Determination of TSNAs in flue gas based on one-step cleanup and liquid chromatography-tandem mass spectrometry

Abstract

The method for determining TSNAs in flue gas based on one-step purification and liquid chromatography-tandem mass spectrometry comprises the following steps: using Cambridge filter discs to collect the particulate matter in the mainstream smoke of cigarettes, transferring it to a centrifuge tube, adding internal standard mixed solution and water at the same time, and vortexing and extracting at room temperature; centrifuging, transferring the extract to a centrifuge tube, adding dichloromethane at the same time, vortexing and centrifuging again, taking the lower layer of dichloromethane clear liquid to another centrifuge tube, transferring the centrifuge tube to a water bath to evaporate the dichloromethane; adding water to re-dissolve the extract and transferring the solution to an injection vial; and then performing chromatography-tandem mass spectrometry analysis. The present invention develops a simple method for analyzing TSNAs in mainstream cigarette smoke. The method uses water to extract TSNAs in the particulate matter in the smoke, and uses a one-step liquid-liquid extraction method to purify the water extract. The method achieves better purification effect and better analytical sensitivity, is simple to operate, has good purification effect, and is widely applicable.

Description

Technical Field

[0001] The invention belongs to the field of tobacco chemistry, and specifically relates to a method for determining TSNAs in particulate matter in mainstream cigarette smoke. Background Art

[0002] It is well known that tobacco and tobacco products pose health risks. Many carcinogens and mutagens have been identified in tobacco products. Among them, tobacco-specific nitrosamines (TSNAs) are the most concerning potential carcinogens in tobacco. TSNAs are widely present in tobacco products such as cigarettes, cigars, and snus. Among them, NNK and NNN have been defined as Class I carcinogens by the International Agency for Research on Cancer (IARC), while NAB and NAT have been defined as Class III carcinogens.

[0003] Since TSNAs are directly related to health, the analysis of TSNAs in tobacco and tobacco products has always attracted much attention. In 1974, Hoffmann et al. first reported the use of gas chromatography to analyze the NNN content in cigarette smoke. Subsequently, methods such as gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-tandem mass spectrometry were reported one after another. The method of connecting thermal energy analyzer (TEA) with gas chromatography or liquid chromatography was used to identify and quantitatively analyze TSNAs. However, this method was gradually eliminated due to its inability to distinguish different nitrosamine substances and high detection limit. Liquid chromatography-tandem mass spectrometry has a lower detection limit, better specificity and a wider linear dynamic range than gas chromatography-thermal energy analyzer. At present, the method based on ammonium acetate aqueous solution extraction and liquid chromatography-tandem mass spectrometry has become the CORESTA recommended method and international standard method. However, this method also has certain defects. First, this method often fails to detect low-content compounds such as NAB when analyzing flue-cured tobacco smoke. Secondly, since the extract is directly subjected to liquid chromatography-tandem mass spectrometry analysis without purification, the ion source and chromatographic column of the mass spectrometer are easily contaminated, resulting in the drift of the chromatographic retention time and the attenuation of the mass spectrometric response during batch sample analysis. Therefore, the CORESTA recommended method and the international standard method both recommend that the extract be subjected to solid phase extraction purification before liquid chromatography-tandem mass spectrometry analysis to reduce contamination. However, solid phase extraction purification involves multiple steps such as extraction column activation, sample loading, cleaning, target elution, and the preparation and use of various reagents. The whole process is relatively lengthy, which seriously affects the analysis throughput and data stability. Liu Xin et al. reported a method for purifying TSNAs in samples using online solid phase extraction. Compared with manual solid phase extraction, this method saves time and manpower, and also reduces the various uncertainties of manual solid phase extraction. However, this method requires the use of dedicated online solid phase extraction equipment, and it is also necessary to fill in dedicated solid phase extraction columns and equip them with relevant reagents, which makes the method difficult to popularize. Summary of the invention

[0004] In order to solve the problems of the prior art that low-content compounds are often unable to be detected, the process is lengthy, the analysis throughput is low, the data is unstable, the cost is high, the operation is inconvenient and it is not easy to popularize, the present invention provides a method for determining TSNAs in the particulate matter of mainstream cigarette smoke based on one-step purification and liquid chromatography-tandem mass spectrometry.

