CN112578069A - Analysis method for simultaneously measuring 11 components in electronic smoke sol - Google Patents
Analysis method for simultaneously measuring 11 components in electronic smoke sol Download PDFInfo
- Publication number
- CN112578069A CN112578069A CN202011599980.3A CN202011599980A CN112578069A CN 112578069 A CN112578069 A CN 112578069A CN 202011599980 A CN202011599980 A CN 202011599980A CN 112578069 A CN112578069 A CN 112578069A
- Authority
- CN
- China
- Prior art keywords
- temperature
- filter
- sample
- internal standard
- kept
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004458 analytical method Methods 0.000 title claims abstract description 26
- 239000000779 smoke Substances 0.000 title claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 54
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 54
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims abstract description 53
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 51
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 48
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000000443 aerosol Substances 0.000 claims abstract description 30
- YPFDHNVEDLHUCE-UHFFFAOYSA-N propane-1,3-diol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 29
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 26
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 claims abstract description 21
- 229960002715 nicotine Drugs 0.000 claims abstract description 21
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000011550 stock solution Substances 0.000 claims abstract description 19
- NOOLISFMXDJSKH-UTLUCORTSA-N (+)-Neomenthol Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@@H]1O NOOLISFMXDJSKH-UTLUCORTSA-N 0.000 claims abstract description 18
- NOOLISFMXDJSKH-UHFFFAOYSA-N DL-menthol Natural products CC(C)C1CCC(C)CC1O NOOLISFMXDJSKH-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229940041616 menthol Drugs 0.000 claims abstract description 18
- 229960004063 propylene glycol Drugs 0.000 claims abstract description 18
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical compound OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000004817 gas chromatography Methods 0.000 claims abstract description 14
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 13
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 239000002904 solvent Substances 0.000 claims abstract description 8
- 238000000605 extraction Methods 0.000 claims abstract description 6
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 claims description 28
- SMUQFGGVLNAIOZ-UHFFFAOYSA-N quinaldine Chemical compound C1=CC=CC2=NC(C)=CC=C21 SMUQFGGVLNAIOZ-UHFFFAOYSA-N 0.000 claims description 28
- 239000012159 carrier gas Substances 0.000 claims description 20
- 239000012224 working solution Substances 0.000 claims description 15
- 235000019437 butane-1,3-diol Nutrition 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 239000007789 gas Substances 0.000 claims description 12
- 238000005303 weighing Methods 0.000 claims description 12
- 239000012491 analyte Substances 0.000 claims description 10
- 238000007664 blowing Methods 0.000 claims description 10
- 239000001307 helium Substances 0.000 claims description 10
- 229910052734 helium Inorganic materials 0.000 claims description 10
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 10
- 150000001875 compounds Chemical class 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 7
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 6
- 238000001165 gas chromatography-thermal conductivity detection Methods 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 5
- 238000011109 contamination Methods 0.000 claims description 5
- 238000000769 gas chromatography-flame ionisation detection Methods 0.000 claims description 5
- 239000013076 target substance Substances 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 4
- 239000001257 hydrogen Substances 0.000 claims description 4
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 2
- 239000012086 standard solution Substances 0.000 claims description 2
- 238000002137 ultrasound extraction Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 6
- 239000000523 sample Substances 0.000 description 31
- 229960005150 glycerol Drugs 0.000 description 14
- 238000011084 recovery Methods 0.000 description 6
- 239000003571 electronic cigarette Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 description 4
- 235000013772 propylene glycol Nutrition 0.000 description 4
- DNIAPMSPPWPWGF-VKHMYHEASA-N (+)-propylene glycol Chemical compound C[C@H](O)CO DNIAPMSPPWPWGF-VKHMYHEASA-N 0.000 description 2
- 229940035437 1,3-propanediol Drugs 0.000 description 2
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940093476 ethylene glycol Drugs 0.000 description 2
- 239000003546 flue gas Substances 0.000 description 2
- 229920000166 polytrimethylene carbonate Polymers 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 229940058015 1,3-butylene glycol Drugs 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000010813 internal standard method Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to an analysis method for simultaneously measuring 11 components in electronic smoke sol, which comprises the following steps: (1) filter disc treatment, aerosol collection and sample preparation, wherein a certain volume of isopropanol solvent is used for extracting the filter disc, a certain volume of internal standard stock solution is added, and after oscillating extraction is carried out for 30 minutes, supernatant is taken and transferred to a chromatographic analysis bottle to be detected; (2) taking the supernatant obtained in the step (1) for Gas Chromatography (GC) analysis; (3) and calculating the content of the target object in the sample. The method realizes the simultaneous detection of 11 components such as methanol, ethanol, ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, diethylene glycol, menthol, triethylene glycol, nicotine, water and the like in the electronic smoke sol.
Description
Technical Field
The invention belongs to the technical field of tobacco chemical analysis, and particularly relates to an analysis method for simultaneously determining 11 components in electronic smoke sol.
