CN105445406A - Two-dimensional liquid-gas chromatographic analysis apparatus and analysis method for determining contents of tobacco-specific nitrosamines - Google Patents
Two-dimensional liquid-gas chromatographic analysis apparatus and analysis method for determining contents of tobacco-specific nitrosamines Download PDFInfo
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Abstract
The invention relates to a two-dimensional liquid-gas chromatographic analysis apparatus for determining the contents of tobacco-specific nitrosamines. The two-dimensional liquid-gas chromatographic analysis apparatus comprises a liquid chromatographic analysis apparatus (16) and a gas chromatographic analysis apparatus (8) located at the lower stream of the liquid chromatographic analysis apparatus (16); the gas chromatographic analysis apparatus (8) comprises a PTV sample introduction device (1), a hollow liner tube (2), a pre-separation chromatographic column (3) and a separation chromatographic column (4) connected in sequence, wherein a solvent discharge valve (6) is arranged between the pre-separation chromatographic column (3) and the separation chromatographic column (4); a chromatographic column used in the liquid chromatographic analysis apparatus (16) is an alumina column (162); and outlets, for outflow fractions obtained after on-line cutting and containing to-be-determined components, of both the PTV sample introduction device (1) and the liquid chromatographic analysis apparatus (16) are communicated with each other. The invention also relates to an analysis method for determining the contents of tobacco-specific nitrosamines and application of the analysis apparatus and the analysis method in analysis of urine of smokers.
Description
Technical field
The invention belongs to chemical analysis instrument technical field, being specifically related to a kind of liquid-gas phase Two way chromatograms analytical equipment for measuring tobacco-specific nitrosamine content and analytical approach and they application in smoker's urinalysis.
Background technology
Tobacco-specific nitrosamine (TSNAs) is the objectionable constituent that in cigarette smoke, a class is important, the TSNAs reported at present has 8 kinds, wherein comparatively deep to the research of N-nitrosonornicotine (NNN), 4-(methyl nitrosamino group)-1-(3-pyridine radicals)-1-butanone (NNK), N-nitrosoanatabine (NAT) and N-nitroso-anabasine (NAB) 4 kinds.The research of forefathers shows: NNN, NNK have 1 grade of carcinogenic activity, and NAB, NAT have 3 grades of carcinogenic activities.In cigarette smoking process, TSNAs likely can enter human body with cigarette smoke, thus the health of smoker is impacted, the tobacco-specific nitrosamine content therefore in Accurate Determining smoker urine is significant with healthy relation for understanding smoking further in depth.
Current commercial gas chromatographicanalyzer device is for storing the normally kapillary of testing sample after injection port, and its capacity is less, thus limits the volume entering testing sample in gas chromatography.For the testing sample that some component concentrations to be measured are low, often need through the pre-treatment step such as concentrated, transfer, constant volume, sample preparation Manual operation link is loaded down with trivial details and the reappearance of measurement result can be caused poor.During current gas Chromatographic Determination tobacco-specific nitrosamine, sample pre-treatments adopts Solid-Phase Extraction or column chromatography for separation purification, these methods purification sample needs concentrated, transfer repeatedly, not only complex operation, but also can because of with an organic solvent contaminated environment a large amount of in open environment, large to the health hazard of experiment operator.Therefore need very urgently to find easier, sample-pretreating method fast.
Online liquid-gas phase Two way chromatograms is the new analytical technology grown up in recent years, and sample is first through liquid chromatography isolation of purified in the art, then the outflow cut cutting on line containing component to be measured is entered gas chromatography analysis.The on-line coupling of liquid chromatography and gas chromatography can give full play to the complementarity of two-stage chromatogram, the Gao Zhuxiao of liquid chromatography substantially improves the clean-up effect of sample on the one hand, being separated of trace composition to be measured and interfering material in complicated substrate can be realized, between Two way chromatograms, online switching also can reduce the concentrated and transfer in sample pretreatment process on the other hand, decrease artificial analytical error, make precision of analysis, precision improves greatly.
