Application of polyamino alkane compound as tobacco alkaloid gas chromatographic analysis protective agent and determination method of tobacco alkaloid

Abstract

The invention belongs to the technical field of tobacco chemical analysis and detection, and particularly relates to application of a polyamino alkane compound as a tobacco alkaloid gas chromatographic analysis protective agent and a tobacco alkaloid determination method. According to the application of the polyamino alkane compound as the tobacco alkaloid gas chromatographic analysis protective agent, the polyamino alkane compound is selected from one or more of compounds with a structural formula as shown in a formula I; in the formula I, n, m and z are respectively and independently selected from natural numbers; the molecular weight of the compound with the structural formula as shown in the formula I is not higher than 500Da. A flexible polyamine group of the polyamino alkane compound is preferentially adsorbed to active sites such as silicon hydroxyl and metal to form a passivation protection layer, and a long-chain alkyl structure can block contact between target alkaloid and the active sites, so that the detection sensitivity and the response strength are improved, and the quantitative accuracy is improved.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of tobacco chemical analysis and detection, in particular to application of a polyaminoalkane compound as a tobacco alkaloid gas chromatography analysis protective agent and a tobacco alkaloid determination method. BACKGROUND

[0002] Tobacco alkaloid compounds mainly include nicotine, nornicotine, anatabine, anabasine and the like, have an important influence on tobacco quality, and are key indicators for evaluating tobacco quality.

[0003] In traditional tobacco, nicotine is mg / g level, and the rest of the alkaloid substances are ppm level or below. In recent years, low-nicotine tobacco products have emerged in an endless stream, and nicotine is also ppm level. Due to the high activity of tobacco alkaloid compounds, in the commonly used gas chromatography-mass spectrometer, when the content is ppm level or below, due to the serious matrix effect, the tobacco alkaloid compounds strongly interact with the active sites in the instrument, thereby causing serious adsorption, signal tailing, weak response and the like. For a long time, the accurate quantification of tobacco alkaloid substances has been one of the difficulties in the analysis and testing field.

[0004] At present, the compensation methods for the matrix effect mainly include a matrix matching standard solution method, an isotope internal standard method, a plurality of internal standard correction methods, an injection technology improvement method, a sample multiple purification method and an analysis protective agent adding method and the like. Researches show that adding a matrix modifier (i.e. a chromatographic analysis protective agent) has a certain degree of compensation for the matrix effect, so that the accuracy and precision of the quantitative analysis of the measured substances are improved, the peak shape and intensity of the measured substance chromatographic peak are improved, and the method has the advantages of simple operation, reduction of instrument maintenance and the like.

[0005] A kind of analysis protective agent for compensating cigarette mainstream smoke analysis matrix effect is disclosed in Chinese patent CN113567571B authorized on February 20, 2024, specifically a kind of compound with one end of molecule being polar amine group and the other end being non-polar alkyl chain, and the two ends of the molecule are connected by carbon-carbon single bond in the middle of the molecule. The analysis protective agent is added to the smoke sample extract liquid for gas chromatography-mass spectrometry analysis. The analysis protective agent improves the peak shape and intensity of the chromatographic peak, improves the linearity of the standard curve, effectively reduces or eliminates the interference of the complex matrix from the smoke on the determination of the components of the cigarette mainstream smoke, so that the quantitative result is accurate and reliable.

[0006] However, the quantitative analysis result of the above-mentioned analysis protective agent for tobacco alkaloid chromatographic analysis is still not accurate. SUMMARY

[0007] The purpose of the present invention is to provide a polyaminoalkane compound used as a protective agent for gas chromatography analysis of tobacco alkaloids, so as to solve the problem of poor accuracy of quantitative analysis results of existing protective agents.

[0008] The second object of the present invention is to provide a method for determining tobacco alkaloids, which solves the problem of poor accuracy of quantitative analysis results caused by the analytical protectants used in existing determination methods.

[0009] In order to solve the above technical problems, the technical solution of the present invention for using the polyaminoalkane compound as a protective agent for gas chromatography analysis of tobacco alkaloids is as follows:

[0010] A use of a polyaminoalkane compound as a protective agent for gas chromatography analysis of tobacco alkaloids, wherein the polyaminoalkane compound is selected from one or more compounds of the structural formula shown in Formula I;

[0011]

[0012] In Formula I, n, m, and z are independently selected from natural numbers; the molecular weight of the compound represented by the structural formula of Formula I is not higher than 500 Da.

