CN104122357A

CN104122357A - Method for detecting specific nitrosamine (TSNAs) in tobacco under condition of Canada depth suction

Info

Publication number: CN104122357A
Application number: CN201410348978.7A
Authority: CN
Inventors: 张凤梅; 朱瑞芝; 司晓喜; 何沛; 张涛; 刘春波; 向能军; 申钦鹏; 王昆淼; 杨光宇; 刘志华
Original assignee: China Tobacco Yunnan Industrial Co Ltd
Current assignee: China Tobacco Yunnan Industrial Co Ltd
Priority date: 2014-07-22
Filing date: 2014-07-22
Publication date: 2014-10-29

Abstract

The invention provides a method for detecting specific nitrosamine (TSNAs) in tobacco by an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method under the condition of Canada depth suction, and belongs to a method for detecting specific TSNAs in tobacco. The method comprises the steps of adding an ammonium acetate aqueous solution including an internal standard substance to captured total particle matter of cigarette smoke generated after smoking under the condition of Canada depth suction, carrying out ultrasonic extraction, filtering an extract of 1-2 mL through an organic filtering membrane of 0.22 micron, analyzing TSNAs by an UPLC-MS/MS device, and quantifying according to the area ratio of a component peak and an isotope internal standard substance peak. The method provided by the invention can be used for detecting TSNAs in main smoke of flue-cured and blended type tobacco under the condition of Canada depth suction, and has the advantages that the pre-processing is simple, the quantification is exact, the instrument analysis speed is high, the detection limit is low, the sample processing throughput is high and the like.

Description

The detection method of tobacco-specific nitrosamine under a kind of Canadian degree of depth puffing regimens

Technical field

The invention belongs to technical field of analytical chemistry, relate to a kind of method for detecting tobacco-specific nitrosamines of simple efficient and sensible, be specifically related to the detection method of tobacco-specific nitrosamine under a kind of Canadian degree of depth puffing regimens.

Background technology

Tobacco-specific nitrosamine (TSNAs) is the abundantest class nitrosamine material of N-content of nitrosamines in tobacco, is only present in tobacco, tobacco product and cigarette smoke, and the content of TSNAs is significant to correct evaluation smoking healthhazard.IARC (IARC) shows the toxicity assessment result of objectionable constituent in Canadian government list, and 4-(N-methyl-N-nitrosamine)-1-(3-pyridine radicals)-butanone (NNK) and N-nitroso-nornicotine (NNN) pure chemistry composition are the carcinogenic composition of questionable person's body.In order to reduce tobacco-specific nitrosamine to smoker and the healthy possible injury of involuntary smoker, control their content in tobacco and tobacco product, become an important problem.Therefore, the TSNAs in Accurate Determining tobacco and tobacco product, the research and development that contribute to for correct evaluating smoking healthhazard, promote low hazard cigarette provide reliable foundation.The comparatively deep TSNAs of research is N-nitroso-nornicotine (NNN), N-nitroso-anabasine (NAB), N-nitroso-anatabine (NAT) and 4-(N-methyl-N-nitrosamine)-1-(3-pyridine radicals)-butanone (NNK) at present.

Vapor-phase chromatography is just applied to the analysis of tobacco-specific nitrosamine for 1973, the GC-TEA method that the TSNAs method of testing Main Basis GB/T23228-2008 using at present specifies, the method has specified the mensuration of tobacco-specific nitrosamine in cigarette mainstream smoke total particulate matter.Its principal feature is the TPM that each sample need to trap 20 cigarette, point 3 extractions, and the alkali alumina post of filling activation, the nearly 30min of each sample analysis time, every day, sample size can not exceed 8.Therefore, there is complex pretreatment, solvent-oil ratio is large, analysis time is long, TEA detecting device can not be distinguished TSNAs and is total to the problems such as the nitrosamine compound, the dead volume that flow out are large, detection limit is high, be unsuitable for the accurate test of batch samples.

Mao Youan etc. mention a kind of method of liquid chromatography-tandem mass spectrometry detection tobacco-specific nitrosamine in Chinese patent literature CN1616962A.The principal feature of the method is the TPM of 20 cigarette of trapping, uses trimethyl ¹³c ₃mark click coffee, because making interior mark, vibrates to be total to filter disc with citric acid-phosphate buffered solution and cyclohexane and extracts, N ₂the lower concentrated organic phase extract of protection, adds hcl acidifying, after being separated, adds aqueous slkali neutralization toward water, and water is crossed to inverse bonded solid-phase extraction column, finally concentrated.The method is because adopting trimethyl ¹³c ₃mark click coffee is because making interior mark, therefore not strong to the target compound selectivity detecting; Organic phase and water extraction solvent respectively need 30mL, and solvent consumption is large; Extract altogether with organic phase and water vibration, the formation of foam of back extraction process can cause loss and error; Hcl acidifying, NaOH neutralize, are separated, concentrate etc. and causes complex pretreatment; The nearly 30min of each sample analysis time, every day, sample size was restricted.

