CN101354385A - Method for simultaneously analyzing twenty amino acids in tobacco and product thereof - Google Patents
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- CN101354385A CN101354385A CNA2008100276923A CN200810027692A CN101354385A CN 101354385 A CN101354385 A CN 101354385A CN A2008100276923 A CNA2008100276923 A CN A2008100276923A CN 200810027692 A CN200810027692 A CN 200810027692A CN 101354385 A CN101354385 A CN 101354385A
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Abstract
The invention discloses a method used for simultaneously analyzing 20 types of amino acids in tobaccos and tobacco products, adopts the coupling of liquid chromatogram and electrospray tandem mass spectrum, and comprises the following experimental steps: a tobacco sample is preprocessed, extracting solution is obtained and eluted with binary flow phase gradient on a reversed phase chromatography column, two standard curves are produced, and liquid chromatogram-electrospray ion trap tandem mass spectrometry is adopted for analyzing and detecting the 20 types of amino acids in the tobaccos under a positive ion mode. The sampling of the tobacco sample is directly implemented after extraction and filtration and other preprocessing steps including derivation and solid phase extraction are avoided in the method of the invention, thus providing simplicity, convenience and fastness; the method has lowered chromatogram separable degree requirement of detected objects, can effectively and simultaneously solve the co-flow problem and the separated detection problem of amino acid isomers, builds a quickly simultaneous analyzing method of the 20 types of non-derivatized amino acids in the tobaccos, has good reproducibility, high sensitivity and selectivity, greatly simplifies analyzing steps, raises analyzing efficiency, and can be successfully applied into the analytic determination of the amino acids in various tobacco samples.
Description
Technical field
The present invention relates to chemical analysis detection technique field, be specifically related to a kind of method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously.
Background technology
Amino acid is the important component part in the tobacco leaf, is and smoke perfume, cigarette quality and the closely-related tobacco component of brand identity, and amino acid participates in the brown reaction of non-enzyme in the tobacco leaf, i.e. Maillard reaction, and this reaction can produce multiple important aroma component.Therefore, the amino acid of measuring in the tobacco all has crucial meaning to formula development, explained hereafter and quality control.
Because the amino acid kind in the tobacco is more, and have acidity, neutrality and three kinds of different chemical property of alkalescence, add tobacco matrix complexity, therefore in the tobacco several amino acids time mensuration have certain difficulty.At present, liquid chromatography (LC) method is one of determined amino acid method of more use.Because amino acid do not have ultraviolet and the required chromophoric group of fluoroscopic examination,, then must carry out before the post or post-column derivation to amino acid if detect with liquid phase chromatography.The derivatization complex operation step, take time and effort, and the stability of derivative compound is difficult to pass judgment on intuitively, the completeness of some amino acid derived reaction also is difficult to guarantee, derivatization reaction also may generate non-target derivant simultaneously, and these all can influence amino acid whose accurate mensuration.Automatic amino acid analyser is also deposited the same problem of deriving.
Tobacco component is quite complicated, and carrying out amino acid analysis with LC, automatic amino acid analyser, chromatography of ions (IC) and Capillary Electrophoresis methods such as (CE) often needs sample carried out numerous and diverse pre-treatments such as Solid-Phase Extraction, with the minimizing interference.Pre-treatment meetings such as existing Solid-Phase Extraction cause the loss of some amino acid (or amino acid derivativges) to a certain extent, and some disturbs impurity also to be difficult to eliminate by pre-treatment.
In recent years, along with the development of electro-spray ionization (ESI) and tandem mass spectrum (MS/MS) technology, liquid chromatography and esi-msn coupling (LC-ESI-MS/MS) are widely used in the complex compound analysis.LC-MS/MS method highly sensitive, the selectivity excellence.Because MS/MS has the characteristic ion abstraction function, compare with chromatographic process, the not so difficult strictness of chromatographic resolution degree requirement of determinand.Utilize liquid chromatography simultaneously 20 kinds of amino acid whose rapid analysiss of non-derivative in the tobacco not to be seen relevant technology report at present with electron spray ion trap tandem mass spectrometry (LC-ESI-MS/MS) coupling technique.
Summary of the invention
The objective of the invention is to overcome the prior art deficiency, a kind of method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously is provided.
