CN102798683A - Universal total-component quantitative analysis method of gas chromatography-mass spectrometry - Google Patents

Abstract

The invention discloses a universal total-component quantitative analysis method of gas chromatography-mass spectrometry. Firstly, the method employs a gas chromatography-mass spectrometry (GC-MS, full scan mode) method to obtain data information of to-be-analyzed samples, performs mass spectral de-convolution and peak identification on obtained full-scan information with an automated mass spectral deconvolution and identification system (AMDIS), extracts mass spectral information from an original data file according to results of peak identification, calculates characteristic values of compound ions, chooses characteristic ions of the compound, and finally establishes selected ion monitoring quantitative analysis method with the characteristic ions. A core of the method lies in choosing characteristic ions of the compound through the characteristic values of the compound ions. The method can give a plurality of characteristic ions and characteristic values to characteristic evaluation indexes thereof. The given characteristic ions can be used as quantitative ions and quantitative candidate ions.

Description

A kind of general gas chromatography-mass spectrum full constituent quantitative analysis method

Technical field

The present invention relates to analytical chemistry and metabolism group field, be a kind of based on gas chromatography-mass spectrum to all are identified the method that compound carries out quantitative test in the COMPLEX MIXED system.

Background technology

Gas chromatography-mass spectrography (GC-MS) combines the separating power and the mass spectral qualitative function of chromatogram, can realize the qualitative and quantitative analysis to complex mixture.With chromatogram mass spectrum quantitative test complex mixture the time, usually need confirm characteristic ion.Compound characteristic ion under the GC-MS full scan pattern is meant the ms fragment ion of certain target compound, and this ion only is present in this compound, and is not present in (be have on the chromatographic retention overlapping) other compound that flows out altogether with it.If there is not common outflow compound in certain compound, can think that then each fragmention of this compound all is a characteristic ion.If compound exists a plurality of compounds (chromatographic peak is overlapping serious) that flow out altogether perhaps to have the constitutional isomer that flows out altogether, then possibly there is not characteristic ion in this compound.

Characteristic ion has very big effect to the compound qualitative, quantitative based on GC-MS.Compound qualitative aspect, people can carry out the retrieval in mass spectrogram storehouse with peeling off out from overlapping chromatographic peak with the identical fragmention of characteristic ion retention time, thereby realize that compound is qualitative.Aspect the compound quantitative test, characteristic ion can be used as the quota ion under the full scan pattern.Characteristic ion is that certain compound institute is peculiar in the common outflow compound, so use characteristic ion quantitative integration, can avoid overlap peak to quantitative interference.Characteristic ion can also select to gather ion as quantitative data under ion scan (SIM) pattern.Because data that adopt to select the ion detection pattern to obtain have higher sensitivity than full scan pattern, so characteristic ion receives publicity as the effect of SIM ion more.

In recent years, in the research of metabolism group and traditional Chinese medicine fingerprint etc., the chemical fingerprint method that system's multianalysis sample is formed has obtained development fast and has used widely.These methods will be carried out qualitative and quantitative analysis to all compounds of being analyzed (full constituent) usually.And the selection of characteristic ion is very important to the full constituent chemical composition analysis based on GC-MS.The most general pattern of compound ionization was electron impact ionization (EI) pattern during GC-MS analyzed.Under this pattern, the fragmention number that each compound obtains has tens, even hundreds of is individual, and the characteristic ion of searching overlap peak in the chemical fingerprint with manual mode hardly maybe.

Commercially available software LECO ChromaTOF chromatogram mass spectrum workstation has provided it and has analyzed the characteristic ion of overlap peak in the data.But this software is not described its characteristic.Actual conditions lower part eluting peak altogether is not have characteristic ion, but this software can be identified the characteristic ion in peak to all.In addition, generally this software can not be used for analyzing the GC-MS raw data that other workstation produces.

The present invention is a universal method, is applicable to the data that different GC-MS instruments are gathered.Through calculating the eigenwert that chromatogram flows out the compound ions fragment, flow out the characteristic of ion more altogether, filter out the characteristic ion of compound.This method also provides the eigenwert that is filtered out characteristic ion simultaneously, is used to estimate the characteristic of this ion.These characteristic ions finally are used for GC-MS being, and the quota ion that the full constituent on basis analyzes is selected, or is used for selecting based on GC-MS the foundation of " full constituent analysis " method of ion detection.