Test smoke delivery by machine drawn and start from the thirties in 20th century, Bradford in 1936 propose the suction capacity of 35mL/puff first.1969, tobacco and tobacco product ISO (International Standards Organization) under the proposal of DIN office (DIN), are set up, one of work that this tissue is carried out is the earliest exactly the suction parameter of suggestion use " suction capacity is 35mL, and the suction duration is 2s, and suction frequency is 1min ".

1988, ISO proposed to formulate the unified suction mode in the whole world, and entrusts CORESTA to carry out necessary research.CORESTA has successively carried out a series of comprehensive research, and has proposed the revision of ISO pattern in 1991.The widely used ISO4387:2000 in countries in the world is the revision version of ISO4387:1991 at present, it is the basis that each department and country formulate relevant policies and regulation, but also have indivedual countries and regions to use other suction mode, than Canadian suction mode, design parameter is in table 1.

Table 1 ISO and Canadian suction mode parameter

Pattern	Suction capacity (mL)	Duration (s)	Suction interval (s)	Air vent stops up (%)
					ISO	35	2	60	0
Canada	55	2	30	100

Although ISO suction mode does not stop up air vent, when consumer's smoking cigarette, may may cover air hole with finger and lip, thereby cause exhaust gas volumn to increase.

" not smoke delivery and mankind's exposed amount of the more different trade mark cigarette of energy effective ratio of existing iso standard of test cigarette burst size, can not be used for effective control " thought by tobacco product control group of the World Health Organization (WHO) (TobReg).

For tackling new situation, the invention provides the detection method of tobacco-specific nitrosamine under a kind of Canadian degree of depth puffing regimens.

Summary of the invention

The object of this invention is to provide the method for detecting tobacco-specific nitrosamines of a kind of efficient and sensible under Canadian degree of depth suction mode, the each sample of the method only need to trap the TPM of less cigarette, pre-treatment is simple, each sample analysis time is short, sample processing throughput is high, is applicable to batch samples and accurately tests.

To achieve these goals, the technical solution used in the present invention is as follows:

Method for detecting tobacco-specific nitrosamines under a kind of Canadian degree of depth puffing regimens, comprises the following steps:

Step (1), with the TPM in 2～3 sample cigarette mainstream flue gas of 1 cambridge filter collection;

Step (2), step (1) is obtained filter disc put into 50mL conical flask, add the ammonium acetate solution of 10～15mL containing interior target 0.1～0.2mol/L, then under the condition of 30～40 DEG C of temperature, power 30～40W, ultrasonic extraction 25～30min, leave standstill again 5～7min, be extracted liquid;

Step (3), the extract that step (2) is obtained is crossed organic filter membrane of 0.22 μ m, and filtrate enters Liquid Chromatography-tandem Mass instrument, selects ion scan to detect, Isotopically labelled internal standard.

Liquid phase chromatogram condition in step in technical scheme of the present invention (3) is as follows:

Chromatographic column: C18 post, 1.7 μ m, 2.1 × 100mm; Mobile phase A: methyl alcohol; Mobile phase B: 0.1～0.2% v/v aqueous formic acid; Flow velocity: 0.2～0.4mL/min; Column temperature: 50～60 DEG C; Sample size: 4～6 μ L;

Elution program is as shown in table 1:

Table 1

Time min	Mobile phase A %	Mobile phase B %
			0	10	90
2.5	100	0
			3	10	90

Mass spectrum condition in step in technical scheme of the present invention (3) is as follows:

Ionization mode: electron spray; Scan pattern: positive ion; Spray voltage: 4500～5500V; Ion source temperature: 550～650 DEG C; Gas1:50MPa; Gas2:70MPa; DP:35V.

The MS detection parameters in step in technical scheme of the present invention (3) is as shown in table 2:

Table 2

The type of the sample cigarette described in technical solution of the present invention is fire-cured tobacco type or mixed type.

Sample cigarette described in technical solution of the present invention is by national relevant industries standard, processes through balance 48h under 22 DEG C ± 1 DEG C of temperature and relative humidity 60 ± 2% conditions.

This method is applicable to detect the TSNAs in cigarette mainstream smoke total particulate matter under Canadian degree of depth puffing regimens.

