CN115326994A - A method, system and method for simultaneous analysis of multiple types of smoke exposure biomarkers - Google Patents

Abstract

The invention provides a method for simultaneous analysis of multiple types of smoke exposure biomarkers, comprising the following steps: adding an internal standard sample to urine, hydrolyzing, freeze-drying, concentrating, and reconstituting to obtain a sample solution by centrifugation; A multi-heart-cutting two-dimensional liquid chromatography-tandem mass spectrometry system was used for accurate quantification of smoke exposure biomarkers by internal standard method. The invention also provides a multi-dimensional liquid chromatography-mass spectrometry analysis system and a method for using the same. The invention effectively removes the interference of impurities through the first-dimensional separation, and improves the sensitivity and accuracy of target detection in combination with the enrichment effect of the trapping column. Comprehensive optimization of pretreatment conditions, mobile phase, chromatographic column and mass spectrometry parameters, etc., can realize the simultaneous analysis of multiple types of acid-base compounds with large differences in polarity and content, and provide a method for human smoke exposure evaluation. More efficient and convenient high-throughput analysis methods.

Description

A method, system and application for simultaneous analysis of multi-category smoke exposure biomarkers method

technical field

The invention belongs to the field of smoke exposure biomarker analysis and relates to a method for simultaneous analysis of multi-category smoke exposure biomarkers.

Background technique

With the full implementation of Healthy China and consumers' increasing attention to the issue of "smoking and health", the health risks of tobacco products have become a hot spot of public concern. At present, the chemical evaluation method of international tobacco product risk assessment is gradually transitioning from the analysis of the release of harmful components of smoking machines to the analysis of smoke exposure level with smokers as the evaluation object, and the evaluation is carried out by using smoke exposure biomarkers.

In the analysis of smoke exposure, the metabolites of nicotine, tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons and aromatic amines are currently the most representative markers, which can reflect the smoke exposure level of smokers. Because there are many types of smoke exposure biomarkers, and their contents and chemical properties vary greatly, different methods need to be used to analyze different types of smoke exposure biomarkers. For example, the content of nicotine metabolites in the urine of smokers is relatively high, which may Direct analysis by GC-MS or LC-MS; tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, and aromatic amine markers are extremely low, and liquid-liquid extraction, solid-phase extraction columns, or functional nanomaterials need to be used to detect the nitrosamines in body fluids. The target object is purified and enriched before detection. The pretreatment cost is high, the process is cumbersome, and it is easy to cause sample loss or contamination. The development of a method for the simultaneous analysis of multiple types of smoke exposure biomarkers can greatly improve the detection efficiency and make the evaluation of human smoke exposure more efficient and convenient.

Multidimensional liquid chromatography has become a research hotspot in recent years because of its characteristic of optimizing the combination of two or more liquid chromatography with different separation principles. It has high selectivity and sensitivity and is an ideal choice for analyzing complex matrix samples. , has been widely used in the analysis of biological samples, proteomics and natural products. Multidimensional liquid chromatography has a variety of separation modes, including single heart-cutting and multiple heart-cutting, etc. Compared with single heart-cutting method, multi-heart-cutting can analyze more types of targets, with greater difference in properties, and a wider range of applications.

Contents of the invention

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a method for simultaneous analysis of multi-category smoke exposure biomarkers, which is used to solve the problem of high processing cost, cumbersome process and easy sample loss in the prior art. Or pollution defects, the present invention can realize the simultaneous analysis of multiple smoke exposure biomarkers in human urine.

In order to achieve the above and related purposes, the present invention provides a method for simultaneous analysis of multi-category smoke exposure biomarkers, comprising the following steps:

A1) Add internal standard sample to urine, hydrolyze, freeze-dry, concentrate, and centrifuge to obtain sample solution after reconstitution;

A2) The sample solution was analyzed by the constructed multiple heart-cutting two-dimensional liquid chromatography-tandem mass spectrometry system, and the smoke exposure biomarkers were accurately quantified by the internal standard method.

Preferably, the volume ratio of the urine:internal standard solution=(1:0.001)-(1:0.060).

Preferably, in step A1), the hydrolysis is enzymatic hydrolysis or acid hydrolysis.

Preferably, in step A1), the redissolving solvent is deionized water, methanol or acetonitrile, preferably, the redissolving solvent is deionized water.

Preferably, the enzyme used in the enzymatic hydrolysis is at least one of β-glucuronidase and arylsulfatase.

Preferably, the volume ratio of urine:β-glucuronidase=(1:0.001)-(1:0.1).

Preferably, the acid used in the acidolysis is hydrochloric acid.

Preferably, a buffer is also added to the urine. More preferably, the buffer is sodium acetate-acetic acid buffer.

Preferably, the volume ratio of urine: sodium acetate-acetic acid buffer = (1:0.2)-(1:4).

Further preferably, the sodium acetate-acetic acid buffer has a concentration of 5-500 mM.

Further preferably, the pH of the sodium acetate-acetic acid buffer is 4.0-6.0.

Preferably, the centrifugal speed is 5000-13000rpm.

Preferably, the centrifugation time is 5-20min.

Preferably, the multi-category smoke exposure biomarkers are selected from cotinine, N-nitrosobasine, N-nitrosoannicotine, 4-(methylnitrosamine)-1- (3-pyridyl)-1-butanol, 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, 4-aminobiphenyl, 1-hydroxynaphthalene, 2-hydroxynaphthalene, 1-hydroxypyrene, 1 - one or more of hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 2-hydroxyfluorene or 3-hydroxyfluorene.

Preferably, in step A1), the internal standard is selected from the group consisting of d ₃ -cotinine (d ₃ -COT), d ₄ -N-nitrosobasine (d ₄ -NAB), d ₄ -N- Nitrosoanenicotine (d ₄ -NAT), 4-(methyl-d ₃ -nitrosoamino)-1-(3-pyridyl)-1-butanol (d ₃ -NNAL), d ₇ - 1-aminonaphthalene (d ₇ -1-NA), d ₇ -2-aminonaphthalene (d ₇ -2-NA), d ₉ -3-aminobiphenyl (d ₉ -3-ABP), d _9- 4 -aminobiphenyl (d ₉ -4-ABP), d ₉ -1-hydroxypyrene (d ₉ -1-OHPyr), ¹³ C ₆ -3-hydroxyphenanthrene ( ¹³ C ₆ -3-OHPhe), ¹³ C ₆ - one or more of 3-hydroxyfluorene ( ¹³ C ₆ -3-OHFlu), d ₇ -2-hydroxynaphthalene (d ₇ -2-OHNap).

