NL2032578A - Method for Determining Purity of Nicotine by Use of GCMS-GC Method - Google Patents
Method for Determining Purity of Nicotine by Use of GCMS-GC Method Download PDFInfo
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- G—PHYSICS
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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
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Abstract
The present invention discloses a method for determining a purity of nicotine by use of a GCMS-GC (gas chromatography mass spectrometry-gas chromatography) method, 5 including: Step A: preliminarily determining a purity (nic%) of the nicotine by use of a GC/MS method; Step B: determining a moisture content (wat%) of the nicotine by use of a GC method; and Step C: carrying out correction to obtain an exact purity (nic%) of the nicotine, wherein a correction formula is expressed as NIC% = nic%*(100% - wat%). The present invention establishes a method for determining a purity of nicotine 10 by use of a gas chromatography mass spectrometry combined with a gas chromatography. Compared with a gravimetric method using a tungstosilicic acid widely used at present, because in this method, after a modern chromatographic technique is utilized to efficiently separate a sample, the molecular weight (one of inherent physicochemical constants) of nicotine is taken as a detection target, the method 15 is high in specificity for nicotine detection and uneasy to be interfered by other impurities, especially alkaline impurities (alkaloids, inorganic bases) in the sample, and accordingly, when being provided for carrying out purity detection on nicotine samples, especially those samples laid long and with high impurity contents, the method is easy to operate, has a strong separation capacity and produces reliable qualitative and 20 quantitative results. Thus, the method may be promoted as a rapid detection method for nicotine purity.
Description
Method for Determining Purity of Nicotine by Use of GCMS-GC Method Technical Field The present invention relates to the technical field of quality evaluation and determination of tobacco and tobacco products, in particular to a method for determining a purity of nicotine by use of a GCMS-GC method. Background Nicotine (also known as "Niguding" or nicotine in English) sequentially has excitatory and paralyzing effects on vegetative nerves and nervous centralis, it is one of important constituents of tobaccos and tobacco products, and also an important index of measurement in quality control of tobaccos and tobacco products; at the same time, the accuracy of nicotine purity calibration not only concerns the quality control of tobaccos and tobacco products, ensures the accuracy of analysis on the determination of the nicotine content of total particulate matters in mainstream smoke in cigarette smoke and the determination of the total vegetable alkaloid content of tobaccos and tobacco products, but also is of great significance to the comprehensive utilization of waste and inferior tobacco leaves and the further development and utilization of nicotine and derivatives thereof.
At present, the determination of nicotine purity is implemented mainly by use of a gravimetric method using a tungstosilicic acid (Zobacco and Tobacco Products - Determination of Nicotine Purity - Gravimetric Method Using Tungstosilicic Acid (ISO 13276:1997)), because reference reagents and data introduced by volumetric containers are generally not used in this method, analysis results are relatively accurate, thus, the method is the No. 39 CORESTA recommended method, and it is also the method adopted by some European and American national standards and the tobacco industry standards of the People's Republic of China (YC/T 247-2008). The gravimetric method using a tungstosilicic acid played an important role during the immature development of instrumental analysis technologies: based on a principle of azeotropy of nicotine and water vapor, under alkaline conditions, water vapor carries nicotine out, then the obtained object and a tungstosilicic acid (SiOz 12W03-26H20) form crystalline precipitates, the precipitates are placed in an oven and burned at 120°C until a constant weight is obtained, and finally, the nicotine content is calculated by use of the gravimetric method. The method may get more accurate results, but has some limitations: the analysis time is long; nicotine and nornicotine cannot be distinguished, and a determination result is the sum of the two substances; and when a solution contains inorganic salts such as ammonium salt, precipitates are not easy to form, which will interfere with detection results. However, the gravimetric method using a tungstosilicic acid also has some problems such as complicated operation, long time consumption, large artificial errors, and determination easily interfered by other alkaloids and nicotine degradation products. Therefore, studies on new methods for the determination of nicotine purity have significant scientific research and application values.
Besides the gravimetric method using a tungstosilicic acid, common methods for nicotine content determination in literature reports also include spectrophotometric methods and chromatographic methods such as HPLC, GC, LC-MS/MS methods, etc., as well as IPC, IC, SPE and titration methods for special uses. Chromatographic methods have a good separation effect on constituents in samples to be detected, especially methods implemented by taking MS as detection means. However, a LC method in which UV or fluorescent light are taken as a detector has no response to moisture; meanwhile, due to a water removal function of a MS detector itself, nicotine purity measured by use of GC, LC/MS, GC/MS methods and the like implemented by taking MS as a detector is a water deduction result, that is, there is a relatively large method error in a measured value.