This method is under Canadian degree of depth puffing regimens after smoking cigarette, in the filter disc of trapping cigarette smoke TPM, add the ammonium acetate solution containing internal standard compound, after ultrasonic extraction, get 1～2mL extract and cross organic filter membrane of 0.22 micron, with UPLC-MS/MS instrument analysis TSNAs, quantitative according to the Area Ratio at component peaks and Isotopic Internal Standard thing peak.

The present invention compared with prior art, its beneficial effect is: under the present invention Canada degree of depth puffing regimens, method for detecting tobacco-specific nitrosamines can detect the TSNAs in fire-cured tobacco type and blended type cigarette main flume, each sample only need to trap the TPM of 2～3 cigarette, pre-treatment is simple, only need direct injected after ultrasonic extraction), detection limit is low, wherein, NNN, NNK, the detection limit of NAT and NAB is respectively 0.06ng/cig, 0.01ng/cig, 0.02ng/cig and 0.03ng/cig, each sample analysis time is short, be only 3min, sample processing throughput is high, every day, sample size exceeded 200, so being applicable to batch samples accurately tests.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.

Embodiment 1

Step (1), by national relevant industries standard, makes fire-cured tobacco type sample cigarette balance 48h under 21 DEG C of temperature and relative humidity 58% condition, with the TPM in 2 cigarette mainstream flue gas of 1 cambridge filter collection;

Step (2), step (1) is obtained filter disc put into 50mL conical flask, add 10mL to contain the ammonium acetate solution of interior target 0.1mol/L, then under the condition of 30 DEG C of temperature, power 30W, ultrasonic extraction 25min, then leave standstill 5min, be extracted liquid;

Step (3), the extract that step (2) is obtained is got 1mL and crosses organic filter membrane of 0.22 μ m, and filtrate enters Liquid Chromatography-tandem Mass instrument, selects ion scan to detect, Isotopically labelled internal standard.

Wherein, the liquid phase chromatogram condition in step (3) is as follows:

Chromatographic column: C18 post, 1.7 μ m, 2.1 × 100mm; Mobile phase A: methyl alcohol; Mobile phase B: 0.1% v/v aqueous formic acid; Flow velocity: 0.2mL/min; Column temperature: 50 DEG C; Sample size: 4 μ L;

Elution program is as shown in table 3:

Table 3

Time min	Mobile phase A %	Mobile phase B %
			0	10	90
2.5	100	0

Wherein, the mass spectrum condition in step (3) is as follows:

Ionization mode: electron spray; Scan pattern: positive ion; Spray voltage: 4500V; Ion source temperature: 550 DEG C; Gas1:50MPa; Gas2:70MPa; DP:35V.

The MS detection parameters in step (3) is as shown in table 4:

Table 4

According to the method for the above embodiment of the present invention, replication three times.The TSNAs content recording in fire-cured tobacco type sample cigarette mainstream smoke total particulate matter is respectively: NNN14.25ng/cig, NAT29.28ng/cig, NAB2.86ng/cig, NNK12.10ng/cig.

Embodiment 2

Step (1), by national relevant industries standard, makes mixed type sample cigarette balance 48h under 22 DEG C DEG C of temperature and relative humidity 60% condition, with the TPM in 2 cigarette mainstream flue gas of 1 cambridge filter collection;

Step (2), step (1) is obtained filter disc put into 50mL conical flask, add 12mL to contain the ammonium acetate solution of interior target 0.15mol/L, then under the condition of 35 DEG C of temperature, power 36W, ultrasonic extraction 28min, then leave standstill 6min, be extracted liquid;

Step (3), the extract that step (2) is obtained is got 2mL and crosses organic filter membrane of 0.22 μ m, and filtrate enters Liquid Chromatography-tandem Mass instrument, selects ion scan to detect, Isotopically labelled internal standard.

Wherein, the liquid phase chromatogram condition in step (3) is as follows:

Chromatographic column: C18 post, 1.7 μ m, 2.1 × 100mm; Mobile phase A: methyl alcohol; Mobile phase B: 0.17% v/v aqueous formic acid; Flow velocity: 0.3mL/min; Column temperature: 56 DEG C; Sample size: 5 μ L;

Elution program is as shown in table 5:

Table 5

Time min	Mobile phase A %	Mobile phase B %
			0	10	90
2.5	100	0
			3	10	90

Wherein, the mass spectrum condition in step (3) is as follows:

Ionization mode: electron spray; Scan pattern: positive ion; Spray voltage: 5000V; Ion source temperature: 600 DEG C; Gas1:50MPa; Gas2:70MPa; DP:35V.