Preferably, the multi-class heart-cutting two-dimensional liquid chromatography-tandem mass spectrometry is used to measure multi-category smoke exposure biomarkers, comprising the following steps:

B1) Preparation of standard samples: take cotinine, N-nitrosobasine, N-nitrosoaneonicotine, 4-(methylnitrosamine)-1-(3-pyridyl)-1 -Butanol, 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, 4-aminobiphenyl, 1-hydroxynaphthalene, 2-hydroxynaphthalene, 1-hydroxypyrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene , 3-Hydroxyphenanthrene, 4-Hydroxyphenanthrene, 9-Hydroxyphenanthrene, 2-Hydroxyfluorene and 3-Hydroxyfluorene standard samples, add internal standard sample, then add methanol to constant volume, prepare into a standard solution;

B2) Sample detection: the standard sample prepared in step B1) and the sample to be tested after sample pretreatment were analyzed by a multi-heart-cutting two-dimensional liquid chromatography-tandem mass spectrometry system, and the first-dimensional liquid chromatography was used to separate and remove impurities. The multi-category smoke exposure biomarkers are divided into multiple groups according to the retention time of the first-dimensional separation, and then separated by the second-dimensional liquid chromatography, and measured by mass spectrometry to determine the multi-category smoke exposure in the sample to be tested. Content of biomarker components.

Preferably, in step B2), the first dimension chromatographic column: an ion exchange column, a C18 column or a CN column; preferably, a CN column.

Preferably, in step B2), the compensation pump mobile phase: at least one of deionized water, methanol, acetonitrile, phosphate buffer, acetate buffer, formate buffer, ammonia solution; preferably, Deionized water.

Preferably, in step B2), trapping column: at least one of C18 column, HILIC column, PAH column, NH2 column, PFP column, Amino column, CN column, Phenyl column.

Preferably, in step B2), the second-dimensional chromatographic column: C18 column, PAH column, PFP column or HILIC column; further preferably, the second-dimensional chromatographic column is a C18 column; still more preferably, the second Dimension C18 chromatographic column is T3 or RP18 chromatographic column.

Preferably, the one-dimensional chromatographic conditions are:

One-dimensional pump mobile phase A phase: ammonium formate-water solution, mobile phase B; methanol or acetonitrile solution;

The first dimension column temperature: 25-45°C, detection wavelength: 230-400nm; injection volume: 0.5-20μL;

Flow rate: 0-30min flow rate is 0.2-0.4mL/min, 31-65min flow rate is 0.0-0.4mL/min;

Mobile phase of compensation pump: deionized water; compensation flow rate: 00-900μL/min;

Gradient elution program: 0-3min, 3-5%B; 10-15min, 85-95%B; 21-30min, 3-5%B;

Preferably, the two-dimensional chromatographic conditions are:

Two-dimensional pump mobile phase A: formic acid-water solution; mobile phase B: acetonitrile or methanol solution;

The temperature of the second dimension chromatographic column is 25-45°C;

Gradient elution: 0-12min, 3-5%B; 15-18min, 85-95%B; 18.1-23min, 3-5%B; 30-32min, 85-95%B; 32.1-37min, 3- 5% B; 37.1-52min, 35-50%B; 54-59min, 85-95%B; 59.1-65min, 3-5%B.

Preferably, the mass spectrometry conditions are:

Mass spectrometry: triple quadrupole tandem mass spectrometry, using electrospray ionization source (ESI), multiple reaction monitoring (MRM) mode; ion source temperature: 500-600 ℃; ion pair residence monitoring time: 20-50ms; nebulizer gas and Auxiliary gas pressure: 50-60psi; air curtain gas pressure: 10-25psi; when scanning in positive ion mode, electrospray voltage: 4000-5500V; when scanning in negative ion mode, electrospray voltage: -4500 to -5500V.

The second aspect of the present invention also provides a multi-dimensional liquid chromatography-mass spectrometry analysis system, including a first-dimensional liquid chromatography, a compensation pump, a three-way interface, a multi-way valve, a trapping column unit, and a second-dimensional liquid chromatography; The first-dimensional liquid chromatography includes a sample injector, a first-dimensional column thermostat, a one-dimensional pump, a one-dimensional chromatographic column, and a detector; the trapping column unit includes a trapping column and a multi-chromatographic column selection switching valve ; The second-dimensional liquid chromatography includes a two-dimensional pump, a second-dimensional column thermostat, a two-dimensional chromatographic column, and a mass spectrometer; the one-dimensional pump is connected to the one-dimensional chromatographic column, and the outlet of the one-dimensional chromatographic column passes through The pipeline is connected with the detector, and the outlet of the two-dimensional chromatographic column is connected with the mass spectrometer through the pipeline; The multi-way valve is respectively connected with the two-dimensional pump and the inlet of the two-dimensional chromatographic column through pipelines, the multi-column selection switching valve is connected with the multi-way valve, and the trapping column is connected with the multi-column selection switching valve.

Preferably, the trapping column unit includes a first trapping column, a second trapping column, a third trapping column, a fourth trapping column, a fifth trapping column, a sixth trapping column and multi-column selection Switching valve, the two ends of the first trapping column are respectively connected to the first inlet of the multi-chromatographic column selection switching valve and the first outlet of the multi-chromatographic column selection switching valve through pipelines, and the two ends of the second trapping column are connected to each other through pipelines. The pipelines are respectively connected to the second inlet of the multi-column selective switching valve and the second outlet of the multi-chromatographic column selective switching valve; The three inlets are connected to the third outlet of the multi-column selective switching valve; the two ends of the fourth trapping column are respectively connected to the fourth inlet of the multi-chromatographic column selective switching valve and the fourth outlet of the multi-chromatic column selective switching valve through pipelines The two ends of the fifth trapping column are respectively connected to the fifth inlet of the multi-chromatographic column selective switching valve and the fifth outlet of the multi-chromatographic column selective switching valve through pipelines; the two ends of the sixth trapping column are respectively connected through pipelines It is connected with the sixth inlet of the multi-column selection switching valve and the sixth outlet of the multi-column selection switching valve.