Background
Electronic cigarettes are products for smokers to smoke nicotine after atomization, and the components in the aerosol are always concerned. 1, 2-propylene glycol and glycerol are widely used solvents of electronic cigarettes, ethanol and water are widely used auxiliary solvents or additives, methanol, diethylene glycol, ethylene glycol, triethylene glycol, 1, 3-propylene glycol and the like are possibly taken as impurities of the 1, 2-propylene glycol and the glycerol or other ways to be brought into aerosol, so that adverse effects on human health are brought, and the like are widely concerned, and menthol is the most widely used flavoring agent in electronic cigarette aerosol. Currently, the main methods for measuring such substances in electronic cigarettes are gas chromatography, gas chromatography-mass spectrometry, continuous flow method, raman spectroscopy, liquid chromatography-tandem mass spectrometry and the like, but the throughput of such methods is low, and substances such as diethylene glycol, triethylene glycol, water, methanol, ethanol, menthol and the like are rarely or not researched.
The invention content is as follows:
the invention aims to establish an analysis method (methanol, ethanol, ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, diethylene glycol, menthol, triethylene glycol, nicotine and water) for simultaneously measuring 11 components in the electronic smoke sol based on the technical defects, and is suitable for the general survey analysis of the electronic smoke sol sample.
The purpose of the invention is realized by the following technical scheme:
an analysis method for simultaneously measuring 11 components in electronic smoke sol comprises the following steps:
(A) and (3) filter disc treatment: prior to weighing, the filter should be placed in the environment corresponding to the time of weighing for at least 24 hours. For all operations, the operator should wear gloves of suitable material to prevent finger contamination. During operation, a filter support cover may be installed to prevent moisture loss or absorption. The filter should be disposed of as soon as possible after collection of the analyte to prevent moisture uptake or loss;
collection of aerosols and sample preparation: an electronic smoke sol collection system was set up using the method recommended by CORESTA at N ° 81 and the aerosol was collected on a cambridge filter. The trapping parameters, such as the number of aspirations or aspirations, depend on the actual situation. During the experiment it should be ensured that the collection of aerosol is less than the capacity of the filter disc. When the filter is removed, the side with the aerosol adsorbed is folded twice against the inside of the fold using forceps, during which the edges of the filter are carefully handled. The interior of the front of the filter holder was then wiped with two unused filter discs and placed into a flask to collect residual aerosol;
extracting the filter disc by using a certain volume of isopropanol solvent, adding a certain volume of internal standard stock solution, performing oscillation extraction for 30 minutes, taking supernate, transferring the supernate into a chromatographic analysis bottle to be tested, and storing the supernate in a dark place;
(B) performing Gas Chromatography (GC) analysis on the supernatant obtained after the extraction in the step (A);
(C) and calculating the content of the target object in the sample.
Preferably, in step (A), the internal standard solution is an isopropanol solution of 1, 3-butanediol and 2-methylquinoline at concentrations of 100mg/mL and 10mg/mL, respectively.
Preferably, in step (a), 1, 3-butanediol is an internal standard for compounds other than nicotine and 2-methylquinoline is an internal standard for nicotine.
Preferably, in step (A), the volume of the isopropanol extractant is 18.8mL (using 44mm Cambridge filter, while adding 200. mu.L of internal standard stock solution) or 49.5mL (using 92mm Cambridge filter, while adding 500. mu.L of internal standard stock solution)
Preferably, in the step (A), the time of ultrasonic extraction is 30-60 min, preferably 30 min.
Preferably, in step (B), the chromatographic conditions of the gas chromatographic analysis comprise:
methanol, ethanol, ethylene glycol, 1, 2-propanediol, 1, 3-propanediol, glycerol, diethylene glycol, menthol, triethylene glycol, nicotine were separated using a DB-ALC1 capillary column (30m × 0.32mm, 1.8 μm), and analyzed by GC-FID;
adopting a temperature programming mode: the initial temperature is 90 ℃, the temperature is kept for 1min, the temperature is increased to 120 ℃ at the speed of 15 ℃/min, the temperature is kept for 3min, the temperature is increased to 250 ℃ at the speed of 40 ℃/min, the temperature is kept for 3min, and the total running time is 12.25 min;
carrier gas: helium gas; the flow rate of the carrier gas is 1.8 mL/min;
tail blowing: 20 mL/min; air: 450 mL/min; hydrogen gas: 40 mL/min;
sample inlet temperature: 250 ℃; detector temperature: 275 ℃; sample introduction amount: 1 mu L of the solution; the split ratio is as follows: 20:1.
② the water is separated by HP-PLOT/Q capillary chromatographic column (30 m.times.0.53 mm, 40 μm), and analyzed by GC-TCD;
adopting a temperature programming mode: the initial temperature is 100 ℃, the temperature is kept for 1min, the temperature is increased to 220 ℃ at the speed of 15 ℃/min, the temperature is kept for 6min, and the total operation time is 15 min;
carrier gas: helium gas; the flow rate of the carrier gas is 4.0 mL/min;
tail blowing: 8 mL/min; reference gas: 20 mL/min;
sample inlet temperature: 250 ℃; detector temperature: 250 ℃; sample introduction amount: 2 mu L of the solution; the split ratio is as follows: 5:1.