This area is in the urgent need to a kind of liquid-gas phase Two way chromatograms analytical equipment for measuring tobacco-specific nitrosamine content and analytical approach.
Summary of the invention
The first object of the present invention is to provide a kind of liquid-gas phase Two way chromatograms analytical equipment for measuring tobacco-specific nitrosamine content and analytical approach; Second object is to provide above-mentioned analytical equipment and the application of analytical approach in smoker's urinalysis.
First aspect present invention relates to a kind of liquid-gas phase Two way chromatograms analytical equipment, it gas chromatographic analysis device 8 comprising liquid chromatography system 16 and be positioned at its downstream;
Wherein said gas chromatographic analysis device 8 comprise be linked in sequence PTV sampling device 1, for storing the hollow bushing pipe 2 of testing sample, preseparation chromatography post 3 and separation chromatography post 4, the emptying pipeline of solvent is provided with between described preseparation chromatography post 3 and described separation chromatography post 4, the emptying pipeline of described solvent has solvent drain valve 6, described solvent drain valve 6 is discharged for the solvent in the testing sample that the pre-separation of described preseparation chromatography post 3 gone out, remains component to be measured and enters in described separation chromatography post 4 to analyze with carrier gas and detect;
The chromatographic column adopted in wherein said liquid chromatography system 16 is alumina column 162;
The outlet of the outflow cut containing component to be measured after wherein said PTV sampling device 1 and liquid chromatography system 16 cutting on line.
In analytical equipment of the present invention, the transport path of carrier gas is identical with component to be measured, namely successively through described hollow bushing pipe 2, preseparation chromatography post 3 and separation chromatography post 4.
In a preferred embodiment of the present invention, numerical control six-way valve 9 is provided with between described liquid chromatography system 16 and gas chromatographic analysis device 8, its first interface flows into for the outflow cut of described liquid chromatography system 16, 5th interface flows into for the second mobile phase 18 be used for the outflow cut containing component to be measured is brought in described gas chromatographic analysis device 8, second and the 4th interface be connected to respectively trapping coil pipe 7 two ends, trap by the component to be measured that this trapping coil pipe 7 pairs of first interfaces flow into, 3rd interface is connected to the PTV sampling device 1 of described gas chromatographic analysis device 8, 6th interface is waste liquid escape hole 15.
In a preferred embodiment of the present invention, described hollow bushing pipe 2 is quartzy hollow bushing pipe, and its volume is 0.5-2mL.
In a preferred embodiment of the present invention, described alumina column 162 is ball-aluminium oxide chromatographic column, and this post is formed by the ball-aluminium oxide filling of 3 ~ 5 μm.Wherein, the interior diameter of post and length can set as required, and the interior diameter of such as post is 3.5mm, and length is 150mm.
In a preferred embodiment of the present invention, this analytical equipment downstream connects mass spectrometer.
Second aspect present invention relates to a kind of analytical approach using liquid-gas phase Two way chromatograms analytical equipment described according to a first aspect of the present invention, and it comprises the following steps:
A. testing sample is separated through described alumina column 162 by described liquid chromatography system 16, and obtains the outflow cut containing component to be measured through cutting on line;
B. the above-mentioned outflow cut containing component to be measured is passed through described PTV sampling device 1 sample introduction in described gas chromatographic analysis device 8, store in described hollow bushing pipe 2;
C. the described outflow cut containing component to be measured is through described pre-separation post 3, carries out initial gross separation by described containing the solvent in the outflow cut of component to be measured and component to be measured;
D. the described solvent after initial gross separation is discharged by described solvent drain valve 6, and residue enters analysis detection in described separating column 4 containing the outflow cut of component to be measured with carrier gas, adopts internal standard method to obtain the content of component to be measured.
In a preferred embodiment of the present invention, in step a, described cutting on line carries out as follows: use numerical control six-way valve 9 to carry out cut cutting to the effluent of described liquid chromatography system 16 according to retention time, to be captured in described trapping coil pipe 7 by the outflow cut containing component to be measured with specific retention time.