The third aspect of the present invention also provides a method for using a multidimensional liquid chromatography-mass spectrometry analysis system, comprising the following steps:

E1) Capture stage:

E11) adopting the trapping mode of the multidimensional liquid chromatography-mass spectrometry analysis system, trapping the first target component by the second trapping column;

E12) Switch the capture mode to the analysis mode, and cut the impurity components between the first and second target components into the waste liquid;

E13) When the second target component is eluted from the one-dimensional chromatographic column, switch back to the trapping mode; the multi-chromatographic column selection switching valve is synchronously switched to the third trapping column for trapping the second target component;

E14) After the capture of the second target component is completed, the capture mode is switched to the analysis mode, and the impurity component between the second and the third target component is cut into the waste liquid;

E15) When the third target component is eluted from the one-dimensional chromatographic column, switch back to the trapping mode; the multi-chromatographic column selection switching valve is synchronously switched to the fourth trapping column for trapping the third target component;

E16) After the capture of the third target component is completed, the capture mode is switched to the analysis mode, and the impurity component between the third and the fourth target component is cut into the waste liquid;

E17) When the fourth target component is eluted from the one-dimensional chromatographic column, switch back to the trapping mode; the multi-chromatographic column selection switching valve is synchronously switched to the fifth trapping column for trapping the fourth target component;

E18) After the capture of the fourth target component is completed, the capture mode is switched to the analysis mode, and the impurity component between the fourth and the fifth target component is cut into the waste liquid;

E19) When the fifth target component is eluted from the one-dimensional chromatographic column, switch back to the trapping mode; the multi-chromatographic column selection switching valve is synchronously switched to the sixth trapping column for trapping the sixth target component to Make all the target components trapped on the trapping column;

E2) Analysis stage:

Switching to the analysis mode, the flow of the one-dimensional pump relative to the one-dimensional chromatographic column performs impurity elution and chromatographic column rebalancing, and the flow of the two-dimensional pump (6) performs elution and analysis of all target components on the trapping unit in sequence.

Preferably, the capture mode includes the following steps:

F1) Driven by the mobile phase of the one-dimensional pump, the sample solution flows into the one-dimensional chromatographic column for preliminary separation to obtain multiple target components. The first target component flows into the first interface of the three-way interface through the detector. The introduced compensation mobile phase flows into the second interface of the three-way interface to mix the target component with the compensation mobile phase;

F2) After the target component obtained in step F1) flows out through the third interface of the three-way interface, it enters from the first inflow interface of the multi-way valve, enters the second interface of the multi-way valve through the first interface of the multi-way valve, and passes through the multi-way valve. The "IN" inlet of the column selection switching valve enters, flows out through the second inlet of the multi-column selection switching valve, and enters the second trapping column for trapping, and the mobile phase flows out from the second trapping column through the second outlet of the column selection switching valve. Then flow out from the "OUT" outlet of the multi-column selection switching valve, enter the fifth port of the multi-way valve, flow out from the sixth port of the multi-way valve, and enter the waste liquid to complete the capture of the first target component;

F3) After entering from the "IN" inlet of the multi-column selection switching valve, it flows out through the third inlet of the multi-column selection switching valve, and enters the third trapping column for trapping, and the mobile phase passes through the multi-column selection from the third trapping column Flow out from the third outlet of the switching valve, and then flow out from the "OUT" outlet of the multi-column selection switching valve. capture;

F4) After entering from the "IN" inlet of the multi-column selection switching valve, it flows out through the fourth inlet of the multi-column selection switching valve, and enters the fourth trapping column for trapping, and the mobile phase passes through the multi-column selection from the fourth trapping column It flows out from the fourth outlet of the switching valve, and then flows out from the "OUT" outlet of the multi-column selection switching valve. After entering the fifth port of the multi-way valve, it flows out from the sixth port of the multi-way valve and enters the waste liquid to complete the separation of the third target component. capture;

F5) After entering from the "IN" inlet of the multi-column selection switching valve, it flows out through the fifth inlet of the multi-column selection switching valve, and enters the fifth trapping column for trapping, and the mobile phase passes through the multi-column selection from the fifth trapping column Flow out from the fifth outlet of the switching valve, and then flow out from the "OUT" outlet of the multi-column selection switching valve, enter the fifth port of the multi-way valve, flow out from the sixth port of the multi-way valve, and enter the waste liquid to complete the second target component capture;

F6) After entering from the "IN" inlet of the multi-column selection switching valve, it flows out through the sixth inlet of the multi-column selection switching valve, and enters the sixth trapping column for trapping, and the mobile phase is selected from the sixth trapping column through the multi-chromatographic column It flows out from the sixth outlet of the switching valve, and then flows out from the "OUT" outlet of the multi-column selection switching valve. After entering the fifth port of the multi-way valve, it flows out from the sixth port of the multi-way valve and enters the waste liquid to complete the separation of the second target component. Trapping; the preliminary separation in step F1) to obtain multiple target components on the trapping column to complete the trapping.

Preferably, the analysis mode includes the following steps:

G1) The eluent of the one-dimensional chromatographic column flows into the first interface of the three-way interface driven by the mobile phase introduced by the one-dimensional pump, and at the same time, the compensation mobile phase introduced by the compensation pump flows into the second interface of the three-way interface;

G2) After the mixed liquid obtained in step G1) flows out through the third interface of the three-way interface, it enters through the first interface of the multi-way valve, and discharges the waste liquid through the sixth interface of the multi-way valve (7);

G3) The mobile phase introduced by the two-dimensional pump flows in through the third port of the multi-way valve, flows out from the second port of the multi-way valve after being switched, enters from the "IN" inlet of the multi-column selection switching valve, and passes through the multi-column selection switching valve It flows out from the second inlet and enters the second trapping column (10) for eluting. The eluent flows out through the second outlet of the multi-column selection switching valve, and then flows out from the "OUT" outlet of the multi-column selection switching valve and enters the multi-way valve. The fifth interface flows out from the fourth interface of the multi-way valve, enters the two-dimensional chromatographic column for further separation, and the eluent flows into the mass spectrometer for determination;

G4) The mobile phase introduced by the two-dimensional pump flows in through the third port of the multi-way valve, flows out from the second port of the multi-way valve after being switched, enters from the "IN" inlet of the multi-column selection switching valve, and passes through the multi-column selection switching valve It flows out from the second inlet and enters the second trapping column for eluting. The eluent flows out through the second outlet of the multi-column selection switching valve, and then flows out from the "OUT" outlet of the multi-column selection switching valve, and enters the fifth port of the multi-way valve. , flows out from the fourth port of the multi-way valve, enters the two-dimensional chromatographic column for further separation, and the eluent flows into the mass spectrometer for determination;

G5) The mobile phase introduced by the two-dimensional pump flows in through the third port of the multi-way valve, flows out from the second port of the multi-way valve after being switched, enters from the "IN" inlet of the multi-column selection switching valve, and passes through the multi-column selection switching valve It flows out from the third inlet and enters the third trapping column for eluting. The eluent flows out through the third outlet of the multi-column selection switching valve, and then flows out from the "OUT" outlet of the multi-column selection switching valve, and enters the fifth port of the multi-way valve. , flows out from the fourth port of the multi-way valve, enters the two-dimensional chromatographic column for further separation, and the eluent flows into the mass spectrometer for determination;