Preferably, in step (C), the content of the target in the sample is quantitatively calculated according to an internal standard curve method which is: preparing a series of standard working solutions containing a target substance, adding internal standards (1, 3-butanediol and 2-methylquinoline), and establishing a standard curve by taking the concentration (mg/mL) of each target compound as a horizontal coordinate and taking the peak area ratio of an analyte to the internal standard substance as a vertical coordinate; substituting the analysis result of the step (B) into a standard working curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
Preferably, the concentration ranges of each target in the series of standard working solutions are as follows: 0.25-10 mg/mL of 1, 2-propylene glycol and glycerol, 0.025-1 mg/mL of methanol, ethanol, ethylene glycol, 1, 3-propylene glycol, diethylene glycol, menthol, triethylene glycol and nicotine, and 0-1 mg/mL of water.
In a specific embodiment, the method comprises the steps of:
(A) and (3) filter disc treatment: prior to weighing, the filter should be placed in the environment corresponding to the time of weighing for at least 24 hours. For all operations, the operator should wear gloves of suitable material to prevent finger contamination. During operation, a filter support cover may be installed to prevent moisture loss or absorption. The filter should be disposed of as soon as possible after collection of the analyte to prevent moisture uptake or loss.
Collection of aerosols and sample preparation: an electronic smoke sol collection system was set up using the method recommended by CORESTA at N ° 81 and the aerosol was collected on a cambridge filter. The trapping parameters, such as the number of aspirations or aspirations, depend on the actual situation. During the experiment it should be ensured that the collection of aerosol is less than the capacity of the filter disc. When the filter is removed, the side with the aerosol adsorbed is folded twice against the inside of the fold using forceps, during which the edges of the filter are carefully handled. The interior of the front of the filter holder was then wiped with two unused filter discs and placed into a flask to collect residual aerosol.
A certain volume of 18.8mL of solvent (44 mm Cambridge filter is used, 200. mu.L of internal standard stock solution (10 mg/mL and 1mg/mL of isopropanol solution of 1, 3-butanediol and 2-methylquinoline) or 49.5mL of internal standard stock solution (10 mg/mL and 1mg/mL of isopropanol solution of 1, 3-butanediol and 2-methylquinoline) is simultaneously added when 92mm Cambridge filter is used) is used for extracting the filter, after shaking for 30 minutes, the supernatant is taken and transferred to a chromatographic bottle for testing, and the filter is protected from light.
(B) Taking the supernatant obtained in the step (A) to perform Gas Chromatography (GC) analysis, wherein the analysis conditions are as follows:
methanol, ethanol, ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, diethylene glycol, menthol, triethylene glycol, nicotine were separated using DB-ALC1 capillary chromatography column (30m × 0.32mm, 1.8 μm), and analyzed by GC-FID; the programmed heating mode is that the initial temperature is 90 ℃, the temperature is kept for 1min, the temperature is increased to 120 ℃ at the speed of 15 ℃/min, the temperature is kept for 3min, the temperature is increased to 250 ℃ at the speed of 40 ℃/min, the temperature is kept for 3min, and the total running time is 12.25 min; the carrier gas is helium; the flow rate of the carrier gas is 1.8 mL/min; tail blowing 20 mL/min; air 450 mL/min; hydrogen is 40 mL/min; the temperature of a sample inlet is 250 ℃; the detector temperature was 275 ℃; the sample injection amount is 1 mu L; the split ratio was 20: 1.
The water was separated using an HP-PLOT/Q capillary chromatography column (30 m.times.0.53 mm, 40 μm), analyzed by GC-TCD; the programmed heating mode is that the initial temperature is 100 ℃, the temperature is kept for 1min, the temperature is increased to 220 ℃ at the speed of 15 ℃/min, the temperature is kept for 6min, and the total operation time is 15 min; the carrier gas is helium; the flow rate of the carrier gas is 4.0 mL/min; blowing 8mL/min at the tail; 20mL/min of reference gas; the temperature of a sample inlet is 250 ℃; the temperature of the detector is 250 ℃; the sample injection amount is 2 mu L; the split ratio was 5: 1.
(C) Quantitatively calculating the content of the target object in the sample by adopting an internal standard curve method; wherein, the internal standard curve method is as follows: preparing a series of standard working solutions containing a target substance, adding internal standards (1, 3-butanediol and 2-methylquinoline), and establishing a standard curve by taking the concentration (mg/mL) of each target compound as a horizontal coordinate and taking the peak area ratio of an analyte to the internal standard substance as a vertical coordinate; substituting the analysis result of the step (B) into the standard curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
Compared with the prior art, the invention has at least the following beneficial technical effects:
the invention establishes an analysis method for simultaneously measuring 11 components in the electronic flue gas sol based on GC technology, realizes the simultaneous analysis of methanol, ethanol, ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, diethylene glycol, menthol, triethylene glycol, nicotine and water, has the advantages of high flux, good sensitivity, repeatability, recovery rate and the like, and is suitable for the detection of a large number of electronic flue gas sol samples.