[0005] The technical solution of the present invention is as follows:

[0006] The method for determining TSNAs in flue gas based on one-step purification and liquid chromatography-tandem mass spectrometry is characterized by comprising the following steps:

[0007] The particulate matter in the mainstream smoke of cigarettes was collected using Cambridge filters and transferred to a centrifuge tube. An internal standard mixture and water were added and vortexed for extraction at room temperature. The extract was centrifuged and transferred to a centrifuge tube. Dichloromethane was added and vortexed for centrifugation again. The lower layer of dichloromethane clear liquid was transferred to another centrifuge tube. The centrifuge tube was transferred to a water bath and the dichloromethane was evaporated. Water was added to re-dissolve the extract and the solution was transferred to an injection vial. Then, chromatography-tandem mass spectrometry analysis was performed.

[0008] Preferably, the standard solution preparation process is as follows:

[0009] Weigh 5.0 mg each of NNN, NNK, NAT, NAB and d4-NNN, d4-NNK, d4-NAT, and d4-NAB into their respective 10 mL volumetric flasks, make up to volume with methanol to prepare 0.5 mg / mL single standard stock solutions; take 2 mL of NNN, NNK, NAT, and NAB single standard stock solutions into the same 10 mL volumetric flask, make up to volume with water to prepare 100 100 μg / mL mixed standard solutions; take 2 mL of d4-NNN, d4-NNK, d4-NAT, and d4-NAB single standard stock solutions into the same 100 mL volumetric flask, make up to volume with water to prepare 10 μg / mL internal standard mixed solutions.

[0010] Further preferably, according to the national standard GB / T 19609-2004, a 92mm Cambridge filter is used to collect the mainstream smoke particulate matter of 20 cigarettes; the captured smoke particulate matter is transferred to a 50mL centrifuge tube, and 1mL of 0.1μg / mL internal standard mixture and 29mL of water are added at the same time; vortex extraction is performed at 2500rpm at room temperature for 10min; centrifugation is performed, 10mL of the extract is transferred to a 15mL centrifuge tube, and 2mL of dichloromethane is added at the same time; vortexing for 1min and then centrifuging for 1min, 500μL of the lower dichloromethane clear liquid is taken to a new 1.5mL centrifuge tube; the centrifuge tube is transferred to a 40℃ water bath for about 5min to evaporate the dichloromethane; 300μL of water-reconstituted extract is added and the solution is transferred to a 2mL injection vial with an inner liner.

[0011] Further preferably, the chromatographic conditions are: separation using 2.1mm×150mm×1.8μm Waters HSS T3; mobile phase A is water with 0.8% formic acid added, and B is methanol; gradient elution program: 0-1min, mobile phase B is maintained at 10%; 1-8min, B increases from 10% to 30%; 8-8.01min, mobile phase B is switched to 95%; 8.01-9min, mobile phase B is maintained at 95%; 9-9.01, mobile phase B is switched to 10%; 9.01-10min, mobile phase B is maintained at 10%; column temperature: 30°C, injection volume: 2μL, flow rate: 0.25mL / min. .

[0012] Further preferably, the mass spectrometry conditions are: electrospray ionization ion source; positive ion scanning mode; spray voltage 5500V; curtain gas pressure 35Psi; ion source temperature 650°C; auxiliary gas 1 pressure 55psi; auxiliary gas 2 pressure 60Psi; declustering voltage 50V.

