports of the type are not found in the current published documents.
Disclosure of Invention
Aiming at the defects in the technology, the invention provides the method for determining the content of free nicotine and protonated nicotine by ultra-high performance phase-combining chromatography, which can effectively and quickly detect the content of free nicotine and protonated nicotine.
In order to achieve the above object, the present invention provides a method for determining free and protonated nicotine content by ultra-high performance phase chromatography, comprising:
(1) preparation of nicotine standard solution: weighing nicotine reference substances, performing ultrasonic dissolution through chromatographic methanol to fix the volume, then performing liquid transfer, fixing the volume with dichloromethane, and filtering to obtain a series of standard solutions with different concentrations;
(2) preparing a free nicotine solution of a sample to be tested: accurately weighing a sample, fixing the volume by using ultrapure water, moving the sample to a centrifuge tube with a plug, extracting the sample for multiple times by using dichloromethane, combining dichloromethane layers obtained by each extraction, fixing the volume, and filtering to obtain filtrate I to be detected.
(3) Preparation of the protonated nicotine solution for the sample to be tested: and (3) adding saturated sodium hydroxide into the water phase of the centrifuge tube with the plug after extraction in the step (2), extracting for multiple times by using dichloromethane, combining dichloromethane layers obtained by each extraction, fixing the volume, and filtering to obtain a filtrate II to be detected.
(4) Precisely sucking the standard solution obtained in the step (1) and injecting the standard solution into an ultra-high performance phase-combination chromatograph for detection; and (4) precisely sucking the filtrate I to be detected and the filtrate II to be detected obtained in the step (2) and the step (3) and injecting the filtrates into an ultra-high performance phase-combining chromatograph for detection.
Wherein, the chromatographic conditions of the ultra-high performance phase-matching chromatograph are as follows: the mobile phase is supercritical CO 2 The auxiliary solvent is one of methanol, ethanol, isopropanol and acetonitrile.
Wherein the standard working solution configuration comprises:
(1) weighing 100mg of nicotine reference substance in a 50ml volumetric flask, and performing ultrasonic dissolution with chromatographic methanol to a constant volume to obtain a concentration of 2000 mg.L -1 Standard mother liquor;
(2) accurately transferring the standard mother liquor obtained in the step (1) into volumetric flasks of 0.1mL, 0.2mL, 0.5mL, 0.8mL and 1 mL-10 mL by using a pipette, fixing the volume of dichloromethane, and filtering to obtain 20 mg.L -1 、40mg·L -1 、100mg·L -1 、160mg·L -1 、200mg·L -1 A series of standard solutions with different concentrations.
Wherein, in the step (2), the preparation of the free nicotine solution of the sample to be tested comprises the following steps:
(1) accurately weighing an electronic cigarette oil sample in a 10mL volumetric flask, metering the volume with ultrapure water, and transferring the electronic cigarette oil sample into a 50mL centrifuge tube with a plug;
(2) adding 5-20mL of dichloromethane into the centrifuge tube with the plug in the step (1), shaking up and down, standing for 1-5min, and taking the lower layer (dichloromethane layer) into a volumetric flask;
(3) and (3) repeating the step (2) for 1-3 times, combining the lower layers obtained each time, fixing the volume by using dichloromethane, and filtering to obtain a filtrate I to be detected.
Wherein, in the step (2), the preparation of the free nicotine solution of the sample to be tested comprises the following steps:
(1) accurately weighing an electronic cigarette oil sample in a 10mL volumetric flask, metering the volume with ultrapure water, and transferring the electronic cigarette oil sample into a 50mL centrifuge tube with a plug;
(2) adding 10mL of dichloromethane into the centrifuge tube with the plug in the step (1), shaking up and down, standing for 1-5min, and taking the lower layer (dichloromethane layer) into a volumetric flask;
(3) and (3) repeating the step (2), combining the lower layers obtained twice, fixing the volume by using dichloromethane, and filtering to obtain a filtrate I to be detected.
Wherein, in the step (3), the preparation of the protonated nicotine solution for the sample to be tested comprises: and (3) adding 10-500 mu L of saturated sodium hydroxide solution into the water phase of the centrifuge tube with the plug after extraction in the step (2), shaking up and standing for 1-10min, extracting twice with dichloromethane, combining the lower layers of the dichloromethane, fixing the volume of the dichloromethane, and filtering to obtain a filtrate II to be detected.