Summary of the invention

The objective of the invention is to set up a kind of full constituent quantitative analysis method based on gas chromatography-mass spectrum.The core technology of this method is choosing of compound characteristic quota ion.With respect to present commercially available software LECO ChromaTOF; This method has highly versatile (every GC-MS raw data that can convert the CDF form to can both be used this method); Characteristic ion characteristic clear and definite (weighing with eigenwert) can provide characteristics such as several characteristic ion and eigenwert thereof simultaneously.

The technical scheme that the present invention adopts is following:

A kind of full constituent quantitative analysis method based on gas chromatography-mass spectrum; Adopt GC-MS full scan mode to gather the full scan raw data; Utilize AMDIS software that analyzing samples is carried out peak identification; It is selected with characteristic ion then the chromatographic peak that is extracted to be divided into groups, and last use characteristic ion is set up GC-MS and selected ion scan full constituent analytical approach.

Concrete steps are following,

1) GC-MS chemical fingerprint Data Acquisition: to the characteristics of carrying out full constituent analytic sample (being human serum sample and tobacco sample among this patent embodiment); Treat analyzing samples and carry out corresponding sample preparation, the full scan compartment analysis of the promoting the circulation of qi phase of going forward side by side chromatography-mass spectroscopy.

2) full scan chromatogram peak identification: the analyzing samples raw data file (CDF form) that obtains is imported AMDIS software (v.2.62 or other version all can), carry out peak identification.Deconvolute with the peak identification parameter be: component width setting value is 20, adjacent peak subtraction, resolution, sensitivity, parameters such as shape requirement are set to two respectively, high, low, medium etc.The peak recognition result is checked, removed the repetition recognition result.

3) divide into groups: from AMDIS analysis result file, extract the reservation information (comprising the summit time, the analyzing spot information of initial, summit, end point) of the chromatographic peak of discerning, and utilize this information to divide into groups.Group forming criterion is for to be divided into one group with adjacent one another are and overlapping compound (flowing out compound altogether).The concrete operations of dividing into groups are following: seeks the time window that last chromatographic peak concluding time between two adjacent chromatographic peaks and a back chromatographic peak zero-time differ by more than 0.1min (can revise according to actual conditions).Packet node is defined as:

Ti+1＝Tend x+(Tini x+1-Tend x)×0.5

In the formula, Ti+1 is the zero-time (also being the concluding time of i group) of i+1 group, and Tend x is the concluding time of x chromatographic peak, and Tini x+1 is the zero-time of x+1 chromatographic peak.

4) selection of characteristic ion:, from the CDF raw data, extract the mass spectrogram quantitative information of all chromatographic peak summits according to the reservation information that is identified compound (comprising the summit time, the analyzing spot information of initial, summit, end point).If certain compound is eluting peak altogether, then choose absolute value less than the maximum mass spectrum ion of 3 abundance of threshold value (Agilent 5975 instruments select 100000 usually) as quota ion.For there being certain chromatographic peak that flows out altogether, compare the adjacent mass spectrum quantitative information that flows out the chromatographic peak summit altogether of its summit with front and back, calculate the eigenwert (can represent the value of this ion characteristic property) of ion, find out its corresponding characteristic mass spectrum ion.The specific algorithm of eigenwert is following:

The first step: chromatographic peak summit ms fragment strength information to be calculated is sorted, filter out the ms fragment of 10% before the intensity ordering, as candidate's eigenvalue calculation ion.More weak fragmention is not counted in the eigenvalue calculation scope because measuring error own is bigger.

Second step: computation of characteristic values.