Drawings
FIG. 1 is a chromatogram of a target and an internal standard in a standard working solution (internal standard 1: 1, 3-butanediol; internal standard 2: 2-methylquinoline);
FIG. 2 is a chromatogram of a target and an internal standard in a typical sample solution (internal standard 1: 1, 3-butanediol; internal standard 2: 2-methylquinoline).
Detailed Description
The technical solution of the present invention will be further described below with reference to specific embodiments.
Example 1: analysis method for simultaneously measuring 11 components in electronic smoke sol
(1) Instruments and reagents
The instrument comprises the following steps: the gas chromatograph is Bruker 456GC and is provided with a hydrogen flame detector (FID) and a Thermal Conductivity Detector (TCD), the sample inlet has a split-flow sample introduction mode, and the sample inlet, the column box and the detector are respectively provided with an independently controllable heating unit; an oscillator (YY-5A, Riwa instruments manufacturing Co., Ltd.); analytical balance (AE163, Mettler, Switzerland, sensory: 0.1 mg).
Reagent consumables: methanol, ethanol from Merck (analytical grade); diethylene glycol, 1, 3-propylene glycol, glycerol, 1, 3-butylene glycol, menthol and ethylene glycol are purchased from Alfa Aesar (the purity is more than or equal to 99 percent); 1, 2-propanediol from Sigma-Aldrich (purity ≥ 99.5%); triethylene glycol was purchased from TCI (purity > 99%), nicotine was purchased from TRC (purity > 99%); 2-methylquinoline from damas-beta (> 99% pure); the water is deionized water.
(2) Working conditions of the apparatus
Chromatographic conditions are as follows: methanol, ethanol, ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, diethylene glycol, menthol, triethylene glycol, nicotine were separated using DB-ALC1 capillary chromatography column (30m × 0.32mm, 1.8 μm), and analyzed by GC-FID; the programmed heating mode is that the initial temperature is 90 ℃, the temperature is kept for 1min, the temperature is increased to 120 ℃ at the speed of 15 ℃/min, the temperature is kept for 3min, the temperature is increased to 250 ℃ at the speed of 40 ℃/min, the temperature is kept for 3min, and the total running time is 12.25 min; the carrier gas is helium; the flow rate of the carrier gas is 1.8 mL/min; tail blowing 20 mL/min; air 450 mL/min; hydrogen is 40 mL/min; the temperature of a sample inlet is 250 ℃; the detector temperature was 275 ℃; the sample injection amount is 1 mu L; the split ratio was 20: 1.
The water was separated using an HP-PLOT/Q capillary chromatography column (30 m.times.0.53 mm, 40 μm), analyzed by GC-TCD; the programmed heating mode is that the initial temperature is 100 ℃, the temperature is kept for 1min, the temperature is increased to 220 ℃ at the speed of 15 ℃/min, the temperature is kept for 6min, and the total operation time is 15 min; the carrier gas is helium; the flow rate of the carrier gas is 4.0 mL/min; blowing 8mL/min at the tail; 20mL/min of reference gas; the temperature of a sample inlet is 250 ℃; the temperature of the detector is 250 ℃; the sample injection amount is 2 mu L; the split ratio was 5: 1.
(3) Sample pretreatment
Filter sheet treatment
Prior to weighing, the filter should be placed in the environment corresponding to the time of weighing for at least 24 hours. For all operations, the operator should wear gloves of suitable material to prevent finger contamination. During operation, a filter support cover may be installed to prevent moisture loss or absorption. The filter should be disposed of as soon as possible after collection of the analyte to prevent moisture uptake or loss.
Collecting aerosol and preparing sample
An electronic smoke sol collection system was set up using the method recommended by CORESTA at N ° 81 and the aerosol was collected on a cambridge filter. The trapping parameters, such as the number of aspirations or aspirations, depend on the actual situation. During the experiment it should be ensured that the collection of aerosol is less than the capacity of the filter disc. When the filter is removed, the side with the aerosol adsorbed is folded twice against the inside of the fold using forceps, during which the edges of the filter are carefully handled. The interior of the front of the filter holder was then wiped with two unused filter discs and placed into a flask to collect residual aerosol.
③ using 18.8mL (when using 44mm Cambridge filter, adding 200 muL internal standard stock solution) or 49.5mL (when using 92mm Cambridge filter, adding 500 muL internal standard stock solution) of solvent with certain volume to extract the filter, oscillating and extracting for 30 minutes, taking the supernatant, transferring the supernatant into a chromatographic analysis bottle to be tested, and storing the supernatant in a dark place.
(4) Standard working solution preparation
Preparation of internal standard stock solution: accurately weighing 10g of 1, 3-butanediol and 1g of 2-methylquinoline in a 100mL brown volumetric flask (accurate to 0.1mg), adding isopropanol to dissolve and fix the volume to a scale, thus obtaining an internal standard stock solution.