[0013] The beneficial technical effects of the present invention are as follows:

[0014] The present invention develops a simple method for analyzing TSNAs in mainstream cigarette smoke. The method uses water to extract TSNAs in the particulate matter of smoke, and uses a one-step liquid-liquid extraction method to purify the water extract. The purified sample can be directly subjected to liquid chromatography-tandem mass spectrometry analysis after solvent replacement. Extraction with water can avoid the extraction of fat-soluble compounds in smoke, and liquid-liquid extraction and purification of the extract with dichloromethane can remove water-soluble impurities in the water extract. The method achieves better purification effect and better analytical sensitivity than direct sampling after ammonium acetate solution extraction or ammonium acetate solution extraction-solid phase extraction purification. The method is simple to operate, has good purification effect, and is widely applicable. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Comparison of ultrasonic and vortex extraction effects at different extraction times;

[0016] Figure 2 The chromatograms of flue gas sample analysis in Example 1 are compared with those in Prior Art 1 and Prior Art 2. DETAILED DESCRIPTION

[0017] The present invention will be further described in detail below through specific embodiments, but it is not intended to limit the technical solution of the present invention. All changes or equivalent substitutions made based on the present invention should fall within the protection scope of the present invention.

[0018] Example 1

[0019] This example is a method for determining TSNAs in mainstream cigarette smoke based on one-step purification and liquid chromatography-tandem mass spectrometry.

[0020] Sample preparation:

[0021] According to the national standard GB / T 19609-2004, 20 ordinary cigarette mainstream smoke particulates were collected using a 92mm Cambridge filter. The captured smoke particulates were transferred to a 50mL centrifuge tube, and 1mL of internal standard mixture (0.1μg / mL) and 29mL of water were added. Vortex (speed 2500rpm) and extract for 10min at room temperature. Centrifuge, transfer 10mL of the extract to a 15mL centrifuge tube, and add 2mL of dichloromethane. Vortex for 1min and centrifuge for another 1min, take 500μL of the lower layer (dichloromethane) and transfer it to a new 1.5mL centrifuge tube. Transfer the centrifuge tube to a 40℃ water bath for about 5min to evaporate the dichloromethane. Add 300μL of water to reconstitute the extract and transfer the solution to a 2mL injection vial with an inner liner.

[0022] Instrumental analysis:

[0023] Equipment, reagents, standards and solutions:

[0024] Ultra-high performance liquid chromatography (UPLC) instrument from Waters, USA, AB SCIEX 5500 triple quadrupole mass spectrometer from AB, USA. Milli-Q water purifier from Millipore, USA, SM 100 sample grinder from Lach, Germany, multi-tube vortex vortexer from Talboys, USA, Eppendorf 5804 high-speed centrifuge from Germany, Sartorius analytical balance from Germany, sensitivity 0.0001g, RM20H turntable smoking machine from Borgwaldt, Germany.

[0025] Acetonitrile and methanol were purchased from Fisher scientific (Fair Lawn, NJ, USA). Formic acid (chromatographic grade) and dichloromethane (chromatographic grade) were purchased from J&K Reagents. Ultrapure water (18.2 MΩ cm) was produced by Millipore Mili-Q system. NNK, NNN, NAB, NAT standards and related deuterated internal standard compounds (d4-NNK, d4-NNN, d4-NAB, d4-NAT) were purchased from Toronto Research Chemicals, Inc., Canada (North York, ON, Canada). Glass fiber filter (92 mm Cambridge filter) was purchased from Borgwaldt KC (Hamburg, Germany).

[0026] Preparation of standard solution: Weigh 5.0 mg of each of NNN, NNK, NAT, NAB and d4-NNN, d4-NNK, d4-NAT, and d4-NAB into their respective 10 mL volumetric flasks, dilute to volume with methanol, and prepare 0.5 mg / mL single standard stock solution. Take 2 mL of NNN, NNK, NAT, and NAB single standard stock solutions into the same 10 mL volumetric flask, dilute to volume with water, and prepare 100 μg / mL mixed standard solution. Take 2 mL of d4-NNN, d4-NNK, d4-NAT, and d4-NAB single standard stock solutions into the same 100 mL volumetric flask, dilute to volume with water, and prepare 10 μg / mL internal standard mixed solution.