Computation of characteristic values is the prerequisite of carrying out the characteristic ion screening accurately.For the mass chromatogram of certain ion channel m, have the list of feature values of the chromatographic peak a of overlap peak b interference to be shown:

$S_a^m=\frac{I_a^m-I_b^m}{I_a^m}=1-\frac{I_b^m}{I_a^m}---\left(1\right)$

is the eigenwert of chromatographic peak a in ion channel m in the formula;

is the mass spectrum response intensity of peak a when its retention time ,

be the mass spectrum response intensity of Interference Peaks b when its retention time . can pass through formula (2-3) and calculate:

$I_a^m=I_{t_{R_a}}^m-I_{b_{t_{R_a}}}^m-I_{\mathrm{baseline}}^m---\left(2\right)$

$I_b^m=I_{t_{R_b}}^m-I_{a_{t_{R_b}}}^m-I_{\mathrm{baseline}}^m---\left(3\right)$

With formula 2,3 substitution formula 1, obtain

$S_a^m=\frac{I_{t_{R_a}}^m-I_{b_{t_{R_a}}}^m-I_{t_{R_b}}^m+I_{a_{t_{R_b}}}^m}{I_{t_{R_a}}^m-I_{b_{t_{R_a}}}^m-I_{\mathrm{baseline}}^m}---\left(4\right)$

In the formula

For peak b at retention time t _RaThe time the mass spectrum response intensity,

For peak a at retention time t _RbThe time the mass spectrum response intensity.

and

these two variablees can not directly obtain from the GC-MS raw data, can obtain through the analog computation to chromatographic peak.

is the mass spectrum response intensity of baseline.

Under the ideal situation, the chromatogram eluting peak can be represented (accompanying drawing 1) with gauss of distribution function, and available mathematical expression is represented as follows:

$I\left(t\right)=I_{t_R}\×e^{-0.5{\left(\frac{t-t_R}{o}\right)}^2}---\left(5\right)$

I in the following formula (t) is the compound mass spectrum response intensity when time t,

For compound at its retention time t _RThe mass spectrum response intensity, t _RBe chromatographic retention, σ is the standard deviation of chromatographic peak.Because under the actual conditions, chromatogram peak-to-peak type is not complete symmetry, the front end of chromatographic peak can be modeled as:

$I\left(t\right)=I_{t_R}\×e^{-4.5{\left(\frac{t-t_R}{t_{R_i}-t_R}\right)}^2}---\left(6\right)$

is the zero-time of chromatographic peak in the formula.The rear end of chromatographic peak can be modeled as:

$I\left(t\right)=I_{t_R}\×e^{-4.5{\left(\frac{t-t_R}{t_{R_t}-t_R}\right)}^2}---\left(7\right)$

is the concluding time of chromatographic peak in the formula.At ion channel m; For overlapping chromatographic peak a and b, then but approximate treatment be:

$I_{a_{t_{R_b}}}^m\&ap;I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{a_t}}-t_{R_a}}\right)}^2}---\left(8\right)(t_{R_a}<t_{R_b})$

$I_{a_{t_{R_b}}}^m\&ap;I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_a}-t_{R_b}}{t_{R_{a_i}}-t_{R_a}}\right)}^2}---\left(9\right)(t_{R_a}>t_{R_b})$

In like manner,

but approximate treatment be:

$I_{b_{t_{R_a}}}^m\&ap;I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{b_i}}-t_{R_b}}\right)}^2}---\left(10\right)(t_{R_a}<t_{R_b})$

$I_{b_{t_{R_a}}}^m\&ap;I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_a}-t_{R_b}}{t_{R_{b_t}}-t_{R_b}}\right)}^2}---\left(11\right)(t_{R_a}>t_{R_b})$

Mass chromatogram for certain ion channel m; According to Interference Peaks with investigate peak retention time relation, the calculating of

can be divided into Interference Peaks before investigating the peak, Interference Peaks after investigating the peak, Interference Peaks is present in the front-end and back-end of investigating the peak simultaneously, do not have four kinds of situation such as Interference Peaks.

First kind of situation: Interference Peaks is before investigating the peak (

sees accompanying drawing 2).Overlapping situation according to the peak starting point between investigation peak a and the Interference Peaks b, summit, end point etc.; With formula 8,10 substitution formula 4, the eigenwert

that gets peak a monic side at the peak is:

$S_{a_i}^m\&ap;1-\frac{I_{t_{R_b}}^m-I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{a_i}}-t_{R_a}}\right)}^2}}{I_{t_{R_a}}^m-I_{t_{R_b}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{b_t}}-t_{R_b}}\right)}^2}}---\left(12\right)$

Because formula 8～11 is an approximate formula; What simultaneously formula 12 had carried out also that baseline value ignores is approximate, these approximate treatment meetings at

perhaps

value than hour bringing than mistake.At this moment; The calculated value of second molecule of formula 12 possibly occur less than 0 the time; This be since influence and the peak a of baseline to not in full conformity with the Gaussian peak model; The calculated value that makes

is bigger slightly than actual value; And under this ion channel; Peak b is in just not response; Promptly

equals 0, and this ion is the characteristic ion of the relative peak b of peak a.Therefore; Give second molecule assignment this moment is 0;

calculated value is 1, and m is the eigenwert of the relative b of a.