In a preferred embodiment of the present invention, when described testing sample is the urine of smoker, it also comprises following pre-treatment step: by the urine sample of smoker methylene chloride or the chloroformic solution extraction that contain the corresponding internal standard compound of component to be measured; Get organic layer after stratification to filter, obtain testing sample.Described extraction can adopt various known extracting process in prior art, preferably adopts whirlpool extraction.Described filtration can adopt various known filter method in prior art, preferably adopts syringe filters to filter.
Third aspect present invention relates to liquid-gas phase Two way chromatograms analytical equipment described according to a first aspect of the present invention for measuring the purposes of tobacco-specific nitrosamine content.
Fourth aspect present invention relates to analytical approach described according to a second aspect of the present invention for measuring the purposes of tobacco-specific nitrosamine content.
Fifth aspect present invention relates to liquid-gas phase Two way chromatograms analytical equipment described according to a first aspect of the present invention for measuring the purposes of tobacco-specific nitrosamine content in smoker's urine.
Relative to prior art, the present invention has the following advantages:
1, in the liquid chromatography system in Two way chromatograms analytical equipment of the present invention, 3 ~ 5 μm of spherical alkali aluminas are adopted independently to load high-efficiency column, and adopt this high-efficiency column to purify testing sample, instead of normally used gel column in existing instrument, compare with standard method (GB/T23228-2008 or CORESTAN ° 63), the effect of sample purification improves greatly, and the apparatus and method after improvement can meet the analysis requirement of super low loading tobacco-specific nitrosamine in smoker's urine.
2, the gas chromatographic analysis device improved in Two way chromatograms analytical equipment of the present invention, due to the existence of hollow bushing pipe and solvent drain valve, each analysis sample introduction can reach 200 μ L, compared with conventional gas phase chromatogram maximum sample size 2.0 μ L, sample size expands more than 100 times, and sensitivity for analysis obtains and significantly improves.And due to the significantly raising of sample size, can directly enter gas chromatographic analysis device of the present invention by after the large volume cut cutting after liquid chromatography purification.
3, Two way chromatograms analytical equipment of the present invention and analytical approach avoid the pre-treatment step such as concentrated, transfer, constant volume in sample pretreatment process, decrease experimental error, because sample preparation Manual operation link greatly reduces, the reappearance of method is also improved significantly.
4, Two way chromatograms analytical equipment of the present invention and analytical approach are unrestricted for volatilization, half volatile component, all can realize the present invention.
5, adopt spherical alkali alumina post in liquid chromatography in Two way chromatograms analytical equipment of the present invention, for the mode of chromatography purification existing in prior art, can greatly save organic mobile phase.
Accompanying drawing explanation
Fig. 1 is online liquid-gas phase Two way chromatograms analytical equipment structural representation of the present invention; Wherein the direction of arrow represents material throughput direction.
Fig. 2 is the gas chromatographic analysis device structural representation in online liquid-gas phase Two way chromatograms analytical equipment of the present invention.
Fig. 3 is liquid chromatography alumina column structural representation.
Fig. 4 is the ball-aluminium oxide scanning electron microscopic picture of filling in alumina column.
Fig. 5 is tobacco-specific nitrosamine liquid chromatography separate colors spectrogram.
Fig. 6 four to grow tobacco unique nitrosamine mass spectrogram.
Fig. 7 four to grow tobacco unique nitrosamine Selective ion mode chromatogram.
Fig. 8 is analytical approach schematic flow sheet of the present invention.
Wherein each Reference numeral has following implication:
1-PTV sampling device; 2-hollow bushing pipe; 3-pre-separation post; 4-separating column; 5-post web member; 6-solvent drain valve; 7-traps coil pipe; 8-gas chromatographic analysis device; 9-numerical control six-way valve; 10-second valve B; 11-first pump; 12-second pump; 13-first degasser; 14-second degasser; 15-waste liquid escape hole; 16-liquid chromatography system; 161-column oven; 162-alumina column 163-UV-detector; 164-injector; 17-first mobile phase; 18-second mobile phase.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further illustrated, but limited the present invention never in any form, and any conversion done based on training centre of the present invention or replacement, all belong to protection scope of the present invention.