Purpose of the present invention is achieved by the following technical programs:
A kind of method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously is provided, adopts liquid chromatography and esi-msn coupling, may further comprise the steps:
(1) tobacco sample obtains extract through pre-treatment;
(2) extract on reverse-phase chromatographic column with two-phase eluent gradient wash-out;
(3) typical curve of two kinds of variable concentrations series of making adopts liquid chromatography-electron spray ion trap tandem mass spectrometry to carry out the analyzing and testing of 20 seed amino acids in the tobacco under positive ion mode.
The described pre-treatment of step (1) comprises adopts that to contain norvaline concentration be that the HCl solution of 1.7 μ M is extraction solution, extracts offal under ultrasonic concussion; Extract filters.Described HCl solution concentration is 0.03～0.1M, and preferred concentration is 0.03M.
The ratio of described extraction offal and extraction solution is 0.1～0.2g: 20ml; The extraction time is 15min; Described filtration is to adopt 0.2 μ m membrane filtration.
The described reverse-phase chromatographic column of step (2) is HyPURITY C
18Described binary moving phase is 99% water-1% acetonitrile-0.1% 9 fluorine valeric acid and 10% water-90% acetonitrile-0.1% 9 fluorine valeric acid.
The typical curve of two kinds of concentration series of the described making of step (3) is:
(1) mark norvaline concentration was the amino-acid mixed and standard solution I of 1.7 μ M in preparation contained, and wherein the concentration of aspartic acid, serine, glutamine, threonine, glutamic acid, cystine, γ-An Jidingsuan, nipecotic acid, valine, dl-methionine, histidine, tyrosine, lysine, isoleucine, arginine, leucine, phenylalanine and tryptophane is the 0.03M HCl solution of 50 μ M;
(2) mark norvaline concentration was the amino-acid mixed and standard solution II of 1.7 μ M in preparation contained, and wherein the concentration of proline and asparagine is respectively the 0.03M HCl solution of 500 μ M;
(3) be that the 0.03M HCl of 1.7 μ M is made into series concentration to standard solution I and standard solution II, the production standard curve with containing norvaline concentration.
The condition determination of the described liquid chromatography of step (3)-electron spray ion trap tandem mass spectrometry is: nitrogen pressure: 0.64MPa; Helium pressure: 0.38MPa; Polarity: positive ionization; Scanning form: full scan; Acquisition time: 40min; Activationary time: 30ms; Sweep gas: 0LTQ unit; Spray voltage: 5kV; Capillary temperature: 350 ℃; Sweep limit: standard; Sweep speed: standard; Segmentation.Described segmentation is to be divided into 4 sections, and wherein the sheath gas of the 3rd segmentation and auxiliary gas are respectively 38LTQ unit and 0LTQ unit, and the sheath gas and the auxiliary gas of all the other segmentations are respectively 46LTQ unit and 19LTQ unit.
The invention has the beneficial effects as follows:
(1) tobacco sample has extracted and has filtered the back direct injected, need not to derive and other pre-treatments such as Solid-Phase Extraction, has simplified analytical procedure greatly, has improved analysis efficiency;
(2) compare with simple chromatogram analysis method, so strict to the chromatographic resolution degree requirement of determinand, can effectively solve common outflow problem;
(3) the present invention utilizes method for separating liquid phase chromatography, solved the separation detection problem of amino acid isomer, set up simultaneously to 20 kinds of amino acid whose rapid analysiss of non-derivative in the tobacco, favorable reproducibility, sensitivity and selectivity height can be successfully applied in the amino acid whose assay determination of various tobacco samples.
In sum, the present invention has set up the amino acid whose assay method of non-derivative of a kind of easy, efficient, high sensitivity and high selectivity.The present invention is by chromatographic resolution and mass spectral The ion extraction, simplified sample pre-treatments greatly, made things convenient for amino acid whose qualitative and quantitative analysis to be measured, effectively solved the more scabrous problem that flows out altogether of liquid-phase chromatography method, the method good stability, can directly be used for the amino acid whose analysis that respectively grows tobacco, for formulating, product design, explained hereafter and quality control provide important amino acid reference data as the conventional sense method.The LC-MS method can easily realize analyzing several amino acids the time in the tobacco, as the piperidines of the L-in the tobacco-2-carboxylic acid (L-Pip), has not yet to see and is measuring amino acid whose other analytical approachs of measuring L-Pip simultaneously of common tobacco.