Preparing standard stock solutions of methanol, ethanol, ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, diethylene glycol, menthol, triethylene glycol and nicotine: accurately weighing 5g of 1, 2-propylene glycol, glycerol and 0.5g of methanol, ethanol, ethylene glycol, 1, 3-propylene glycol, diethylene glycol, menthol, triethylene glycol and nicotine respectively, putting the materials into a 50mL brown volumetric flask, adding isopropanol to dissolve and fix the volume to a scale.
Preparing standard working solution of methanol, ethanol, ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, diethylene glycol, menthol, triethylene glycol and nicotine: and respectively transferring 25 mu L, 50 mu L, 100 mu L, 250 mu L, 500 mu L and 1000 mu L of standard stock solutions into a 10mL brown volumetric flask, respectively and accurately adding 100 mu L of internal standard stock solutions, and diluting with isopropanol to fix the volume to a scale, thus obtaining six standard working solutions.
Preparation of water standard working solution
And respectively transferring 0 mu L, 1 mu L, 2 mu L, 4 mu L, 7 mu L and 10 mu L of aqueous solution into a 10mL volumetric flask, respectively and accurately adding 100 mu L of internal standard stock solution, diluting with isopropanol, and fixing the volume to the scale to obtain six standard working solutions.
(5) Sample assay
Samples were tested according to the instrumental test conditions and the test was repeated twice for each sample. While a set of blanks was made for each sample batch.
And (3) respectively carrying out GC analysis on the standard working solution obtained in the step (4) and the sample solution obtained in the step (3), wherein chromatograms of the target substance in the standard working solution and the internal standard thereof are shown in figures 1 and 2. Quantifying by adopting an internal standard method, and establishing a standard curve by taking the concentration (mg/mL) of each target compound as a horizontal coordinate and taking the peak area ratio of an analyte to an internal standard substance as a vertical coordinate; and (4) substituting the analysis result of the step (3) into the standard curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
(6) Method verification
According to the lowest-level standard working solution, calculating the detection Limit (LOD) of the method according to the signal-to-noise ratio of 3 times, calculating the quantification Limit (LOQ) of the method according to the signal-to-noise ratio of 10 times, calculating the standard addition recovery rate according to the low, medium and high 3 addition levels, adding 5 parallel samples at each level, and calculating the test precision according to the parallel test result. The recovery, detection limit and precision of the method are shown in table 1. As can be seen from the table, the recovery rate of the spiked sample is 97.6-102.8%, the detection limit is 0.003-0.030 mg/mL, and the precision is 0.59-3.57%.
TABLE 1 recovery, detection limit and precision of 11 components in the electronic fume sol
| Serial number | Compound (I) | Recovery (%) | LOD(mg/mL) | LOQ(mg/mL) | Precision (%) |
| 1 | Methanol | 102.4 | 0.007 | 0.023 | 0.88 |
| 2 | Ethanol | 102.5 | 0.004 | 0.012 | 1.28 |
| 3 | Ethylene glycol | 102.8 | 0.008 | 0.025 | 146 |
| 4 | 1, 2-propanediol | 102.5 | 0.010 | 0.032 | 0.89 |
| 5 | 1, 3-propanediol | 100.6 | 0.009 | 0.031 | 1.23 |
| 6 | Glycerol | 102.3 | 0.026 | 0.087 | 1.46 |
| 7 | Diethylene glycol | 99.1 | 0.015 | 0.049 | 0.99 |
| 8 | Menthol | 99.7 | 0.003 | 0.011 | 1.03 |
| 9 | Triethylene glycol | 102.3 | 0.012 | 0.040 | 1.45 |
| 10 | Nicotine | 100.9 | 0.004 | 0.013 | 0.59 |
| 11 | Water (W) | 97.6 | 0.030 | 0.100 | 3.57 |
(7) Analysis of actual samples
The results of 20 representative e-cigarette aerosols were tested according to the above assay method and are detailed in table 2. As can be seen from the table, in these samples, the water content was 9.30 to 54.67mg/100, the nicotine content was 0.44 to 5.24mg/100, the 1, 2-propanediol content was 42.10 to 283.08mg/100, the glycerol content was 48.24 to 318.48mg/100, the ethanol content was 0.44 to 2.71mg/100, and the menthol content was 0.60 to 2.28mg/100, while the other components were not detected.