Embodiment 1
Liquid-gas phase Two way chromatograms analytical equipment as shown in Figure 1, it gas chromatographic analysis device 8 comprising liquid chromatography system 16 and be positioned at its downstream;
As shown in Figure 2, wherein said gas chromatographic analysis device 8 comprise be linked in sequence PTV sampling device 1, for storing the hollow bushing pipe 2 of testing sample, preseparation chromatography post 3 and separation chromatography post 4, the emptying pipeline of solvent is provided with between described preseparation chromatography post 3 and described separation chromatography post 4, the emptying pipeline of described solvent has solvent drain valve 6, described solvent drain valve 6 is discharged for the solvent in the testing sample that the pre-separation of described preseparation chromatography post 3 gone out, remains component to be measured and enters in described separation chromatography post 4 to analyze with carrier gas and detect;
The chromatographic column adopted in wherein said liquid chromatography system 16 is alumina column 162; Because main concurrent (phenols, nicotinoids, the hydro carbons etc.) polarity difference in tobacco-specific nitrosamine and urine is larger, adopts the interfering component of alkali alumina post energy separating most of the present invention, thus realize the purification of sample.
The outlet of the outflow cut containing component to be measured after wherein said PTV sampling device 1 and liquid chromatography system 16 cutting on line.This analytical equipment downstream connects mass spectrometer.Described hollow bushing pipe 2 is quartzy hollow bushing pipe, and its volume is 1.2mL.As shown in Figure 3 and Figure 4, described alumina column 162 is ball-aluminium oxide chromatographic column, and this post is formed by the ball-aluminium oxide filling of 3 ~ 5 μm, and the interior diameter of post is 3.5mm, and length is 150mm.
Numerical control six-way valve 9 is provided with between described liquid chromatography system 16 and gas chromatographic analysis device 8, its first interface flows into for the outflow cut of described liquid chromatography system 16, 5th interface flows into for the second mobile phase 18 be used for the outflow cut containing component to be measured is brought in described gas chromatographic analysis device 8, second and the 4th interface be connected to respectively trapping coil pipe 7 two ends, trap by the component to be measured that this trapping coil pipe 7 pairs of first interfaces flow into, 3rd interface is connected to the PTV sampling device 1 of described gas chromatographic analysis device 8, 6th interface is waste liquid escape hole 15
The principle of work of this device is as follows: first start the first pump 11, makes the first mobile phase 17 enter in liquid chromatography system 16 via the first degasser 13; Testing sample enters in liquid chromatography system 16 through injector 164, and is separated through alumina column 162; Outflow cut from described liquid chromatography system 16 enters in numerical control six-way valve 9 through first interface, and the outflow cut containing component to be measured proceeds in trapping coil pipe 7 through numerical control six-way valve; After having shifted, switch numerical control six-way valve, allow the first pump 11 mobile phase waste liquid and the outflow cut that do not contain component to be measured discharge through waste liquid escape hole 15.Start the second pump 12, make the second mobile phase 18 enter in numerical control six-way valve 9 via the second degasser 14; The outflow cut containing component to be measured of trapping in trapping coil pipe 7 is transported in the described hollow bushing pipe 2 of gas chromatographic analysis device 8 through the 3rd interface discharge of described numerical control six-way valve 9 through the second valve 10 (common valve) by the second mobile phase 18, containing the outflow cut of component to be measured through described pre-separation post 3, carry out initial gross separation by described containing the solvent in the outflow cut of component to be measured and component to be measured; Described solvent after initial gross separation is discharged by described solvent drain valve 6, and the outflow cut containing component to be measured after concentrated enters in described separating column 4 to analyze and detects, and is detected after separating column 4 is separated by mass spectrometer again.