Description of drawings
The extraction ion flow graph of Figure 120 seed amino acid and interior mark norvaline
Ile and Leu extract the ion flow graph before Fig. 2 mark-on
Ile and Leu extract the ion flow graph behind Fig. 3 mark-on
Val and Nva extract the ion flow graph before Fig. 4 mark-on
Val and Nva extract the ion flow graph behind Fig. 5 mark-on
The amino acid whose second order ms figure of Fig. 6～Figure 26
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.
1, instrument, material and reagent
The LC-MS LTQ of U.S. power ﹠ light company liquid chromatography-iontrap mass spectrometer is equipped with automatic sampler, quaternary gradient mass spectrum pump, LTQ ion trap mass spectrometry charged spray ionization source (ESI), Xcalibur 1.4SR1 system controlling software; Italy Claind N2LCMS nitrogen gas generator; France Millipore Element A10 water purification machine; U.S. Cole-Parmer 8892 ultrasonic cleaning machines; Sartorious CP225D electronic balance (Max 220g, d=0.01mg (80g), 0.1mg (220g)); Whatman 0.2 μ m nylon pin type filter; Tobacco powder.
Kilnitamin standard solution (Fluka company) contains 17 seed amino acids, and concentration respectively is 1mM; Internal standard compound L-norvaline (the beautiful pearl east wind in Shanghai Bioisystech Co., Ltd); Asparagine water (Shanghai uncle bio tech ltd difficult to understand); Tryptophane, proline and glutamine (Chemical Reagent Co., Ltd., Sinopharm Group); γ-An Jidingsuan (Sigma company); L-piperidines-2-carboxylic acid, nine fluorine valeric acids (NFPA) (Aldrich company); Methyl alcohol and acetonitrile (MERCK company); The pure concentrated hydrochloric acid of top grade; Milli-Q level water, by Milliore Element A10 preparation, if without special instruction, water is Milli-Q level water in the experiment.
2, experimental technique
2.1 sample preparation
Take by weighing 0.1～0.2g offal and place conical flask, add 20mL and contain the 0.03MHCl solution that norvaline concentration is 1.7 μ M, ultrasonic concussion 15min sways conical flask and makes the solution concentration distributed uniform for a moment, direct injected behind 0.2 μ m membrane filtration.
2.2 the thermoelectric HyPURITYC of chromatographic condition
18Chromatographic column (200mm * 2.1mm, 5 μ m), Agilent Reliance protection column sleeve and Eclipse XDB-C8 guard column (12.5 * 2.1mm, 5 μ m).Input mode: loopful (5 μ L); Flow velocity: 200 μ L/min; Column oven: 25 ℃; Sample disc temperature: 15 ℃; Irrigation with syringe volume (flush volume): 1000 μ L; Wash needle body long-pending (wash volume): 800 μ L.Mobile phase A: 99% water-1% acetonitrile-0.1%NFPA; Mobile phase B: 10% water-90% acetonitrile-0.1%NFPA.Eluent gradient sees Table 1.
Table 1 eluent gradient
2.3 mass spectrum condition nitrogen pressure: 0.64MPa; Helium pressure: 0.38MPa; Polarity: positive ionization; Scanning form: full scan; Acquisition time: 40min; Activationary time: 30ms; Sweep gas: 0LTQ unit; Spray voltage: 5kV; Capillary temperature: 350 ℃; Sweep limit: standard; Sweep speed: standard; Settings such as segmentation, split time, separation width, normalization collision energy see Table 2, and wherein the sheath gas of the 3rd segmentation and auxiliary gas are respectively 38LTQ unit and OLTQ unit, and the sheath gas and the auxiliary gas of all the other segmentations are respectively 46LTQ unit and 19LTQ unit.Other parameters are determined by hands-off tuning or according to the mass spectral default setting of LTQ.