In recent years, instrumental analysis technologies have developed rapidly and made great progress. In particular, the development of chromatography-mass spectrometry provides a powerful technical support for the studies on new methods for nicotine purity determination. Because of the high separation performance of the chromatographic technology, nicotine can be separated from other constituents, thereby reducing the interference of other constituents to the determination of nicotine purity. Meanwhile, because the molecular weight as one of the essential attributes of compounds is determined by mass spectrometry, the detection results are more intuitive and accurate. Therefore, the GC-MS technique has a good application prospect in the determination of nicotine purity. Based on the GC-MS technique, a method for determining a purity of nicotine by use of a GCMS-GC method is proposed in this paper, the purity of nicotine after water deduction is determined efficiently by use of GC/MS, then the water content of a sample is determined by use of a GC internal standard method, and then more accurate purity data of nicotine 1s obtained via data analysis. Compared with currently used detection methods, this method has the characteristics of simpleness, quickness, easiness to operate and good repeatability, and has important scientific research significance and high application values. Summary The purpose of the present invention is to overcome the disadvantages that the gravimetric method using a tungstosilicic acid cannot distinguish nicotine from nornicotine, and when a solution contains inorganic salts such as ammonium salt, precipitates are not easy to form, thereby interfering with detection results, and to provide a method for determining a purity of nicotine by use of a GCMS-GC method. The purpose of the invention is achieved through the following technical solution: a method for determining a purity of nicotine by use of a GCMS-GC method, comprising: Step A: preliminarily determining a purity (nic%) of the nicotine by use of a GC/MS method; Step B: determining a moisture content (wat%) of the nicotine by use of a GC method; and Step C: carrying out correction to obtain an exact purity (nic%) of the nicotine, where a correction formula is expressed as NIC% = nic%*(100% - wat%o). Preferably, chromatographic conditions in Step A are as follows: chromatographic column: HP-5MS elastic quartz capillary chromatographic column (specification: 30 m x 0.25 mm x 0.25 um); injection volume: 1.0 pL, splitless injection; solvent cutting time: 5 min; injection port temperature: 250°C; temperature programmed conditions: an initial temperature is 100°C, then it is increased to 115°C at
0.5C/min, and kept for 5 min; then, the temperature is increased to 200°C at 5°C/min and kept for 2 min; and finally, the temperature is increased to 220°C at 20°C/min, and kept for 10 min; ion source temperature: 230°C; auxiliary area temperature, i.e., interface temperature: 280°C; carrier gas: He, constant flow mode, flow velocity: 1.2 mL/min; ionization mode: electron impact source; ionization energy: 70 eV; scan mode: selective full scan mode; and scan range: 29-500 amu. Preferably, chromatographic conditions in Step B are as follows: chromatographic column: HP-PLOT/Q elastic quartz capillary chromatographic column (specification: 30 m * 0.32 mm = 0.20 pm); injection volume: 1.0 pL, split injection, split ratio: 10:1; solvent cutting time: 5 min; injection port temperature: 250°C; carrier gas: compressed air, 6 psi; temperature programming: constant temperature mode, 170°C, kept for 8 min; and detector temperature: 250°C.
Preferably, before Step A, the preparation of detected samples is also included, and comprises: samples to be detected, after being diluted to a constant volume with a diluent solvent, are uniformly mixed, and then the obtained mixture is fed into a chromatographic bottle for later use.
Preferably, before Step B, after the samples to be detected are diluted to a constant volume with a diluent solvent, the obtained object is uniformly mixed, sealed and stored away from light for later use.
Preferably, the diluent solvent is an isopropanol solution with an ethanol concentration of 5 mL/L.
The present invention establishes a new method for detecting the purity of nicotine by use of a GC/MS-GC technique.
The GC/MS method may quickly, easily and accurately determine the purity of nicotine, and because a MS detector has a characteristic of automatically removing water during a detection process, the purity of nicotine can be obtained without water.
Meanwhile, after the water content in a sample is determined in combination with a mature GC internal standard method, the obtained water content is fitted with data obtained by GC/MS to obtain accurate nicotine purity data.
The method is simple and efficient, and has high sensitivity, small manual errors, low requirements for the number of samples, and accurate detection results.
The method has a good prospect of popularization and application.
Brief Description of the Figures Fig. 1 shows a TIC diagram of nic% determined by GC/MS method after dilution with different solvents.
Fig. 2 shows the analytical mass spectrometry of the nicotine peak identified in the TIC diagram is consistent with the controlled trial identification result of the mass spectrum database.
Fig. 3 shows the comparison between the results of the gravimetric method using a tungstosilicic acid and the GC/MS-GC method.
Description of the Invention In order to make the purpose, technical solution and advantages of the present invention clearer, a clear and complete description of the technical solution of the present invention will be given below in combination with the drawings attached in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiment of the present invention, but not all of the embodiments. The components of embodiment of the present invention usually described and shown herein 5 may be arranged and designed in various configurations.
Therefore, the following detailed description of the embodiment of the present invention provided in the attached drawings is not intended to limit the scope of the present invention requiring protection, but merely represents the selected embodiment of the present invention. In view of the embodiments of the present invention, other all embodiments obtained by common technicians in this field with no creative works contributed belong to the protection range of the present invention.