Wherein, in the step (3), the preparation of the protonated nicotine solution for the sample to be tested comprises: and (3) adding 500 mu L of saturated sodium hydroxide solution into the water phase of the centrifuge tube with the plug after extraction in the step (2), shaking up and standing for 1-10min, extracting twice with dichloromethane, combining the lower layers, fixing the volume with dichloromethane, and filtering to obtain a filtrate II to be detected.
Wherein, the parameter conditions for detecting in the ultra-high performance phase-combination chromatography in the step (4) are specifically as follows: flow rate: 0.5-3 mL/min -1 (ii) a Detection wavelength: 220-300 nm; sample introduction amount: 0.5-10 μ L; temperature of the chromatographic column: 30-80 ℃; system back pressure 1500-; isocratic elution for 2-4 min; the mobile phase is supercritical CO 2 The auxiliary solvent is one of methanol, ethanol, isopropanol and acetonitrile.
Wherein, the parameter conditions for detecting the ultra-high performance phase-combination chromatography in the step (3) are specifically as follows: flow rate: 2.5 mL/min -1 (ii) a Detection wavelength: 259 nm; sample introduction amount: 2 mu L of the solution; temperature of the chromatographic column: 50 ℃; system back pressure 2000 pis; isocratic elution for 2 min: CO 2 2 (90%) methanol as an auxiliary solvent (1)0％)。
Wherein, in step (2), the sample comprises E-liquid and/or homemade nicotine salt.
Wherein, in the steps (1) to (3), the filtering conditions are as follows: filtering through a 0.22 mu m microporous filter membrane.
The invention has the beneficial effects that: compared with the prior art, the method for determining the content of free and protonated nicotine by using the ultra-high performance phase-locked chromatography has the beneficial effects that:
(1) by adopting ultra-high performance combined phase chromatography, rotary evaporation is not needed, and the organic solvent is directly injected, so that the pretreatment time of the sample is greatly shortened;
(2) the supercritical carbon dioxide is used as a main mobile phase, the low-proportion organic solvent is used as an auxiliary solvent, the separation efficiency is high, the solvent is saved, the waste liquid treatment cost is reduced to a great extent, and the method is green and environment-friendly. Meanwhile, the column is not easy to be lost by the mobile phase, so that the cost is saved;
(3) the sample analysis time is short, the peak shape is good, the sample substrate is free of interference, and the method can be used as a rapid, convenient and reliable detection means for quality control of the raw materials of the commercial electronic cigarette oil and nicotine.
Drawings
FIG. 1 is a chromatogram of a solvent of the present invention, methanol, ethanol, isopropanol, and acetonitrile;
FIG. 2 is a chromatogram of a blank solvent, a standard solution and a solution to be tested according to the present invention;
FIG. 3 is a chromatogram of a first, second and third extraction with dichloromethane in accordance with the present invention;
FIG. 4 is a chromatogram of 10, 30 and 50 μ L of sodium hydroxide of the present invention;
fig. 5 is a standard graph of the present invention.
Detailed Description
In order to more clearly describe the present invention, the present invention will be further described with reference to the accompanying drawings.
The invention relates to a method for measuring free state and protonated nicotine content by ultra-high performance combined chromatography, which comprises the following steps:
(1) preparation of nicotine standard solution: weighing nicotine reference substances, performing ultrasonic dissolution through chromatographic methanol to fix volume, then performing liquid transfer, fixing volume with dichloromethane, and filtering to obtain a series of standard solutions with different concentrations;
(2) preparing a free nicotine solution of a sample to be tested: accurately weighing a sample, fixing the volume with ultrapure water, moving to a centrifuge tube with a plug, extracting for multiple times with dichloromethane, combining dichloromethane layers obtained by each extraction, fixing the volume, and filtering to obtain a filtrate I to be detected.
(3) Preparation of the protonated nicotine solution for the sample to be tested: and (3) adding saturated sodium hydroxide into the water phase of the centrifuge tube with the plug after extraction in the step (2), extracting for multiple times by using dichloromethane, combining dichloromethane layers obtained by each extraction, fixing the volume, and filtering to obtain a filtrate II to be detected.
(4) Precisely sucking the standard solution obtained in the step (1) and injecting the standard solution into an ultra-high performance phase-combination chromatograph for detection; and (4) precisely sucking the filtrate I to be detected and the filtrate II to be detected obtained in the step (2) and the step (3) and injecting the filtrates into an ultra-high performance phase-combining chromatograph for detection.