[0013] The present invention improves upon the prior art and provides the use of polyaminoalkane compounds as protective agents for gas chromatography analysis of tobacco alkaloids. By using polyaminoalkane compounds of a certain molecular weight (molecular weight that is too high cannot be vaporized) as analytical protective agents, the flexible polyamine groups of the compounds preferentially adsorb to active sites such as silanol groups and metals, forming a stable passivation protective layer. Simultaneously, the long-chain alkyl structures in the compounds extend outward, effectively blocking contact between the target alkaloids and the active sites. This competitive adsorption mechanism significantly reduces nonspecific adsorption of alkaloids during the analysis process, thereby avoiding problems such as chromatographic peak tailing, reduced signal response, and quantitative deviation.

[0014] In order to further improve the retention time matching between the polyaminoalkane compound and the target alkaloid, preferably, in Formula I, m=2-5, n=2-5, and z=1-4.

[0015] In order to further improve the detection accuracy of alkaloids in cigarette smoke matrix, alkaloids in different retention time windows are dynamically covered in gas chromatography analysis. Preferably, when the tobacco is cigarette, the polyaminoalkane compound includes a compound of the structural formula shown in Formula II and a compound of the structural formula shown in Formula III;

[0016]

[0017] The retention time of spermidine (Formula II) is similar to that of nornicotine, and the retention time of spermine (Formula III) is similar to that of cotinine. By combining two or more polyaminoalkane compounds, alkaloids in different retention time windows can be dynamically covered in gas chromatography analysis, thereby achieving simultaneous and effective protection of alkaloids with a wide range of retention time distribution.

[0018] In order to further improve the accuracy of alkaloid detection in cigarette smoke matrix, preferably, the mass ratio of the compound represented by the structural formula II to the compound represented by the structural formula III is (4-5):(2-3).

[0019] In order to further improve the detection accuracy of alkaloids in the electronic cigarette matrix, preferably, when the tobacco is an electronic cigarette, the polyaminoalkane compound includes a compound of the structural formula represented by formula IV, a compound of the structural formula represented by formula VI, and a compound of the structural formula represented by formula VII;

[0020]

[0021] In order to further improve the accuracy of alkaloid detection in electronic cigarette matrices, preferably, the mass ratio of the compound represented by the structural formula IV, the compound represented by the structural formula VI and the compound represented by the structural formula VII is (10-12):(2-3):(2-3).

[0022] In order to further improve the accuracy of alkaloid detection in the buccal tobacco matrix, preferably, when the tobacco is buccal tobacco, the polyaminoalkane compound includes a compound of the structural formula represented by formula V, a compound of the structural formula represented by formula II, and a compound of the structural formula represented by formula VII;

[0023]

[0024] In order to further improve the detection accuracy of alkaloids in the buccal smoke matrix, preferably, the mass ratio of the compound represented by the structural formula of Formula V, the compound represented by the structural formula of Formula II and the compound represented by the structural formula of Formula VII is (6-8):(5-6):(3-4).

[0025] The technical solution of the method for determining tobacco alkaloids of the present invention is:

[0026] A method for determining tobacco alkaloids comprises the following steps: mixing the extract obtained by extracting alkaloids from tobacco with the polyaminoalkane compound, and performing gas chromatography-mass spectrometry analysis.

[0027] The determination method of tobacco alkaloids provided by the present application can improve the detection sensitivity and response intensity, improve the chromatographic peak shape and separation efficiency, enhance the linearity and accuracy of the calibration curve, improve the accuracy and recovery rate stability of the method, and has high sensitivity, low deviation and strong stability, and is especially suitable for precise analysis of trace alkaloids in complex matrix (such as cigarette extract), and can more truly reflect the actual release level of alkaloids in the sample, and provide more reliable technical support for quality control.

[0028] In order to further improve the passivation protection layer forming ability and block the contact between the target alkaloids and the active sites, preferably, the mixing is mixing of the extract and the polyaminoalkane compound solution, the concentration of the polyaminoalkane compound solution is 1-5 mg / mL, and the volume ratio of the extract and the polyaminoalkane compound solution is (9-10):(1-2).

[0029] In order to further improve the chromatographic separation ability, preferably, the chromatographic conditions in the gas chromatography-mass spectrometry analysis are as follows: chromatographic column: capillary chromatographic column, stationary phase is trifluoropropylmethyl polysiloxane; injection port temperature is 230-240 DEG C; programmed temperature: initial temperature 100 DEG C, holding for 2 min, increasing to 160 DEG C at 10 DEG C / min, increasing to 175 DEG C at 2 DEG C / min, increasing to 280 DEG C at 10 DEG C / min, and then running for 10 min; injection volume is 1 mu L, pulse splitless injection, 50 psi for 0.5 min.

[0030] In order to further improve the quantitative accuracy, preferably, the mass spectrometry conditions in the gas chromatography-mass spectrometry analysis are as follows: ionization mode: electron impact source; ionization energy: 70 eV; ion source temperature: 230 DEG C; transmission line temperature: 280 DEG C; quadrupole temperature: 150 DEG C; solvent delay time: 10 min; scanning mode: selected ion monitoring mode.