It is noted that the embodiments in the present invention and the features in the embodiments may be combined with each other without conflict.
It shall be noted that similar labels and letters indicate similar items in the attached drawings below, so once an item has been defined in one attached drawing; it does not need to be further defined and interpreted in subsequent attached drawings.
1. Material and Method
1.1 Material and Instruments
1.1.1 Reagent: hydrochloric acid (AR, Yantai Shuangshuang Chemical Co., Ltd.); tungstosilicic acid (AR, No. 2 Reagent Factory of Shanghai Chemical Reagent Co., Ltd.); isopropanol (chromatographically pure, TEDIA Company, Inc.); absolute ethyl alcohol (AR, No. 1 Reagent Factory of Shanghai Chemical Reagent Co., Ltd.); water (distilled or deionized water in accordance with the provisions of GB/T 6682 for Grade- I water).
1.1.2 Instrument: Motto-Q water purification system (Millipore Company); AG204 electronic scale (capacity: 0.1 mg, METTLER TOLEDO Company in Switzerland); constant temperature drying oven (101A-2, Shanghai Laboratory Instrument General Factory), GC (7890A, equipped with TCD detector, Agilent Company), GC-MS/MS (7890A-7000B, Agilent Company, single-stage mass spectrometry mode; Agilent 5975C-7890A, Agilent Company); bottle- top dispenser (Brand Company in Germany); pipette (Tripette Company).
1.2 Preparation of Test Sample Preparation of dilute solvent (isopropanol solution of ethanol with a concentration of 5 mL/L): transfer 5.0 mL of ethanol to 1000 mL of volumetric flask, and use isopropanol to reach the scale for constant volume. It needs to weigh 100 mg of the nicotine sample accurately, accurate to 0.1 mg, and place the same in a 10 mL of volumetric flask. The diluent solvent is adopted for constant volume. After the mixture is evenly mixed, the sample is loaded into a chromatographic bottle for later test. Parallel determination shall be carried out for each sample for twice.
1.3 Determination Method
1.3.1 Determination of nicotine content by use of GC/MS method
1.3.1.1 Chromatographic condition Chromatographic column: HP-SMS elastic quartz capillary chromatographic column (30 m = 0.25 mm x 0.25 um); injection volume: 1.0 pL, splitless injection; solvent cutting time: 5 min; injection port temperature: 250°C ; temperature programmed conditions: an initial temperature is 100°C, then it is increased to 115°C at 0.5°C/min, and kept for 5 min; then, the temperature is increased to 200°C at 5°C/min and kept for 2 min; and finally, the temperature is increased to 220°C at 20°C/min, and kept for 10 min; ion source temperature: 230°C; auxiliary area temperature, i.e, interface temperature: 280°C; carrier gas: He (purity: >99.999%), constant flow mode, flow velocity: 1.2 mL/min; ionization mode: electron impact source; ionization energy: 70 eV; scan mode: selective full scan mode (scan); and scan range: 29-500 amu.
1.3.1.2 Calculation of nicotine content Nicotine content in the sample is expressed in percentage, which can be obtained via Formula (1): nic% = Te x 100% (1) wherein: nic% - nicotine content in the sample; Anic - peak area of nicotine; 24; - sum of the areas of all chromatographic peaks except solvent peaks on a chromatogram. The arithmetic mean of the two parallel samples is taken as the detection result of the nicotine content, which is accurate to 0.01%.
1.3.2 Determination of moisture in the nicotine sample by use of GC method
1.3.2.1 Preparation of moisture content determination series of standard working solutions Weigh 0 mg (without water, 1.e., blank solvent), 5.0 mg, 10.0 mg, 20.0 mg, 30.0 mg and 50.0 mg of water respectively, put them into different 100 mL brown volumetric flasks, and use the dilute the solution to reach the scale for constant volume, so as to complete the preparation of Grade-6 standard working solution. If the moisture content of the sample exceeds the coverage range of standard working solution, the coverage range of standard working solution can be appropriately extended. In order to prevent water absorption, the solution shall be sealed. The diluent solvent shall be continuously stirred or oscillated before use to make the moisture distributed evenly. The diluent solvent used for the preparation of standard solution shall be the same as that used for the preparation of test sample. This series of standard working solutions shall be prepared before use.
1.3.2.2 Chromatographic condition chromatographic column: HP-PLOT/Q elastic quartz capillary chromatographic column (specification: 30 m x 0.32 mm x 0.20 um); injection volume: 1.0 pL, split injection, split ratio: 10:1; solvent cutting time: 5 min; injection port temperature: 250°C; carrier gas: compressed air, 6 psi; temperature programming: constant temperature mode, 170°C, kept for 8 min; and detector temperature: 250°C.