Wherein, the chromatographic conditions of the ultra-high performance phase-matching chromatograph are as follows: the mobile phase is supercritical CO 2 The auxiliary solvent is one of methanol, ethanol, isopropanol and acetonitrile.
The preparation method of the standard working solution comprises the following steps:
(1) weighing 100mg of nicotine reference substance in a 50ml volumetric flask, and performing ultrasonic dissolution with chromatographic methanol to a constant volume to obtain a concentration of 2000 mg.L -1 Standard mother liquor;
(2) accurately transferring the standard mother liquor obtained in the step (1) into volumetric flasks of 0.1mL, 0.2mL, 0.5mL, 0.8mL and 1 mL-10 mL by using a pipette, fixing the volume of dichloromethane, and filtering to obtain 20 mg.L -1 、40mg·L -1 、100mg·L -1 、160mg·L -1 、200mg·L -1 A series of standard solutions with different concentrations.
The preparation of the free nicotine solution of the sample to be tested comprises the following steps:
(1) accurately weighing an electronic cigarette oil sample in a 10mL volumetric flask, metering the volume with ultrapure water, and transferring the electronic cigarette oil sample into a 50mL centrifuge tube with a plug;
(2) adding 5-20mL of dichloromethane into the centrifuge tube with the plug in the step (1), shaking up and down, standing for 1-5min, and taking the lower layer (dichloromethane layer) into a volumetric flask;
(3) repeating the step (2) for 1-3 times, combining the lower layers obtained each time, fixing the volume by using dichloromethane, and filtering to obtain a filtrate I to be detected;
in step (3), the preparation of the protonated nicotine solution for the sample to be tested comprises: and (3) adding 10-500 mu L of saturated sodium hydroxide solution into the water phase of the centrifuge tube with the plug after extraction in the step (2), shaking up and standing for 1-10min, extracting twice with dichloromethane, combining the lower layers of the dichloromethane, fixing the volume of the dichloromethane, and filtering to obtain a filtrate II to be detected.
The method comprises the steps of performing liquid-liquid extraction, dissolving a sample by using water, adding an organic solvent, wherein the organic solvent can extract free nicotine, at the moment, free nicotine is in an organic phase, namely the filtrate I to be detected obtained in the step (2), protonated nicotine is in an aqueous phase, and the protonated nicotine in the aqueous phase is completely converted into the free nicotine by using sodium hydroxide, namely the filtrate II to be detected obtained in the step (3), so that the content of the free nicotine and the content of the protonated nicotine are simultaneously determined. The method has the advantages that rotary evaporation is not needed, the organic solvent is directly fed, the sample pretreatment time is greatly shortened, meanwhile, the supercritical carbon dioxide is used as a main mobile phase, the low-proportion organic solvent is used as an auxiliary solvent, the separation efficiency is high, the solvent is saved, the waste liquid treatment cost is reduced to a great extent, and the method is green and environment-friendly. The mobile phase is not easy to lose the column, and the cost is saved. The sample analysis time is short, the peak shape is good, and the sample matrix is free of interference.
The following examples demonstrate the various properties of the present invention, wherein the main reagents, instrumentation and samples to be tested are as follows:
main reagents, instrumentation and samples to be tested:
nicotine control (99.5%, available from Sigma company, usa); methanol (chromatographic grade, available from Merck, germany); ethanol (chromatographic grade, available from Merck, germany); isopropanol (chromatographic grade, available from j.t. baker); acetonitrile (chromatographic grade, from Merck, germany); carbon dioxide (99.9% from Shenzhen Shenchuan industry Co., Ltd.); dichloromethane (analytically pure, purchased from Beijing national drug group); the experimental water was ultrapure water (resistivity 18.2 M.OMEGA.. multidot.cm); sodium hydroxide (analytically pure, purchased from Beijing national drug group); e-liquid (commercially available);
the instrument comprises the following steps: ultra-high performance phase-locked chromatograph, ACQULITY UPC 2 Diode-array detectors (available from waters, usa); chromatographic column, ACQULITY UPC 2 Viridis BEH, 3X 100mm, 1.7 μm (available from waters, USA); ultrasonic instruments (purchased from Shenzhen modern ultrasound industries, Ltd.); electronic balance (ex rittler-toledo, switzerland); ultrapure water system (available from Milli-pore, USA).
A sample to be tested: an electronic cigarette sample and a nicotine salt.