[0031] In order to further improve the extraction efficiency, preferably, when the tobacco is cigarette, the extraction is extracting the filter piece for collecting the mainstream smoke generated by 3-5 cigarettes in 40-50 mL of alkaloid extractant; when the tobacco is a smoke-in-mouth or electronic cigarette liquid, the extraction is mixing the smoke-in-mouth tobacco powder or electronic cigarette liquid with the alkaloid extractant, and 20-30 mL of alkaloid extractant is added for every 1-2 g of smoke-in-mouth tobacco powder or electronic cigarette liquid. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The mass spectrum of the secondary alkaloids in BX1 of Example 1 of the present application on GC-MS. DETAILED DESCRIPTION

[0033] The technical concept of the application of the polyaminoalkane compound as a tobacco alkaloid gas chromatography analysis protective agent is as follows:

[0034] The analysis protective agent disclosed in the prior art for compensating for the matrix effect of cigarette mainstream smoke analysis is a compound with a polar amine group at one end of a carbon-carbon single bond and a nonpolar alkyl chain at the other end, which is suitable for sensitive compounds with active groups, including hydroxyl groups, carbon groups, ester groups, amino groups, amides, unsaturated bonds, and heteroatoms. However, this kind of analysis protective agent has the problem of poor quantitative accuracy for tobacco alkaloid gas chromatography analysis.

[0035] The present application uses a polyaminoalkane compound as a tobacco alkaloid gas chromatography analysis protective agent. The flexible polyamine group of the compound preferentially adsorbs on active sites such as silicon hydroxyl groups and metals to form a passivation protective layer, and the long-chain alkyl structure can block the contact between the target alkaloid and the active sites, thereby improving the detection sensitivity and response strength and improving the quantitative accuracy.

[0036] The method for determining tobacco alkaloids of the present application comprises the following steps:

[0037] 1) Pretreatment

[0038] When the tobacco is a cigarette, refer to GB / T 16450-2004 for cigarette smoking: smoke 1 puff every 60 s, puff volume 35 mL, puff duration 2 s, smoke 3-5 cigarettes under this condition, and use a filter to capture the particulate matter in the cigarette mainstream smoke; use sodium hydroxide solution to free the alkaloids in the filter, then use 40-50 mL of the first alkaloid extractant for first extraction, and obtain the extract after filtration.

[0039] When the tobacco is a chewing tobacco or electronic cigarette liquid, mix the chewing tobacco cut or electronic cigarette liquid with the second alkaloid extractant for second extraction. For every 1-2 g of chewing tobacco cut or electronic cigarette liquid, add 20-30 mL of alkaloid extractant, and obtain the extract after filtration.

[0040] 2) Determination

[0041] Mix the extract obtained after extracting the alkaloids from the tobacco with the polyaminoalkane compound solution, the concentration of the polyaminoalkane compound solution is 1-5 mg / mL, and the volume ratio of the extract and the polyaminoalkane compound solution is (9-10):(1-2), and perform gas chromatography-mass spectrometry analysis.

[0042] It should be noted that the concentration of the polyaminoalkane compound solution is 1-5 mg / mL, which means that the concentration of any one polyaminoalkane compound in the solution is 1-5 mg / mL.

[0043] The chromatographic conditions for the gas chromatography-mass spectrometry analysis are as follows: chromatographic column: capillary chromatographic column, the stationary phase is trifluoropropyl methylpolysiloxane; the injection port temperature is 230-240°C; the programmed temperature is: initial temperature 100°C, maintained for 2 minutes, increased to 160°C at 10°C / min, increased to 175°C at 2°C / min, increased to 280°C at 10°C / min, and then run for 10 minutes; the injection volume is 1 μL, pulsed non-split injection, 50 psi for 0.5 minutes.

[0044] The mass spectrometry conditions during the gas chromatography-mass spectrometry analysis were as follows: ionization mode: electron bombardment source; ionization energy: 70 eV; ion source temperature: 230° C.; transfer line temperature: 280° C.; quadrupole temperature: 150° C.; solvent delay time: 10 min; and scanning mode: selected ion monitoring mode.

[0045] The polyaminoalkane compound is selected from one or more compounds of the structural formula shown in Formula I;

[0046]

[0047] In Formula I, n, m, and z are independently selected from natural numbers; the molecular weight of the compound represented by the structural formula of Formula I is not higher than 500 Da.