1.3.2.3 Making of standard curve for moisture content determination A series of standard working solutions shall be successively injected for gas chromatography, the peak area of moisture and internal standard anhydrous ethanol shall be recorded, and the peak area ratio of moisture in each standard solution to the internal standard substance shall be calculated. The standard working curve shall be drawn with the water concentration as the abscissa and the ratio of the water peak area to the peak area of the internal standard substance as the ordinate. The linear fitting shall be used for checking whether the response of the TCD detector is linear. The correlation coefficient R2 is >0.995, and internal standard method is used for quantification. It shall make standard curves for each batch of inspection. In addition, a medium concentration standard solution shall be injected after the determination for each 20 samples. The whole standard curve shall be remade, if the result is greater than 5% different from the original value. Due to the presence of water in the diluent solvent, the regression curve shall not pass through the far point. If the water content of the diluen solvent exceeds 1.0 mg/mL, this batch of extraction agent shall not be used.
1.3.2.4 Calculation of moisture content: The moisture content in the sample is expressed in percentage, which can be obtained via Formula (2): wat% = “== x 100% (2) wherein: wat% - moisture content of the sample; C - moisture content given by standard work curve, unit: mg-mL!; V - diluent volume, unit: mL; m - mass of sample, unit: mg; The arithmetic mean of the two parallel samples is taken as the detection result of the moisture content in the sample, which is accurate to 0.01%.
1.3.3 Calculation for the determination result of nicotine purity by use of GC/MS-GC method Based on the determination results, it is substituted into the following formula to calculate the nicotine purity (NIC%) of the sample. It is expressed in percentage, which can be obtained via Formula (3): NIC% = nic% x (1 —wat%) 3) wherein: NI('% - nicotine purity of the sample; nic% - nicotine content in the sample; wat? - moisture content of the sample; The nicotine purity NIC% in the sample shall be accurate to 0.01% according to the detection result.
2. Result and Discussion
2.1 Optimization of GC/MS Determination Condition
2.1.1 Selection of solvent cutting time As shown in Fig. 1 and Table 1, the retention time of solvents (RTsonent) and nicotine (RTaic) in the chromatogram of nic% determination by use of a GC/MS method is 1-2 min and 16-17 min respectively after dilution with different solvents. Therefore, the solvent cutting time is set to 5 min to avoid missing impurities with the retention time at 5-16 min during the determination of unknown samples.
Table 1 Spectrogram Information and Determination Results of NIC% by Use of GC/MS method after Dilution with Different Solvents (n=5) “Solvents RTswewmin RTa/min nic% Isopropanol (containing 5 mL/L of anhydrous ethanol) 1.458 16.265 97.20% Isopropanol 1.251 16.259 97.36% Methanol 1.211 16.253 97.02% Acetonitrile 1.464 16.347 97.23% Wherein, n is the number of parallel tests, the same below.
2.1.2 Selection of injection port temperature The boiling point of nicotine is 247°C and the melting point is -79°C. Therefore, the injection port temperature is set to 250°C, which can ensure the rapid passage of the nicotine sample in the injection port and detection pipeline.
2.1.3 Selection of temperature programmed conditions By comparing multiple temperature programmed conditions and taking the detection time and separation degree between nicotine peak and adjacent peak into account comprehensively, the temperature programmed conditions are finally determined as follows: an initial temperature is 100°C, then it is increased to 115°C at
0.5°C/min, and kept for 5 min; then, the temperature is increased to 200°C at 5°C/min and kept for 2 min; and finally, the temperature is increased to 220°C at 20°C/min, and kept for 10 min.
2.1.4 Optimization of mass spectrometry conditions Since the impurities in nicotine and its main constituent - nicotine are alkaloid compounds containing pyridine ring with similar structures, correction factors thereof are approximately equal, so the correction factors can be ignored and the area can be normalized directly to obtain nic%. Therefore, the full scan mode is adopted to obtain the peaks of various constituents including nicotine.
Considering that the impurities in the samples are mostly derivatives or degradation products of nicotine, generally, the molecular weight of the above-mentioned impurities is not very different from the molecular weight 162.23 of the nicotine. No impurities with mass-to-charge ratio #:/z>500 are found in the test. However, the molecular weight of nitrogen is 28. The purpose of setting the minimum scanning mass-to-charge ratio m/z>29 is not to expect the nitrogen to affect the detection. Therefore, the scan range shall be selected as m/z= 29-500.
2.1.5 Selection of diluent solvent As shown in Fig. 1 and Table 1, the retention time and peak areas of nicotine peak are slightly different after dilution with different solvents. When isopropanol (containing 5S mL/L of anhydrous ethanol), isopropanol and acetonitrile are used as diluent solvents, the detected nic% will tend to the median value of the five solvents. However, acetonitrile is more toxic and shall be removed. The diluent solvent of the sample in the test of measuring wat%o in the nicotine sample by use of a GC method is also isopropanol (containing 5 mL/L of anhydrous ethanol), wherein anhydrous ethanol, as the internal standard substance for moisture determination, cannot be omitted. Therefore, isopropanol (containing 5 mL/L of anhydrous ethanol) solvent is also used in the detection process of nic%, that is, the same diluted sample can be used to detect nic% and wat®% respectively, so that the conclusion is more convincing.