G6) The mobile phase introduced by the two-dimensional pump flows in through the third port of the multi-way valve, flows out from the second port of the multi-way valve after being switched, enters from the "IN" inlet of the multi-column selection switching valve, and passes through the multi-column selection switching valve The fourth inlet flows out and enters the fourth trapping column for eluting. The eluent flows out through the fourth outlet of the multi-column selection switching valve, and then flows out from the "OUT" outlet of the multi-column selection switching valve, and enters the fifth port of the multi-way valve. , flows out from the fourth port of the multi-way valve, enters the two-dimensional chromatographic column for further separation, and the eluent flows into the mass spectrometer for determination;

G7) The mobile phase introduced by the two-dimensional pump flows in through the third port of the multi-way valve, flows out from the second port of the multi-way valve after being switched, enters from the "IN" inlet of the multi-column selection switching valve, and passes through the multi-column selection switching valve The eluent flows out from the fifth inlet and enters the fifth trapping column for elution. The eluent flows out through the fifth outlet of the multi-column selection switching valve, and then flows out from the "OUT" outlet of the multi-column selection switching valve, and enters the fifth interface of the multi-way valve. , flows out from the fourth interface of the multi-way valve, enters the two-dimensional chromatographic column for further separation, and the eluent flows into the mass spectrometer for detection;

G8) The mobile phase introduced by the two-dimensional pump flows in through the third port of the multi-way valve, flows out from the second port of the multi-way valve after being switched, enters from the "IN" inlet of the multi-column selection switching valve, and passes through the multi-column selection switching valve The sixth inlet flows out and enters the fifth trapping column for eluting. The eluent flows out through the sixth outlet of the multi-column selection switching valve, and then flows out from the "OUT" outlet of the multi-column selection switching valve, and enters the fifth port of the multi-way valve. , flows out from the fourth port of the multi-way valve, enters the two-dimensional chromatographic column for further separation, and the eluent flows into the mass spectrometer for detection, so that the target analytes trapped on each trapping column are eluted, and then mass spectrometry is performed respectively.

As mentioned above, the present invention has the following beneficial effects:

In view of the complex matrix of urine samples, the variety of smoke exposure biomarkers, and the large difference in properties, the present invention provides a multi-heart-cutting two-dimensional liquid chromatography-tandem mass spectrometry, and further provides a natural isotope method to realize urine Simultaneous quantitative analysis of multiple categories of smoke exposure biomarkers in liquid. The present invention effectively removes impurity interference through the first-dimensional separation, improves the sensitivity and accuracy of target detection, and at the same time comprehensively optimizes pretreatment conditions, mobile phase, chromatographic column, and mass spectrometry parameters, and achieves a large polarity difference. The simultaneous analysis of acidic and basic compounds provides a more efficient and convenient high-throughput analysis method for the evaluation of human smoke exposure.

In the prior art, when the first dimension is eluted, the proportion of the organic phase of the solvent is relatively high, and many compounds cannot be supplemented by a complementary column. The simple processing method of the instrument company is to use a quantitative loop to divide the first dimension into multiple segments. Quantitative loop, stored inside, directly transferred to the second dimension for analysis. As a result, if the sample matrix is complex, there will be many other compounds on the quantitative loop, which will interfere with the second-dimensional analysis, and will be transferred directly. Sometimes the chromatographic peaks will be very broad and the sensitivity will not be high. The quantitative loop system is suitable for systems with relatively high content and relatively uncomplicated matrix. But if the sensitivity of the compound is extremely low, and the matrix interference of the sample is also particularly serious, it is difficult to solve it with a quantitative loop system at this time, and the device of the present invention can well solve the above problems.

Description of drawings

Figure 1 is a schematic diagram of a multiple heart-cutting two-dimensional liquid chromatography-tandem mass spectrometry system.

Figure 2 is the MRM diagram of the COT standard.

Figure 3 is the MRM diagram of NNAL standard.

Figure 4 is the MRM diagram of the NAT standard.

Figure 5 is the MRM chart of NAB standard.

Figure 6 is the MRM diagrams of 2-NA and 1-NA standards.

Fig. 7 is the MRM diagram of 3-ABP and 4-ABP standard products.

Figure 8 is the MRM charts of 2-OHNap and 1-OHNap standards.

Figure 9 is the MRM charts of 2-OHFlu and 3-OHFlu standards.

Figure 10 is the MRM charts of 1-OHPhe, 2-OHPhe, 3-OHPhe, 4-OHPhe and 9-OHPhe standard products.

Figure 11 is the MRM diagram of 1-OHPyr standard.

Figure 12 is the TIC chromatograms of polycyclic aromatic hydrocarbons analyzed by using RP18 chromatographic column and T3 chromatographic column.

Figure 13 is the TIC chromatogram of the actual sample.

Reference signs:

1 one-dimensional pump

2 1D columns

3 detectors

4 three-way interface

41 Tee interface first interface

42 Tee interface Second interface

43 Tee interface third interface

5 compensation pump

6 two-dimensional pump

7 multi-way valve

71 The first port of the multi-way valve

72 The second port of the multi-way valve

73 The third port of the multi-way valve

74 The fourth port of the multi-way valve

75 The fifth port of the multi-way valve

76 The sixth port of the multi-way valve

8 Multi-column selection switching valve

81 Multi-column selection switching valve first inlet

82 The first outlet of multi-column selection switching valve

83 Multi-column selection switching valve second inlet

84 Second outlet of multi-column selection switching valve

85 Multi-column selection switching valve third inlet

86 The third outlet of multi-column selection switching valve

87 The fourth inlet of multi-column selection switching valve

88 The fourth outlet of multi-column selection switching valve

89 Multi-column selection switching valve fifth inlet

810 Multi-Column Selection Switching Valve Fifth Outlet

811 Multi-column selection switching valve sixth inlet

812 Multi-column selection switching valve sixth outlet

813 Multi-Column Selector Switching Valve "IN" Inlet

814 Multi-column selection switching valve "OUT" outlet

9 First trapping column

10 Second trapping column

11 The third trapping column

12 Fourth trapping column

13 fifth trapping column

14 The sixth trapping column

15 2D columns

16 mass spectrometry

Detailed ways

Further set forth the present invention below in conjunction with specific embodiment, should be understood that these embodiments are only used for illustrating the present invention and are not intended to limit protection scope of the present invention.