Condition setting such as table 2 segmentation, split time, separation width, normalization collision energy
2.4 standard curve making adopts inner mark method ration.Mark norvaline concentration was the amino-acid mixed and standard solution I (aspartic acid (Asp) of 1.7 μ M in preparation contained, serine (Ser), glutamine (Gln), threonine (Thr), glutamic acid (Glu), cystine (Cys), γ-An Jidingsuan (Gaba), nipecotic acid (Pip), valine (Val), dl-methionine (Met), histidine (His), tyrosine (Tyr), lysine (Lys), isoleucine (Ile), arginine (Arg), leucine (Leu), the concentration of phenylalanine (Phe) and tryptophane (Try) is the 0.03M HCl solution of 50 μ M); Mark norvaline concentration was the amino-acid mixed and standard solution II (concentration of proline (Pro) and asparagine (Asn) is respectively the 0.03M HCl solution of 500 μ M) of 1.7 μ M in preparation contained.With containing norvaline concentration is that the 0.03M HCl of 1.7 μ M is made into series concentration to standard solution I and standard solution II, the production standard curve.
3, experimental result
3.1 determining of chromatographic condition
3.1.1 amino acid is to the requirement of temperature
When temperature was higher, amino-acid mixed mark reacted easily, and some amino acid content is reduced.Therefore, all amino acid standard solution should in time be kept at below 2 ℃ when not using, and when analytic sample sample disc are set in 15 ℃ of lower temperature.Amino acid may change in post when higher for fear of column temperature, and column oven is set in 25 ℃.Another benefit that suitably reduces column temperature is obviously to increase isomers leucine (Leu) and the degree of separation of isoleucine (Ile) on chromatographic column, sees accompanying drawing 1.But it is low that column temperature should not be set, in order to avoid the post pressure is excessive, also makes Phe and Try go out the peak simultaneously and postpone, and increases analysis time.
3.1.2 chromatographic column and moving phase
At C
18Tested multiple moving phase on the analytical column, the result shows Thermo HyPURITYC
18Cooperate 99% water-1% acetonitrile-0.1%NFPA and 10% water-90% acetonitrile-0.1%NFPA to make moving phase and can obtain good chromatographic peak profile and suitable chromatographic run time, and can make isomers Leu and Ile reach baseline separation, see accompanying drawing 1.NFPA is as moving phase modifier.In addition, NFPA also helps and strengthens electrospray ionization mass spectrum to amino acid whose positive ionization detection signal.
The easy wash-out NFPA of acetonitrile, too much NFPA increases background interference easily, influences detection sensitivity, and therefore, the usage ratio of Mobile phase B is difficult for too high, and experiment below 25%, can reach the effect that good separation detects to the proportional control of Mobile phase B.Be noted that NFPA can constantly gather and slowly change the character of stationary phase in chromatographic column, amino acid whose appearance time is changed.In order to guarantee good retention time reappearance, carry out qualitative and quantitative analysis more easily, behind sample introduction 100 pins,, strip the NFPA that gathers with 100% acetonitrile flushing chromatographic column 30min more, use sample introduction after the moving phase balance chromatographic column again.
3.1.3 the selection of internal standard compound
Norvaline is not stored in the tobacco amino acid, and is stable and can separate fully on chromatogram with its isomers valine under this experiment condition, and price is lower, is more satisfactory internal standard compound.
3.1.4 the discriminating of amino acid isomer
Leucine (Leu) and isoleucine (Ile), valine (Val) and norvaline (Nva) be isomers each other, and second order ms is similar, can't singly be identified with mass spectrum, therefore needs to distinguish in conjunction with chromatographic resolution.Experiment adds a certain amount of Ile and Val respectively peak sequence is judged by the mark-on method.After adding Ile or Val standard, the chromatographic peak height that flows out previously obviously increases, and peak area obviously increases, and can judge that Ile and Val flow out earlier than Leu and Nva respectively, sees that accompanying drawing 2～accompanying drawing 5 extracts the ion flow graph.
3.2 determining of mass spectrum condition
Mass spectral condition appropriately be provided with to amino acid whose accurately and high-sensitivity detection very important.Experiment has contrasted secondary full scan (Full MS2) and has selected two kinds of scan patterns of ion monitoring (SIM).The secondary full scan can reduce background interference effectively, obtains higher sensitivity, and selectivity is better than SIM, and it is preferable to detect effect, so experimental selection secondary full scan carries out quantitative test.In the second order ms of 21 seed amino acids, see accompanying drawing 6～Figure 26, except that Arg and Cys, the main fragment ion of all the other amino acid is decarboxylation, dehydroxylation or deamination base peak, i.e. [M-HCOOH+H]
+, [M-H
2O+H]
+Or [M-N
2H+H]
+The accurate choice relation of quota ion is to the quantitative feature selecting of mass spectrum.Quota ion is preferentially chosen the bigger feature daughter ion of relative abundance, to obtain sensitivity preferably.For Glu, the characteristic of factor ion m/z 130 is not strong, has Interference Peaks overlapping, therefore selects relative abundance time strong decarboxylation base peak m/z102.