[0048] It is understood that z in Formula I can be composed of different z1, z2, z3...zx; similarly, m can be composed of different m1, m2, m3...mx, and n can be composed of different n1, n2, n3...nx. The details are as follows:

[0049]

[0050] Among them, x is selected from natural numbers; z = z1+z2+z3…+zx; m = m1+m2+m3…+mx; n = n1+n2+n3…+nx; z1, z2, z3…zx, m1, m2, m3…mx, n1, n2, n3…nx are all independently selected from natural numbers.

[0051] In a specific embodiment, the concentration of the sodium hydroxide solution in step 1) is 10-15%; the filter disc is a Cambridge filter disc, and the diameter of the Cambridge filter disc is 40-50 mm; and 4-5 mL of sodium hydroxide solution is added to each Cambridge filter disc.

[0052] In a specific embodiment, the first alkaloid extractant in step 1) is 0.01-0.05% of amylamine-methyl tert-butyl ether.

[0053] In the specific embodiment, the first extraction in step 1) is oscillation extraction, and the rotation speed of the oscillation extraction is 200-300 r / min.

[0054] In the specific embodiment, the filtration in step 1) is all carried out by using a 0.45 μm nylon membrane, and anhydrous sodium sulfate is used for water removal during the filtration.

[0055] In the specific embodiment, the second alkaloid extraction agent in step 1) is isopropyl alcohol.

[0056] In the specific embodiment, when the tobacco is an electronic cigarette liquid, the second extraction in step 1) is oscillation extraction, and the rotation speed of the oscillation extraction is 100-200 rpm; when the tobacco is a smoke-in-mouth cigarette, the second extraction in step 1) is vortex extraction, and the rotation speed of the vortex extraction is 200-300 rpm.

[0057] The technical solutions of the present application are further described in combination with examples. The chemicals used in the following examples are all commercially available conventional products unless otherwise specified.

[0058] I. A specific embodiment of the application of the polyaminoalkane compound as a tobacco alkaloid gas chromatography analysis protective agent and a tobacco alkaloid determination method

[0059] Example 1

[0060] The application of the polyaminoalkane compound as a tobacco alkaloid gas chromatography analysis protective agent in this embodiment is illustrated by a tobacco alkaloid determination method in cigarette mainstream smoke, which comprises the following steps:

[0061] (1) Collection of particulate matter in cigarette mainstream smoke: the cigarette smoking conditions refer to GB / T 16450-2004, i.e. 1 puff every 60 s, puff volume 35 mL, and puff duration 2 s. Under these conditions, 5 cigarettes are smoked, and the particulate matter in the cigarette mainstream smoke is collected by a Cambridge filter.

[0062] (2) Pretreatment of the Cambridge filter: the Cambridge filter with a diameter of 44 mm, which has collected the particulate matter in the cigarette mainstream smoke in step (1), is placed in a 100 mL conical flask, 4 mL of 10% NaOH is added, and the mixture is allowed to stand for 10 min. Then, 40 mL of 0.01% pentylamine-methyl tert-butyl ether is added, and the mixture is subjected to extraction by mechanical oscillation at 220 r / min for 2 h. After the extraction is completed, 4 g of anhydrous sodium sulfate is added to a filter with a 0.45 μm nylon membrane, and 3-4 mL of the upper layer of the obtained solution without debris is added to the filter, and then filtered into a chromatographic flask to obtain an extraction solution.

[0063] (3) Preparation and addition of analytical protective agent (AP): Since the retention times of spermidine and spermine are close to those of nornicotine and cotinine, respectively, they completely cover the target compounds. Among all the APs mentioned above, the experimental results show that it is the optimal combination in the smoke matrix. The specific configuration is as follows: 40 mg of spermidine (Formula II) and 20 mg of spermine (Formula III) are weighed into a 20 mL volumetric flask, and isopropanol solvent is added to dilute the concentration of spermidine to 2 mg / mL and the concentration of spermine to 1 mg / mL to obtain the analytical protective agent (AP) solution. Accurately take 900 μL of the extract into a brown chromatographic bottle, add 100 μL of AP solution, cover the lid, shake gently, and then inject.

[0064]

[0065] In formula II, z=z1=1; m=m1=4; n=n1=3.

[0066]

[0067] In formula III, z=z1=2; m=m1=2; n=n1=2.

[0068] When injecting a series of standard working solutions, AP solution also needs to be added. The procedure is as follows: take 900 μL of standard working solution into a brown chromatographic bottle, add 100 μL of AP solution, cover the bottle, shake gently and then inject.

[0069] (4) Determination:

[0070] Gas chromatography conditions:

[0071] Chromatographic column: capillary column, the stationary phase is trifluoropropyl methylpolysiloxane, the specifications are [30m (length) × 0.25mm (inner diameter) × 1μm (film thickness)];

[0072] The injection port temperature should be 230°C.

[0073] Program temperature rise: initial temperature 100℃, hold for 2 min, increase to 160℃ at 10℃ / min, increase to 175℃ at 2℃ / min, increase to 280℃ at 10℃ / min and run for 10 min.