2.1.2 Optimization of calculation formula of nic% in samples detected by use of GC/MS The area normalization method is used for measuring the peak area of each component in the sample and the total chromatographic peak area except the solvent peak. The percentage of nicotine peak area in the total peak area is calculated to obtain the nicotine content. The sum of the contents of all components in the sample 1s set at 100%, and the method for calculating a certain component is as follows: xi% = fiAE (fA) ¥100% ® In the formula, x; is the percentage of component i in the test sample; fi is the correction factor of component i; Aiis the peak area or peak height of component i. In this method, the peak area is substituted into the formula for calculation; Since the impurities in nicotine and its main constituent - nicotine are alkaloid compounds containing pyridine ring with similar structures, correction factors thereof are approximately equal, so the correction factors can be ignored and the area can be normalized directly, which can be calculated according to the following formula: x% = AVZAi*100% @ Formula ©) is used for calculating the percentage of nicotine peak area in the total peak area, and the nicotine content can be obtained, as shown in Formula (1).
2.1.3 Analysis of possible main impurities in nicotine sample by use of a
GC/MS method The spectrogram collected by GC/MS is qualitatively analyzed by Mass Hunter workstation. The main constituent of the sample is nicotine (Fig. 2), and the main impurities are alkaloids commonly found in the tobacco, such as nornicotine, etc. (Table 2) Table 2 Possible Alkaloids in the Nicotine Sample SN Conctituay Molcular Molocular Structural Retention Matching Formula Weight Formula Time Degree \ ates HN 1 Nicotine CoH aN 162.1 Go in 97.77% (B)
97.68% (C)
91.55% (A) 2 Myosmine CoHiN: 146.1 oO ne 90.87% (B)
91.02% (C) \ ¢ 3 Nicotyrine CioHioN2 158.1 CS] HE 77.7% (A) en ON 45.5-45.6 29.02% (A) 4 Anabasne CuHieN: 176.1 C= in 83.02% (B)
86.46% (C) Note: impurities in samples A, B and C are different in matching degree.
2.2 Optimization of GC Determination Conditions
2.2.1 Selection of determination method for water content in nicotine samples Chromatography has a good separation effect on the components in the samples to be detected, but there are some limitations of the detector's own nature: liquid chromatography (LC) equipped with ultraviolet (UV) detector or fluorescence detector (FLD) does not respond to water; meanwhile, chromatographs such as GC, LC/MS and GC/MS equipped with mass spectrometry (MS) detector are used for protecting the MS detector because the samples to be detected will be dehydrated before entering into the MS detector. Therefore, the nicotine content determined by these methods cannot reflect the influence of water on the determination results, that is, there is a large method error in the measured value. Long placed nicotine will absorb moisture, resulting in the increase of water content, and the decrease of nicotine purity. When the moisture content is greater than
0.1%, the influence thereof on the purity of nicotine cannot be ignored. Therefore, it is necessary to determine the water content in the nicotine, and to modify the nicotine content value determined by use of a GC/MS method, and the water content in nicotine samples is determined by use of a GC internal standard method. So, it needs to weigh 100 mg of the nicotine sample accurately, accurate to 0.1 mg, and place the same in a 10 mL of volumetric flask. Isopropanol extractant containing 5 mL/L of anhydrous ethanol as the internal standard is adopted for constant volume. After the mixture is evenly mixed, the sample is loaded into a chromatographic bottle to determine the water contained in the nicotine sample.
2.2.2 Selection of chromatographic column It is proved that the stationary phase as bonded polystyrene-diethylbenzene HP- PLOT/Q capillary chromatographic column (Agilent, 30 m x 0.32 mm x 0.20 um) is suitable.
2.3 Optimization of Sample Quantity for Parallel Detection
2.3.1 Parallel detection for five times Table 3 Determination Results of Nicotine Moisture Content (wat%) by Use of GC Method 1* 2 3# 4% 5% c(mgmL) 1025 1050 1023 988 1012 m (mg) 0.399 0.413 0.387 0.346 0.366 3.75% 4.86% wat% 3.89% 3.94% 3.78% 3.50% 3.62%
2.3.2 Parallel detection for twice Table 4 Detection Results of Nicotine Purity NIC% Obtained by Detection for Two Parallel Samples OE GuMmsGCMethed Serial Number of Parallel Determination - nic% wat%o NIC% 2 97.54% 3.81% 93.82% Average value/ % 97.58% 3.77% 93.90% RAD 008% 212% 017%
2.3.3 Determination for the times of parallel detection For the same sample, 5 parallel samples and 2 parallel samples shall be detected respectively according to the above detection steps, and the detection results are compared. There is no significant difference between the detection results of 5 parallel samples and 2 parallel samples (RAD<1%) (See the table below). Table 5 Comparison of Detection Results of Nicotine Purity NIC% Obtained by Detection for Different Quantities of Parallel Samples - TT GomMmsGCMethed Serial number of Parallel Determination Bene nic%o wat% NIC% Detection with five parallel samples ~~ 97.51% 3.75% 93.85% Detection with two parallel samples 97.58% 3.77% 93.90% Average value/ % 97.55% 3.76% 93.88% RADM 00% 033% 005% Combined with the above verification, the precision of this method is good (RSD < 5%). In addition, considering that the usual chromatographic detection methods refer to the analysis of two parallel samples, the arithmetic mean thereof is the detection result. Therefore, twice shall be selected as the sample detection times.