In like manner; As

hour; Denominator in second also may occur less than 0 situation,

has serious interference much larger than

peak b to peak a at this moment.Therefore remove in the denominator second;

calculated value is less than 0, and m is not the characteristic ion of the relative peak b of peak a.

Second kind of situation: Interference Peaks is after investigating the peak (

sees accompanying drawing 3).Overlapping situation according to the peak starting point between investigation peak a and the Interference Peaks b, summit, end point etc.; With formula 9,11 substitution formula 4, must peak a be in the eigenwert

of tail of the peak one side:

$S_{a_t}^m\&ap;1-\frac{I_{t_{R_b}}^m-I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{a_t}}-t_{R_a}}\right)}^2}}{I_{t_{R_a}}^m-I_{t_{R_b}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{b_i}}-t_{R_b}}\right)}^2}}---\left(13\right)$

Formula 13 is done the regulation of formula 12 equally, and promptly second molecular computing value was less than 0 o'clock, and assignment is 0.The denominator calculated value omitted second of denominator less than 0 o'clock.

The third situation: Interference Peaks is present in the front-end and back-end of investigating the peak simultaneously.Also calculated

and

value.

The 4th kind of situation: do not have Interference Peaks.Characteristic ion is defined as the peak response ion of absolute value less than threshold value.The purpose that limits threshold value is that the ion that prevents high concentration (response is big) transships on detecting device.For Agilent5975 GC-MS instrument, this value can be made as 100000.

In the 3rd step, eigenwert ordering and characteristic ion are confirmed.The eigenwert that calculates is sorted, and (eigenwert is more near 1, and then this ion characteristic property is good more, and this is worth visual concrete condition and changes greater than 0.8 to screen and list eigenwert.When having

and

simultaneously, according to both average orderings) ion and response intensity on mass spectrum and eigenwert.The selected characteristic value sorts forward ion as characteristic ion.

5) selected characteristic ion is enrolled in the gas chromatography-mass spectrum selectivity ion scan method, carry out quantitative data collection and quantitative test.

The effect that the present invention has is: a kind of full constituent quantitative analysis method based on gas chromatography-mass spectrum is provided.

Because this method is based on universal data layout (CDF), this method does not rely on different GC-MS instrument workstations, can handle the GC-MS data that all can convert the CDF form to.For example the workstation data of instruments such as Agilent 5975, Shimadzu 2010, LECO Pegasus 4D can convert the CDF form to.

Because this method is given characteristic ion and eigenwert thereof simultaneously, with respect to the given characteristic ion of commercially available software LECOChromaTOF workstation, the characteristic ion characteristic that this method obtained is obvious.Because LECO ChromaTOF workstation has just provided all and has been identified the characteristic ion of compound.And in fact some has the overlapping isomers compound of identical mass spectrogram and chromatogram top and not has characteristic ion.Our in this case rule can not provide characteristic ion.For the characteristic ion that provides, this method provides its eigenwert simultaneously, the quality of handled easily person judging characteristic property.

Because this method is according to eigenwert and response intensity ordering; Provide all characteristic ions and eigenwert thereof simultaneously, the characteristic ion that this method obtains can be used for quota ion and the auxiliary quota ion based on a plurality of compounds of complex sample of selecting ion detection are measured simultaneously or full constituent is analyzed.

Description of drawings

The Gaussian distribution simulation synoptic diagram of Fig. 1 chromatographic peak;

Fig. 2 is total to the synoptic diagram of the retention time of eluting peak b less than peak a retention time;

Fig. 3 is total to the synoptic diagram of the retention time of eluting peak b greater than the peak retention time of peak a;

Fig. 4 human serum sample's GC-MS full scan finger-print;

The GC-MS full scan finger-print of Fig. 5 flue-cured tobacco sample;

The chromatogram that has similar mass spectrogram in Fig. 6 actual sample is total to eluting peak.