(I) testing and analyzing the preparation conditions under the conditions of the method
Example 1
(1) Preparing a nicotine standard test solution: weighing 100mg of nicotine reference substance in a 50ml volumetric flask, and performing ultrasonic dissolution with chromatographic methanol to a constant volume to obtain a concentration of 2000 mg.L -1 Standard mother liquor; accurately transferring the obtained standard mother liquor into volumetric flasks of 0.1mL, 0.2mL, 0.5mL, 0.8mL and 1 mL-10 mL by using a pipette, metering the volume of dichloromethane, and filtering by using a 0.22 mu m microporous membrane to obtain 20 mg.L -1 、40mg·L -1 、100mg·L -1 、160mg·L -1 、200mg·L -1 And a series of standard solutions with different concentrations.
(2) And (3) testing: the solubility obtained in the step (1) is 100 mg.L -1 The standard solution is used for detecting by an ultra-high performance phase-combination chromatograph; wherein, the chromatographic conditions of the ultra-high performance phase-combination chromatograph are as follows: flow rate: 2.5 mL/min -1 (ii) a Detection wavelength: 259 nm; sample introduction amount: 2 mu L of the solution; temperature of the chromatographic column: 50 ℃; system back pressure 2000 pis; isocratic elution for 2 min: CO 2 2 (90%) the mobile phase is supercritical CO 2 The auxiliary solvent is methanol.
Example 2
(1) Preparing a nicotine standard test solution: same as in step (1) of example 1;
(2) the solubility obtained in the step (1) is 100 mg.L -1 Detecting the standard solution by an ultra-high performance phase-matching chromatograph; wherein, the ultra-efficient phase-combination chromatographThe chromatographic conditions of (A) are as follows: flow rate: 2.5 mL/min -1 (ii) a Detection wavelength: 259 nm; sample introduction amount: 2 mu L of the solution; temperature of the chromatographic column: 50 ℃; system back pressure 2000 pis; isocratic elution for 2 min: CO 2 2 (90%) the mobile phase is supercritical CO 2 The auxiliary solvent is ethanol.
Example 3
(1) Preparing a nicotine standard test solution: same as in step (1) of example 1;
(2) the solubility obtained in the step (1) is 100 mg.L -1 The standard solution is used for detecting by an ultra-high performance phase-combination chromatograph; wherein, the chromatographic conditions of the ultra-high performance phase-combination chromatograph are as follows: flow rate: 2.5 mL/min -1 (ii) a Detection wavelength: 259 nm; sample introduction amount: 2 mu L of the solution; temperature of the chromatographic column: 50 ℃; system back pressure 2000 pis; isocratic elution for 2 min: CO 2 2 (90%) the mobile phase is supercritical CO 2 The auxiliary solvent is isopropanol.
Example 4
(1) Preparing a nicotine standard test solution: same as in step (1) of example 1;
(2) the solubility obtained in step (1) of the example was 100 mg. L -1 The standard solution is used for detecting by an ultra-high performance phase-compatible chromatograph; wherein, the chromatographic conditions of the ultra-high performance phase-combination chromatograph are as follows: flow rate: 2.5 mL/min -1 (ii) a Detection wavelength: 259 nm; sample introduction amount: 2 mu L of the solution; temperature of the chromatographic column: 50 ℃; system back pressure 2000 pis; isocratic elution for 2 min: CO 2 2 (90%) the mobile phase is supercritical CO 2 And the auxiliary solvent is acetonitrile.
The chromatogram obtained in examples 1 to 4 is shown in FIG. 1, and it is seen from FIG. 1 that when the auxiliary solvent is methanol, the peak shape is good, the peak appearance time is fast, the ethanol peak shape is short and fat, isopropanol is used as the auxiliary solvent, the peak shape is trailing, acetonitrile is used as the solvent, and nicotine does not appear, so that when methanol is used as the auxiliary solvent, the chromatographic conditions of ultra-high phase-closure chromatography are the best conditions.
Example 5
(1) Preparing a blank solvent test solution: taking a dichloromethane solvent into a volumetric flask of 5ml, and filtering the dichloromethane solvent through a microporous filter membrane of 0.22 mu m to obtain a blank solvent test solution;
(2) preparing a free nicotine sample solution to be tested: accurately weighing an electronic cigarette oil sample in a 10mL volumetric flask, fixing the volume by ultrapure water, transferring to a 50mL centrifuge tube with a plug, adding 10mL dichloromethane, shaking up and down, standing for 4min, taking the lower layer in a 25mL volumetric flask, adding dichloromethane to fix the volume, and filtering by a 0.22 mu m microporous membrane to obtain the free nicotine solution 1.