The MS detection parameters in step (3) is as shown in table 6:

Table 6

According to the method for the above embodiment of the present invention, replication three times.The TSNAs content recording in mixed type sample cigarette mainstream smoke total particulate matter is respectively: NNN478.01ng/cig, NAT383.10ng/cig, NAB34.95ng/cig, NNK47.35ng/cig.

Embodiment 3

Step (1), by national relevant industries standard, makes fire-cured tobacco type sample balance 48h under 23 DEG C of temperature and relative humidity 62% condition, with the TPM in 3 cigarette mainstream flue gas of 1 cambridge filter collection;

Step (2), step (1) is obtained filter disc put into 50mL conical flask, add 15mL to contain the ammonium acetate solution of interior target 0.2mol/L, then under the condition of 40 DEG C of temperature, power 40W, ultrasonic extraction 30min, then leave standstill 7min, be extracted liquid;

Step (3), the extract that step (2) is obtained is got 1.4mL and crosses organic filter membrane of 0.22 μ m, and filtrate enters Liquid Chromatography-tandem Mass instrument, selects ion scan to detect, Isotopically labelled internal standard.

Wherein, the liquid phase chromatogram condition in step (3) is as follows:

Chromatographic column: C18 post, 1.7 μ m, 2.1 × 100mm; Mobile phase A: methyl alcohol; Mobile phase B: 0.2% v/v aqueous formic acid; Flow velocity: 0.4mL/min; Column temperature: 60 DEG C; Sample size: 6 μ L;

Elution program is as shown in table 7:

Table 7

Time min

Mobile phase A %

Mobile phase B %

0	10	90
			2.5	100	0
3	10	90

Wherein, the mass spectrum condition in step (3) is as follows:

Ionization mode: electron spray; Scan pattern: positive ion; Spray voltage: 5500V; Ion source temperature: 650 DEG C; Gas1:50MPa; Gas2:70MPa; DP:35V.

The MS detection parameters in step (3) is as shown in table 8:

Table 8

According to the method for the above embodiment of the present invention, replication three times.The TSNAs content recording in fire-cured tobacco type sample cigarette mainstream smoke total particulate matter is respectively: NNN49.25ng/cig, NAT52.00ng/cig, NAB6.45ng/cig, NNK18.38ng/cig.

Claims

1. a method for detecting tobacco-specific nitrosamines under Canadian degree of depth puffing regimens, is characterized in that comprising the following steps:

Step (1), with 2 ~ 3 sample cigarette mainstream smoke total particulate matters of 1 cambridge filter collection;

Step (2), step (1) is obtained filter disc put into 50mL conical flask, add 10 ~ 15mL containing the ammonium acetate solution of interior target 0.1 ~ 0.2mol/L, then under the condition of 30 ~ 40 DEG C of temperature, power 30 ~ 40W, ultrasonic extraction 25 ~ 30min, then leave standstill 5 ~ 7min, be extracted liquid;

2. method for detecting tobacco-specific nitrosamines under Canadian degree of depth puffing regimens according to claim 1, is characterized in that the liquid phase chromatogram condition in step (3) is as follows:

Chromatographic column: C18 post, 1.7 μ m, 2.1 × 100mm; Mobile phase A: methyl alcohol; Mobile phase B: 0.1 ~ 0.2% v/v aqueous formic acid; Flow velocity: 0.2 ~ 0.4mL/min; Column temperature: 50 ~ 60 ° of C; Sample size: 4 ~ 6 μ L;

Elution program is as follows:

Method for detecting tobacco-specific nitrosamines under Canadian degree of depth puffing regimens according to claim 1, is characterized in that the mass spectrum condition in step (3) is as follows:

Ionization mode: electron spray; Scan pattern: positive ion; Spray voltage: 4500 ~ 5500V; Ion source temperature: 550 ~ 650 DEG C; Gas1:50MPa; Gas2:70MPa; DP:35V.

3. method for detecting tobacco-specific nitrosamines according to claim 1, is characterized in that the MS detection parameters in step (3) is as follows:

Method for detecting tobacco-specific nitrosamines under Canadian degree of depth puffing regimens according to claim 1, is characterized in that: the type of described sample cigarette is fire-cured tobacco type or mixed type.

4. method for detecting tobacco-specific nitrosamines under Canadian degree of depth puffing regimens according to claim 1, is characterized in that: described sample cigarette is processed through balance 48h under 22 DEG C ± 1 DEG C of temperature and relative humidity 60 ± 2% conditions.