[0027] Preparation of standard curve solution: The concentration range of standard curve for all target TSNAs was 1-100 ng / mL, and the concentration of internal standard substance in all standard curve dilution points was 10 ng / mL.

[0028] Chromatographic conditions: Flue gas analysis was separated by Waters HSS T3 (2.1 mm × 150 mm × 1.8 μm); mobile phase A was water (added with 0.8% formic acid), and mobile phase B was methanol. Gradient elution program: 0-1 min, mobile phase B was maintained at 10%; 1-8 min, B increased from 10% to 30%; 8-8.01 min, mobile phase B was switched to 95%; 8.01-9 min, mobile phase B was maintained at 95%; 9-9.01, mobile phase B was switched to 10%; 9.01-10 min, mobile phase B was maintained at 10%. Column temperature: 30 ° C. Injection volume: 2 μL. Flow rate: 0.25 mL / min.

[0029] Mass spectrometry conditions: electrospray ionization source; positive ion scan mode; spray voltage 5500 V; curtain gas pressure 35 Psi; ion source temperature 650 ° C; auxiliary gas 1 pressure 55 psi; auxiliary gas 2 pressure 60 Psi; declustering voltage 50 V. The parent ions and fragment ions of TSNAs and their deuterated internal standards are shown in Table 1.

[0030] Table 1 TSNAs analysis mass spectrometry ion pairs

[0031]

[0032]

[0033] Compared with existing methods:

[0034] The most widely used method for analyzing TSNAs in smoke is to extract TSNAs in cigarette smoke with a 0.1 mol / L ammonium acetate aqueous solution and directly perform LC-MS / MS analysis on the extract (prior art 1) or perform LC-MS / MS analysis after solid phase extraction (prior art 2). It has now become the latest CORESTA recommended method and ISO standard method. The analysis method established in this example (water extraction + dichloromethane purification + LC-MS / MS analysis) was compared with the above method for analyzing flue-cured tobacco samples. The results showed that for flue-cured tobacco smoke samples, compared with the above method, the analysis method established in this example had purer chromatographic peaks and higher response of target compounds ( Figure 2 ). For flue-cured tobacco smoke samples, the chromatographic peaks of NAB in the methods of prior art 1 and prior art 2 are no longer identifiable, the chromatographic peak of NNN in the method of document A is very weak, and NNN is invisible in the method of document B. Compared with the existing methods, this experimental method has significant improvements in impurity interference and response intensity, and the four target compounds can be clearly identified and integrated.

[0035] In addition, from Figure 2 It can also be seen that there is no significant difference in the chromatographic peaks obtained by extraction with water and extraction with 0.1 mol / L ammonium acetate. After extraction with water and then liquid-liquid extraction and purification with dichloromethane, the chromatographic peak response intensities of the four target compounds are significantly improved. The reason is that the liquid-liquid extraction removes a large amount of matrix that inhibits the mass spectrometry response of TSNAs.

[0036] Selection of extraction time and extraction method:

[0037] The experiment compared the extraction effects of ultrasonic-assisted extraction and high-speed vortex oscillation extraction on flue gas TSNAs at different extraction times. Figure 1 The results showed that there was no significant difference in the TSNAs content obtained by the two extraction methods. From the 5th minute onwards, the extraction time also had no significant effect on the extraction effect of TSNAs. Finally, this experiment chose vortex oscillation extraction, and the extraction time was selected as 10 minutes.

[0038] Optimization of chromatographic conditions:

[0039] The experiment compared the separation effect and sensitivity of various types of chromatographic columns of 10cm and 15cm. The results showed that C18 and T3 columns have better separation and sensitivity than phenyl columns, amine columns, HILIC columns, etc. In the flue gas sample, the content of TSNAs is reduced due to combustion and pyrolysis, and the sample matrix becomes more complex. At this time, the separation effect of the 15cm long T3 column is better ( Figure 2). It is worth noting that the use of a longer chromatographic column will cause a certain separation of the cis and trans isomers of NNN, resulting in a bifurcated NNN peak. Adding 0.8% formic acid to the aqueous phase can reduce the bifurcation of the NNN chromatographic peak, but it cannot be completely avoided.