Different segmentation (Segment) can be called independently tuning file, helps improving the sensitivity of detection.The retention time of experimental basis chromatographic peak divides 4 segmentations at interval.Suitable sweep limit helps reducing background noise and impurity disturbs, and improves the sensitivity and the accuracy that detect, and experiment has been carried out optimization setting to each amino acid whose sweep limit, is shown in Table 2.
3.3 determining of pre-treatment condition
3.3.1 different influences of extracting solution to chromatographic peak profile
Experiment is extracted the amino acid in the tobacco with the acid solution of variable concentrations, comprises formic acid, acetate and hydrochloric acid solution.Experiment finds that 1mM acetate extracts solution and makes amino acid whose chromatographic peak bifurcated cracking such as Val, Nva, His, Ile and Leu, and along with the raising of concentration, and the phenomenon of chromatographic peak bifurcated cracking is more for obviously.Formic acid extracts solution also similar phenomenon.HCl extracts solution then can be at C
18Obtain peak shape preferably on the post.
3.3.2 the different extraction effects that extract solution
Experiment has contrasted several extraction effect of extraction solution commonly used, i.e. 70% methanol solution, 80% ethanolic solution and 0.1M HCl.Take by weighing the tobacco sample of equivalent,, come the extraction effect of three kinds of solution of comparison by contrast amino acid chromatographic peak area.By table 3 as seen, the amino acid content of 80% alcohol extract is minimum, and several amino acids content such as the Val of 70% methanol extraction, His, Tyr, Lys, Phe and Try all are starkly lower than uses 0.1M HCl to extract.Relatively comprehensive, the extraction effect of the hydrochloric acid solution of 0.1M is better.
The different extraction effect contrasts of extracting solution of table 3
3.3.3 the extraction effect of the HCl solution of variable concentrations
Take by weighing equivalent offal sample, the extraction effect that contrast variable concentrations HCl extracts solution sees Table 4.0.1M HCl is relatively poor to the extraction effect of Ser, Glu, Lys and Arg, the extraction effect to Lys and Arg of 0.05MHCl is all bad.0.03M HCl obviously increases the extraction effect of Lys.Relatively comprehensive, select for use 0.03MHCl as extracting solution.
The extraction effect contrast of the HCl solution of table 4 variable concentrations
3.3.4 the extraction time
Take by weighing the 0.1g offal, add the 20mL extraction solution, under ultrasonic concussion, extract 15min, 30min, 45min and 60min respectively, cross the laggard LC-MS of 0.2 μ m filter membrane and analyze.Each sample advances two pins, and peak area is averaged.Experiment finds that the extraction time of 15min can reach extraction effect.Because sample weighting amount is less, the extraction solvent amount substantially exceeds sample weighting amount, and amino acid can be dissolved in hydrochloric acid solution faster, therefore can reach dissolution equilibrium faster.
3.4 method validation
20 kinds of amino acid whose content in tobacco to be measured differ greatly, and what have is lower than detectability, and as Cys, the then content that has is up to more than 0.2%, as Pro.In order to measure 20 seed amino acids simultaneously, the typical curve of two kinds of variable concentrations series has been set up in experiment, i.e. the typical curve that the higher Pro of content and Asn are set up a kind of concentration series is set up the typical curve of another kind of concentration series to other amino acid.Like this, a sample single injected sampling just can draw all amino acid whose test datas, reaches to save time the purpose of raising the efficiency.