Embodiment

Embodiment 1

1. sample

This example is sample with the human serum.Blood serum sample is provided by Dalian City No.6 People's Hospital.

2. analytical approach

2.1 the collection of sample TIC

(1) sample pretreatment: with 100 μ L serum after room temperature is thawed; Successively add 400 μ L methyl alcohol and 20 μ L inner mark solutions (methanol solution of sorbic acid, tridecylic acid and nonadecanoic acid is 50 μ g/mL), vortex 30s; Centrifugal (15000g) 15min under 4 ℃ gets 370 μ L supernatant freeze-drying.In the freeze-drying sample, add 50 μ L pyridines and 100 μ L BSTFA (1%TMCS), 30min derives under 80 ℃ of water-baths.The sample sample introduction analysis in 24h of deriving.

(2) GC-MS analysis condition: chromatographic column: DB-5 (30m * 250 μ m * 0.25 μ m, J& W Scientific, Folsom, CA), carrier gas: helium (99.9995%), constant current mode, flow velocity 1.2mL/min.Sampling volume: 2 μ L, injector temperature: 290 ℃, transmission line: 280 ℃.Heating schedule: 70 ℃ of initial temperatures (keeping 3min) rise to 320 ℃ (keeping 5min) with the speed of 10 ℃/min.230 ℃ of ion source temperatures, detector voltage: 1600V.Solvent 430s clipping time, scanning of the mass spectrum scope: 33-600, sweep speed: 5Hz

The laboratory sample chromatogram is seen accompanying drawing 4.

2.2 peak identification

The analyzing samples raw data file (CDF form) that obtains is imported AMDIS software (v.2.62 or other version all can), carry out peak identification.The parameter of deconvoluting is: component width setting value is 20, adjacent peak subtraction, and resolution, sensitivity, parameters such as shape requirement are set to two respectively, high, low, medium etc.Operation AMDIS obtains the peak recognition result.Deletion repetition recognition result finally confirms to identify 275 compounds.

2.3 overlap peak is confirmed (grouping)

From AMDIS analysis result file, extract the reservation information (comprising the summit time, the analyzing spot information of initial, summit, end point) of the chromatographic peak of discerning, and utilize this information to divide into groups.Group forming criterion is for to be divided into one group with adjacent one another are and overlapping compound (flowing out compound altogether).The concrete operations of dividing into groups are following: seeks the time window that last chromatographic peak concluding time between two adjacent chromatographic peaks and a back chromatographic peak zero-time differ by more than 0.1min (can revise according to actual conditions).Packet node is defined as:

Ti+1＝Tend x+(Tini x+1-Tend x)×0.5

In the formula, Ti+1 is the zero-time (also being the concluding time of i group) of i+1 group, and Tend x is the concluding time of x chromatographic peak, and Tini x+1 is the zero-time of x+1 chromatographic peak.

The purpose of dividing into groups is to specify to flow out chromatographic peak altogether, is used for characteristic ion and searches.This example extracts 109 compounds existence altogether and flows out phenomenon altogether.

2.4 the calculating of eigenwert and characteristic ion extract

After confirm dividing into groups, according to the reservation information that is identified compound (comprising the summit time, the analyzing spot information of initial, summit, end point), the mass spectrum strength information of chromatographic peak in the extraction group from the CDF raw data.For certain specific chromatographic peak, compare the mass spectrum strength information of the adjacent chromatographic peak summit of its summit with front and back, calculate the eigenwert of ms fragment, and filter out characteristic ion, as the quota ion of quantitative test through eigenwert.If certain compound is eluting peak altogether, then choose absolute value less than the maximum mass spectrum ion of 3 abundance of threshold value (selecting 100000 usually) as quota ion.For there being the chromatographic peak that flows out phenomenon altogether, the eigenwert of its fragmention adopts following formula to calculate:

$S_{a_i}^m\&ap;1-\frac{I_{t_{R_b}}^m-I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{a_i}}-t_{R_a}}\right)}^2}}{I_{t_{R_a}}^m-I_{t_{R_b}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{b_t}}-t_{R_b}}\right)}^2}}(t_{R_a}>t_{R_b})---\left(1\right)$

$S_{a_t}^m\&ap;1-\frac{I_{t_{R_b}}^m-I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{a_t}}-t_{R_a}}\right)}^2}}{I_{t_{R_a}}^m-I_{t_{R_b}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{b_i}}-t_{R_b}}\right)}^2}}(t_{R_a}<t_{R_b})---\left(2\right)$

In the formula; and is respectively the eigenwert of and the tail of the peak side first at the peak of chromatographic peak a among the ion channel m, and

and

represents the summit mass spectrum response intensity of peak a and peak b among the ion channel m.

respectively, peak a and peak b start and end vertices and retention time.Eigenwert is exported greater than 0.8 ion, as characteristic ion.Each compound is chosen two characteristic ions (subsequent use).