Embodiment 2
Adopt liquid-gas phase Two way chromatograms analytical equipment as described in Example 1 to analyze to measure the method for the peculiar nitrous acid content of wherein tobacco to smoker's urine, it comprises the following steps:
A. get the urine sample 50mL of smoker in the test tube of 100mL, the dichloromethane solution 10mL added wherein containing the deuterated nitrous acid of internal standard compound (concentration is 50ng/L) extracts; With high-speed homogenization machine 20000 turns/min whirlpool concussion 5min, take off the dichloromethane layer clear liquid 1.0mL of layer after stratification, filter through 0.25 μm of filter, obtain testing sample; Testing sample is separated through described alumina column 162 by described liquid chromatography system 16, and obtains the outflow cut containing component to be measured through cutting on line.Test compares oscillation extraction, the method for ultrasonic extraction and high-speed homogenization whirlpool concussion extraction extracts tobacco-specific nitrosamine in sample, result shows that the method that high-speed homogenization whirlpool shakes has the highest efficiency, with 20000 turns/min whirlpool concussion 5min sample can be allowed fully to mix and make tobacco-specific nitrosamine total transfer to methylene chloride mutually in, time expand, the recovery on tobacco-specific nitrosamine significantly do not affect, so this experimental selection 20000 turns/min whirlpool concussion 5min extracts sample.
B. the above-mentioned outflow cut containing component to be measured is passed through described PTV sampling device 1 sample introduction in described gas chromatographic analysis device 8, store in described hollow bushing pipe 2;
C. the described outflow cut containing component to be measured is through described pre-separation post 3, carries out initial gross separation by described containing the solvent in the outflow cut of component to be measured and component to be measured;
D. the described solvent after initial gross separation is discharged by described solvent drain valve 6, and residue enters analysis detection in described separating column 4 containing the outflow cut of component to be measured with carrier gas, adopts internal standard method to obtain the content of component to be measured.
In step a, described cutting on line carries out as follows: use numerical control six-way valve 9 to carry out cut cutting to the effluent of described liquid chromatography system 16 according to retention time, to be captured in described trapping coil pipe 7 by the outflow cut containing component to be measured with specific retention time.
After wherein the cutting of liquid chromatography cut enters the hollow bushing pipe of gas chromatography, after solvent need being got rid of completely, vapor-phase chromatography analysis could be started.Liquid chromatography preferably adopts volatile mobile phase, the liquid chromatogram mobile phase selected in the present embodiment is methylene chloride and acetone, be easy volatile solvent, when the ratio of methylene chloride and acetone has good clean-up effect for four unique nitrosamines that grow tobacco time (9:1), therefore this mobile phase is selected in experiment.Experimental result shows, gas chromatographic column case is warmed up to 50 DEG C, vacuumizes 6.0min solvent can be allowed to discharge completely from solvent escape hole.Discharge completely in order to ensure each solvent of analyzing, discharge solvent time in this experiment and extend 2.0min, being chosen as efflux time is 8.0min.
In above-mentioned analytical approach, actual conditions is as follows:
Liquid chromatography separation condition: liquid chromatography band UV-detector, determined wavelength is 254nm; Liquid chromatography separation column is ball-aluminium oxide post (this post is formed by the ball-aluminium oxide filling of 3 ~ 5 μm, and the interior diameter of post is 2.5mm, and length is 150mm), controls column temperature 30 DEG C of constant temperature by column oven.Liquid chromatogram mobile phase is dichloromethane-acetone (90/10, v/v), flow velocity 0.30mL/min; Double pump system, the first pump 11 is for controlling liquid-phase chromatographic column flow, and the second pump 12 is for controlling the switching between liquid chromatography (HPLC) and gas chromatography (GC).Liquid chromatography sampling volume is 200 μ L.Under this condition tobacco-specific nitrosamine go out peak between 6.0 – 8.4min (Fig. 5), collect as far as possible completely to make the component to be measured in sample, avoid retention time to fluctuate and cause loss, in the present invention, 5.8 – 8.6min being flowed out cut proceeds in trapping coil pipe, because flow velocity is 0.30mL/min, the volume that liquid chromatography cutting enters gas chromatography is 0.84mL.
