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Brief introduction of mass spectrometry technology

Directory 1 pinyin 2 English reference 3 overview 4 definition of mass spectrometry 5 mass spectrometer 5. 1 sampling system 5. 1 direct sampling 5. 1.2 gas chromatography-mass spectrometry (GCMS) 5. 1. 3 liquid chromatography-mass spectrometry (LCMS) 5. 1.4 supercritical fluid chromatography-mass spectrometry (SFCMS) 5.2 ionization MALDI 5.2. 1 electron bombardment ionization (EI) 5.2.2 chemical ionization (Cl) 5.2.3 fast atom bombardment (FAB) or fast ion bombardment ionization (fab). 5.2.5 Electrospray Ionization (ESI) 5.2.6 Atmospheric Pressure Chemical Ionization (APCI) 5.3 Mass Analyzer 5.3.4 Time-of-flight Analyzer (TOF) 5.3.5 Fourier Transform Analyzer (FTMS) 5.4 Tandem Mass Spectrometry 5.5 Signal Detection and Data Acquisition 6 Reference 1 Pinyin zhì pǔ fǎ ǔ

2 English reference mass spectrometry [WS/T 455—20 14 health monitoring and evaluation terms]

Summarize that mass spectrometry is a method that bombards gas molecules with electron current, knocks out electrons in the molecules, forms molecular ions with positive charges, and then splits them into a series of fragment ions. Then, positive ions with different mass-to-charge ratios are separated by magnetic field, their relative intensities are recorded, and mass spectrograms are drawn, so as to carry out element analysis, molecular weight determination, molecular formula determination and molecular structure inference. Mass spectrometry has become one of the important methods commonly used in structural identification of chemical components of traditional Chinese medicine.

Mass spectrometry is an analytical method that generates gaseous ions from the compounds to be detected, and then separates and detects the ions according to the mass-to-charge ratio (m/z). The detection limit can reach1015 ~10/2? Molar order of magnitude. Mass spectrometry can provide the information of molecular mass and structure, and the quantitative determination can adopt internal standard method or external standard method.

4 Definition of mass spectrometry Mass spectrometry refers to the method of analyzing a sample according to the mass number and relative abundance of ions formed after ionization of the sample [1].

The main components of the mass spectrometer are shown in the figure. At 103 ~ 106 is maintained by water pump? In the vacuum state of Pa, various positive ions (or negative ions) generated by ion source are accelerated, separated by mass analyzer and then detected by detector. Computer system is used to control instruments, record, process and store data. When equipped with standard library software, the computer system can compare the measured mass spectrum with the spectrum in the standard library to obtain the composition and structure information of possible compounds.

Figure? Main components of mass spectrometer

5. 1 The sampling system shall not affect the vacuum degree of the mass spectrometer. The choice of injection mode depends on the nature, purity and ionization mode of the sample.

5. 1. 1 Direct injection Under normal temperature and pressure, neutral molecules of gaseous or liquid compounds enter the ion source through a controllable leakage system. Volatile compounds adsorbed on solid or dissolved in liquid can be extracted and enriched by headspace analysis, desorbed by programmed temperature, and then introduced into mass spectrometer by capillary.

The volatile solid sample can be placed in a small crucible at the top of the sampling rod and heated and gasified in a high vacuum near the ion source. Using desorption ionization technology, samples with unstable heat and difficult volatilization can be ionized at the same time of gasification.

Many separation techniques have been combined with mass spectrometry. The separated components to be detected can be introduced into the mass spectrometer for analysis through an appropriate interface.

5. 1.2 The effluent separated by gas chromatography-mass spectrometry (GCMS) is gaseous, and the molecular size of the compounds to be detected is also suitable for mass spectrometry analysis. In the case of using capillary gas chromatography column and high-capacity mass spectrometry vacuum pump, chromatographic effluent can be directly introduced into mass spectrometer.

5. 1.3 liquid chromatography-mass spectrometry (LCMS) needs a special interface to separate the compounds to be detected from the chromatographic effluent and form gas molecules or ions suitable for mass spectrometry analysis. Particle beam (PBI), moving band (MBD) and atmospheric pressure ionization (API) are available interfaces for liquid chromatography-mass spectrometry. In order to reduce pollution and avoid chemical noise and ionization suppression, buffer salts or additives contained in the mobile phase should usually be volatile and their dosage is limited.