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

Reagents and experimental utensils used in the following examples are routinely used reagents and experimental utensils, all of which can be purchased from the market. The specific reagents and instruments used are as follows:

1. Reagents

Acetonitrile, methanol (HPLC grade, U.S. TEDIA company); formic acid (≥98%, Germany Merck company); ammonium formate (U.S. sigma company); β-glucuronidase (U.S. Sigma company); peak); cotinine (COT), N-nitrosobasine (NAB), N-nitrosoannicotine (NAT), 4-(methylnitrosamine)-1-(3- pyridyl)-1-butanol (NNAL), d ₃ -cotinine (d ₃ -COT), d ₄ -N-nitrosobasine (d ₄ -NAB), d ₄ -N- Nitroneonicotinoid (d ₄ -NAT), 4-(methyl-d ₃ -nitrosoamino)-1-(3-pyridyl)-1-butanol (d ₃ -NNAL), 1-aminonaphthalene (1-NA), 2-aminonaphthalene (2-NA), 3-aminobiphenyl (3-ABP), 4-aminobiphenyl (4-ABP), d ₇ -1-aminonaphthalene (d ₇ -1 -NA), d ₇ -2-aminonaphthalene (d ₇ -2-NA), d ₉ -3-aminobiphenyl (d ₉ -3-ABP), d ₉ -4-aminobiphenyl (d ₉ -4 -ABP), 1-hydroxynaphthalene (1-OHNap), 2-hydroxynaphthalene (2-OHNap), 1-hydroxypyrene (1-OHPyr), 1-hydroxyphenanthrene (1-OHPhe), 2-hydroxyphenanthrene (2 -OHPhe), 3-hydroxyphenanthrene (3-OHPhe), 4-hydroxyphenanthrene (4-OHPhe), 9-hydroxyphenanthrene (9-OHPhe), 2-hydroxyfluorene (2-OHFlu), 3-hydroxyfluorene (3 -OHFlu), d ₉ -1-hydroxypyrene (d ₉ -1-OHPyr), ¹³ C ₆ -3-hydroxyphenanthrene ( ¹³ C ₆ -3-OHPhe), ¹³ C ₆ -3-hydroxyfluorene ( ¹³ C ₆ -3-OHFlu), d ₇ -2-hydroxynaphthalene (d ₇ -2-OHNap), etc. were purchased from Canada TRC Reagent Company.

2. Instrument

1290 liquid chromatograph of American Agilent Company, equipped with autosampler, quaternary mixing pump, binary pump, one-yuan pump, column thermostat, two-position multi-way valve (7), G4234A/C fast switching valve, diode array Detector (3) (DAD); electronic balance (accuracy: 0.0001g, Mettler Toledo, Switzerland); SW12H ultrasonic instrument (Sono Swiss, Switzerland); Milli-Q pure water instrument (Millpore, USA); Eppendorf 5810R high-speed centrifuge And ultra-low temperature refrigerator (Eppendorf, Germany); API 5500 triple quadrupole mass spectrometer (16) (SCIEX, USA); lyophilizer (LABCONCO, USA).

3. The MRM parameters of each analyte and internal standard in the mass spectrometry (16) detection are as shown in Table 1:

Table 1 MRM parameters of analyte and internal standard

Example 1

1 Sample pretreatment

Thaw the collected urine sample at room temperature, take 5mL sample in a 100mL beaker, add 10mL sodium acetate-acetic acid buffer (10mM, pH 5.1), 50μL internal standard solution and 50μL β-glucuronidase in sequence, mix well and seal The membrane was sealed and placed in a constant temperature water bath at 37°C for 16 hours in the dark for enzymatic hydrolysis. The enzymatically hydrolyzed samples were frozen in a -80°C ultra-low temperature refrigerator, then vacuum freeze-dried (shelf temperature 30°C), reconstituted with 500 μL deionized water, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken for analysis.

Calculation of isotope target content of 2COT

The natural abundances of ¹² C and ¹³ C are 98.89% and 1.11%, respectively, and COT contains 10 carbon atoms. When using MRM to analyze COT, this method selects m/z 178.0 as the quantitative parent ion of COT, which is a natural The [M+H] ⁺ ion content of ¹³ C-COT is 11.1% of the [M+H] ⁺ ion content of COT, which can meet the detection requirements of the method.

3 analysis conditions

The mobile phase A of the one-dimensional pump is 10mM ammonium formate-water solution, and B is methanol. Angela Venusil XBP CN column (2.1mm×100mm, 5μm) is used as the first-dimensional separation column. The column temperature is 30°C and the DAD detection wavelength The sample size is 260nm, the injection volume is 20μL, the flow rate is 0.3mL/min for 0-30min, and 0mL/min for 31-65min. Gradient elution conditions are: 0-3min, 3%B; 10-15min, 95%B; 21-30min, 3%B. The mobile phase of the compensation pump (5) is deionized water, and the compensation flow rate is 600 μL/min.

The mobile phase A of the two-dimensional pump is 0.1% formic acid-water solution, B is acetonitrile, using XBridge C18 chromatographic column (4.6mm × 30mm, 5 μm) as the trapping column, Atlantis T3 chromatographic column (2.1mm × 150mm, 3.0 μm) Analytical column as the second dimension. The column temperature is 30°C, the flow rate is 0.1 mL/min for 0-11 min, and 0.3 mL/min for 11.2-65 min. Gradient elution conditions are 0-12min, 5%B; 15-18min, 95%B; 18.1-23min, 5%B; 30-32min, 95%B; 32.1-37min, 5%B; 37.1-52min, 40 %B; 54-59min, 95%B; 59.1-65min, 5%B.

The mass spectrometer adopts electrospray ionization source (ESI); the detection method is multiple reaction monitoring (MRM); the ion source temperature is 600°C; the ion pair dwell time (Dwell time) is 50ms; the nebulizer gas and auxiliary gas pressure are 50psi; The air curtain gas pressure is 10psi; the collision gas level is Medium; when scanning in positive ion mode, the electrospray voltage is 5500V; when scanning in negative ion mode, the electrospray voltage is -4500V.

Example 2

Figures 2 to 11 are the MRM diagrams of the second-dimensional separation of 18 analyte standard samples. It can be seen that, except for 3-/4-ABP, 2-/3-OHFlu, 2-/3-OHPhe, 1-/4 Except several isomers such as -OHPhe, the rest of the compounds were effectively separated.

Example 3

The prepared standard working solution was analyzed, with the concentration ratio of the standard sample and the internal standard as the abscissa, and the peak area ratio as the ordinate, and the standard working curve of each target compound was obtained after linear regression. The standard working solution with the lowest concentration of each target was measured in parallel for 10 times, and the standard deviation was calculated, with 10 times the standard deviation as the quantitative limit of the method and 3 times the standard deviation as the detection limit of the method. The results are shown in Table 2. The linearity of the method is good (R ² >0.993); the detection limit of COT is 87.0pg/mL, and the limit of quantification is 290.0pg/mL; the detection limit of other target analytes is 0.8-13.1 pg/mL, the limit of quantification is 2.7-43.7pg/mL, all of which meet the requirements of quantitative detection.