2.4 Precision Verification
2.4.1 nic% in the sample detected by GC/MS The nicotine content of nicotine sample A (long placed sample), sample B (newly prepared sample) and another long placed sample C is determined by use of the GC-MS method, and the results are shown in the table below. Table 6 Determination Results of Nicotine Content nic% by Use of GC/MS Method Sample No. mew eo Average Value RSD 1 2 3 4 5 A 97.66% 97.56% 97.56% 97.34% 9738% 97.50% 0.14% B 99.79% 99.70% 99.69% 99.68% 99.73% 99.72% 0.04% C 97.96% 98.12% 97.97% 97.96% 98.24% 98.05% 0.13% The determination result RSD of nic% by use of GC/MS method is 0. 10%-0. 12%, all less than 1%, which indicates good precision.
2.4.2 Wat % in the sample detected by GC/MS Five diluted solutions shall be prepared for nicotine samples A, B and C according to the above method, and the nicotine moisture content thereof shall be determined by use of the GC method respectively. The results are as shown in the table below.
Table 7 Determination Results of Nicotine Moisture Content (wat%) by Use of GC Method Sample Sample Volume Content Water Content _ /mg /mg % Average Value RSD /mg-mL-!
101.9 0.212 2.12 2.08% A 102.8 0.221 2.21 2.15% 2.08% 2.96%
103.2 0.208 2.08 2.02%
99.8 0.202 2.02 2.02% 1007 0020 020 020%
103.4 0.021 0.21 0.20% B 99.1 0.018 0.18 0.18% 0.19% 4.45%
99.8 0.020 0.20 0.20%
99.4 0.019 0.19 0.19%
100.6 0.175 1.75 1.74% C 99.2 0.172 1.72 1.73% 1.76% 3.72%
100.8 0.170 1.70 1.69%
100.4 0.182 1.82 1.81% The determination result RSD of wat% by use of GC method is 2.96%-3.72%, all less than 5%, which indicates good precision.
2.5 Repetition and Reproducibility The same nicotine sample is treated by the above method and the content is determined to evaluate the repeatability and reproducibility of the sample. The intra-day repeatability is measured by calculating the relative standard deviation (RSD) of the same sample repeatedly measured for five times a day for three consecutive days. The daytime reproducibility is expressed by measuring five groups of samples each day for three consecutive days and calculating the relative standard deviation. The test results are shown in Table 8. The results show that the relative standard deviation (RSD) of intra-day repeatability of nicotine purity measured by this method is 0.07%-0.10%, and the relative standard deviation (RSD) of daytime reproducibility is 0.09%-0.12%, respectively, which are all less than 5%, indicating that this method has good repeatability and stability, and can meet the quantitative requirements.
Table 8 Intra-day Repeatability (n=15) and Daytime Repeatability (n=15) Determined for Nicotine Purity "Intraday Repeatability (n=15) Daytime Reproducibility and Nicotine Repeatability (n=15) Measured Average Measured Average Value Value! % RSD Value/% Value/ % RSD Dal 9546 95s
95.54 95.47
95.63 95.56 0.07 95.68 95.58 0.09
95.59 95.64
93.56 95.58 Dav2 9558 955%
95.44 95.48
95.62 95.58 0.09 95.56 93.55 0.11
93.59 95.42
95.68 95.68 Dy3 986
95.42 95.58
95.54 95.54 0.10 95.62 95.53 0.12
95.48 95.43
95.65 93.39
2.6 Comparison Verification
2.6.1 Detection results by classical methods The purity of nicotine samples A, B and C shall be determined according to the Tobacco and Tobacco Products - Determination of Nicotine Purity - Gravimetric Method Using Tungstosilicic Acid (ISO 13276:1997), and the results are as shown in Table 9. Table 8 Determination Results of Nicotine Purity by Use of Gravimetric Method Using a Tungstosilicic Acid © NIC% (Gravimetric Method Using a Tungstosilicic Acid) sample 0 0000000 1* 2 3% 4* 5# EVA RSD A 9637% 95.44% 9593% 9521% 95.94% 95 78% 048% B 99.80% 99.91% 99.12% 99.64% 99.23% 99.54% 0.35% C 97.23% 96.78% 96.49% 96.21% 97.67% 96.88% 0.61%
2.6.2 Comparison between the results of the gravimetric method using a tungstosilicic acid and the GC/MS-GC method Based on the determination results in Table 6 and Table 7, it is substituted into Formula (3) to calculate the nicotine purity (NIC%) of the sample, and the calculation results are as shown in Table 10. Table 9 Determination Results of Nicotine Purity (NIC%) by Use of GC/MS-GC Method “Sample nich wa% NIC% A 9780% 2.08% 95.47% B 99.72% 0.19% 99.52% C 98.03% 1.76% 96.32% The determination results of nicotine purity by use of the gravimetric method using a tungstosilicic acid and this method (GC/MS-GC method) are similar, and the relationship between the two methods 1s directly reflected in Fig. 3. It can be seen from Table 11 that the relative average deviation (RAD) of nicotine purity results obtained by the two methods is less than 1%, indicating the reliability of nicotine purity determination by use of this method.