(1) particle beam interface? After the effluent of liquid chromatography is atomized and desolvated in the desolventizing chamber, only the neutral molecules of the compound to be detected are introduced into the mass spectrometry ion source. The particle beam interface is suitable for the analysis of weakly polar compounds with molecular weight less than 1000. The measured mass spectra can be produced by electron bombardment ionization or chemical ionization. Electron bombardment ionization mass spectrometry contains abundant structural information.

(2) Mobile belt interface? Evenly drop the liquid chromatography effluent with the flow rate of 0.5 ~ 65438 0.5 ml/min on the moving belt. After evaporation and solvent removal, the compound to be detected is introduced into the mass spectrometry ion source. The moving band interface is not suitable for the analysis of polar or thermally unstable compounds, and the measured mass spectrum can be produced by electron bombardment ionization, chemical ionization or fast atom bombardment ionization.

(3) Atmospheric pressure ionization interface? Electrospray ionization and atmospheric pressure chemical ionization are widely used atmospheric pressure ionization interface technologies in liquid chromatography-mass spectrometry. Because of their ionization function, these interfaces are also called atmospheric pressure ion sources and will be introduced in ionization mode.

5. 1.4 Supercritical fluid chromatography-mass spectrometry (SFCMS) At present, supercritical fluid chromatography-mass spectrometry mainly adopts atmospheric pressure chemical ionization or electrospray ionization interface, and the chromatographic effluent is converted into gas through a heating restrictor located between the chromatographic column and the ion source, and then enters the mass spectrometer for analysis. 5. Capillary electrophoresis-mass spectrometry (CEMS)

Almost all modes of capillary electrophoresis can be combined with mass spectrometry. When selecting the interface, we should pay attention to the low-speed characteristics of capillary electrophoresis and use volatile buffer. Electrospray ionization is the most commonly used interface technology for capillary electrophoresis and mass spectrometry.

5.2 Ionization mode According to the properties of the compound to be tested and the type of information to be obtained, different ionization modes can be selected to make the compound to be tested generate gaseous positive ions or negative ions for further mass spectrometry analysis. In some cases, injection and ionization are completed in the same process, and it is difficult to distinguish them clearly.

5.2. 1 electron bombards gaseous compound molecules in the ionization (EI) ion source, and bombards them with electron beams with energy (usually 70eV) greater than their ionization energy. Mass spectrometry analysis often contains molecular ions of compounds to be tested and fragment ions with structural characteristics of compounds to be tested. Electron bombardment ionization is suitable for the ionization of thermally stable and volatile compounds, and is the most commonly used ionization method in gas chromatography-mass spectrometry. Electron bombardment ionization can also be used in liquid chromatography-mass spectrometry when the interface such as particle beam or moving belt is used.

5.2.2 Reaction gas molecules (such as methane, isobutane and ammonia) in chemical ionization (Cl) ion source are ionized by high-energy electron bombardment, and further ion-molecule reaction occurs to generate stable reaction gas ions, and then the compounds to be detected are ionized. Chemical ionization can generate (M+H)+ or (MH) characteristic ions of the compound to be tested (M) or addition ions generated by the compound to be tested and reagent gas molecules. Compared with electron bombardment ionization mass spectrometry, chemical ionization mass spectrometry has fewer fragment ions and is suitable for the analysis of compounds whose molecular mass information cannot be obtained by electron bombardment ionization.

5.2.3 Fast atom bombardment (FAB) or fast ion bombardment (LSIMS) ionizes high-energy neutral atoms (such as argon) or high-energy cesium ions, so as to ionize the compounds to be tested which are placed on the metal surface and dispersed in an inert viscous matrix (such as glycerol), and generate (M+H)+ or (MH) characteristic ions or addition ions of the compounds to be tested and the matrix molecules. Fast atom bombardment or fast ion bombardment ionization is very suitable for molecular mass determination and structural characterization of various polar and thermally unstable compounds, and is widely used in the analysis of polypeptides, antibiotics, nucleotides, lipids, organometallic compounds and surfactants with molecular weight as high as 10000.