Gas chromatography and Mass Spectrometry Conditions are optimized respectively, GC conditions is mainly optimized chromatographic column and heating schedule, compare the separating effect of different chromatographic column and heating schedule, with four grow tobacco unique nitrosamine be separated completely and as far as possible shorten analysis time for target.Further, reach the highest response for target with four unique nitrosamines that grow tobacco, Mass Spectrometry Conditions is optimized.
Analytical conditions for gas chromatography: after sample enters gas chromatography completely, closes liquid chromatography passage, removes the solvent in quartzy hollow bushing pipe by solvent drain valve in gas chromatography.Rear closedown solvent escape hole got rid of completely by solvent, starts gas chromatography intensification and carries out gas chromatographic analysis.Column cap band can get rid of the gas chromatograph (see Fig. 2) of the quartz liner of solvent, and the volume of quartzy hollow bushing pipe is 1.2mL, and pre-separation post specification is 5m × 0.25mmi.d × 0.25 μm df, DB-5ms (J & W); Separating column specification is: AgilentDB-35MSUI capillary chromatographic column (30m × 250 μm × 0.25 μm); Carrier gas is helium, adopts constant current mode; Flow velocity is 1.2mL/min; Injector temperature: 290 DEG C; Chromatographic column heating schedule: initial temperature 50 DEG C (keeping 1.0min), rises to 170 DEG C with 30 DEG C/min speed, then rise to 230 DEG C with 5 DEG C/min speed, then rise to 290 DEG C with 30 DEG C/min speed.
Mass spectrophotometry condition: ionization mode: EI source; Ionizing energy: 30eV; Ionogenic temperature: 250 DEG C; Level Four bar temperature: 120 DEG C; Scan pattern: Select ion monitor (SIM) pattern, transmission line temperature 250 DEG C, quality of scanning scope 50-300amu, sweep spacing 0.5s.According to its mass spectrogram (Fig. 6), adopt the method extracting isolated son from total ion current figure to carry out quantitatively, the quota ion of NNK, NNN, NAT, NAB is respectively m/z177,178,190,192; The quota ion of its four kinds of Isotopic Internal Standards is respectively m/z191,182,194,196; Under conditions selected, the four Selective ion mode chromatograms growing tobacco unique nitrosamine are shown in Fig. 7.
Embodiment 3-assay method working curve, detection limit and quantitative limit
The preparation of standard solution:
Prepared by standard reserving solution: the NNN taking 0.1g respectively, NNK, NAT, NAB (being accurate to 0.1mg) is in 100mL volumetric flask, dissolve and constant volume with methylene chloride, being mixed with each concentration of component is 1.0mg/mL hybrid standard storing solution, and sealing stored protected from light is under-20 DEG C of conditions.
Inner mark solution is laid in: the d4-NNN taking 100mg respectively, d4-NNK, d4-NAT, d4-NAB (being accurate to 0.1mg) is in 100mL volumetric flask, dissolve with methylene chloride and be settled to scale, being mixed with each concentration of component is mark storing solution in 0.1mg/mL mixing, and sealing stored protected from light is under-20 DEG C of conditions.
Standard working solution: obtain 6 grade standard working solutions with chloroform stepwise dilution standard reserving solution, the concentration of corresponding each component to be measured is respectively 0.01ng/mL, 0.05ng/mL, 0.1ng/mL, 0.5ng/mL, 1.0ng/mL, 5.0ng/mL, the concentration of internal standard compound is 0.05ng/mL.
Sample extraction solution: be mixed with the dichloromethane solution containing 0.05ng/mL internal standard compound with interior mark storing solution.
(internal standard compound concentration is 0.05ng/mL to prepare the standard solution of a series of different TSNAs (comprising NNN, NNK, NAT and NAB) concentration, analyze by instrument condition selected in embodiment 2, carry out linear regression with standard specimen peak area (A) and concentration (C), obtain equation of linear regression; The results are shown in Table 1.By the standard solution continuous sample introduction 10 times of least concentration, measure its standard deviation, using 3 times of standard deviations as detection limit, using 10 times of standard deviations as quantitative limit; Concrete outcome is in table 1.