2.5 compare with the characteristic ion result of calculation of commercially available software LECO ChromaTOF

The GC-TOFMS data that experiment is obtained are carried out characteristic ion calculating and result's output with LECO workstation software ChromaTOF and this patent method respectively, and institute's result calculated is compared.The result finds, exists in the overlapping compound at 109 that are investigated, and the characteristic ion that adopts ChromaTOF software and this method to obtain all has 106 to be correct.In two kinds of methods, the wrongheaded compound of characteristic ion has nothing in common with each other.But be response weak (absolute strength is less than 2000), the ion that baseline interference is strong.

Embodiment 2

1. sample collecting:

This example is an example with the flue-cured tobacco sample.Gather flue-cured tobacco middle part tobacco leaf sample, 40 ℃ are baked to constant weight.Tobacco leaf is removed stalk respectively, pulverizes, cross 40 mesh sieves, seal up for safekeeping, be stored in 4 ℃ of refrigerators with self-styled polybag.

2. analytical approach

2.1 the collection of sample TIC

(1) extraction and concentrate: accurate weighing 4.00g offal (crossing 40 mesh sieves), join 11mL stainless steel abstraction pool, add 200 μ L inner mark solutions (the 2-methylnaphthalene, 2mg/mL).Abstraction pool and receiving bottle are loaded onto the accelerated solvent extraction pond.Extraction solvent, methylene chloride.100 ℃ of extraction temperature, extracting pressure 1500psi, extraction cycle number of times 3 times, 5 minutes static extracting time, abstraction pool volume 11mL.Be concentrated into 1mL under the extract normal pressure.

(2) chromatographic condition: chromatographic column is DB-5MS (30m * 0.25mm * 0.25 μ m).The temperature programme condition: 50 ℃ (stopping 1min), 8 ℃/min rises to 220 ℃ (stopping 7min), and 10 ℃/min rises to 280 ℃ (stopping 20min).Carrier gas, helium.Flow velocity, 1.2mL/min.Sample size, 1 μ L.

(3) mass spectrum condition: electron impact ionization (EI); Ion source temperature, 230 ℃; Detector voltage: 1047V.Scanning of the mass spectrum scope: 33-500

The laboratory sample chromatogram is seen accompanying drawing 5.

2.2 peak identification

The analyzing samples raw data file (CDF form) that obtains is imported AMDIS software (v.2.62 or other version all can), carry out peak identification.The parameter of deconvoluting is: component width setting value is 20, adjacent peak subtraction, and resolution, sensitivity, parameters such as shape requirement are set to two respectively, high, low, medium etc.Operation AMDIS obtains the peak recognition result.Deletion repetition recognition result finally confirms to identify 331 compounds.

2.3 overlap peak is confirmed (grouping)

From AMDIS analysis result file, extract the reservation information (comprising the summit time, the analyzing spot information of initial, summit, end point) of the chromatographic peak of discerning, and utilize this information to divide into groups.Group forming criterion is for to be divided into one group with adjacent one another are and overlapping compound (flowing out compound altogether).The concrete operations of dividing into groups are following: seeks the time window that last chromatographic peak concluding time between two adjacent chromatographic peaks and a back chromatographic peak zero-time differ by more than 0.1min (can revise according to actual conditions).Packet node is defined as:

Ti+1＝Tend x+(Tini x+1-Tend x)×0.5

In the formula, Ti+1 is the zero-time (also being the concluding time of i group) of i+1 group, and Tend x is the concluding time of x chromatographic peak, and Tini x+1 is the zero-time of x+1 chromatographic peak.

Overlap peak is added up, and this example extracts 151 compounds existence altogether and flows out phenomenon altogether.

2.4 the calculating of eigenwert and characteristic ion extract

After confirm dividing into groups, according to the reservation information that is identified compound (comprising the summit time, the analyzing spot information of initial, summit, end point), the mass spectrum strength information of chromatographic peak in the extraction group from the CDF raw data.For certain specific chromatographic peak, compare the mass spectrum strength information of the adjacent chromatographic peak summit of its summit with front and back, calculate the eigenwert of ms fragment, and filter out characteristic ion, as the quota ion of quantitative test through eigenwert.If certain compound is eluting peak altogether, then choose absolute value less than the maximum mass spectrum ion of 3 abundance of threshold value (selecting 100000 usually) as quota ion.For there being the chromatographic peak that flows out phenomenon altogether, the eigenwert of its fragmention adopts following formula to calculate:

$S_{a_i}^m\&ap;1-\frac{I_{t_{R_b}}^m-I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{a_i}}-t_{R_a}}\right)}^2}}{I_{t_{R_a}}^m-I_{t_{R_b}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{b_t}}-t_{R_b}}\right)}^2}}(t_{R_a}>t_{R_b})---\left(1\right)$

$S_{a_t}^m\&ap;1-\frac{I_{t_{R_b}}^m-I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{a_t}}-t_{R_a}}\right)}^2}}{I_{t_{R_a}}^m-I_{t_{R_b}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{b_i}}-t_{R_b}}\right)}^2}}(t_{R_a}<t_{R_b})---\left(2\right)$

In the formula;

and

is respectively the eigenwert of and the tail of the peak side first at the peak of chromatographic peak a among the ion channel m, and

and

represents the summit mass spectrum response intensity of peak a and peak b among the ion channel m.

respectively, peak a and peak b start and end vertices and retention time.Eigenwert is exported greater than 0.8 ion, as characteristic ion.Each compound is chosen two characteristic ions (subsequent use).

2.5 compare with the characteristic ion result of calculation of commercially available software LECO ChromaTOF

Similar with embodiment 1.The GC-TOFMS data that experiment is obtained are carried out characteristic ion calculating and result's output with LECO workstation software ChromaTOF and this patent method respectively, and institute's result calculated is compared.The result finds, exists in the overlapping chromatographic peak at 151 that are investigated, and it is correct (promptly eluting peak does not have overlapping together) that the characteristic ion that adopts ChromaTOF software to obtain has 107 characteristic ions, and the characteristic ion of 44 erroneous judgements is arranged in addition.Adopting the employed method of this patent that 125 characteristic ions are arranged is correct (comprising that 26 compound right judgement are not for existing characteristic ion), and the characteristic ion of 13 erroneous judgements is arranged, and 13 are judged as by error and do not have characteristic ion.Finally, the accuracy rate that ChromaTOF software features ion is judged is 70.9%, and the accuracy rate that this method is judged is 82.8%.The reason that ChromaTOF software accuracy rate is lower is it and has also given characteristic ion for the compound (this example is 26, accompanying drawing 6) that does not have characteristic ion.The method that this patent adopts is then directly judged some compound and is not had characteristic ion, though also can there be some mistakes (this example has 13 compounds) in the process of this judgement.

In a word, the present invention has provided a kind of full constituent quantitative analysis method of general gas chromatography-mass spectrum.This method is applicable to the quantitative test of complex mixture.For the relative simple sample of composition (like embodiment 1) of analytic target, the effect of the characteristic ion that this method is chosen and present only commercially available software ChromaTOF is suitable; For forming the result that characteristic ion that relatively more complicated this method of analytic target (like embodiment 2) chooses is superior to present commercially available software.In addition, compare with commercially available software, this method has versatility, is applicable to the data from different GC-MS analytical instrument.

Claims (4)

1. general gas chromatography-mass spectrum full constituent quantitative analysis method is characterized in that:

Adopt gas chromatography-mass spectrum full scan mode to obtain the raw data of sample to be analyzed; Utilize mass spectrum to deconvolute automatically and peak recognition system (AMIDS, Automated Mass Spectral Deconvolutionand Identification System) is carried out peak identification to gas chromatography-mass spectrum full scan data; It is selected then the chromatographic peak of being discerned to be carried out eigenvalue calculation and characteristic ion, and the most selected characteristic ion is used for the full constituent quantitative test.

2. according to the said method of claim 1, it is characterized in that: the concrete steps that described gas chromatography-mass spectrum full scan chromatogram carries out peak identification are following,

1) gas chromatography-mass spectrum full scan Data Acquisition:, treat analyzing samples and carry out corresponding sample preparation, the full scan compartment analysis of the promoting the circulation of qi phase of going forward side by side chromatography-mass spectroscopy to the characteristics of carrying out the full constituent analytic sample;

2) the gas chromatography-mass spectrum full scan chromatogram peak of sample identification: the gas chromatography-mass spectrum full scan raw data that obtains is changed into the CDF form, import AMDIS software, the AMDIS software version is 2.62 or higher; Deconvolute and chromatographic peak identification, the peak identification parameter that deconvolutes is: component width setting value is 20, adjacent peak subtraction; Resolution; Sensitivity, parameters such as shaperequirement are set to two respectively, high; Low, medium etc.; The peak recognition result is checked, removed the repetition recognition result.

3. according to the said method of claim 1, it is characterized in that: the chromatographic peak to identifying divides into groups, and chromatographic peak in organizing is carried out adjacent peak-to-peak characteristic ion search, and concrete steps are following:

1) divide into groups: from AMDIS analysis result file, extract the reservation information of the chromatographic peak of discerning, reservation information comprises the summit time, the analyzing spot information of initial, summit, end point, and utilize this information to divide into groups;

Group forming criterion is for to be divided into one group with adjacent one another are and overlapping compound (flowing out compound altogether);

The concrete operations of dividing into groups are for seeking the time window that last chromatographic peak concluding time and back one chromatographic peak zero-time between two adjacent chromatographic peaks differ by more than 0.1min, and time phase difference also can be made amendment by technological general knowledge according to actual conditions;

Packet node is defined as:

T _i+1＝T _end x+(T _{ini x+1}-T _{end x})×0.5

In the formula, T _I+1Being the zero-time of i+1 group, also is the concluding time of i group; T _{End x}Be the concluding time of x chromatographic peak, T _{Ini x+1}It is the zero-time of x+1 chromatographic peak;

2) selection of characteristic ion in the group: after confirming to divide into groups, according to the reservation information that is identified compound, reservation information comprises the summit time, the analyzing spot information of initial, summit, end point, the mass spectrum strength information of chromatographic peak in the extraction group from the CDF raw data;

For certain specific chromatographic peak, compare the mass spectrum strength information of the adjacent chromatographic peak summit of its summit with front and back, calculate the eigenwert of ms fragment, and filter out characteristic ion, as the quota ion of quantitative test through eigenwert; If certain compound is eluting peak altogether, then choose absolute value less than the maximum mass spectrum ion of 3 abundance of threshold value (Agilent 5975 instruments select 100000 usually) as quota ion;

In there being the chromatographic peak that flows out phenomenon altogether, the eigenwert of its fragmention adopts following formula to calculate:

$S_{a_i}^m\&ap;1-\frac{I_{t_{R_b}}^m-I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{a_i}}-t_{R_a}}\right)}^2}}{I_{t_{R_a}}^m-I_{t_{R_b}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{b_t}}-t_{R_b}}\right)}^2}}(t_{R_a}>t_{R_b})---\left(1\right)$

$S_{a_t}^m\&ap;1-\frac{I_{t_{R_b}}^m-I_{t_{R_a}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{a_t}}-t_{R_a}}\right)}^2}}{I_{t_{R_a}}^m-I_{t_{R_b}}^m\&times;e^{-4.5{\left(\frac{t_{R_b}-t_{R_a}}{t_{R_{b_i}}-t_{R_b}}\right)}^2}}(t_{R_a}<t_{R_b})---\left(2\right)$

Where, and

, respectively peak ion channel m in the first peak and peak in a caudal eigenvalues, and

represents the peak ion channel m a and b, the peak intensity of the peak mass response;

respectively, peak a and peak b start and end vertices and retention time; the eigenvalues greater than 0.8 ion output, as the characteristic ions; Select two characteristic ions for each compound, one spare.

4. according to the said method of claim 1, it is characterized in that: with the quantitative test ion of the characteristic ion of choosing, enroll gas chromatography-mass spectrum and select the ion database, carry out the quantitative test of compound full constituent as compound.

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