When fast atom bombardment (FAB) or fast ion bombardment ionization is used in LC-MS, it is necessary to add 1% ~ 10% glycerol to the chromatographic mobile phase, and the flow rate must be kept very low (1 ~ 10μ l/min).

5.2.4 Matrix-assisted laser desorption ionization (MALDI) Coat the sample dissolved in a proper matrix on a metal target and irradiate it with high-intensity ultraviolet or infrared pulsed laser to ionize the compound to be detected. Matrix-assisted laser desorption ionization is mainly used for the analysis of biological macromolecules with molecular weight above 100000, and is suitable for use in combination with time-of-flight analyzer.

5.2.5 Electrospray ionization (ESI) ionization is carried out at atmospheric pressure. The solution to be detected (such as liquid chromatography effluent) enters the ion source through a capillary with a high voltage of several thousand volts at the end, and the solvent in the generated tiny droplets is removed by gas-assisted atomization to form gaseous ions with single charge or multiple charges. These ions are then transferred from atmospheric pressure to the high vacuum of the mass spectrometer through the step-by-step decompression zone. Electrospray ionization can be carried out at the flow rate of 1 μ l/min ~ 1 ml/min, which is suitable for studying polar compounds and biological macromolecules with molecular weight as high as 100000. It is the most successful interface technology for liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry.

Solvents commonly used in reversed-phase high performance liquid chromatography, such as water, methanol, acetonitrile, etc., are very beneficial to electrospray ionization, but pure water or pure organic solvent as mobile phase is not conducive to solvent removal or ion formation; Under the condition of high flow rate, the mobile phase contains a small amount of water or at least 20% ~ 30% organic solvent, which is helpful to obtain high analytical sensitivity.

5.2.6 Atmospheric pressure chemical ionization (APCI) has the same principle as chemical ionization, but ionization is carried out at atmospheric pressure. The mobile phase is atomized into a gaseous state under the action of heat and nitrogen flow, and ionized when passing through a discharge electrode with a high voltage of several thousand volts, and the generated reagent gas ions react with the molecules of the compound to be detected to form single charge ions. Positive ions are usually (M+H)+ and negative ions are (MH). Atmospheric pressure chemical ionization can be carried out at a flow rate as high as 2ml/min, which is one of the important interfaces of liquid chromatography-mass spectrometry.

Vacuum interface is often used for electrospray ion source and atmospheric pressure chemical ion source, which is convenient for mutual replacement. When choosing electrospray ionization or atmospheric pressure chemical ionization, analysts should not only consider the nature, composition and flow rate of the solution (such as liquid chromatography mobile phase), but also the chemical properties of the compounds to be tested are very important. Electrospray ionization is more suitable for polar compounds that are easily ionized in solution, as well as compounds and biological macromolecules (such as protein, peptides, etc. ) an electrospray ion source can be used. Atmospheric pressure chemical ionization is often used to analyze small molecules or weakly polar compounds (such as sterols and carotenoids) with molecular weight less than 1500, which mainly produce (M+H)+ or (MH) ions, with few fragment ions.

Relatively speaking, electrospray ionization is more suitable for thermally unstable samples, while atmospheric pressure chemical ion source is easy to be combined with normal phase liquid chromatography. Many neutral compounds are suitable for electrospray ionization and atmospheric pressure chemical ionization at the same time, and they all have quite high sensitivity. Whether electrospray ionization or atmospheric pressure chemical ionization, the choice of positive ion or negative ion ionization mode mainly depends on the properties of the compound to be tested.

5.3 Mass analyzer In a high vacuum state, the mass analyzer separates ions according to the mass-to-charge ratio. Quality range and resolution are two main performance indexes of quality analyzer. Mass range refers to the range of mass-to-charge ratio that can be measured by mass spectrometer, and resolution refers to the ability of mass spectrometer to distinguish adjacent peaks with little mass difference. Commonly used mass analyzers include sector magnetic field analyzer, quadrupole analyzer, ion trap analyzer, time-of-flight analyzer and Fourier transform analyzer.