[0040] Method evaluation:

[0041] The accuracy of the method was evaluated by spike recovery. It was found that the recovery rate of TSNAs in flue gas was between 95.7% and 107.7%, indicating that the method has good accuracy. The precision of the method was evaluated by the repeatability of flue-cured tobacco sample analysis. It was found that the intra-batch repeatability in flue gas samples was between 3.2% and 4.3%, and the inter-batch repeatability was between 4.1% and 6.1%, indicating that the method has good analytical precision. The stepwise diluted d4-NNN, d4-NNK, d4-NAT, and d4-NAB were added to the flue gas extract, and the established chromatography-mass spectrometry analysis method was used for analysis, with 3 times the signal-to-noise ratio and 10 times the signal-to-noise ratio of the chromatographic peak as the detection limit and quantification limit of the target. It was found that the detection limit in the flue gas sample was between 0.1-0.3ng / stick, and the quantification limit was between 0.2-0.6ng / stick. The method quantification limit was lower than the content of the corresponding TSNAs in the flue-cured tobacco samples, indicating that the TSNAs in the flue-cured tobacco samples can be measured. The linear response of TSNAs on LC-MS / MS was also evaluated and it was found that the first-order linear response factors (r 2 ) were all greater than 0.99, indicating that the method had a good linear response to the measured TSNAs.

[0042] Table 2 Recovery of TSNAs in the particulate matter of mainstream cigarette smoke

[0043]

[0044] Note: The spike recovery in smoke is added and calculated based on the content measured in each filter of the flue-cured tobacco cigarette sample (mainstream smoke particulate matter of 20 cigarettes).

[0045] Note: Intra-batch repeatability is the relative standard deviation calculated by processing 5 copies of the same sample at one time and analyzing them continuously. Inter-batch repeatability is the relative standard deviation calculated by processing and analyzing 5 copies of the same sample daily for 3 consecutive days.

[0046] Table 3 Repeatability of TSNAs analysis in particulate matter in mainstream cigarette smoke

[0047]

[0048] Note: Intra-batch repeatability is the relative standard deviation calculated by processing 5 copies of the same sample at one time and analyzing them continuously. Inter-batch repeatability is the relative standard deviation calculated by processing and analyzing 5 copies of the same sample daily for 3 consecutive days.

[0049] Table 4 Detection limit and quantification limit of TSNAs in flue gas

[0050]

[0051] Note: The detection limit and quantification limit are the concentrations of d4-NNN, d4-NNK, d4-NAT, and d4-NAB at 3-fold and 10-fold signal-to-noise ratios obtained by serial dilution in the sample matrix.

[0052] Example 2

[0053] Cinnabar tobacco is a mutant strain of flue-cured tobacco, most of its nicotine is converted into demethylnicotine. Since TSNAs are nitrosylated products of tobacco alkaloids, changes in the alkaloid form may change the content of TSNAs in its smoke. The method of the present invention is used to analyze TSNAs in the smoke of Cinnabar tobacco and normal flue-cured tobacco.

[0054] According to Table 5, in the mainstream smoke samples, it was found that the NNN content of cinnabar smoke was significantly higher than that of normal flue-cured tobacco, the contents of NNK, NAT, and NAB were significantly reduced, and there was no significant difference in the total content of TSNAs. The smoke data showed that the morphology of TSNAs in cinnabar smoke changed significantly, but the total amount did not change significantly. The experimental results also show that the TSNAs analysis method in smoke established by the present invention can be well adapted to the analysis of flue-cured tobacco samples.