Table 1: assay method working curve, related coefficient, detection limit and quantitative limit
The sensitivity of determining instrument is weighed with its detectability (LOD) and quantitative limit (LOQ), and wherein said detection limit refers to minimum concentration or the content of certain element required for analytic signal that can reliably be detected; Wherein said quantitative limit refers to measured object in sample can by the minimum flow of quantitative measurement.
The value of known detectability is 3 times of standard deviations, and the value of quantitative limit is 10 times of standard deviations.
Result in table 1 shows that the sensitivity of analytical equipment of the present invention is higher.
The embodiment 4-method recovery and repeatability
Withinday precision is: same smoker's urine is interior on the same day under the same conditions to be measured 7 times, and calculates the relative standard deviation (RSD) of 7 parallel determinations, can obtain the RSD of four kinds of TSNAs within the scope of 4.6%-5.2%; Illustration method precision is good.
Day to day precision is: smoker's urine that every day, amount of smoking frequency was consistent is measured 1 every day under the same conditions, totally 7 days, and calculate the relative standard deviation (RSD) of 7 measurement results, the RSD of four kinds of TSNAs can be obtained within the scope of 5.3%-5.8%; Illustrate and to measure in different time, method precision is still good.
During mensuration, every sample TSNAs (comprising NNN, NNK, NAT and NAB) takes same sample 4 parts, and wherein 1 part does not add standard items, another 3 parts of TSNAs adding known quantity respectively, addition is respectively 0.03,0.06,0.09ng; Sample by sample pre-treatments condition process selected in embodiment 2, and is analyzed by selected instrument condition sample introduction, deducts the non-mark-on sample amount of measuring (background), then calculate the recovery divided by Standard entertion gauge by the mark-on sample amount of measuring.Obtain the recovery of TSNAs between 76.3% ~ 83.2%, the illustration method recovery is very high.
Embodiment 5-measures the tobacco-specific nitrosamine content in smoker's urine
Adopt the method in embodiment 2 to measure the tobacco-specific nitrosamine in smoker's urine, the results are shown in Table 2.Can find out from analysis result, although have employed analytical equipment and the analytical approach of ultra-high sensitive, but only have tobacco-specific nitrosamine concentration in blended type cigarette smoker urine more than the quantitative limit of analytical approach, the tobacco-specific nitrosamine content major part in Virginian-type cigarette smoker urine is still below quantitative limit.No matter be blended type cigarette smoker or Virginian-type cigarette smoker, the tobacco-specific nitrosamine content in its urine is all far below concentration level human body being produced to harm.
TSNAs assay result (ng/L) in table 2 representative sample
Remarks: "-" represents that content is lower than quantitative limit.
Claims (9)
1. a liquid-gas phase Two way chromatograms analytical equipment, is characterized in that, it gas chromatographic analysis device (8) comprising liquid chromatography system (16) and be positioned at its downstream;
Wherein said gas chromatographic analysis device (8) comprises the PTV sampling device (1) be linked in sequence, for storing the hollow bushing pipe (2) of testing sample, preseparation chromatography post (3) and separation chromatography post (4), the emptying pipeline of solvent is provided with between described preseparation chromatography post (3) and described separation chromatography post (4), the emptying pipeline of described solvent has solvent drain valve (6), described solvent drain valve (6) is discharged for the solvent in the testing sample that the pre-separation of described preseparation chromatography post (3) gone out, remain component to be measured and enter analysis detection in described separation chromatography post (4) with carrier gas,
The chromatographic column adopted in wherein said liquid chromatography system (16) is alumina column (162);
The outlet of the outflow cut containing component to be measured after wherein said PTV sampling device (1) and liquid chromatography system (16) cutting on line.