[0074] Carrier gas: helium, constant flow mode, flow rate 1 mL / min.

[0075] Injection volume and split ratio: The injection volume was 1 μL, pulsed splitless injection was performed at 50 psi for 0.5 min.

[0076] Mass spectrometry conditions:

[0077] Ionization method: electron impact source (EI).

[0078] Ionizing energy: 70 eV.

[0079] Ion source temperature: 230 °C.

[0080] Transfer line temperature: 280 °C.

[0081] Quadrupole temperature: 150 °C.

[0082] Solvent delay time: 10 min.

[0083] Scan mode: Selected ion monitoring mode (SIM), see Table 1 for scan parameters for each compound.

[0084] Since the nicotine concentration is much higher than that of the secondary alkaloids (more than 90% of the total alkaloid content), it needs to be injected separately. The injection mode is split injection, and the split ratio is 20:1, and the rest remains unchanged.

[0085] Table 1 Retention time, quantitative and qualitative ions and residence time of alkaloids and their internal standards on VF200 MS column

[0086]

[0087] (5) Preparation of standard working curve: According to the above gas chromatography-mass spectrometry analysis conditions, a series of standard working solutions were determined, and the internal standard method was used for quantitative analysis. The concentrations of the series of standard working solutions are shown in Table 2. The ratio of the quantitative ion peak area of the 7 alkaloids in the standard working solution to the quantitative ion peak area of the internal standard was taken as the ordinate, and the ratio of the concentration of each alkaloid to the concentration of the internal standard was taken as the abscissa. The standard working curve was drawn, and the linear correlation coefficient R 2 not less than 0.99.

[0088] A standard working curve should be prepared for each test. A standard working solution of moderate concentration should be added after every 20 sample determinations, and if the measured value differs from the original value by more than 10%, the standard working curve should be prepared again.

[0089] Table 2 Concentrations of series of standard working solutions

[0090]

[0091] (6) Result analysis:

[0092] The quantitative analysis was performed using the selected ion scanning mode, and the quantitative ion was used for quantitative analysis. When determining the sample, the peak area of the target substance in the sample and the corresponding internal standard was used to calculate the concentration of the 8 alkaloids (nicotine, nornicotine, myosmine, pseudotropine, anatabine, anabasine, 2,3'-dipyridyl, and cotinine) in the extract solution according to the standard curve, and then the release amount of the 8 alkaloids in the total particulate matter of the mainstream smoke of the cigarette was calculated.

[0093] The release amount (m) of alkaloids in the cigarette sample is calculated by formula (1):

[0094]

[0095] In formula (1):

[0096] m - the release amount of 8 alkaloids (nicotine, nornicotine, myosmine, anabasine, anatabine, nornicotine, 2,3'-bipyridine, and cotinine) in the total particulate matter of the mainstream smoke of each cigarette, in units of micrograms per cigarette (pg / cig);

[0097] C - the concentration of 8 alkaloids (nicotine, nornicotine, myosmine, anabasine, anatabine, nornicotine, 2,3'-bipyridine, and cotinine) in the extract, in units of micrograms per milliliter (pg / mL).

[0098] V - the added volume of 0.01% pentylamine-methyl tert-butyl ether extractant, in units of milliliters (mL).

[0099] n - the number of cigarettes, in units of cigarettes (cig).

[0100] After the addition of AP, multidimensional data show that it has a significant advantage in the analysis of alkaloids, which is embodied in the following aspects:

[0101] 1) Improve detection sensitivity and response intensity

[0102] The mass spectrum of the secondary alkaloids in BX1 on GC-MS is shown in Figure 1 , where the black line and the red line represent the mass spectrum before and after the addition of AP (the horizontal coordinate is time / time, and the vertical coordinate is normalization), and the response area and tailing factor data of the target alkaloids are shown in Tables 3 and 4. Figure 1 As can be seen from Table 3-4, the response area of all compounds is significantly improved after the addition of AP, with an increase of several times to more than ten times, for example, the response area of nornicotine increases from 14994.4 to 252121.7, with an increase of 16.8 times. At the same time, Table 6 shows that the limit of detection (LOD) and the limit of quantification (LOQ) are generally reduced to about 1 / 3 of the original value (such as the LOD of nornicotine from 39 ng / mL to 13 ng / mL), indicating that AP greatly improves the detection sensitivity, enabling it to more accurately identify and quantify low-concentration target substances.

[0103] 2) Improve chromatographic peak shape and separation efficiency

[0104] After the addition of AP, the tailing factor was significantly reduced (for example, the tailing factor of nornicotine decreased from 5.4 to 1.5), and the peak shape became more symmetrical (Tables 3 and 4). This suggests that AP may have optimized the separation process by reducing the interaction between the target compound and the active sites on the chromatographic column, thereby reducing peak tailing, improving resolution, and reducing analytical errors.