(3) Placing the blank solvent test solution obtained in the step (1) into an ultra-high performance phase-matching chromatograph for detection; and (3) detecting the free nicotine solution obtained in the step (2) by using an ultra-high performance phase-combining chromatograph, wherein the chromatographic conditions of the ultra-high performance phase-combining chromatograph are as follows: flow rate: 2.5 mL/min -1 (ii) a Detection wavelength: 259 nm; sample introduction amount: 2 mu L of the solution; temperature of the chromatographic column: 50 ℃; system back pressure 2000 pis; isocratic elution for 2 min: CO 2 2 (90%) the mobile phase is supercritical CO 2 The auxiliary solvent is methanol.
The chromatogram obtained in example 1 and the chromatogram obtained in step (3) in example 5 are shown in FIG. 2, wherein the first peak in a blank solvent-dichloromethane in the chromatogram is the solvent peak; in the nicotine reference substance in the synthetic chromatogram, a solvent peak appears firstly, and nicotine appears secondly; in the combined phase chromatogram sample, a solvent peak appears firstly, an impurity peak appears secondly, and a free nicotine peak appears finally, the nicotine peak is good in shape, and the blank solvent and the standard solution have no interference on nicotine analysis.
Example 6
(1) 10mL of dichloromethane was added into the centrifuge tube with a stopper extracted in the step (2) of example 5, shaken up and down, left to stand for 4min, and then the lower layer was taken out of a 25mL volumetric flask, added with dichloromethane to a constant volume, and filtered through a 0.22 μm microporous membrane to obtain a free nicotine solution 2.
(2) And (3) detecting the free nicotine solution obtained in the step (1) by using an ultra-high performance phase-combination chromatograph, wherein the chromatographic conditions of the ultra-high performance phase-combination chromatograph are the same as those of the step (3) in the example 1.
Example 7
(1) 10mL of dichloromethane was added to the centrifuge tube with a stopper extracted in the step (1) of example 6, the mixture was shaken up and down, and after standing for 4min, the lower layer was taken out of a 25mL volumetric flask, and then dichloromethane was added to the mixture to a constant volume, and the mixture was filtered through a 0.22 μm microporous membrane to obtain a free nicotine solution 3.
(2) And (3) detecting the free nicotine solution obtained in the step (1) by using an ultra-high performance phase-combination chromatograph, wherein the chromatographic conditions of the ultra-high performance phase-combination chromatograph are the same as those of the step (3) in the example 1.
Analysis of results of ultra performance phase-compatible chromatography tests for examples 5-7: in the step (3) of the embodiment 1, the free nicotine solution obtained in the step (2) is used for detection by an ultra high performance phase combination chromatograph, the chromatogram obtained after the ultra high performance phase combination chromatograph test of the embodiments 5-7 is shown in the attached figure 3, the analysis result obtained by putting the free nicotine 1-3 obtained in the embodiments 5-7 into the ultra high performance phase combination chromatograph ACQULITY UPLC H-class detection is shown in the attached figure 3, and the quantitative analysis is carried out in the two modes to obtain the analysis result shown in the table 1
Table 1 shows the effect of the number of extractions with methylene chloride
As can be seen from table 1 and fig. 3, no peak is shown in fig. 3 during the third extraction, which indicates that free nicotine was completely extracted in the first two extractions, so the optimal number of extractions is 2, and the analysis result of the method is substantially consistent with that of the international standard Ultra Performance Liquid Chromatography (UPLC) test, which indicates that the method of the present invention is reliable.
Example 8
(1) Extracting the water phase in the centrifuge tube with the plug after extraction in the step (1) in the example 6 twice by using dichloromethane, wherein the first extraction is the same as the step (1) in the example 1, the mode and the conditions of the second extraction are the same as the step (1) in the example 2, combining the lower layers (dichloromethane layers) of the two extractions, using dichloromethane to fix the volume, and filtering by using a 0.22 mu m microporous membrane to obtain a protonated nicotine sample solution 0 to be detected.
(2) And (3) testing: and (3) subjecting the protonated nicotine sample solution 1 to be detected obtained in the step (1) to detection by an ultra-high performance phase-locked chromatograph, wherein the chromatographic conditions of the ultra-high performance phase-locked chromatograph are the same as those of the step (3) in the example 1.