Table 2 The linear equation, correlation coefficient, detection limit and quantification limit of the target

name linear equation R² Detection limit (pg/ml) Limit of quantitation (pg/ml) NNAL Y＝1.4e^-2X+1.8e^-2 0.9999 2.5 8.4 COT Y＝9.5e^-2X-7.6e^-1 0.9999 87.0 290.0 NAT Y＝7e^-3X-9e^-3 0.9988 1.0 3.3 NAB Y＝9e^-3X+7e^-3 0.9998 0.9 3.0 2-NA Y＝5e^-3X+4e^-3 0.9995 1.4 4.7 1-NA Y＝4.5e^-1X+1.8e⁰ 0.9994 2.7 9.0 3/4-ABP Y＝2e^-3X+2e^-3 0.9998 0.8 2.7 2-OH Nap Y＝5e^-5X+1e^-3 0.9998 11.3 37.7 1-OH Nap Y＝4e^-5X-8e^-5 0.9989 13.1 43.7 2/3-OHFlu Y＝2e^-6X-8e^-5 0.9985 7.9 26.5 2/3-OHPhe Y＝8e^-5X+1e^-3 0.9997 5.3 17.5 9-OHPhe Y＝1e^-4X+4e^-4 0.9938 3.1 10.3 1/4-OHPhe Y＝6e^-5X-9e^-5 0.9986 3.4 11.2 1-OHPyr Y＝5e^-5X-3e^-4 0.9995 2.9 9.7

Example 4

Select a urine sample from a non-smoker, add standard solutions at three levels of 1/2 (low), 1 (medium) and 2 (high) times the average content of the target analyte in smokers, and quantify by internal standard method , Determination of spike recovery. For targets with low average content such as NAT and NAB, increase the amount of spiked, so that the low-level spiked concentration is greater than the limit of quantification, which meets the needs of accurate quantification. The results are shown in Table 3, except that the partial recoveries of 9-OHPhe, 1-/4-OHPhe and 1-OHPyr are around 80%, most of the target analytes have low, medium and high levels of spiked recoveries Between 90-110%. The intraday and interday precisions of a urine sample were measured 5 times each, and the intraday and interday precisions of the target analytes were measured between 1.00-5.41% and 2.35-5.99%, respectively. It shows that this method has good recovery and precision, meets the detection requirements, and can be used for the quantitative analysis of trace smoke exposure biomarkers in urine.

Table 3 Spike recoveries, intra-day and inter-day precision of target substances in urine samples

Example 5

1 Sample pretreatment

Thaw the collected urine sample at room temperature, take 5mL sample in a 100mL beaker, add 10mL sodium acetate-acetic acid buffer (10mM, pH 5.1), 50μL internal standard solution and 50μL β-glucuronidase in sequence, mix well Seal with a parafilm and place in a constant temperature water bath at 37°C for 16 hours in the dark for enzymatic hydrolysis. The enzymatically hydrolyzed samples were frozen in a -80°C ultra-low temperature refrigerator, then vacuum freeze-dried (shelf temperature 30°C), reconstituted with 500 μL deionized water, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken for analysis.

Calculation of isotope target content of 2COT

The natural abundances of ¹² C and ¹³ C are 98.89% and 1.11%, respectively, and COT contains 10 carbon atoms. When using MRM to analyze COT, this method selects m/z 178.0 as the quantitative parent ion of COT, which is a natural The [M+H] ⁺ ion content of ¹³ C-COT is 11.1% of the [M+H] ⁺ ion content of COT, which can meet the detection requirements of the method.

3 analysis conditions

The mobile phase A of the one-dimensional pump is 10mM ammonium formate-water solution, and B is methanol. Angela Venusil XBP CN column (2.1mm×100mm, 5μm) is used as the first-dimensional separation column. The column temperature is 30°C and the DAD detection wavelength The sample size is 260nm, the injection volume is 20μL, the flow rate is 0.3mL/min for 0-30min, and 0mL/min for 31-65min. Gradient elution conditions are: 0-3min, 3%B; 10-15min, 95%B; 21-30min, 3%B. The mobile phase of the compensation pump was deionized water, and the compensation flow rate was 600 μL/min.

The mobile phase A of the two-dimensional pump (6) is 0.1% formic acid-water solution, and B is acetonitrile. The XBridge C18 chromatographic column (4.6mm × 30mm, 5 μm) is used as the Trap trap column, and the Symmetry Shield RP18 chromatographic column (2.1mm × 150mm, 3.0μm) as the second dimension analysis column. The column temperature is 30°C, the flow rate is 0.1 mL/min from 0-11 min, and 0.3 mL/min from 11.2-65 min. The gradient elution condition is 0-12min, 5% B; 15-18min, 95% B; 18.1-23min, 5% B; 30-32min, 95% B; 32.1-37min, 5% B; 37.1-52min, 35 %B; 54-59min, 95%B; 59.1-65min, 5%B.

The mass spectrometer adopts electrospray ionization source (ESI); the detection method is multiple reaction monitoring (MRM); the ion source temperature is 600°C; the ion pair dwell time (Dwell time) is 50ms; the nebulizer gas and auxiliary gas pressure are 50psi; The air curtain gas pressure is 10psi; the collision gas level is Medium; when scanning in positive ion mode, the electrospray voltage is 5500V; when scanning in negative ion mode, the electrospray voltage is -4500V.

4 Results Analysis

The choice of second dimension column greatly affects the sensitivity and resolution of the final target analytes. Figure 12 is the TIC diagram of separation of polycyclic aromatic hydrocarbons by the RP18 column and the T3 column during the second-dimensional column selection verification. It can be seen that under the same chromatographic conditions, the RP18 column has a better resolution for polycyclic aromatic hydrocarbons, while The T3 column is more sensitive to PAHs, and both can get better analytical results.

At the same time, in Example 5, the Symmetry Shield RP18 chromatographic column was used as the analytical column in the second-dimensional separation, and a good retention effect was also obtained for 18 compounds. The detection results are shown in Figure 13. Figure 13 is the TIC chromatogram obtained by analyzing the actual sample using multiple heart-cutting-two-dimensional liquid chromatography-tandem mass spectrometry system. As can be seen from Figure 13, the detection of cotinine using the "natural carbon isotope" method, even if the urine sample is enriched and concentrated, the signal response of COT can be greatly weakened, and the simultaneous detection of other trace metabolites can be achieved, so this method It can be applied to the simultaneous detection of compounds with large content differences to reduce analysis costs and workload.

In summary, the present invention establishes a multi-heart-cutting two-dimensional liquid chromatography-tandem mass spectrometry method, which realizes multi-category smoke exposure of nicotine, tobacco-specific nitrosamines, aromatic amines and polycyclic aromatic hydrocarbons in urine. Simultaneous analysis of biomarkers. The method has high sensitivity, good reproducibility, effective removal of impurities and good separation of components, and has broad application prospects in the simultaneous analysis and detection of multiple types of substances in complex biological samples.

Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial application value.