[0105] 3) Enhanced calibration curve linearity and accuracy

[0106] Table 5 shows that after adding AP, the correlation coefficients R 2 All increased to above 0.999 (such as nornicotine R 2 The AP value increased from 0.989 to 1.000, and the intercept approached zero (for example, the Mysmin intercept decreased from 0.130224 to -0.001790). This indicates that AP optimizes the linear range, reduces baseline interference, and significantly enhances the reliability of the calibration curve, providing a more accurate basis for quantitative analysis.

[0107] 4) Improve method accuracy and recovery stability

[0108] As shown in Table 7, the addition of AP brought recoveries closer to the theoretical value (100%) at all concentration levels (low, medium, and high), and the fluctuation range narrowed. For example, the recovery of nornicotine at high concentrations decreased from 155.3% to 104.6%, while the recovery of anabasine at low concentrations improved from 145.8% to 101.9%. This demonstrates that AP effectively reduces matrix effects or interferences, improving the accuracy of the method.

[0109] 5) Optimize precision

[0110] Table 8 shows that both intra-day precision (RSD%) and inter-day precision decreased significantly after AP. For example, the inter-day precision of nornicotine decreased from 18.6% to 9.8%, and the intra-day precision of cotinine decreased from 8.2% to 4.9%. This demonstrates that AP significantly improved the precision of the method by stabilizing analytical conditions, significantly reducing the variability of the results.

[0111] 6) The practical significance of comprehensive performance improvement

[0112] The introduction of AP makes the overall performance of the method reach a higher standard: R 2The linearity of ≥0.999, the detection limit of 7-592 ng / mL, the recoveries of 89.9-113.6%, and the intra-day precision of 3.7-5.4% demonstrate that this method combines high sensitivity, low bias, and strong stability, making it particularly suitable for the accurate analysis of trace alkaloids in complex matrices such as cigarette extracts. Without the addition of AP, the adsorption of alkaloids in the standard solutions is significantly stronger than in the sample solutions, resulting in biased sample test results, as shown in the recoveries in Table 7 and the measured values ​​in Table 8. Therefore, the addition of AP more accurately reflects the actual release levels of alkaloids in the samples, providing more reliable technical support for quality control.

[0113] Table 3 Response area and tailing factor of BX1 before and after adding AP

[0114]

[0115] Table 4 Response area and tailing factor of cigarette sample extract before and after adding AP

[0116]

[0117] Table 5 Linearity and intercept of the series of standard curves before and after adding AP

[0118]

[0119]

[0120] Table 6 Detection limit and quantification limit before and after adding AP

[0121]

[0122] Table 7 Recovery before and after adding AP

[0123]

[0124]

[0125] Table 8 Precision before and after adding AP

[0126]

[0127] Example 2

[0128] The application of the polyaminoalkane compound of this embodiment as a protective agent for gas chromatography analysis of tobacco alkaloids is illustrated by a method for determining alkaloids in electronic cigarette liquid, which includes the following steps:

[0129] (1) Electronic cigarette liquid pretreatment: take 1 g of electronic cigarette liquid in a 50 mL conical flask, add 20 mL of isopropyl alcohol solution, oscillate at 150 rpm for 10 min for extraction, filter the membrane, and then take 900 μL of the solution in a brown chromatographic flask.

[0130] (2) Preparation and addition of analysis protectant (AP): Due to the significant difference between the electronic cigarette liquid matrix and the cigarette smoke matrix, there are more volatile substances with low boiling points, and in addition, the hydroxyl group in the target substance can be replaced with nortropine. The AP optimization results show that the optimal combination of AP suitable for electronic cigarette liquid is diethylene triamine (RT 8.6 min, covering the front part of the target), tetraethylenepentamine (RT 18.2 min, covering the middle part of the target), and pentaethylene hexamine (RT 24.1 min, covering the rear part of the target). The configuration process is as follows: respectively take 100 mg of diethylene triamine (cas number: 111-40-0, formula IV), 20 mg of tetraethylenepentamine (cas number: 112-57-2, formula VI), and 20 mg of pentaethylene hexamine (cas number: 4067-16-7, formula VII) in a 20 mL volumetric flask, add isopropyl alcohol solvent to dilute to a concentration of 5 mg / mL for diethylene triamine, a concentration of 1 mg / mL for tetraethylenepentamine, and a concentration of 1 mg / mL for pentaethylene hexamine. Accurately take 900 μL of the sample filtrate into a brown chromatographic flask, add 100 μL of AP solution, cover the cap, gently shake, and then inject.