Example 9
(1) Adding 10 mu L of saturated sodium hydroxide solution into the water phase in the centrifuge tube with the plug after extraction in the step (1) in the example 6, extracting twice by using dichloromethane, wherein the first extraction is the same as the step (1) in the example 1, the second extraction mode and conditions are the same as the step (1) in the example 2, combining the lower layers (dichloromethane layers) of the two extractions, using dichloromethane to fix the volume, and filtering by using a 0.22 mu m microporous membrane to obtain the protonated nicotine sample solution 1 to be tested.
(2) And (3) testing: and (3) subjecting the protonated nicotine sample solution 1 to be detected obtained in the step (1) to detection by an ultra-high performance phase-locked chromatograph, wherein the chromatographic conditions of the ultra-high performance phase-locked chromatograph are the same as those of the step (3) in the example 1.
Example 10
(1) Adding 10 mu L of saturated sodium hydroxide solution into the water phase in the centrifuge tube with the plug after extraction in the step (1) in the example 9, extracting twice by using dichloromethane, wherein the first extraction is the same as the step (1) in the example 1, the second extraction mode and conditions are the same as the step (1) in the example 2, combining the lower layers (dichloromethane layers) of the two extractions, using dichloromethane to fix the volume, and filtering by using a 0.22 mu m microporous membrane to obtain the protonated nicotine sample solution 2 to be tested.
(2) And (3) testing: and (3) subjecting the protonated nicotine sample solution 1 to be detected obtained in the step (1) to detection by an ultra-high performance phase-locked chromatograph, wherein the chromatographic conditions of the ultra-high performance phase-locked chromatograph are the same as those of the step (3) in the example 1.
Example 11
(1) Adding 30 mu L of saturated sodium hydroxide solution into the water phase in the centrifuge tube with the plug after extraction in the step (1) in the example 10, extracting twice by using dichloromethane, wherein the first extraction is the same as the step (1) in the example 1, the second extraction mode and conditions are the same as the step (1) in the example 2, combining the lower layers (dichloromethane layers) of the two extractions, using dichloromethane to fix the volume, and filtering by using a 0.22 mu m microporous membrane to obtain a protonated nicotine sample solution to be tested 3.
(2) And (3) testing: and (3) subjecting the protonated nicotine sample solution 1 to be detected obtained in the step (1) to detection by an ultra-high performance phase-locked chromatograph, wherein the chromatographic conditions of the ultra-high performance phase-locked chromatograph are the same as those of the step (3) in the example 1.
Example 12
(1) Adding 50 mu L of saturated sodium hydroxide solution into the water phase in the centrifuge tube with the plug after extraction in the step (1) in the example 11, extracting twice by using dichloromethane, wherein the first extraction is the same as the step (1) in the example 1, the second extraction mode and conditions are the same as the step (1) in the example 2, combining the lower layers (dichloromethane layers) of the two extractions, using dichloromethane to fix the volume, and filtering by using a 0.22 mu m microporous membrane to obtain the protonated nicotine sample solution 4 to be tested.
(2) And (3) testing: and (3) subjecting the protonated nicotine sample solution 1 to be detected obtained in the step (1) to detection by an ultra-high performance phase-locked chromatograph, wherein the chromatographic conditions of the ultra-high performance phase-locked chromatograph are the same as those of the step (3) in the example 1.
Example 13
(1) Adding 900 mu L of saturated sodium hydroxide solution into the water phase in the centrifuge tube with the plug after extraction in the step (1) in the example 12, extracting twice by using dichloromethane, wherein the first extraction is the same as the step (1) in the example 1, the second extraction mode and conditions are the same as the step (1) in the example 2, combining the lower layers (dichloromethane layers) of the two extractions, using dichloromethane to fix the volume, and filtering by using a 0.22 mu m microporous membrane to obtain the protonated nicotine sample solution 5 to be tested.
(2) And (3) testing: and (3) subjecting the protonated nicotine sample solution 1 to be detected obtained in the step (1) to detection by an ultra-high performance phase-locked chromatograph, wherein the chromatographic conditions of the ultra-high performance phase-locked chromatograph are the same as those of the step (3) in the example 1.