5.3. 1 The ions generated in the ion source of the sector magnetic field analyzer are accelerated by the accelerating voltage (V) and focused into the sector magnetic field (magnetic field strength B). Under the action of magnetic field, ions with different mass-to-charge ratios deflect and move according to their respective radii of curvature (r):

m/zB2r2/2V

By changing the magnetic field strength, ions with different mass-to-charge ratios can have the same radius of curvature (r), and then reach the detector through the slit outlet.

The sector magnetic field analyzer can detect single-charge ions with molecular weight as high as 15000. When it is combined with electrostatic field analyzer to form a dual-focus sector magnetic field analyzer, the resolution can reach 105.

5.3.2 Quadrupole Analyzer This analyzer consists of four metal rod electrodes arranged in parallel. Direct current voltage (DC) and radio frequency voltage (RF) act on the electrodes to form a high-frequency oscillating electric field (quadrupole field). Under certain DC voltage and RF voltage conditions, only ions with a certain mass-to-charge ratio can stably cross the quadrupole field and reach the detector. Changing the magnitude of DC voltage and RF voltage, but keeping their ratio unchanged, can realize mass spectrometry scanning.

The upper limit of detectable molecular weight of quadrupole analyzer is usually 4000, and the resolution is about 103.

5.3.3 Ion trap analyzer The quadrupole ion trap (QIT) consists of two end cap electrodes and an annular electrode located between them. The end cap electrode is at ground potential, while the ring electrode is applied with radio frequency voltage (RF) to form a three-dimensional quadrupole field. The quadrupole field can store all ions with a mass-to-charge ratio greater than a certain value at an appropriate RF voltage. By adopting the mode of "unstable mass selection" and increasing the RF voltage, ions can be ejected from the ion trap in the order of high mass to low mass. The ionization and mass analysis of volatile compounds can be completed in the same quadrupole field. By setting the time series, a single quadrupole ion trap can realize the function of multistage mass spectrometry (MSn).

Linear ion trap (LIT) is a two-dimensional quadrupole ion trap, which is equivalent to a quadrupole mass analyzer in structure, but its working mode is similar to that of a three-dimensional ion trap. Quadrupole linear ion trap has better ion storage efficiency and storage capacity, improved ion injection efficiency, faster scanning speed and higher detection sensitivity.

The biomacromolecule ions produced by electrospray ionization or matrix-assisted laser desorption ionization can enter the ion trap analyzer for analysis by ion guidance. The upper mass limit of ion trap analyzer and quadrupole analyzer is similar, and the resolution is 103 ~ 104.

5.3.4 The time-of-flight analyzer (TOF) has the same kinetic energy but different mass, and the ions are separated due to different flight speeds. When the flight distance is fixed, the time required for ions to fly is proportional to the square root of the mass-to-charge ratio, and ions with small mass reach the detector in a short time. In order to determine the time of flight, ions are introduced into the mass analyzer in discontinuous groups to determine the initial time of flight. Ion groups can be generated by pulsed ionization (such as matrix-assisted laser desorption ionization), or continuous ion flow can be introduced into the flight tube at a given time through the gate control system.

Modern time-of-flight analyzer has the characteristics of wide mass analysis range (the upper limit of molecular weight is about 15000), high ion transmission efficiency (especially fast spectrogram acquisition), various detection capabilities, simple instrument design and operation, high mass resolution (about 104) and so on, and has become the mainstream technology of biomacromolecules analysis.

5.3.5 Fourier transform analyzer (FTMS) ions gyrate in a magnetic field with a certain intensity, and their orbits change with the alternating electric field of vibration. When the frequency of the alternating electric field is the same as the ion cyclotron frequency, the ion accelerates steadily, the orbital radius becomes larger and larger, and the kinetic energy increases continuously. When the alternating electric field is turned off, the ions in the orbit generate alternating mirror current on the electrode. The computer converts the mirror current signal into a spectral signal by Fourier transform to obtain a mass spectrum.

Ionization and mass analysis of the compounds to be detected can be completed in the same analyzer. Fourier transform analyzer is suitable for compounds with molecular weight higher than 10000, with resolution as high as 106 and accurate determination of mass-to-charge ratio to one thousandth.