[0131]

[0132] In formula IV, z = z1 = 1; m = m1 = 2; n = n1 = 2.

[0133]

[0134] In formula VI, z1 = 1, m1 = 2, n1 = 1; z2 = 1, m2 = 1, n2 = 1; z3 = 1, m3 = 1, n3 = 2; z = 3, m = 4, n = 4.

[0135]

[0136] In formula VII, z1 = 1, m1 = 2, n1 = 1; z2 = 1, m2 = 1, n2 = 1; z3 = 1, m3 = 1, n3 = 1; z4 = 1, m4 = 1, n4 = 2; z = 4, m = 5, n = 5.

[0137] When the series of standard working solution is injected, AP solution also needs to be added, and the method is as follows: take 900 μL of the standard solution in a brown chromatographic flask, add 100 μL of AP solution, cover the cap, gently shake, and then inject.

[0138] (3) Determination: the determination method is the same as step (4) in Example 1.

[0139] The response area and content test results of the e-liquid extract before and after adding AP are shown in Table 9. The retention time (min), quantitative ion (m / z) and qualitative ion (m / z) of the newly added target substance dienonnicotine (cas number: 494-98-4) are 16.5, 144 and 117, respectively, and those of trans-hydroxygitaline (cas number: 34834-67-8) are 25.5, 192 and 106, respectively. 4-piperidyl 4-pyridine and deuterated gitaline are used as internal standards for the two substances, respectively.

[0140] Table 9. Response area and content test results of e-liquid extract before and after adding AP

[0141]

[0142] As can be seen from Table 9, after adding AP, the response area of alkaloids in the e-liquid matrix can be significantly improved. Similar to Example 1, the test results more truly reflect the content level of alkaloids in the sample.

[0143] Example 3

[0144] The application of the polyaminoalkane compound of the present example as a tobacco alkaloid gas chromatography analysis protective agent is illustrated by a method for determining alkaloids in a chewing gum, which comprises the following steps:

[0145] (1) Pretreatment of chewing gum sample: 1 g of gum-based chewing gum is frozen and ground in 50 mL conical flask with liquid nitrogen, 20 mL of isopropyl alcohol solution is added, and extraction is performed by vortexing at a frequency of 200 rpm for 10 min. After filtering the membrane, 900 μL of the solution is taken into a brown chromatographic flask.

[0146] (2) Analysis of the preparation and addition of protective agent (AP): Because the base of the chewing gum type of oral tobacco is significantly different from the cigarette smoke and the liquid base of the electronic cigarette, it has more polar components, which can easily lead to the enhancement of the matrix effect of polar target substances such as nornicotine. To this end, the AP combination is re-optimized. The optimization results show that the optimal combination of AP suitable for the chewing gum type of oral tobacco is triethylenetetramine (RT 10.4 min, covering the front section of the target substance), spermidine (RT 11.2 min, covering the front and middle sections of the target substance), and pentaethylenehexamine (RT 24.1 min, covering the rear section of the target substance). The configuration process is as follows: weigh 60 mg of triethylenetetramine (cas number: 112-24-3, formula V), 50 mg of spermidine (formula II), and 30 mg of pentaethylenehexamine (cas number: 4067-16-7, formula VII) into a 20 mL volumetric flask, and dilute with isopropyl alcohol to a concentration of 3 mg / mL for triethylenetetramine, 2.5 mg / mL for spermidine, and 1.5 mg / mL for pentaethylenehexamine. Add 100 μL of AP solution to the chromatographic bottle containing the existing extraction liquid, cover the cap, and gently shake before injection.

[0147]

[0148] In formula V, z1 = 1, m1 = 2, n1 = 1; z2 = 1, m2 = 1, n2 = 2; z = z1 + z2 = 2, m = m1 + m2 = 3, n = n1 + n2 = 3.

[0149]

[0150] In formula II, z = z1 = 1; m = m1 = 4; n = n1 = 3.

[0151]

[0152] In formula VII, z1 = 1, m1 = 2, n1 = 1; z2 = 1, m2 = 1, n2 = 1; z3 = 1, m3 = 1, n3 = 1; z4 = 1, m4 = 1, n4 = 2; z = 4, m = 5, n = 5.

[0153] When the series of standard working solutions are injected, the AP solution also needs to be added, and the method is as follows: take 900 μL of the standard solution in a brown chromatographic bottle, add 100 μL of the AP solution, cover the cap, and gently shake before injection.

[0154] (3) Determination: The determination method is the same as step (4) in Example 1.

[0155] The retention time (min), quantitative ion (m / z), and qualitative ion (m / z) of the newly added target substance N-formyl nornicotine (cas number: 3000-81-5) are 25.5, 147, and 176, respectively, and deuterated cotinine is selected as the internal standard.