2. liquid-gas phase Two way chromatograms analytical equipment according to claim 1, it is characterized in that, numerical control six-way valve (9) is provided with between described liquid chromatography system (16) and gas chromatographic analysis device (8), its first interface flows into for the outflow cut of described liquid chromatography system (16), 5th interface flows into for being used for the second mobile phase (18) brought in described gas chromatographic analysis device (8) by the outflow cut containing component to be measured, second and the 4th interface be connected to respectively trapping coil pipe (7) two ends, trap coil pipe (7) with this component to be measured that first interface flows into is trapped, 3rd interface is connected to the PTV sampling device (1) of described gas chromatographic analysis device (8), 6th interface is waste liquid escape hole (15).
3. liquid-gas phase Two way chromatograms analytical equipment according to claim 1, is characterized in that, described hollow bushing pipe (2) is quartzy hollow bushing pipe, and its volume is 0.5-2mL.
4. liquid-gas phase Two way chromatograms analytical equipment according to claim 1, is characterized in that, described alumina column (162) is ball-aluminium oxide chromatographic column, and this post is formed by the ball-aluminium oxide filling of 3 ~ 5 μm.
5. liquid-gas phase Two way chromatograms analytical equipment according to claim 1, is characterized in that, this analytical equipment downstream connects mass spectrometer.
6. use an analytical approach for the liquid-gas phase Two way chromatograms analytical equipment according to any one of claim 1-5, it is characterized in that, it comprises the following steps:
A. testing sample is separated through described alumina column (162) by described liquid chromatography system (16), and obtains the outflow cut containing component to be measured through cutting on line;
B. the above-mentioned outflow cut containing component to be measured is passed through described PTV sampling device (1) sample introduction in described gas chromatographic analysis device (8), store in described hollow bushing pipe (2);
C. the described outflow cut containing component to be measured is through described pre-separation post (3), carries out initial gross separation by described containing the solvent in the outflow cut of component to be measured and component to be measured;
D. the described solvent after initial gross separation passes through described solvent drain valve (6) discharge, and residue enters analysis detection in described separating column (4) containing the outflow cut of component to be measured with carrier gas, adopts internal standard method to obtain the content of component to be measured.
7. analytical approach according to claim 6, it is characterized in that, in step a, described cutting on line carries out as follows: use numerical control six-way valve (9) effluent to described liquid chromatography system (16) to carry out cut cutting according to retention time, to be captured in described trapping coil pipe (7) by the outflow cut containing component to be measured with specific retention time.
8. analytical approach according to claim 6, it is characterized in that, when described testing sample is the urine of smoker, it also comprises following pre-treatment step: by the urine sample of smoker methylene chloride or the chloroformic solution extraction that contain the corresponding internal standard compound of component to be measured; Get organic layer after stratification to filter, obtain testing sample.
9. liquid-gas phase Two way chromatograms analytical equipment according to claim 1 or analytical approach according to claim 6 are for measuring the purposes of tobacco-specific nitrosamine content in smoker's urine.
Priority Applications (1)
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| CN109085270A (en) * | 2018-08-28 | 2018-12-25 | 上海烟草集团有限责任公司 | Method that is a kind of while measuring a variety of tobacco-specific nitrosamines in human plasma |
| CN109781875A (en) * | 2019-01-11 | 2019-05-21 | 云南中烟工业有限责任公司 | A method of utilizing TSNAs in the Soxhlet extraction device detection smoker urine with Solid Phase Extraction function |
| CN110398606A (en) * | 2019-07-24 | 2019-11-01 | 云南中烟工业有限责任公司 | One kind being used for the multiple sample pretreatment devices of Continuous Flow Analysis and its application method |
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| CN109781875A (en) * | 2019-01-11 | 2019-05-21 | 云南中烟工业有限责任公司 | A method of utilizing TSNAs in the Soxhlet extraction device detection smoker urine with Solid Phase Extraction function |
| CN110398606A (en) * | 2019-07-24 | 2019-11-01 | 云南中烟工业有限责任公司 | One kind being used for the multiple sample pretreatment devices of Continuous Flow Analysis and its application method |
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