TABLE 2 results of actual sample analysis (unit: mg/100-mouth)
Note: "ND" means not detected
Claims (6)
1. An analysis method for simultaneously measuring 11 components in electronic smoke sol comprises the following steps:
(A) and (3) filter disc treatment: before weighing, the filter should be placed in the environment to which it corresponds at the time of weighing for at least 24 hours, for all operations, the operator should wear gloves of suitable material to prevent finger contamination, during the operation, a filter holder cover can be installed to prevent water loss or absorption, the filter should be handled as soon as possible after collection of the analyte to prevent water uptake or loss;
collection of aerosols and sample preparation: setting an electronic smoke sol collecting system by using a method N DEG 81 recommended by CORESTA, collecting the aerosol on a Cambridge filter, and trapping parameters, such as suction times or suction times, according to actual conditions; the collection amount of aerosol is ensured to be less than the capacity of the filter disc in the experimental process; when the filter disc is taken out, the filter disc is taken out by using tweezers, one surface adsorbed with the aerosol is folded towards the inner side of the fold twice, and the edge of the filter disc needs to be carefully treated in the process; the interior of the front of the filter holder was then wiped with two unused filter discs and placed into a flask to collect residual aerosol;
extracting the filter disc by using a certain volume of isopropanol solvent, adding a certain volume of internal standard stock solution, performing oscillation extraction for 30 minutes, taking supernate, transferring the supernate into a chromatographic analysis bottle to be tested, and storing the supernate in a dark place;
(B) performing Gas Chromatography (GC) analysis on the supernatant obtained after the extraction in the step (A);
(C) and calculating the content of the target object in the sample.
2. The method according to claim 1, wherein in step (a), the internal standard solution is an isopropanol solution of 1, 3-butanediol and 2-methylquinoline at a concentration of 1mg/mL and 0.1mg/mL, respectively;
in the step (A), 1, 3-butanediol is used as an internal standard of other compounds except nicotine, and 2-methylquinoline is used as an internal standard of nicotine;
the volume of the isopropanol extractant is 18.8mL, and when a 44mm Cambridge filter disc is used, 200 mu L of internal standard stock solution is added at the same time; or 49.5ml, when using 92mm Cambridge filter, adding 500 μ L internal standard stock solution;
the ultrasonic extraction time is 30-60 min.
3. The method according to claim 1 or 2, wherein in step (B), the gas chromatography conditions comprise:
methanol, ethanol, ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, diethylene glycol, menthol, triethylene glycol, nicotine were separated using a DB-ALC1 capillary column, 30m × 0.32mm, 1.8 μm, and analyzed by GC-FID;
② the water was separated using HP-PLOT/Q capillary chromatography column, 30 m.times.0.53 mm, 40 μm, analyzed by GC-TCD.
4. The method of claim 3, wherein in step (B), the gas chromatographic conditions further comprise:
firstly, a GC-FID program heating mode is that the initial temperature is 90 ℃, the temperature is kept for 1min, the temperature is increased to 120 ℃ at the speed of 15 ℃/min, the temperature is kept for 3min, the temperature is increased to 250 ℃ at the speed of 40 ℃/min, and the temperature is kept for 3 min; the total running time is 12.25 min;
carrier gas: helium gas; the flow rate of the carrier gas is 1.8 mL/min;
tail blowing: 20 mL/min; air: 450 mL/min; hydrogen gas: 40 mL/min;
sample inlet temperature: 250 ℃; detector temperature: 275 ℃; sample introduction amount: 1 mu L of the solution; the split ratio is as follows: 20: 1;
secondly, the GC-TCD temperature programming mode is that the initial temperature is 100 ℃, the temperature is kept for 1min, the temperature is increased to 220 ℃ at the speed of 15 ℃/min, and the temperature is kept for 6 min; the total running time is 15 min;
carrier gas: helium gas; the flow rate of the carrier gas is 4.0 mL/min;
tail blowing: 8 mL/min; reference gas: 20 mL/min;
sample inlet temperature: 250 ℃; detector temperature: 250 ℃; sample introduction amount: 2 mu L of the solution; the split ratio is as follows: 5:1.
5. The method according to claim 4, wherein in step (C), the content of the target in the sample is quantitatively calculated according to an internal standard curve method which is: preparing a series of standard working solutions containing a target substance, adding internal standards (1, 3-butanediol and 2-methylquinoline), and establishing a standard curve by taking the concentration (mg/mL) of each target compound as a horizontal coordinate and taking the peak area ratio of an analyte to the internal standard substance as a vertical coordinate; substituting the analysis result of the step (B) into a standard working curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample;
in the series of standard working solutions, the concentration ranges of the targets are as follows: 0.25-10 mg/mL of 1, 2-propylene glycol and glycerol, 0.025-1 mg/mL of methanol, ethanol, ethylene glycol, 1, 3-propylene glycol, diethylene glycol, menthol, triethylene glycol and nicotine, and 0-1 mg/mL of water.