5.4 Tandem Mass Spectrometry Tandem Mass Spectrometry (MSMS) is a quality analysis that combines more than two levels in time or space. Spatial tandem is composed of more than two mass analyzers, such as three-stage quadrupole tandem mass spectrometry. The precursor ions selected by the first-stage mass analyzer (MS 1) enter the collision chamber for activation and decomposition, and the generated fragment ions are analyzed by the second-stage mass analyzer (MS2) to obtain MSMS spectrum. In time series mass spectrometry, the selection, fragmentation and fragmentation of precursor ions are completed in the same mass analyzer (such as quadrupole ion trap analyzer). The splitting of precursor ions can be achieved by metastable splitting, collision-induced splitting, surface-induced splitting and laser-induced splitting.

Tandem mass spectrometry is not limited to two-stage mass spectrometry, and multi-stage mass spectrometry experiments are often expressed as MSn. In practical application, tandem mass spectrometry can obtain data through product scanning, precursor scanning, neutral loss scanning and selective reaction monitoring (SRM), but it is worth noting that time tandem mass spectrometry can not perform precursor ion scanning and neutral loss scanning.

Tandem mass spectrometry has great advantages in the structural analysis of unknown compounds, the identification of compounds to be detected in complex mixtures, the clarification of cracking pathways and the quantitative analysis of low-concentration biological samples. It also has many applications in the medical field. For example, the structural information of precursor ions of drugs, impurities or pollutants can be obtained by product ion scanning, which is helpful for the identification of unknown compounds; The product ion scanning can also be used to detect the amino acid sequences of peptides and protein fragments. When mass spectrometry is combined with gas chromatography or liquid chromatography, if the compounds cannot be completely separated by chromatography, tandem mass spectrometry can selectively determine the characteristic precursor ions of a component, thus obtaining the structure and quantity information of the component without interference from the existing components.

In the study of drug metabolism, tandem mass spectrometry can be used to find metabolites with the same structural characteristics. Because metabolites may contain the same groups as neutral fragments (for example, carboxylic acids tend to lose neutral carbon dioxide molecules), all possible metabolites can be found by neutral loss scanning. If the missing fragment is an ion, the precursor ion scanning method can help to find all the precursor ions of the missing fragment ions.

Selective reactive ion detection (SRM) can eliminate the interference of biological matrix on the quantitative analysis of low concentration compounds to be detected. For example, in the pharmacokinetic study, the ion signal of the drug to be tested may be masked by the ion signal of other compounds in the matrix. By selectively detecting characteristic precursor ions and product ions with MS 1 and MS2, the specific and sensitive analysis of the substance to be detected can be realized.

5.5 Signal detection and data acquisition The ion beam from the mass analyzer is converted into an electrical signal by the detector, amplified, stored by the data processing system and displayed as a mass spectrogram. Mass spectrometry can realize qualitative and quantitative analysis of samples by measuring the mass-to-charge ratio and relative abundance of compounds to be detected.

A neutral molecule loses or captures an electron, that is, it forms a molecular ion with the same mass as the parent molecule. The molecular composition and molecular weight information of the compound to be tested can be obtained by high-resolution mass spectrometer (resolution >: 104) or by peak matching determination of the control compound. Molecular ions break different bonds to produce various fragment ions, and the fracture mode (or fragment mode) is related to the molecular structure. By measuring the mass and relative abundance of fragment ions, the cracking characteristics can be obtained, and the molecular structure of the compounds to be tested can be inferred or confirmed.

Mass spectrometry can realize quantitative analysis of high-tech properties by measuring the abundance of one or more specific ions and comparing with the response of known reference materials. External standard method and internal standard method are commonly used quantitative methods in mass spectrometry, and the accuracy of internal standard method is higher. The internal standard compounds used in mass spectrometry can be structural analogues or stable isotope markers of the compounds to be detected. The former has the advantage of lower cost, but using stable isotopes (such as 2H, 13C, 15N) can obtain higher analytical precision and accuracy, especially when using FAB or LCMS ionization technology (such as electrospray ionization). Stable isotope labels are indicators that always retain isotope labels during sample preparation, separation and ionization.