The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Any person familiar with this technology can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims (12)

1. A method for simultaneous analysis of multi-class smoke exposure biomarkers comprising the steps of:

a1 Adding an internal standard sample into urine, hydrolyzing, freeze-drying, concentrating, re-dissolving, and centrifuging to obtain a sample solution;

a2 The sample solution is analyzed by adopting a constructed multi-center cutting two-dimensional liquid chromatography-tandem mass spectrometry system, and the smoke exposure biomarker is accurately quantified by an internal standard method.

2. The method for the simultaneous analysis of multiple-class smoke exposure biomarkers according to claim 1, wherein in step A1),

the hydrolysis is enzymolysis or acidolysis;

and/or the redissolution solvent is deionized water, methanol or acetonitrile, preferably the redissolution solvent is deionized water.

3. The method for simultaneously analyzing the multi-class smoke exposure biomarkers according to claim 2, wherein the enzyme adopted by the enzymolysis is at least one of β -glucuronidase and arylsulfatase;

and/or the acid adopted for acidolysis is hydrochloric acid.

4. The method of claim 1, comprising one of the following technical features:

1) The multi-class smoke exposure biomarker is selected from one or more of cotinine, N-nitrosoanabasine, N-nitrosoneonicotine, 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanol, 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, 4-aminobiphenyl, 1-hydroxynaphthalene, 2-hydroxynaphthalene, 1-hydroxypyrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 2-hydroxyfluorene or 3-hydroxyfluorene;

2) In step A1), the internal standard is selected from d ₃ -cotinine (d) ₃ -COT)、d ₄ -N-nitrosoanabasine (d) ₄ -NAB)、d ₄ -N-nitrosoneonicotinoid (d) ₄ NAT), 4- (methyl-d ₃ -nitrosamino) -1- (3-pyridyl) -1-butanol (d) ₃ -NNAL)、d ₇ -1-aminonaphthalene (d) ₇ –1-NA)、d ₇ -2-aminonaphthalene (d) ₇ -2-NA)、d ₉ -3-aminobiphenyl (d) ₉ -3-ABP)、d ₉ -4-aminobiphenyl (d) ₉ -4-ABP)、d ₉ -1-hydroxypyrene (d) ₉ -1-OHPyr)、 ¹³ C ₆ -3-hydroxyphenanthrene (b) ¹³ C ₆ -3-OHPhe)、 ¹³ C ₆ -3-hydroxyfluorene(s) (iii) ¹³ C ₆ -3-OHFlu)、d ₇ -2-hydroxynaphthalene (d) ₇ -2-OHNap)).

5. The method of simultaneous multi-class smoke exposure biomarker analysis according to claim 1, wherein the multi-class smoke exposure biomarker assay using multi-center cut two-dimensional liquid chromatography-tandem mass spectrometry comprises the steps of:

b1 Preparation of standard samples: taking any one or more of standard samples of cotinine, N-nitrosoanabasine, N-nitrosoneonicotin, 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanol, 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, 4-aminobiphenyl, 1-hydroxynaphthalene, 2-hydroxynaphthalene, 1-hydroxypyrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 2-hydroxyfluorene and 3-hydroxyfluorene components, adding an internal standard sample, adding methanol for constant volume, and preparing a standard solution; b2 Sample testing: respectively analyzing the standard sample prepared in the step B1) and the sample to be detected after sample pretreatment by adopting a multi-center cutting two-dimensional liquid chromatography-tandem mass spectrometry system, separating and removing impurities through a first-dimensional liquid chromatography, collecting the multi-class smoke exposure biomarkers into multiple groups according to the retention time of the first-dimensional separation, separating through a second-dimensional liquid chromatography, and determining the content of the multi-class smoke exposure biomarkers in the sample to be detected through mass spectrometry.

6. The method for simultaneous analysis of multiple-class smoke exposure biomarkers according to claim 5, wherein in step B2), the analysis conditions of the multicenter cutting two-dimensional liquid chromatography-tandem mass spectrometry system comprise one or several of the following technical features:

c1 A first dimension chromatography column: an ion exchange column, a C18 column or a CN column; preferably, a CN column.

C2 Compensated pump mobile phase: at least one of deionized water, methanol, acetonitrile, phosphate buffer solution, acetate buffer solution, formate buffer solution and ammonia water solution; preferably, deionized water.

C3 Capture column: at least one of C18 column, HILIC column, PAH column, NH2 column, PFP column, amino column, CN column and Phenyl column;

c4 A second dimension chromatography column: a C18 column, PAH column, PFP column or HILIC column; further preferably, the second dimension chromatographic column adopts a C18 column; still further preferably, the second dimension C18 chromatography column is a T3 or RP18 chromatography column.

7. The method of simultaneous analysis of multiple-class smoke exposure biomarkers according to claim 5, wherein the conditions of said multicenter-cut two-dimensional liquid chromatography-tandem mass spectrometry system analysis comprise one or several of the following technical features:

d1 One-dimensional chromatographic conditions were:

one-dimensional pump mobile phase a: ammonium formate-water solution, mobile phase B; methanol or acetonitrile solution;

column temperature of the first dimension chromatographic column: 25-45 ℃, detection wavelength: 230-400nm; sample injection amount: 0.5-20 μ L;

flow rate: the flow rate is 0.2-0.4mL/min at 0-30min, and 0.0-0.4mL/min at 31-65 min;

the mobile phase of the compensation pump is deionized water; compensating the flow rate: 500-900 mu L/min;

gradient elution procedure: 0-3min, 3-5%; 10-15min,85-95% by weight B;21-30min, 3-5%;

d2 Two-dimensional chromatographic conditions were:

two-dimensional pump mobile phase a: formic acid-water solution; and (3) mobile phase B: acetonitrile or methanol solution;

the temperature of the second dimension chromatographic column is 25-45 ℃;

gradient elution: 0-12min,3-5% by weight B;15-18min,85-95% by weight B;18.1-23min, 3-5%; 30-32min,85-95% by weight B;32.1-37min,3-5% by weight of B;37.1-52min,35-50% by weight B;54-59min,85-95% by weight B;59.1-65min,3-5% of water, and B.

D3 ) mass spectrometry conditions were:

mass spectrum: triple quadrupole tandem mass spectrometry, using electrospray ionization (ESI), multiple Reaction Monitoring (MRM) mode; ion source temperature: 500-600 ℃; ion pair residence monitoring time: 20-50ms; atomizing gas and auxiliary gas pressure: 50-60psi; air curtain pressure: 10-25psi; electrospray voltage during positive ion mode scan: 4000-5500V; during scanning in the negative ion mode, electrospray voltage: -4500 to-5500V.