6. Method according to claim 1, characterized in that it comprises the following steps:
(A) and (3) filter disc treatment: before weighing, the filter should be placed in the environment to which it corresponds at the time of weighing for at least 24 hours, for all operations, the operator should wear gloves of suitable material to prevent finger contamination, during the operation, a filter holder cover can be installed to prevent water loss or absorption, the filter should be handled as soon as possible after collection of the analyte to prevent water uptake or loss;
collection of aerosols and sample preparation: setting an electronic smoke sol collecting system by using a method N DEG 81 recommended by CORESTA, collecting aerosol on a Cambridge filter, trapping parameters such as suction times or suction times, and ensuring that the collection amount of the aerosol is less than the capacity of the filter in the experiment process depending on actual conditions, when the filter is taken out, taking out by using tweezers, folding the side adsorbed with the aerosol towards the inner side of the fold twice, carefully processing the edge of the filter in the process, wiping the inner part of the front part of the filter support by using two unused filters, and putting the filter into a flask to collect residual aerosol;
using a certain volume of 18.8mL of solvent, and adding 200 mu L of internal standard stock solution when using a 44mm Cambridge filter; or 49.5ml, when 92mm Cambridge filter disc is used, 500 mu L of internal standard stock solution is added at the same time, the filter disc is extracted, after shaking extraction for 30 minutes, supernatant is taken and transferred to a chromatographic analysis bottle to be tested, and the supernatant is stored in a dark place;
(B) taking the supernatant obtained in the step (A) to perform Gas Chromatography (GC) analysis, wherein the analysis conditions are as follows:
methanol, ethanol, ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, glycerol, diethylene glycol, menthol, triethylene glycol, nicotine were separated using DB-ALC1 capillary chromatography column (30m × 0.32mm, 1.8 μm), and analyzed by GC-FID; the programmed heating mode is that the initial temperature is 90 ℃, the temperature is kept for 1min, the temperature is increased to 120 ℃ at the speed of 15 ℃/min, the temperature is kept for 3min, the temperature is increased to 250 ℃ at the speed of 40 ℃/min, the temperature is kept for 3min, and the total running time is 12.25 min; the carrier gas is helium; the flow rate of the carrier gas is 1.8 mL/min; tail blowing 20 mL/min; air 450 mL/min; hydrogen is 40 mL/min; the temperature of a sample inlet is 250 ℃; the detector temperature was 275 ℃; the sample injection amount is 1 mu L; the split ratio is 20: 1;
the water was separated using an HP-PLOT/Q capillary chromatography column, 30 m.times.0.53 mm, 40 μm, analyzed by GC-TCD; the programmed heating mode is that the initial temperature is 100 ℃, the temperature is kept for 1min, the temperature is increased to 220 ℃ at the speed of 15 ℃/min, the temperature is kept for 6min, and the total operation time is 15 min; the carrier gas is helium; the flow rate of the carrier gas is 4.0 mL/min; blowing 8mL/min at the tail; 20mL/min of reference gas; the temperature of a sample inlet is 250 ℃; the temperature of the detector is 250 ℃; the sample injection amount is 2 mu L; the split ratio is 5: 1;
(C) quantitatively calculating the content of the target object in the sample by adopting an internal standard curve method; wherein, the internal standard curve method is as follows: preparing a series of standard working solutions containing a target substance, adding internal standards of 1, 3-butanediol and 2-methylquinoline, and establishing a standard curve by taking the concentration mg/mL of each target compound as a horizontal coordinate and taking the peak area ratio of an analyte to the internal standard substance as a vertical coordinate; substituting the analysis result of the step (B) into the standard curve to obtain the content of the target object in the solution to be detected, and further calculating to obtain the content of each target object in the sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011599980.3A CN112578069A (en) | 2020-12-29 | 2020-12-29 | Analysis method for simultaneously measuring 11 components in electronic smoke sol |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011599980.3A CN112578069A (en) | 2020-12-29 | 2020-12-29 | Analysis method for simultaneously measuring 11 components in electronic smoke sol |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112578069A true CN112578069A (en) | 2021-03-30 |
Family
ID=75144194
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011599980.3A Pending CN112578069A (en) | 2020-12-29 | 2020-12-29 | Analysis method for simultaneously measuring 11 components in electronic smoke sol |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112578069A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113720937A (en) * | 2021-08-30 | 2021-11-30 | 深圳市真味生物科技有限公司 | Method for measuring content of cooling agent in electronic smoke sol |
| CN114994200A (en) * | 2022-05-25 | 2022-09-02 | 深圳市艾溹技术研究有限公司 | Method for detecting organic alcohol in aerosol solution and aerosol solution |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104614465A (en) * | 2015-02-16 | 2015-05-13 | 国家烟草质量监督检验中心 | Method for simultaneously measuring content of nicotine, 1,2-propylene glycol and glycerol in electronic smoke sol |