Table 10 Comparison of Detection Results of Nicotine Sample Purity NIC% Obtained by Different Methods (n=5) “Sample No.
Nicotine purity ¥% Nicotine purity % RADM% A 9547 95.78% 032% B 99.52% 99.54% 0.02% C 96.32% 96.88% 0.58% Note: a is obtained by use of a GC/MS-GC method, and b is obtained by use of a gravimetric method using a tungstosilicic acid.
As shown in Tables 12-17, t - inspection is carried out on nicotine samples A, B and C, respectively according to the results determined by this method and the gravimetric method using a tungstosilicic acid.
P;>0.05 is obtained, indicating that there is no significant difference in the determination results, which proves the accuracy and feasibility of this method.
Table 11 Detection Results of Purity of Nicotine Sample A Obtained by Different Methods (n=5) “Sample No.
GC/MS-GC Method Gravimetric
Method Using a nic% wat% NIC% Tungstosilicic Acid, NIC% 2 97.56% 2.08% 95.53% 95.44% 3 97.56% 2.13% 95.46% 95.93% 4 97.34% 2.02% 95.38% 95.21%
97.38% 2.02% 95.41% 95.94% Average value 97.50% 2.08% 93.47% 95.78% SD 0.0013 0.0006 0.0008 0.0046 Table 12 Comparison of Significant Difference in Detection Results of Purity of Nicotine Sample A Obtained by Different Methods (n=5) "Comparison of Significant Differences Between Methods Items ~~ Gravimetric Method Using a GC/MS-GC Method Tungstosilicic Acid F-inspection ~~ F=003 ~~ Ps=00028 Conclusion Pg<0.05, double samples have heteroscedasticity. t-inspection ~~ t=278 PB =021 Conclusion P>0.05, no significant difference 5
Table 13 Detection Results of Purity of Nicotine Sample B Obtained by Different Methods (n=5) TO GOMSGCmethod Gravimetie Sample No. Method Usmg ° nic% wat% NIC% Tungstosilicic Acid, NIC% 2 99.70% 0.20% 99.50% 99.91% 3 99.69% 0.18% 99.51% 99.12% 4 99.68% 0.20% 99.48% 99.64%
99.73% 0.19% 99.54% 99.23% Average value 99.72% 0.19% 99.52% 99.54% SD 0.0004 0.0001 (4.0004 0.0035 Table 14 Comparison of Significant Difference in Detection Results of Purity of 5 Nicotine Sample B Obtained by Different Methods (n=5) TT Comparison of Significant Differences Between Methods Items ~~ Gravimetric Method Using a GC/MS-GC Method Tungstosilicic Acid F-inspection ~~ F=002 ~~ Pg=0.0007 Conclusion Pr<0.05, double samples have heteroscedasticity. t-inspection t=278 P=092 Conclusion P0.05, no significant difference Table 1516 Detection Results of Purity of Nicotine Sample C Obtained by Different Methods (n=5) TO GOMS-GCMethod Gravimetrie Sample No. Method Using ° nic% wat% NIC% Tungstosilicic Acid, NIC% 2 98.12% 1.74% 96.41% 96.78% 3 97.97% 1.73% 96.27% 96.49% 4 97.96% 1.69% 96.31% 96.21% 5 98.24% 1.81% 96.46% 97.67% Average value 98.03% 1.76% 96.32% 96.88%
SD 0.0013 0.0007 0.0012 0.0058 Table 17 Comparison of Significant Difference in Detection Results of Purity of Nicotine Sample C Obtained by Different Methods (n=5) © Comparison of Significant Differences Between Methods Items EL Gravimetric method using a GC/MS-GC method tungstosilicic acid F-inspection ~~ F=004 ~~ Pe=01050 Conclusion Pr>0.05, double samples have equal variance. t-inspection t=27% p=021 Conclusion P>0.05, no significant difference
2.6.3 Comparison test of nicotine standard substance purity The purity of nicotine standard substance D (sample lot No.: 140609) provided by the National Tobacco Quality Supervision and Inspection Center is 99.4%. The purity obtained by use of this method on different instruments is 99.27% and 99.15%, respectively. The relative average deviation (RAD) between the two and the purity calibration values provided by the National Center for Tobacco Quality Supervision and Inspection is 0.11% and 0.24%, respectively (see Table 18), indicating that the detection results of this method are reliable. Table 18 Detection Results of Purity of Nicotine Sample D Obtained from Different Units “Serial number of parallel GC/MS-GC method ~~ GCMS-GC method? determination nic% ~~ wat% NIC% nic% wat% NIC% 2 99.36 0.12 99.24 99.33 0.19 99.14 Average value/ % 99.38 0.13 99.25 99.35 0.20 99.16 RAD% 004 553 003 005 513 00 Notes: instrument a: Agilent 7890A, Agilent 7890A-7000B; instrument b: Agilent 7890A, Agilent 5975C-7890A.