[0156] The response area and content test results of the snuff extract before and after adding AP are shown in Table 10.

[0157] Table 10 Response area and content test results of snuff extract before and after adding AP

[0158]

[0159] As can be seen from Table 10, after adding AP, the response area of alkaloids in the snuff matrix can be significantly improved. Similar to Example 1, the test results more truly reflect the content level of alkaloids in the sample.

[0160] Comparative Example 1

[0161] Taking the determination of alkaloids in mainstream cigarette smoke as an example, the analysis protective agent combination method reported in the existing patent was used, in which the mass concentration of 1-amino-undecane and dihexylamine was both 20 mg / mL. After 1 mL of standard solution or sample solution was removed, 50 μL of the above analysis protective agent combination solution was added and shaken. The response area and tailing factor before and after adding AP are shown in Table 11, and the recovery rate before and after adding AP is shown in Table 12.

[0162] As can be seen, compared with the results of Example 1, the protection effect of this AP combination on alkaloids is weaker, and the matrix benefit compensation effect is not ideal. Except that the tailing of nicotine is slightly improved, the improvement of the rest of the compounds and the recovery rate is small, which may be related to the structure of the compounds of this AP combination, which only contains one primary amine and one secondary amine, respectively. In addition, the retention time of the compounds is 8.3 min and 8 min, respectively, which is greatly different from the retention time of the target substance.

[0163] Table 11 Response area and tailing factor of BX1 before and after adding AP

[0164]

[0165]

[0166] Table 12 Recovery rate before and after adding AP

[0167]

[0168] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features, as long as they are within the spirit and principles of the present application. Any modification, equivalent replacement, improvement, etc. made within the scope of the present application should be included in the protection scope of the present application.

Claims

1. A polyaminoalkane compound as a protective agent for gas chromatography analysis of tobacco alkaloids, characterized in that: The polyaminoalkane compound is selected from one or more compounds of the structural formula shown in Formula I; In Formula I, n, m, and z are independently selected from natural numbers; the molecular weight of the compound represented by the structural formula of Formula I is not higher than 500 Da.

2. The use of the polyaminoalkane compound as claimed in claim 1 as a protective agent for gas chromatography analysis of tobacco alkaloids, characterized in that: In formula I, m=2-5, n=2-5, z=1-4.

3. Use of the polyaminoalkane compound according to claim 1 or 2 as a protective agent for gas chromatography analysis of tobacco alkaloids, characterized in that: When the tobacco is cigarettes, the polyaminoalkane compound includes a compound of the structural formula shown in Formula II and a compound of the structural formula shown in Formula III; 4. The use of the polyaminoalkane compound as claimed in claim 3 as a protective agent for gas chromatography analysis of tobacco alkaloids, characterized in that: The mass ratio of the compound represented by the structural formula of formula II to the compound represented by the structural formula of formula III is (4-5):(2-3).

5. Use of the polyaminoalkane compound according to claim 1 or 2 as a protective agent for gas chromatography analysis of tobacco alkaloids, characterized in that: When the tobacco is an electronic cigarette, the polyaminoalkane compound includes a compound of the structural formula represented by Formula IV, a compound of the structural formula represented by Formula VI, and a compound of the structural formula represented by Formula VII; 6. Use of the polyaminoalkane compound according to claim 5 as a protective agent for gas chromatography analysis of tobacco alkaloids, characterized in that: The mass ratio of the compound represented by the structural formula IV, the compound represented by the structural formula VI and the compound represented by the structural formula VII is (10-12):(2-3):(2-3).

7. Use of the polyaminoalkane compound according to claim 1 or 2 as a protective agent for gas chromatography analysis of tobacco alkaloids, characterized in that: When the tobacco is oral tobacco, the polyaminoalkane compound includes a compound of the structural formula represented by formula V, a compound of the structural formula represented by formula II, and a compound of the structural formula represented by formula VII; 8. Use of the polyaminoalkane compound according to claim 7 as a protective agent for gas chromatography analysis of tobacco alkaloids, characterized in that: The mass ratio of the compound represented by formula V, the compound represented by formula II and the compound represented by formula VII is (6-8):(5-6):(3-4).

9. A method for determining tobacco alkaloids, characterized in that: The following steps are involved: The extract obtained by extracting alkaloids from tobacco is mixed with the polyaminoalkane compound according to any one of claims 1 to 4, and subjected to gas chromatography-mass spectrometry analysis.

10. The method for determining tobacco alkaloids according to claim 9, wherein The mixing is performed by mixing the extract and the polyaminoalkane compound solution. The concentration of the polyaminoalkane compound solution is 1-5 mg / mL, and the volume ratio of the extract to the polyaminoalkane compound solution is (9-10):(1-2).

Images