The detection limit of method is determined with S/N=3 by the amino acid standard model of practical measurement series low concentration.The method detection limit is good, between 0.01～0.05 μ M (S/N=3), and related coefficient (r
2) all greater than 0.9977, precision is between 0.78～4.93RSD%.The recovery of method is determined in experiment by standard addition method.Accurately take by weighing offal 204.09mg, extract, replicate determination 5 times, the foundation of averaging and calculating as the recovery with the 20mL extract.Take by weighing three parts of offals then, be respectively 191.60mg, 209.80mg and 159.33mg, the sample treatment according to 2.1, mark-on makes mark-on concentration be respectively 1 μ M, 10 μ M and 20 μ M respectively.Wherein, content is lower than the amino acid of 1 μ M, only carries out 1 μ M mark-on and reclaim experiment, content is lower than the amino acid of 1.5 μ M, carry out the experiment of 1 μ M and 10 μ M mark-ons,, carry out the experiment of 10 μ M and 20 μ M mark-ons for content higher Asn and Pro.Between 81～108%, the recovery of Ile and Arg is lower substantially for the amino acid recovery, is respectively 75% and 53%.The detection limit of 20 seed amino acids, quantitative limit, the range of linearity, related coefficient, precision and the recovery see Table 5.
The detection limit of table 520 seed amino acid (LOD), the range of linearity, related coefficient (r
2), precision
Claims (9)
1, a kind of method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously is characterized in that adopting liquid chromatography and esi-msn coupling, may further comprise the steps:
(1) tobacco sample obtains extract through pre-treatment;
(2) extract on reverse-phase chromatographic column with binary eluent gradient wash-out;
(3) typical curve of two kinds of variable concentrations series of making adopts liquid chromatography-electron spray ion trap tandem mass spectrometry to carry out the analyzing and testing of 20 seed amino acids in the tobacco under positive ion mode.
2,, it is characterized in that the described pre-treatment of step (1) comprises that it is extraction solution as interior target HCl solution that employing contains norvaline, extracts offal under ultrasonic concussion according to the described method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously of claim 1; Extract is filtered.
3, according to the described method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously of claim 2, the ratio that it is characterized in that described extraction offal and extraction solution is 0.1～0.2g:20ml; The extraction time is 15min.
4, according to the described method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously of claim 2, it is characterized in that described HCl solution concentration is 0.03～0.1M, preferred concentration is 0.03M.
5,, it is characterized in that described filtration is to adopt 0.2 μ m membrane filtration according to the described method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously of claim 2.
6, according to the described method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously of claim 1, it is characterized in that the described reverse-phase chromatographic column of step (2) is HyPURITY C
18Described binary moving phase is 99% water-1% acetonitrile-0.1% 9 fluorine valeric acid and 10% water-90% acetonitrile-0.1% 9 fluorine valeric acid.
7,, it is characterized in that the typical curve of two kinds of variable concentrations series of the described making of step (3) is according to the described method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously of claim 1:
(1) mark norvaline concentration was the amino-acid mixed and standard solution I of 1.7 μ M in preparation contained, and wherein the concentration of aspartic acid, serine, glutamine, threonine, glutamic acid, cystine, γ-An Jidingsuan, nipecotic acid, valine, dl-methionine, histidine, tyrosine, lysine, isoleucine, arginine, leucine, phenylalanine and tryptophane is the 0.03M HCl solution of 50 μ M;
(2) mark norvaline concentration was the amino-acid mixed and standard solution II of 1.7 μ M in preparation contained, and wherein the concentration of proline and asparagine is respectively the 0.03M HCl solution of 500 μ M;
(3) be that the 0.03M HCl of 1.7 μ M is made into series concentration to standard solution I and standard solution II, the production standard curve with containing norvaline concentration.
8, according to the described method of analyzing tobacco and goods 20 seed amino acids thereof simultaneously of claim 1, it is characterized in that the condition determination of the described electron spray ion trap tandem mass spectrometry of step (3) is: nitrogen pressure: 0.64MPa; Helium pressure: 0.38MPa; Polarity: positive ionization; Scanning form: full scan; Acquisition time: 40min; Activationary time: 30ms; Sweep gas: 0LTQ unit; Spray voltage: 5kV; Capillary temperature: 350 ℃; Sweep limit: standard; Sweep speed: standard; Segmentation detects.
9, the mass spectrometric analysis method of described according to Claim 8 tobacco and product amino acid thereof, it is characterized in that described segmentation is to be divided into 4 sections, wherein the sheath gas of the 3rd segmentation and auxiliary gas are respectively 38LTQ unit and 0LTQ unit, and the sheath gas and the auxiliary gas of all the other segmentations are respectively 46LTQ unit and 19LTQ unit.
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