[0055] Table 5 Distribution of TSNAs content in mainstream smoke of normal flue-cured tobacco and cinnabar tobacco

[0056]

[0057]

[0058] In summary, the present invention utilizes the characteristics that water can well release TSNAs from flue gas, while dichloromethane can dissolve TSNAs better than water, and develops a TSNAs analysis method based on one-step dichloromethane purification and liquid chromatography-mass spectrometry detection. The method simultaneously avoids the influence of fat-soluble and water-soluble interferences on TSNAs, solves the matrix inhibition problem during TSNAs analysis and the operation complexity problem based on solid phase extraction purification, and has positive significance for improving the sensitivity, accuracy, and analysis throughput of TSNAs detection methods based on LC-MS / MS.

Claims

1. A method for determining TSNAs in flue gas based on one-step purification and liquid chromatography-tandem mass spectrometry, characterized in that The following steps are involved: The particulate matter in the mainstream smoke of cigarettes was collected using Cambridge filter discs, transferred to centrifuge tubes, and internal standard mixed solution and water were added at the same time, and vortexed for extraction at room temperature; centrifuged, the extract was transferred to a centrifuge tube, and dichloromethane was added at the same time, vortexed and centrifuged again, the lower layer of dichloromethane clear liquid was taken to another centrifuge tube, and the centrifuge tube was transferred to a water bath to evaporate the dichloromethane; water was added to re-dissolve the extract and the resulting solution was transferred to a sample injection vial; and then liquid chromatography-tandem mass spectrometry analysis was performed. Among them, TSNAs analyzes the mass spectrometry ion pairs: 。 2. The method according to claim 1, characterized in that The standard solution preparation process is as follows: Weigh 5.0 mg each of NNN, NNK, NAT, NAB and d4-NNN, d4-NNK, d4-NAT, and d4-NAB into their respective 10 mL volumetric flasks, make up to volume with methanol to prepare 0.5 mg / mL single standard stock solutions; take 2 mL of NNN, NNK, NAT, and NAB single standard stock solutions into the same 10 mL volumetric flask, make up to volume with water to prepare 100 μg / mL mixed standard solutions; take 2 mL of d4-NNN, d4-NNK, d4-NAT, and d4-NAB single standard stock solutions into the same 100 mL volumetric flask, make up to volume with water to prepare 10 μg / mL internal standard mixed solutions.

3. The method according to claim 2, characterized in that According to the national standard GB / T 19609-2004, a 92 mm Cambridge filter was used to collect the mainstream smoke particulate matter of 20 cigarettes; the captured smoke particulate matter was transferred to a 50 mL centrifuge tube, and 1 mL of 0.1 μg / mL internal standard mixture and 29 mL of water were added; vortex extraction was performed at 2500 rpm at room temperature for 10 min; centrifugation was performed, and 10 mL of the extract was transferred to a 15 mL centrifuge tube, and 2 mL of dichloromethane was added; vortexing was performed for 1 min and then centrifuged for another 1 min, and 500 μL of the lower dichloromethane clear solution was taken to a new 1.5 mL centrifuge tube; the centrifuge tube was transferred to a 40°C water bath for 5 min to evaporate the dichloromethane; 300 μL of water was added to reconstitute and the resulting solution was transferred to a 2 mL injection vial with an inner liner.

4. The method according to claim 3, characterized in that Chromatographic conditions were as follows: separation was performed using Waters HSS T3 (2.1 mm × 150 mm × 1.8 μm); mobile phase A was water with 0.8% formic acid added, and mobile phase B was methanol; gradient elution program: 0-1 min, mobile phase B was maintained at 10%; 1-8 min, B increased from 10% to 30%; 8-8.01 min, mobile phase B was switched to 95%; 8.01-9 min, mobile phase B was maintained at 95%; 9-9.01 min, mobile phase B was switched to 10%; 9.01-10 min, mobile phase B was maintained at 10%; column temperature: 30 °C, injection volume: 2 μL, flow rate: 0.25 mL / min.

5. The method according to claim 3, characterized in that The mass spectrometry conditions were as follows: electrospray ionization source; positive ion scan mode; spray voltage 5500V; curtain gas pressure 35 Psi; ion source temperature 650 °C; auxiliary gas 1 pressure 55 psi; Auxiliary gas 2 pressure 60 Psi; declustering voltage 50V.

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