Analysis of results of ultra performance compatible chromatography tests for examples 8-13: after the ultrahigh performance phase combination chromatography test is performed on the examples 8 to 13, calculation is performed, the example 8 is marked as 0, and the analogized results that the examples 9 to 13 are 1 to 5 are obtained, and fig. 4 is an analysis result obtained by placing the protonated nicotine sample solution 0 to 4 obtained in the examples 8 to 13 into an ultrahigh performance phase chromatography apparatus acquality UPLC H-class test, and quantitative analysis is performed in two ways, as can be seen from fig. 4, in the example 8, the free nicotine is completely extracted before NaOH is added, only protonated nicotine remains in the aqueous phase, and after NaOH is added for the fourth time, the target substance in the extraction liquid does not peak, which indicates that the addition of NaOH for the previous three times is performed on the aqueous phase, and the protonated nicotine is completely converted into the free NaOH addition amount, so that the optimal addition amount of NaOH is 50 μ L.
Method for determining lower bound and linear range
Subjecting the series of standard solutions with different concentrations obtained in example 1 to sample injection analysis under the optimal phase-matching chromatographic condition, and drawing a standard curve of nicotine by using the mass concentration of nicotine as abscissa and the peak area of nicotine under the corresponding concentration as ordinate, as shown in figure 5, which shows that the nicotine concentration is 0-200 mg.L -1 The linear relation within the range is good; then, according to the traditional signal-to-noise ratio calculation method, under the condition of optimal phase-combination chromatography, the detection limit and the quantitative limit of the measurement method are determined to obtain a linear equation, and the result is shown in Table 2
TABLE 2 Linear equation and related parameters for nicotine in ultra-high performance phase chromatography
The results showed that the detection limit (LODs, n ═ 3) was 0.061 mg. multidot.L -1 The limit of quantitation (LOQs, n is 10) is 0.189 mg. L -1 。
(III) recovery and precision
Taking a sample to be detected as a sample matrix, and respectively setting the standard concentration to be 50 mg.L -1 、100mg·L -1 、150mg·L -1 The blank samples were tested, six groups of samples were run in parallel for each spiked concentration, and the average recovery and relative standard deviation were analyzed by ultra-high performance co-chromatograph, as shown in table 3.
TABLE 3 mean recovery and relative standard deviation of nicotine determined by ultra performance chromatography
The result shows that the nicotine standard recovery rate in the blank sample is 97.9%, the relative standard deviation is less than 5.0%, and the standard recovery rate and the precision are both good and meet the detection requirements.
Example 14
Determination of actual samples
6 different electronic cigarette products and 6 nicotine samples were purchased, prepared by the step (2) of example 2 to obtain electronic tobacco tar 1# -6#, protonated nicotine and free nicotine of nicotine salts A-E, and the electronic tobacco tar 1# -6#, protonated nicotine of nicotine salts A-E and free nicotine were tested by ultra-high performance gel chromatography to obtain Table 4
TABLE 4 measurement results of E-liquid and home-made nicotine salt samples
As can be seen from Table 4, the method can meet the requirement of rapid analysis of free state and protonated nicotine content in the electronic cigarette oil and nicotine raw material, and plays a certain safety guarantee role in the quality of the electronic cigarette oil sold in the market.
The invention has the advantages that:
(1) by adopting ultra-high performance combined phase chromatography, rotary evaporation is not needed, and the organic solvent is directly injected, so that the pretreatment time of the sample is greatly shortened;
(2) the supercritical carbon dioxide is used as a main mobile phase, the low-proportion organic solvent is used as an auxiliary solvent, the separation efficiency is high, the solvent is saved, the waste liquid treatment cost is reduced to a great extent, and the method is green and environment-friendly. Meanwhile, the column is not easy to be lost by the mobile phase, so that the cost is saved;
(3) the sample analysis time is short, the peak shape is good, the sample substrate is free of interference, and the method can be used as a rapid, convenient and reliable detection means for quality control of the raw materials of the commercial electronic cigarette oil and nicotine.
The above disclosure is only one or more specific embodiments of the present invention, but the present invention is not limited thereto, and any variations that can be made by those skilled in the art are intended to fall within the scope of the present invention.