8. A multi-dimensional liquid chromatography-mass spectrometry combined analysis system is characterized by comprising a first-dimensional liquid chromatography, a compensation pump (5), a three-way interface (4), a multi-way valve (7), a trapping column unit and a second-dimensional liquid chromatography; the first-dimensional liquid chromatogram comprises a sample injector, a one-dimensional column incubator, a one-dimensional pump (1), a one-dimensional chromatographic column (2) and a detector (3); the trapping column unit comprises a trapping column and a multicolor spectrum column selection switching valve (8); the second-dimension liquid chromatogram comprises a two-dimension pump (6), a two-dimension column incubator, a two-dimension chromatographic column (15) and a mass spectrum (16); the one-dimensional pump (1) is connected with a one-dimensional chromatographic column (2), the outlet of the one-dimensional chromatographic column (2) is connected with the detector (3) through a pipeline, and the outlet of the two-dimensional chromatographic column (15) is connected with the mass spectrum (16) through a pipeline; the three-way interface (4) is respectively connected with an outlet of the detector (3), the compensation pump (5) and the multi-way valve (7) through pipelines, the multi-way valve (7) is respectively connected with the two-dimensional pump (6) and the two-dimensional chromatographic column (15) through pipelines, the multi-color chromatographic column selection switching valve (8) is communicated with the multi-way valve (7), and the trapping column is communicated with the multi-color chromatographic column selection switching valve (8).

9. The multidimensional liquid chromatography-mass spectrometry combined analysis system according to claim 8, wherein the trapping column unit comprises a first trapping column (9), a second trapping column (10), a third trapping column (11), a fourth trapping column (12), a fifth trapping column (13), a sixth trapping column (14) and a polychromatic column selection switching valve (8), wherein two ends of the first trapping column (9) are respectively connected with a first inlet (81) of the polychromatic column selection switching valve and a first outlet (82) of the polychromatic column selection switching valve through pipelines, and two ends of the second trapping column (10) are respectively connected with a second inlet (83) of the polychromatic column selection switching valve and a second outlet (84) of the polychromatic column selection switching valve through pipelines; two ends of the third capturing column (11) are respectively connected with a third inlet (85) of the multicolor column selection switching valve and a third outlet (86) of the multicolor column selection switching valve through pipelines; two ends of the fourth trapping column (12) are respectively connected with a fourth inlet (87) of the multicolor column selection switching valve and a fourth outlet (88) of the multicolor column selection switching valve through pipelines; two ends of the fifth capturing column (13) are respectively connected with a fifth inlet (89) of the multicolor column selection switching valve and a fifth outlet (810) of the multicolor column selection switching valve through pipelines; and two ends of the sixth trapping column (14) are respectively connected with a sixth inlet (811) and a sixth outlet (812) of the multi-color spectrum column selection switching valve through pipelines.

10. The method of claim 8, wherein the method comprises the steps of:

e1 Capture stage):

e11 Capturing the first target component by a second capturing column (10) by adopting a capturing mode of a multidimensional liquid chromatography-mass spectrometry combined analysis system;

e12 Switching the capture mode to an analysis mode to cut an impurity component between the first and second target components into the waste stream;

e13 Switching back to the trapping mode when the second target component is eluted from the one-dimensional chromatographic column (2); the multi-color spectrum column selection switching valve (8) is synchronously switched to the next capturing column for capturing the next target component;

e14 Steps E11) to E13 are repeated on a third trap column (11), a fourth trap column (12), a fifth trap column (13), and a sixth trap column (14), respectively, so that trapping of all the target components on the trap columns is completed;

e2 Analysis phase):

and switching to an analysis mode, wherein the flow of the one-dimensional pump (1) is subjected to impurity elution and chromatographic column rebalancing relative to the one-dimensional chromatographic column (2), and the flow of the two-dimensional pump (6) is subjected to elution analysis relative to all target components on the trapping unit in sequence.

11. The method of claim 10, wherein the trapping mode comprises the steps of:

f1 The sample solution flows into a one-dimensional chromatographic column (2) to be primarily separated under the driving of a mobile phase of a one-dimensional pump (1) to obtain a plurality of target components, the first target component flows into a first connector (41) of a three-way connector through a detector (3), and meanwhile, a compensation mobile phase introduced by a compensation pump (5) flows into a second connector (42) of the three-way connector to mix the target components with the compensation mobile phase;

f2 The target component obtained IN the step F1) flows OUT through the third interface (43) of the three-way interface, enters from the first interface (71) of the multi-way valve, enters from the second interface (72) of the multi-way valve through the first interface (71) of the multi-way valve, enters from the inlet (813) of the multi-chromatographic column selection switching valve, flows OUT from the second inlet (83) of the multi-chromatographic column selection switching valve, enters the second trapping column (10) for trapping, flows OUT from the second trapping column (10) through the second outlet (84) of the chromatographic column selection switching valve, flows OUT from the outlet (814) of the multi-chromatographic column selection switching valve, enters the fifth interface (75) of the multi-way valve, flows OUT from the sixth interface (76) of the multi-way valve, and enters waste liquid;

f3 F2, F3) is repeated after entering from an inlet (813) of the multi-chromatographic column selection switching valve 'IN', the inflow interface, the trapping column and the outflow interface of the multi-chromatographic column selection switching valve (8), and a plurality of target components obtained by preliminary separation IN the step F1) are trapped on the trapping column.

12. The method of claim 10, wherein the analysis mode comprises the steps of:

g1 Eluent of the one-dimensional chromatographic column (2) flows into a first interface (41) of the three-way interface under the driving of a mobile phase introduced by a one-dimensional pump (1), and meanwhile, a compensation mobile phase introduced by a compensation pump (5) flows into a second interface (42) of the three-way interface;

g2 The mixed liquid obtained in the step G1) flows out through a third connector (43) of the three-way connector, enters through a first connector (71) of the multi-way valve, and is discharged through a sixth connector (76) of the multi-way valve;

g3 The mobile phase introduced by the two-dimensional pump (6) flows IN through a multi-way valve third interface (73), flows OUT through a multi-way valve second interface (72) after being switched, enters from a multi-chromatographic column selection switching valve 'IN' inlet (813), flows OUT through a multi-chromatographic column selection switching valve second inlet (83), enters a second trapping column (10) for elution, flows OUT through a multi-chromatographic column selection switching valve second outlet (84), flows OUT from a multi-chromatographic column selection switching valve 'OUT' outlet (814), enters a multi-way valve fifth interface (75), flows OUT from a multi-way valve fourth interface (74), enters a two-dimensional chromatographic column (15) for further separation, and flows into a mass spectrum (16) for determination;

g4 C) entering from an inlet (813) of a multi-chromatographic column selection switching valve, switching an inflow interface, a trapping column and an outflow interface of the multi-chromatographic column selection switching valve (8), repeating the operation step G3), eluting the target analytes trapped on the respective trapping columns, and performing mass spectrometry.

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