3. Conclusion As shown in Figs. 1-3 and Tables 6-17, there is no significant difference between the results determined in Embodiments 1-3 and those determined by the gravimetric method using a tungstosilicic acid, which proves the accuracy and feasibility of the method described in the present invention. Careful comparison for each group of data shows that: when the nicotine content of the newly prepared sample (sample B) is determined, the determination results by use of the gravimetric method using a tungstosilicic acid and the GC/MS-GC method are basically the same, 99.34% and
99.54%, respectively. However, it is found that the results obtained by the gravimetric method using a tungstosilicic acid are lower than those obtained by the GC/MS-GC method during the detection for the long placed samples (samples A and C). In order to analyze the reasons for the differences, we have put analyzed the composition of impurities in the long-aged samples. The results show that the impurities include myosmine, nicotyrine and anabasne, this is due to that during the placement, the nicotine will be in moisture absorption, degradation and isomerization to generate nicotine derivatives and/or other tobacco endemic alkaloids. Based on the above test results, we speculate that due to the relatively high impurity content of nicotine derivatives in the samples, these alkaloids can hinder the binding and precipitation of nicotine and salts with silicotungstate acid. Therefore, when the gravimetric method using a tungstosilicic acid is used for testing the purity, the nicotine cannot completely precipitate into salt due to the interference of other types of alkaloids and/or other impurities, resulting in slightly smaller measured value than the actual value. On the other hand, since the inherent attribute of nicotine - molecular weight is directly detected by use of a GC/MS method, the measurement results thereof are less affected by other alkaloid impurities, so the measured value thereof shall be closer to the actual value, and the value measured by the GC/MS-GC method shall be greater than that measured by the gravimetric method using a tungstosilicic acid. Our test results are also consistent with the above speculation: when the sample is newly prepared (sample B), the values of nicotine contents respectively measured by the two methods are relatively close due to the little deterioration of nicotine in the sample. When the samples (samples A and C) are detected for a long time, the nicotine in the sample is more metamorphic, and other alkaloids rising from this has a certain influence on the determination data by the gravimetric method using a tungstosilicic acid, making it lower than the actual content.
However, since GC/MS-GC method is not disturbed by other alkaloids, the measured values obtained by GC/MS-GC method are closer to the actual values than those obtained by the gravimetric method using a tungstosilicic acid. Therefore, GC/MS-GC method is more simple and accurate than the gravimetric method using a tungstosilicic acid.
Although the present invention is described in detail by referring to the above- mentioned embodiments, technicians in this field shall understand that it may modify the technical solution recorded in each of the above-mentioned embodiments or replace part of the technical features equivalently. Any modification, equivalent substitution, improvement, etc. within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
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| CN104330514B (en) * | 2014-11-05 | 2016-08-24 | 云南省烟草质量监督检测站 | Combined gas chromatography mass spectrometry combines the method for gas chromatography determination nicotine purity |
| CN107255687A (en) * | 2017-08-23 | 2017-10-17 | 国家烟草质量监督检验中心 | It is a kind of at the same determine nicotine in 11 kinds of secondary alkaloid impurity contents method |
| CN107727784A (en) * | 2017-11-07 | 2018-02-23 | 上海烟草集团有限责任公司 | One grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine |
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| CN104330514B (en) * | 2014-11-05 | 2016-08-24 | 云南省烟草质量监督检测站 | Combined gas chromatography mass spectrometry combines the method for gas chromatography determination nicotine purity |
| CN107255687A (en) * | 2017-08-23 | 2017-10-17 | 国家烟草质量监督检验中心 | It is a kind of at the same determine nicotine in 11 kinds of secondary alkaloid impurity contents method |
| CN107727784A (en) * | 2017-11-07 | 2018-02-23 | 上海烟草集团有限责任公司 | One grow tobacco and tobacco product in the chiral analysis method of nicotine and nornicotine |
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