Claims (6)
1. A method for determining free state and protonated nicotine content by ultra-high performance phase-combination chromatography, comprising:
(1) preparation of nicotine standard solution: weighing nicotine reference substances, performing ultrasonic dissolution through chromatographic methanol to fix the volume, then performing liquid transfer, fixing the volume with dichloromethane, and filtering to obtain a series of standard solutions with different concentrations;
(2) preparing a free nicotine solution of a sample to be tested:
(a) accurately weighing an electronic cigarette oil sample in a 10mL volumetric flask, metering the volume with ultrapure water, and transferring the electronic cigarette oil sample into a 50mL centrifuge tube with a plug;
(b) adding 5-20mL of dichloromethane into the centrifuge tube with the plug in the step (a), shaking up and down, standing for 1-5min, and taking a dichloromethane layer into a volumetric flask;
(c) repeating the step (b) for 1-3 times, combining the lower layers obtained each time, fixing the volume by using dichloromethane, and filtering to obtain a filtrate I to be detected;
(3) preparation of the protonated nicotine solution for the sample to be tested: adding 10-500 mu L of saturated sodium hydroxide solution into the water phase of the centrifuge tube with the plug extracted in the step (2), shaking up and standing for 1-10min, extracting twice with dichloromethane, combining the lower layers of the dichloromethane, fixing the volume of the dichloromethane, and filtering to obtain a filtrate II to be detected;
(4) precisely sucking the standard solution obtained in the step (1) and injecting the standard solution into an ultra-high performance phase-combination chromatograph for detection; precisely sucking the filtrate I and the filtrate II to be detected obtained in the steps (2) and (3) and injecting the filtrate I and the filtrate II into an ultra-high performance phase-combining chromatograph for detection;
wherein, in the step (2), the sample is electronic cigarette oil or self-made nicotine salt; the detection parameter conditions of the ultra-high efficiency phase-combination chromatograph are specifically as follows: the chromatography column is ACQULITY UPC 2 Viridis BEH, 3X 100mm, 1.7 μm; flow rate: 0.5-3 mL/min -1 (ii) a Detection wavelength: 259 nm; sample introduction amount: 0.5-10 μ L; temperature of the chromatographic column: 50 ℃; system back pressure 1500-; isocratic elution for 2-4 min; the mobile phase is 90% CO 2 The auxiliary solvent is 10% methanol or 10% ethanol.
2. The method of determining free and protonated nicotine content using ultra-high performance phase-locked chromatography as claimed in claim 1, wherein the standard working solution configuration comprises:
weighing 100mg of nicotine reference substance in a 50ml volumetric flask, and performing ultrasonic dissolution with chromatographic methanol to a constant volume to obtain a concentration of 2000 mg.L -1 Standard mother liquor;
accurately transferring the standard mother liquor into volumetric flasks of 0.1mL, 0.2mL, 0.5mL, 0.8mL and 1 mL-10 mL by using a pipette, metering the volume of dichloromethane, and filtering to obtain 20 mg.L -1 、40 mg·L -1 、100 mg·L -1 、160 mg·L -1 、200 mg·L -1 A series of standard solutions with different concentrations.
3. The method for determining the content of free nicotine and protonated nicotine by ultra-high performance phase chromatography according to claim 1, wherein in the step (2), the preparation of the free nicotine solution of the sample to be tested comprises:
(a) accurately weighing an electronic cigarette oil sample in a 10mL volumetric flask, metering the volume with ultrapure water, and transferring the electronic cigarette oil sample into a 50mL centrifuge tube with a plug;
(b) adding 10mL of dichloromethane into the centrifuge tube with the plug in the step (a), shaking up and down, standing for 1-5min, and taking a dichloromethane layer in a volumetric flask;
(c) and (c) repeating the step (b), combining the lower layers obtained in the two steps, fixing the volume by using dichloromethane, and filtering to obtain a filtrate I to be detected.
4. The method for determining the free-state and protonated nicotine content through ultra-high performance phase-combining chromatography according to claim 1, wherein in step (3), the preparation of the protonated nicotine solution of the sample to be tested comprises: and (3) adding 500 mu L of saturated sodium hydroxide solution into the water phase of the centrifuge tube with the plug after extraction in the step (2), shaking up and standing for 1-10min, extracting twice with dichloromethane, combining the lower layers, fixing the volume with dichloromethane, and filtering to obtain a filtrate II to be detected.
5. The ultra-high performance phase-locked chromatography method for determining the content of free nicotine and protonated nicotine according to claim 1, wherein the parameter conditions for the ultra-high performance phase chromatography in step (3) are as follows: flow rate: 2.5 mL/min -1 (ii) a Sample introduction amount: 2 mu L of the solution; system back pressure 2000 pis; isocratic elution for 2 min: the mobile phase is 90% CO 2 The auxiliary solution was 10% methanol.
6. The method for determining the free-state and protonated nicotine content by ultra-high performance phase chromatography according to claim 1, wherein in the steps (1) to (3), the filtering conditions are as follows: filtering by a 0.22 mu m microporous filter membrane.
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