| CN104698117A (en) * | 2015-03-20 | 2015-06-10 | 中国烟草总公司郑州烟草研究院 | Simultaneous determination method for nicotine, 1,2-propylene glycol and glycerol in electronic cigarette smoke |
| CN105067728A (en) * | 2015-09-14 | 2015-11-18 | 浙江中烟工业有限责任公司 | Method for measuring solvent compositions in nicotine liquid by combining gas chromatography and mass spectrometry |
| CN110895231A (en) * | 2019-12-26 | 2020-03-20 | 贵州中烟工业有限责任公司 | Method for measuring moisture in aerosol of heated non-combustible cigarette |
| CN111272938A (en) * | 2020-03-19 | 2020-06-12 | 中国烟草总公司郑州烟草研究院 | Method for simultaneously measuring moisture, nicotine, propylene glycol and glycerol in electronic cigarette liquid |
| WO2020232970A1 (en) * | 2019-05-17 | 2020-11-26 | 江南大学 | Method for determining short chain fatty acid content in intestinal contents or excrement |
-
2020
- 2020-12-29 CN CN202011599980.3A patent/CN112578069A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104614465A (en) * | 2015-02-16 | 2015-05-13 | 国家烟草质量监督检验中心 | Method for simultaneously measuring content of nicotine, 1,2-propylene glycol and glycerol in electronic smoke sol |
| CN104698117A (en) * | 2015-03-20 | 2015-06-10 | 中国烟草总公司郑州烟草研究院 | Simultaneous determination method for nicotine, 1,2-propylene glycol and glycerol in electronic cigarette smoke |
| CN105067728A (en) * | 2015-09-14 | 2015-11-18 | 浙江中烟工业有限责任公司 | Method for measuring solvent compositions in nicotine liquid by combining gas chromatography and mass spectrometry |
| WO2020232970A1 (en) * | 2019-05-17 | 2020-11-26 | 江南大学 | Method for determining short chain fatty acid content in intestinal contents or excrement |
| CN110895231A (en) * | 2019-12-26 | 2020-03-20 | 贵州中烟工业有限责任公司 | Method for measuring moisture in aerosol of heated non-combustible cigarette |
| CN111272938A (en) * | 2020-03-19 | 2020-06-12 | 中国烟草总公司郑州烟草研究院 | Method for simultaneously measuring moisture, nicotine, propylene glycol and glycerol in electronic cigarette liquid |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113720937A (en) * | 2021-08-30 | 2021-11-30 | 深圳市真味生物科技有限公司 | Method for measuring content of cooling agent in electronic smoke sol |
| CN114994200A (en) * | 2022-05-25 | 2022-09-02 | 深圳市艾溹技术研究有限公司 | Method for detecting organic alcohol in aerosol solution and aerosol solution |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102012407B (en) | Method for detecting tobacco-specific nitrosamines | |
| CN112578069A (en) | Analysis method for simultaneously measuring 11 components in electronic smoke sol | |
| CN101793880B (en) | Method for measuring benzo(a)pyrene in total particle phase matters in cigarette smoke gas | |
| Zhang et al. | Fully automated analysis of four tobacco-specific N-nitrosamines in mainstream cigarette smoke using two-dimensional online solid phase extraction combined with liquid chromatography–tandem mass spectrometry | |
| CN107271584B (en) | Method for trapping carbonyl compounds and tobacco-specific nitrosamines in cigarette mainstream smoke, extraction method and determination method | |
| CN108303477B (en) | Method for detecting alcohols and other compounds in mainstream smoke of cigarette without burning during heating | |
| CN104698117A (en) | Simultaneous determination method for nicotine, 1,2-propylene glycol and glycerol in electronic cigarette smoke | |
| CN105675757A (en) | Method for determining tobacco-specific N-nitrosamine and polycyclic aromatic hydrocarbons in mainstream smoke of cigarettes simultaneously | |
| CN102353741A (en) | Method for measuring contents of four kinds of tobacco-specific nitrosamines in cigarette mainstream smoke | |
| CN109085270A (en) | Method that is a kind of while measuring a variety of tobacco-specific nitrosamines in human plasma | |
| CN104142374A (en) | Method for measuring content of carbonyl compound in electronic tobacco juice by adopting direct derivation/high performance liquid chromatography | |
| CN105527356B (en) | The peculiar N nitrosamine of tobacco and assay method while polycyclic aromatic hydrocarbon in a kind of cigarette mainstream flue gas based on suction nozzle micro-extraction | |
| CN101113972A (en) | Method for detecting cigarette filter tip entrapping ammine with ion chromatograph conductance | |
| CN111272938A (en) | Method for simultaneously measuring moisture, nicotine, propylene glycol and glycerol in electronic cigarette liquid | |
| CN106442753B (en) | A kind of method of TSNAs content in measurement cigarette mainstream flue gas | |
| CN111679008A (en) | GC-MS-MS method for simultaneously detecting volatile and semi-volatile acids, alcohols and phenols in tobacco leaves and cut tobacco | |
| CN108548880B (en) | Method for measuring various components in electronic cigarette smoke | |
| CN112505227A (en) | Analysis method for simultaneously determining 11 components in electronic cigarette liquid | |
| CN108931596A (en) | Method that is a kind of while detecting acetic acid and nicotine in cigarette mainstream flue gas | |
| Borras et al. | Characterisation of polycyclic aromatic hydrocarbons in atmospheric aerosols by gas chromatography–mass spectrometry | |
| CN109975443B (en) | Method for detecting aldehydes in cigarette filter | |
| CN107490642A (en) | Method that is a kind of while determining 12 kinds of alkaloids in cigarette smoke granule phase substance | |
| CN111983111B (en) | Method for measuring retention rate of cigarette filter stick on hydroxypyridine | |
| CN115201365A (en) | Method for detecting tobacco specific nitrosamine | |
| CN112881556A (en) | Method for determining volatile and semi-volatile organic acids in cigarette mainstream smoke |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |