CN110082422B - Method for measuring internal energy difference of monovalent positive ions obtained by electrospray ionization and electrospray extraction ionization - Google Patents

Method for measuring internal energy difference of monovalent positive ions obtained by electrospray ionization and electrospray extraction ionization Download PDF

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CN110082422B
CN110082422B CN201810787865.5A CN201810787865A CN110082422B CN 110082422 B CN110082422 B CN 110082422B CN 201810787865 A CN201810787865 A CN 201810787865A CN 110082422 B CN110082422 B CN 110082422B
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张小平
钟达财
刘建川
陈焕文
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East China Institute of Technology
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Abstract

The invention provides a method for measuring the internal energy difference of monovalent positive ions obtained by electrospray ionization and electrospray extraction ionization, which comprises the following steps: using a tandem mass spectrum, respectively ionizing target molecules in a sample solution by two ion sources of electrospray ionization and electrospray extraction ionization to form monovalent positive ion parent ions, entering an ion trap under the action of capillary transmission, and obtaining fragment ions by a collision induced dissociation technology; in the process, the ionization condition is kept consistent with the transmission condition, the collision energy is changed to lead the parent ions to collide and induce dissociation, and when the strengths of the same fragment ions obtained in the two modes are equal, the energy difference in the collision induced dissociation process is the internal energy difference of the obtained univalent positive ions. The invention adopts the collision induced dissociation technology, can select, activate, collide and the like parent ions, and effectively avoids the interference of other ions; the calculation method is simple in principle and simple and convenient to operate; the internal energy difference calculated by the method is 11.5 +/-0.2 eV, and the method is accurate in result, high in stability and strong in reliability.

Description

Method for measuring internal energy difference of monovalent positive ions obtained by electrospray ionization and electrospray extraction ionization
Technical Field
The invention belongs to the field of analytical chemistry, relates to the technical field of mass spectrometry, and particularly relates to a method for measuring the internal energy difference of monovalent positive ions obtained by electrospray ionization and electrospray extraction ionization.
Background
The electrospray ionization mass spectrometry (ESI-MS) technology and the electrospray extraction ionization mass spectrometry (EESI-MS) technology can be used for detecting macromolecules such as small molecules, polypeptides, proteins, nucleic acids and the like, and have important application in the fields of chemistry, biology, pharmacy, medicine, environment and the like. ESI and EESI both belong to soft ionization technology, but the working principle of the ESI and EESI is different from and related to the analysis performance. In ESI, a high-voltage electric field is directly applied to a sample solution flowing through a narrow capillary (inner diameter 100 μm), an object to be measured is directly exposed to a high-intensity electric field (e.g., +3kV/m), flows out of the capillary after being acted for a long time (e.g.,10s, calculated by taking the flow rate as 5 μ L/min and the length of the capillary as 10 cm) and is ionized under the action of the electric field and auxiliary gasAnd atomization, and the produced charged liquid drops form ions for subsequent mass spectrum detection through processes such as desolventizing and the like. EESI differs significantly from ESI in that a sample (typically containing a complex matrix) is pneumatically atomized under electrically neutral conditions (i.e., no electric field) to form tiny droplets, which are then combined with reagent droplets (methanol/water) generated by ESI in an open large volume (about 200 cm)3) The cross, collision and fusion in the space are accompanied by the liquid-liquid micro-extraction process (about 50ms) in the open environment to complete the energy charge transfer function in the three-dimensional space and form target molecular ions. It is worth noting that in the EESI process, molecules or ions of the analyte are not in direct contact with a strong electric field all the time, but rather, the molecules or ions are bathed in "charge rain" consisting of charged droplets generated by ESI to obtain charges, and then the ions are formed through processes such as desolvation and the like for subsequent mass spectrometric detection. Clearly, under the same experimental conditions, the EESI process delivers a lower energy to the target ion than the ESI. Studies have demonstrated that EESI is able to maintain the conformation and biological activity of proteins, whereas proteins under ESI lose most of their biological activity through the influence of ionization. This suggests that EESI is a milder soft ionization technique than ESI, and also suggests that EESI differs significantly from ESI in performance (internal energy supplied to the parent ion, etc.). However, how much difference in energy content they specifically exist is not clear. So far, no method for measuring the difference of internal energy of ions generated by the two ion sources by using a mass spectrometer is reported. Therefore, it is necessary to establish a measurement of the difference in internal energy of ESI and EESI.
The self-generated ions are in an excited state and contain certain internal energy, and whether fragment ions are formed or not and different fragmentation modes of the parent ions are determined by the internal energy of the parent ions. The initial internal energy of the ions is from the ion source. The different types of ion sources impart different energies to the same ion, resulting in different internal energies of the ions formed. For example, electron bombardment ionization sources have energies of up to 70eV, which act directly on the sample molecules to produce radical cations with high internal energies, the remaining energy further causing them to fragment to produce fragment ions. If a mild soft ionization source is used, fragment ions are rarely produced, but can be induced by changing the parameters of the ion source. In recent years, a number of methods have been reported for measuring the ion internal energy in ion sources, such as thermometer ion method (int. J. Mass. Spectrum. ion Processes,1987,75(2): 181-. The thermoionic method is used to estimate the internal energy distribution stored in the parent ion, and is mainly used to thermodynamically characterize the continuous fragmentation process of the ion. Deconvolution is a method of determining the internal energy of an ion. The ion survival rate method relates the fragmentation degree of molecules to the internal energy, and is mainly applied to soft ionization. The method is based on two assumptions that ions with the same degree of freedom have similar internal energy distributions, and the internal energy of the ions is not fragmented above the dissociation threshold of the ions themselves. However, most of these methods use an in-source collision induced dissociation (in-source CID) technique, and calculate the distribution of energy in ions by adjusting the acceleration voltage (cone-hole voltage) during ion transport. The disadvantage of using the in-source CID technique is that there is no selection and enrichment process of parent ions, all ions generated during ionization are activated by collision, and because the energy of the in-source CID is lower than that of the CID, the time scale of fragmentation is prolonged, the direct fragmentation dissociation rate is smaller, so that rearrangement reaction is easy to occur, and finally the measured average internal energy value is often lower than that measured by the high-energy CID technique. Meanwhile, the ion survival rate method does not consider dynamic displacement, fragment ions can be obtained only when internal energy higher than critical energy is required to be obtained, and the average internal energy distribution value measured by the method is smaller than the actual value. The CID technology can select and activate parent ions, and the like, thereby avoiding the interference of other ions. Thus, the technique is suitable for analyzing energy content differences of different ion sources. Currently, there is no method for calculating the energy content difference of different ion sources by using CID technology.
Disclosure of Invention
According to the invention, through the research of a collision induced dissociation technology, the influence of different collision energy on the molecular cracking behavior is examined, a method for measuring the internal energy difference of EESI and ESI ion source univalent positive ions is established, and the internal energy difference of EESI and ESI is quantitatively measured. The method has the characteristics of rapidness, simplicity, convenience, easy operation and the like, and can provide reference basis for understanding mass spectrum behaviors of ions generated by different ion sources.
The invention provides a method for measuring the internal energy difference of univalent positive ions obtained by electrospray ionization and electrospray extraction ionization, which comprises the following steps: by using a tandem mass spectrum, target molecules in a sample solution are ionized to form monovalent positive ion parent ions by two ion sources of electrospray ionization and electrospray extraction ionization respectively, enter an ion trap under the action of capillary transmission, and are subjected to a collision induced dissociation technology to obtain abundant fragment ions; in the process, the transmission conditions of electrospray ionization and electrospray extraction ionization are kept consistent with the collision conditions, the collision energy in the ion trap is changed, so that parent ions are collided and induced to dissociate, and under two modes of electrospray ionization tandem mass spectrometry and electrospray extraction ionization tandem mass spectrometry, the collision induced dissociation energy is respectively regulated and controlled so that the intensities of the obtained same fragment ions are equal, and the energy difference in the collision induced dissociation process is the internal energy difference obtained by univalent positive ions.
In tandem mass spectrometry, the total internal energy changes affecting ions mainly include three components, namely, the energy of the ionization process, the energy of the ion transport process, and the collision energy in the ion trap. To form the same fragment ions of the same strength, the total internal energy obtained by the parent ion is constant. The ionization condition and the ion transmission condition are kept consistent, the fragment ion peak intensity is obviously different only by changing the collision energy in the ion trap, namely the obtained fragment ion abundance difference is very obvious, and the reason for generating the fragment ion abundance difference is the energy difference in the ionization process, namely the energy difference transmitted between EESI and ESI to target molecules, so that the energy difference in the process of collision induced dissociation of monovalent positive ions can be measured to calculate the energy difference of monovalent positive ions obtained by electrospray ionization and electrospray extraction ionization.
Further, collision energy is increased from zero, and when the intensity of fragment ions obtained after the collision induced dissociation of the parent ions obtained by the electrospray extraction ionization is equal to the intensity of fragment ions obtained after the collision induced dissociation of the parent ions obtained by the electrospray ionization, the difference of the collision energy in the collision induced dissociation process of the two parent ions is the internal energy difference provided by the two ion sources for the parent ions.
Further, the collision energy value is 0% -100%.
Further, the method for calculating the energy content difference is as follows:
definition EionTotal internal energy obtained for the parent ion, EsouInternal energy provided to the parent ion by the ion source, EtranIs the internal energy, E, obtained by the parent ion in the ion transport process after ionization of the samplecolFor the energy obtained by the parent ion during CID, equation (1) can be derived.
Eion＝Esou+Etran+Ecol (1)
If the collision energy and the conditions of the transmission process are kept consistent, and different ionization sources are changed, the total internal energy obtained by the parent ions in different ion sources is different, and the internal energy difference between the different ion sources is the difference of the internal energies provided by the two ion sources for the parent ions, as shown in formula (2). Definition of
Figure BDA0001734094750000041
The internal energy provided to the parent ions for an electrospray ionization (ESI) source,
Figure BDA0001734094750000042
internal energy provided to the parent ions for an electrospray extraction ionization (EESI) source.
Figure BDA0001734094750000043
Keeping the ionization condition and the transmission condition consistent, and slowly increasing the collision energy in the electrospray ionization tandem mass spectrum from zero, wherein the collision energy is set as
Figure BDA0001734094750000044
In the ionization process of the electrospray ionization tandem mass spectrum, parent ions begin to form fragment ions, and the intensity of the fragment ions is
Figure BDA0001734094750000045
Slowly increasing collision energy in the electrospray extraction ionization tandem mass spectrometry to gradually increase the intensity of fragment ions, and forming the intensity of corresponding fragment ions in the ionization process of the electrospray extraction ionization tandem mass spectrometry
Figure BDA0001734094750000046
When the corresponding collision energy is
Figure BDA0001734094750000051
The difference between the two collision energies
Figure BDA0001734094750000052
I.e., the difference in internal energy provided to the parent ion by the two ion sources, as in equation (3):
Figure BDA0001734094750000053
to form the same fragment ions with the same intensity, the total internal energy obtained by the parent ion is constant, and then combining equation (2) and equation (3) can obtain equation (4):
Figure BDA0001734094750000054
further, the calculation formula of the collision energy is as follows:
Ecol＝NCE×(isolation center)×(charge factor)÷500 (5)
where NCE is the set value of collision energy, (isolation center) is the mass-to-charge ratio of the parent ion, and (charge factor) is the charge factor, and if it is singly charged, the value of the charge factor is 1. The formula (6) can be obtained by combining the formulas (3) to (5):
ΔEion＝(NCE2-NCE1)×(isolation center)×(charge factor)÷500 (6)
wherein NCE2The collision energy in the electrospray extraction ionization tandem mass spectrum is
Figure BDA0001734094750000055
Setting value of time, NCE1For the electrospray ionization tandem mass spectrometry the collision energy is
Figure BDA0001734094750000056
The set value of the time of day,
Figure BDA0001734094750000057
Figure BDA0001734094750000058
further, the change in collision energy increases with a gradient of 1%.
Thus, the difference between the internal energy of the monovalent positive ions obtained by electrospray ionization and electrospray extraction ionization was calculated to be 11.5. + -. 0.2 eV.
Further, the method uses electrospray ionization tandem mass spectrometry and electrospray extraction ionization tandem mass spectrometry employing collision induced dissociation techniques.
The invention has the following beneficial effects:
1. the invention proposes to adopt the Collision Induced Dissociation (CID) technology for the first time, calculate the internal energy difference of univalent positive ions obtained by electrospray ionization and electrospray extraction ionization, compared with the CID technology in the source, the technology adopted by the invention can carry out selective enrichment, activation, collision and the like on the parent ions, other ions can not be activated and collided and dissociated, and the interference of other ions is effectively avoided;
2. the invention adopts the Collision Induced Dissociation (CID) technology, the time scale of the fragmentation process of the parent ions is short, and the rate of forming fragment ions is larger, so that the rearrangement reaction can not occur, and the accuracy of the measurement result can be ensured;
3. the internal energy difference of the two ionization technologies is experimentally measured by adopting the ion abundance behavior difference in the mass spectrum, the method has high efficiency, and the internal energy difference result can be rapidly measured;
4. the principle is simple, the operation is simple and convenient, the calculation process is simple, and a complicated calculation process is not needed;
5. the result is accurate, the stability is high, and the reliability is strong.
Drawings
FIG. 1 is a molecular structure diagram of rhodamine B;
FIG. 2 is a diagram showing the structure of rhodamine B parent ion;
FIG. 3(a) is EESI-MS of rhodamine B2FIG. and (B) is ESI-MS of rhodamine B2A drawing;
FIG. 4(a) is a graph showing the relationship between the peak intensity of the parent ion and the fragment ion and the collision energy, and (b) is a graph showing the relationship between the peak intensity ratio of the fragment ion and the parent ion and the collision energy;
FIG. 5 shows ESI-MS at low collision energy (1% -22%)2(a) And EESI-MS2(b) A trend graph of the absolute intensity of the m/z 413 peak of the middle fragment ion;
FIG. 6 shows ESI-MS at low collision energy (1% -22%)2(a) And EESI-MS2(b) A trend graph of the absolute intensity of the m/z 415 peak of the middle fragment ion;
FIG. 7 shows ESI-MS at low collision energy (1% -22%)2(a) And EESI-MS2(b) A trend graph of the absolute intensity of the m/z 399 peak of the middle fragment ions;
FIG. 8 is a schematic diagram of the molecular structure of arginine;
fig. 9 is a schematic diagram of the molecular structure of clenbuterol.
Detailed Description
The invention provides a method for measuring the internal energy difference of univalent positive ions obtained by electrospray ionization and electrospray extraction ionization, which comprises the following steps: by using a tandem mass spectrum, target molecules in a sample solution are ionized to form monovalent positive ion parent ions by two ion sources of electrospray ionization and electrospray extraction ionization respectively, enter an ion trap under the action of capillary transmission, and are subjected to a collision induced dissociation technology to obtain abundant fragment ions; in the process, the transmission conditions of electrospray ionization and electrospray extraction ionization are kept consistent with the collision conditions, the collision energy in the ion trap is changed, so that parent ions are collided and induced to dissociate, and under two modes of electrospray ionization tandem mass spectrometry and electrospray extraction ionization tandem mass spectrometry, the collision induced dissociation energy is respectively regulated and controlled so that the intensities of the obtained same fragment ions are equal, and the energy difference in the collision induced dissociation process is the internal energy difference obtained by univalent positive ions.
In the tandem mass spectrometry process, high-energy ions accelerated by an electric field have to collide with collision gas molecules to convert the kinetic energy of the ions into energy inside the ions; the high internal energy ions further undergo chemical bond scission to generate fragment ions. Thus, in tandem mass spectrometry, the internal energy changes affecting the ions are mainly composed of three components, namely the energy of the ionization process, the energy of the ion transport process and the collision energy in the ion trap. Theoretically, the same substance (keeping the parameters such as concentration, solvent and the like unchanged) can obtain the same energy in the ion transmission process. The same material obtains the same energy under the same collision energy condition. To form fragment ions of the same strength, the total internal energy obtained by the parent ion is constant. Then keeping the ionization condition consistent with the ion transport condition, only changing the collision energy in the ion trap, the fragment ion peak intensity will have obvious difference, i.e. the obtained fragment ion abundance difference is very obvious, and the reason for generating the fragment ion abundance difference is the energy difference in the ionization process, i.e. the internal energy difference between EESI and ESI transferred to the target molecule. Therefore, the energy difference obtained by the monovalent positive ions can be calculated by measuring the energy difference in the process of collision induced dissociation of the monovalent positive ions.
Further, collision energy is increased from zero, and when the intensity of fragment ions obtained after the collision induced dissociation of the parent ions obtained by the electrospray extraction ionization is equal to the intensity of fragment ions obtained after the collision induced dissociation of the parent ions obtained by the electrospray ionization, the difference value of the two collision energies is the difference of the internal energies provided by the two ion sources for the parent ions.
Further, the collision energy value is 0% -100%.
Further, the method for calculating the energy content difference is as follows:
definition EionTotal internal energy obtained for the parent ion, EsouInternal energy provided to the parent ion by the ion source, EtranIs the internal energy, E, obtained by the parent ion in the ion transport process after ionization of the samplecolFor the energy obtained by the parent ion during CID, equation (1) can be derived.
Eion＝Esou+Etran+Ecol (1)
If the collision energy and the conditions of the transmission process are kept consistent, and different ionization sources are changed, the total internal energy obtained by the parent ions in different ion sources is different, and the internal energy difference between the different ion sources is the difference of the internal energies provided by the two ion sources for the parent ions, as shown in formula (2). Definition of
Figure BDA0001734094750000081
The internal energy provided to the parent ions for an electrospray ionization (ESI) source,
Figure BDA0001734094750000082
internal energy provided to the parent ions for an electrospray extraction ionization (EESI) source.
Figure BDA0001734094750000083
Keeping the ionization condition and the transmission condition consistent, and slowly increasing the electrospray ionization tandem mass spectrum (ESI-MS) from zero2) The collision energy of (1) is
Figure BDA0001734094750000084
ESI-MS (very efficient ESI-MS)2During the ionization process, the parent ion begins to form fragment ions, and the strength of the fragment ions is
Figure BDA0001734094750000085
Slowly increasing electrospray extraction ionization tandem mass spectrometry (EESI-MS)2) The collision energy in (1) is gradually increased when the EESI-MS is used2ProcedureIntensity of corresponding fragment ion formed therein
Figure BDA0001734094750000086
Time, EESI-MS2Corresponding collision energy of
Figure BDA0001734094750000087
The difference between the two collision energies
Figure BDA0001734094750000088
I.e., the difference in internal energy provided to the parent ion by the two ion sources, as in equation (3):
Figure BDA0001734094750000089
to form the same fragment ions with the same intensity, the total internal energy obtained by the parent ion is constant, and then combining equation (2) and equation (3) can obtain equation (4):
Figure BDA0001734094750000091
further, according to a conversion formula between the collision energy and the normalized collision energy provided by Thermo manufacturer, such as formula (5):
Ecol＝NCE×(isolation center)×(charge factor)÷500 (5)
where NCE is the set value of collision energy, (isolation center) is the mass-to-charge ratio of the parent ion, and (charge factor) is the charge factor, and if it is singly charged, the value of the charge factor is 1. The formula (6) can be obtained by combining the formulas (3) to (5):
ΔEion＝(NCE2-NCE1)×(isolation center)×(charge factor)÷500 (6)
wherein NCE2Is EESI-MS2The middle collision energy is
Figure BDA0001734094750000092
Setting value of time, NCE1Is ESI-MS2The middle collision energy is
Figure BDA0001734094750000093
The set value of the time of day,
Figure BDA0001734094750000094
further, the change in collision energy increases with a gradient of 1%.
Thus, the difference between the internal energy of the monovalent positive ions obtained by electrospray ionization and electrospray extraction ionization was calculated to be 11.5. + -. 0.2 eV.
Further, the method uses electrospray ionization tandem mass spectrometry and electrospray extraction ionization tandem mass spectrometry employing collision induced dissociation techniques.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The instrument and device used in the embodiment of the invention are as follows:
the EESI and ESI ion sources are developed and built by a laboratory;
LTQ-XL Linear ion trap Mass spectrometer equipped with an Xcalibur type data processing System (Finnigan, USA).
Main materials and reagents:
ar gas (purity > 99.999%, Jiangxi national Teng gas Co., Ltd.);
rhodamine B (97%), available from carbofuran (shanghai);
arginine (99%), available from carbofuran corporation (shanghai);
clenbuterol, available from the lark wary company (shanghai);
methanol was of HPLC grade and purchased from Sigma-Aldrich (USA).
The experimental process comprises the following steps:
and respectively building an EESI-MS device platform and an ESI-MS device platform, and performing tandem mass spectrometry by regulating and controlling the energy of CID. The linear ion trap mass spectrum (LTQ-MS) adopts a positive ion detection mode, the mass spectrum scanning range is m/z 50-600, the solvent used by ESI and the extracting agent used by EESI are both water, the flow rate is 1 mu L/min, the spraying voltage is 4.0kV, and the spraying gas (N is N2) The pressure is 1.0MPa, the lens voltage is 30V, the capillary temperature is 150 ℃, and the capillary taper hole voltage is 12V. When the tandem mass spectrometry is carried out, the width of an isolation window of the parent ions is set to be 1.0u, the collision time is 30ms, the collision energy is 0-100%, and other parameters are automatically optimized by an LTQ-Tune software system to enable the signal intensity of the parent ions to reach the best. When ESI-MS and EESI-MS experiments are carried out, the conditions of flow, flow rate and the like are precisely adjusted, so that various parameters of the two ion sources are kept consistent.
In examples 1 to 3, rhodamine B is used as a research object, and the internal energy difference of univalent positive ions obtained by electrospray ionization and electrospray extraction ionization is measured. As shown in figures 1 and 2, rhodamine B has a large molecular structure, a conjugated system exists, and the rhodamine B has positive charges and is easy to ionize to form ions. Therefore, rhodamine B is a representative monovalent positive ion subject, and the result is highly accurate.
Example 1:
respectively building EESI-MS and ESI-MS device platforms, performing tandem mass spectrometry on rhodamine B by regulating and controlling CID energy, wherein the mass-to-charge ratio of parent ions formed in a positive ion mode is m/z 443, and obtaining the EESI-MS of the rhodamine B2Graph and ESI-MS2Figure (a). FIG. 3(a) is the EESI-MS of rhodamine B2FIG. 30% collision energy shows that the parent ion m/z 443 mainly loses C respectively2H4、C2H6And CO2Neutral molecules, producing m/z 415, m/z 413 and m/z 399 fragment ions. Under the same collision energy condition, ESI-MS is adopted2The results of experiments on rhodamine B are shown in FIG. 3(B), and m/z 415, m/z 413 and m/z 399 fragment ions, which are in turn in combination with EESI-MS, can also be obtained2The results are consistent. However, comparing FIG. 3(a) and FIG. 3(b), it can be seen that in ESI-MS, the relative abundance of the fragment ions is significantly greater than EEThe high in SI-MS indicates that ionization of rhodamine B by ESI is more complete than fragmentation of its excimer peak m/z 443 by EESI, thus demonstrating that EESI is a softer ionization mode than ESI. The internal energy changes affecting the ions mainly include three components, namely the energy of the ionization process, the energy of the ion transport process and the collision energy in the ion trap. Keeping the ionization condition consistent with the ion transmission condition, and only changing the collision energy in the ion trap, the fragment ion peak intensity will have obvious difference. In the experiment, two different ionization modes of ESI and EESI are adopted, ion transmission conditions and ion trap collision conditions are kept consistent in the two modes, and the obtained fragment ion abundance difference is very obvious. The difference between the two spectra is caused by the difference between the internal energy transmitted by EESI and ESI to the target molecule.
Taking fragment ion m/z 413 as an example, the method of the invention is adopted to calculate the internal energy difference of two ion sources. FIG. 5 shows ESI-MS at low collision energy (1% -22%)2And EESI-MS2And (3) a trend graph of the absolute intensity of the m/z 413 peak of the medium fragment ion. At ESI-MS2In, slowly increasing ESI-MS2When the collision energy is
Figure BDA0001734094750000111
When the precursor ion begins to form fragment ions, the collision energy is totally used for providing internal energy of the precursor ion, and the intensity of the fragment ion m/z 413 is equal to
Figure BDA0001734094750000112
(FIG. 5 (a)). When collision energy is continuously increased, most of the energy transferred to rhodamine B molecules in the collision process is used for reducing the energy of a molecular conjugate system, and a small part of the energy is used for dissociating to form fragments. In EESI-MS2Middle (FIG. 5(b)), EESI-MS is slowly increased2The collision energy in (2) is used for gradually increasing the strength of fragment ions, and when the collision energy is less than 14%, rhodamine B molecules are not coatedActivation, intensity of fragment ion m/z 413
Figure BDA0001734094750000113
Namely, to make ESI-MS2And EESI-MS2The peak intensities of the m/z 413 of the middle fragment ions are the same
Figure BDA0001734094750000121
Corresponding collision energy
Figure BDA0001734094750000122
From the equation (3), the difference between the collision energies in the two tandem mass spectra is:
Figure BDA0001734094750000123
combining the formula (4), it can be known that 13% of the collision energy is the internal energy difference between the two ion sources. From equation (6):
ΔEion＝(NCE2-NCE1)×(isolation center)×(charge factor)÷500＝(14-1)×443×1÷500＝11.5eV
namely, the difference of the internal energy of univalent positive ions obtained by electrospray ionization and electrospray extraction ionization is 11.5 eV.
Example 2:
taking the fragment ion m/z 415 as an example, the method of the invention is adopted to calculate the internal energy difference of the two ion sources. FIG. 6 shows ESI-MS at low collision energy (1% -22%)2And EESI-MS2And (3) a trend graph of the absolute intensity of the m/z 415 peak of the middle fragment ion. At ESI-MS2In, slowly increasing ESI-MS2When the collision energy is
Figure BDA0001734094750000124
% of the total mass of the precursor ions, the precursor ions begin to form fragment ions when the fragment ions m/z 415 have an intensity of
Figure BDA0001734094750000125
In EESI-MS2In (1), the fragment ions are made m/z 415Peak intensity
Figure BDA0001734094750000126
Corresponding collision energy
Figure BDA0001734094750000127
From the equation (3), the difference between the collision energies in the two tandem mass spectra is:
Figure BDA0001734094750000128
combining equation (4), it can be seen that 13% of the collision energy is the difference between the internal energies of the two ion sources. The internal energy difference of the monovalent positive ions obtained by the two ionization sources is calculated to be 11.5eV by the formula (6), and the calculation process is as follows:
ΔEion＝(NCE2-NCE1)×(isolation center)×(charge factor)÷500＝(14-1)×443×1÷500＝11.5eV
example 3:
taking fragment ion m/z 399 as an example, the method of the invention is adopted to calculate the internal energy difference of two ion sources. FIG. 7 shows ESI-MS at low collision energy (1% -22%)2And EESI-MS2And (3) a trend graph of the absolute intensity of the m/z 399 peak of the middle fragment ion. At ESI-MS2When the collision energy is
Figure BDA0001734094750000131
When the parent ion begins to form a fragment ion, the fragment ion m/z 399 has an intensity
Figure BDA0001734094750000132
In EESI-MS2In (1), the peak intensity of fragment ion m/z 399 is adjusted
Figure BDA0001734094750000133
Corresponding collision energy
Figure BDA0001734094750000134
From the equation (3), the difference between the collision energies in the two tandem mass spectra is:
Figure BDA0001734094750000135
combining equation (4), it can be seen that 13% of the collision energy is the difference between the internal energies of the two ion sources. From equation (6):
ΔEion＝(NCE2-NCE1)×(isolation center)×(charge factor)÷500＝(14-1)×443×1÷500＝11.5eV
i.e. the difference in internal energy is 11.5 eV.
Example 4:
as shown in FIG. 8, arginine was used as the subject of the present example. Arginine has a molecular weight of 174, and the parent ion formed in positive ion mode has a mass to charge ratio of m/z 175, and will fragment under CID conditions to form fragment ion m/z 157. At ESI-MS2In the middle, the collision energy is slowly increased when the collision energy is
Figure BDA0001734094750000136
Peak intensity of fragment ion m/z 157
Figure BDA0001734094750000137
In EESI-MS2When the collision energy gradually increased to 34%, the peak intensity of fragment ion m/z 157
Figure BDA0001734094750000138
Only when the peak intensity reaches about 77.2, that is, when the peak intensity of the two is consistent, the required collision energy difference is:
Figure BDA0001734094750000139
therefore, it can be calculated according to equation (6):
ΔEion＝(NCE2-NCE1)×(isolation center)×(charge factor)÷500＝(34-1)×175×1÷500＝11.6eV
namely, the internal energy difference of univalent positive ions obtained by electrospray ionization and electrospray extraction ionization is 11.6 eV.
Example 5:
as shown in fig. 9, the present inventionThe examples were studied on clenbuterol. Clenbuterol has a molecular weight of 277, and forms a parent ion with a mass-to-charge ratio of m/z 278 in positive ion mode, which is fragmented under CID conditions to form fragment ions m/z 259. At ESI-MS2In the middle, the collision energy is slowly increased when the collision energy is
Figure BDA0001734094750000141
Peak intensity of fragment ion m/z 259
Figure BDA0001734094750000142
In EESI-MS2When the collision energy is gradually increased to 22%, the peak intensity of the fragment ion m/z 259
Figure BDA0001734094750000143
The peak intensity of the energy reaches about 3370, namely when the peak intensity of the energy is consistent with the peak intensity of the energy, the required collision energy difference is as follows:
Figure BDA0001734094750000144
therefore, it can be calculated according to equation (6):
ΔEion＝(NCE2-NCE1)×(isolation center)×(charge factor)÷500＝(22-1)×278×1÷500＝11.7eV
namely, the internal energy difference of univalent positive ions obtained by electrospray ionization and electrospray extraction ionization is 11.7 eV.
As shown in Table 1, which is a comparison table of experimental data of examples 1-5, it can be seen that the intensity variations of different species and different fragment ions are calculated according to the method of the present invention, and all the results are consistent. Therefore, the method of the invention has reliable results for calculating the internal energy difference of univalent positive ions obtained by electrospray ionization and electrospray extraction ionization, and the internal energy difference is 11.5 +/-0.2 eV.
TABLE 1
Figure BDA0001734094750000145
Figure BDA0001734094750000151
In conclusion, the invention adopts the ion abundance behavior difference in mass spectrum to carry out experimental determination on the internal energy difference of EESI and ESI of two soft ionization technologies, and the measured value is 11.5 +/-0.2 eV. The method has simple principle, simple and convenient operation and no need of complicated calculation process. The method not only can provide reference basis for comparison of energy difference of different ion sources, but also can provide reference for deep understanding of mass spectrum behaviors of ions generated by different ion sources.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.

Claims (7)

1. A method for measuring the internal energy difference of univalent positive ions obtained by electrospray ionization and electrospray extraction ionization is characterized in that tandem mass spectrometry is used, target molecules in a sample solution are ionized into univalent positive ion parent ions through two ion sources of electrospray ionization and electrospray extraction ionization respectively, the univalent positive ion parent ions enter an ion trap under the action of capillary transmission, and fragment ions are obtained through a collision induced dissociation technology; in the process, the ionization condition is kept consistent with the transmission condition, collision energy is changed to enable parent ions to collide and induce dissociation, collision induced dissociation energy is respectively regulated and controlled to enable the strength of the obtained same fragment ions to be equal under two modes of electrospray ionization tandem mass spectrometry and electrospray extraction ionization tandem mass spectrometry, namely collision energy is increased from zero, when the strength of the fragment ions obtained after the parent ions obtained by electrospray extraction ionization collide and induce dissociation is equal to the strength of the corresponding fragment ions obtained after the parent ions obtained by electrospray ionization collide and induce dissociation, and the difference of the collision energy in the process of collision induced dissociation of the parent ions and the fragment ions is the difference of the internal energy provided for the parent ions by the two ion sources.
2. The method of claim 1, wherein the collision energy is at a value of 0% to 100%.
3. The method of claim 2, wherein the energy content difference is calculated as follows:
definition EionTotal internal energy obtained for the parent ion, EsouInternal energy provided to the parent ion by the ion source, EtranIs the internal energy, E, obtained by the parent ion in the ion transport process after ionization of the samplecolThe energy obtained by the parent ion during collision-induced dissociation yields the formula (1):
Eion＝Esou+Etran+Ecol (1)
keeping the collision condition and the transmission condition consistent, and changing different ionization sources, the difference of the total internal energy obtained by the parent ions is the difference of the internal energy provided by the two ion sources to the parent ions, as shown in formula (2):
Figure FDA0003295079830000011
wherein
Figure FDA0003295079830000012
Provides internal energy for the electrospray ionization source to provide parent ions,
Figure FDA0003295079830000013
providing internal energy for the electrospray extraction ionization source to the parent ions;
keeping the ionization condition and the transmission condition consistent, increasing the collision energy in the electrospray ionization tandem mass spectrum from zero, and setting the collision energy as the time
Figure FDA0003295079830000021
The parent ions begin to form fragment ions in the ionization process of electrospray ionization tandem mass spectrometry, and the intensity of the fragment ions is
Figure FDA0003295079830000022
Increasing collision energy in the electrospray extraction ionization tandem mass spectrometry from zero to gradually increase the intensity of fragment ions, and forming the intensity of corresponding fragment ions in the ionization process of the electrospray extraction ionization tandem mass spectrometry
Figure FDA0003295079830000023
When the corresponding collision energy is
Figure FDA0003295079830000024
The difference in internal energies provided by the two ion sources to the parent ion is:
Figure FDA0003295079830000025
then combining equation (2) and equation (3) yields equation (4):
Figure FDA0003295079830000026
4. a method according to claim 3, characterized in that the collision energy is calculated by the formula:
Ecol＝NCE×(isolationcenter)×(charge factor)÷500 (5)
wherein NCE is a set value of collision energy, (isolation center) is a mass-to-charge ratio of parent ions, and (charge factor) is a charge factor;
the formula (6) can be obtained by combining the formulas (3) to (5):
Δ Eion＝(NCE2-NCE1)×(isolationcenter)×(charge factor)÷500 (6)
wherein NCE2The collision energy in the electrospray extraction ionization tandem mass spectrum is
Figure FDA0003295079830000027
Setting value of time, NCE1For the electrospray ionization tandem mass spectrometry the collision energy is
Figure FDA0003295079830000028
The set value of the time of day,
Figure FDA0003295079830000029
Figure FDA00032950798300000210
5. the method of claim 4, wherein the collision energy is increased in a gradient of 1%.
6. The method of any of claims 2 to 5, wherein the internal energy difference is 11.5 ± 0.2 eV.
7. The method of claim 5, wherein the method uses electrospray ionization tandem mass spectrometry and electrospray extraction ionization tandem mass spectrometry employing collision induced dissociation techniques.
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