CN115326994A - Method and system for simultaneously analyzing multi-class smoke exposure biomarkers and using method - Google Patents
Method and system for simultaneously analyzing multi-class smoke exposure biomarkers and using method Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention provides a method for simultaneously analyzing multi-class smoke exposure biomarkers, which comprises the following steps of: adding an internal standard sample into urine, hydrolyzing, freeze-drying, concentrating, re-dissolving and then centrifuging to obtain a sample solution; and analyzing the sample solution by adopting a constructed multi-center cutting two-dimensional liquid chromatography-tandem mass spectrometry system, and accurately quantifying the smoke exposure biomarker by an internal standard method. The invention also provides a multidimensional liquid chromatography-mass spectrometry combined analysis system and a use method thereof, impurity interference is effectively removed through first-dimension separation, the sensitivity and accuracy of target object detection are improved by combining the enrichment effect of the trapping column, pretreatment conditions, mobile phases, chromatographic columns, mass spectrum parameters and the like are comprehensively optimized, simultaneous analysis of multi-class acid-base compounds with large polarity difference and large content difference can be realized, and a more efficient and convenient high-throughput analysis method is provided for human body smoke exposure evaluation.
Description
Technical Field
The invention belongs to the field of analysis of smoke exposure biomarkers, and relates to a method for simultaneously analyzing multi-class smoke exposure biomarkers.
Background
With the overall implementation of healthy china and the growing concern of consumers about the "smoking and health" problem, the health risks of tobacco products have become a focus of public attention. Currently, the chemical evaluation method for international tobacco product risk assessment gradually transits from the analysis of harmful component release amount of a smoking machine to the analysis of smoke exposure level by taking a smoker as an evaluation object, and adopts a smoke exposure biomarker for evaluation.
In the smoke exposure analysis, metabolites of nicotine, nitrosamine, polycyclic aromatic hydrocarbon and aromatic amine which are specific to tobacco are the most representative markers at present, and can reflect the smoke exposure level of smoking people. Because the smoke exposure biomarkers are various in types and large in content and chemical property difference, different methods are needed to analyze the current smoke exposure biomarkers, for example, the nicotine metabolites in urine of smokers are relatively high in content, and the smoke exposure biomarkers can be directly analyzed by gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry; the content of the specific nitrosamine, polycyclic aromatic hydrocarbon and aromatic amine markers of the tobacco is extremely low, the target objects in body fluid need to be purified and enriched by liquid-liquid extraction, solid-phase extraction columns or functional nano materials and the like and then detected, the pretreatment cost is high, the process is complicated, and the loss or pollution of samples is easily caused. The method for simultaneously analyzing the multi-class smoke exposure biomarkers can greatly improve the detection efficiency and enable the human smoke exposure evaluation to be more efficient and convenient.
The multidimensional liquid chromatography has the characteristic of optimally combining two or more liquid chromatographs with different separation principles, so that the multidimensional liquid chromatography becomes a research hotspot in recent years, has higher selectivity and sensitivity, is an ideal choice for analyzing complex matrix samples, and is widely applied to analysis of biological samples, proteomics and natural products. Multidimensional liquid chromatography has a variety of separation modes, including single-center cutting, multi-center cutting, and the like. Compared with a single-center cutting method, the multi-center cutting method has the advantages of more analyzable target species, larger property difference and wider application range.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention aims to provide a method for simultaneously analyzing multiple classes of smoke exposure biomarkers, which is used to solve the problems of high processing cost, complicated process and easy sample loss or pollution in the prior art.
To achieve the above and related objects, the present invention provides a method for simultaneously analyzing multiple classes of 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.
Preferably, the urine: volume ratio of internal standard solution = (1.
Preferably, in step A1), the hydrolysis is enzymatic or acid hydrolysis.
Preferably, in step A1), the redissolving solvent is deionized water, methanol or acetonitrile, and preferably, the redissolving solvent is deionized water.
Preferably, the enzyme used for enzymolysis is at least one of beta-glucuronidase and arylsulfatase.
Preferably, the urine: volume ratio of β -glucuronidase = (1.
Preferably, the acid used for acid hydrolysis is hydrochloric acid.
Preferably, a buffer is also added to the urine. More preferably, the buffer is a sodium acetate-acetic acid buffer.
Preferably, the urine: the volume ratio of sodium acetate-acetic acid buffer = (1.
Further preferably, the concentration of the sodium acetate-acetic acid buffer is 5 to 500mM.
Further preferably, the pH of the sodium acetate-acetic acid buffer is 4.0 to 6.0.
Preferably, the centrifugal speed is 5000-13000rpm.
Preferably, the centrifugation time is 5-20min.
Preferably, the multi-class smoke exposure biomarker is selected from one or more of cotinine, N-nitrosopseudobases, 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 or 3-hydroxyfluorene.
Preferably, 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-nitrosoneonicotinoids (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)).
Preferably, the multi-class smoke exposure biomarker determination by multi-center cutting two-dimensional liquid chromatography-tandem mass spectrometry comprises the following steps:
b1 Preparing a standard sample: taking any one or more of cotinine, N-nitrosoanabasine, 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 to a constant volume to prepare 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.
Preferably, in step B2), the first dimension chromatographic column: ion exchange column, C18 column or CN column; preferably, a CN column.
Preferably, in step B2), the pump mobile phase is compensated: at least one of deionized water, methanol, acetonitrile, phosphate buffer, acetate buffer, formate buffer and ammonia water solution; preferably, deionized water.
Preferably, in step B2), the trapping column: at least one of C18 column, HILIC column, PAH column, NH2 column, PFP column, amino column, CN column and Phenyl column.
Preferably, in step B2), the second dimension chromatographic column: a C18 column, PAH column, PFP column or HILIC column; further preferably, the second dimension chromatographic column is a C18 column; even more preferably, the second dimension C18 chromatography column is a T3 or RP18 chromatography column.
Preferably, the one-dimensional chromatographic conditions are:
one-dimensional pump mobile phase a: ammonium formate-water solution, mobile phase B; methanol or acetonitrile solution;
first dimension chromatographic column temperature: 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: 00-900 mu L/min;
gradient elution procedure: 0-3min, 3-5%; 10-15min,85-95% by weight of B;21-30min,3-5% by weight B;
preferably, the two-dimensional chromatographic conditions are:
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% of 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.
Preferably, the mass spectrometry conditions are:
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 for positive ion mode scan: 4000-5500V; during scanning in the negative ion mode, electrospray voltage: -4500 to-5500V.
The invention also provides a multi-dimensional liquid chromatography-mass spectrometry combined analysis system, which comprises 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 chromatogram comprises a sample injector, a first-dimensional column incubator, a one-dimensional pump, a one-dimensional chromatographic column and a detector; the trapping column unit comprises a trapping column and a multicolor column selection switching valve; the second-dimensional liquid chromatogram comprises a two-dimensional pump, a second-dimensional column incubator, a two-dimensional chromatographic column and a mass spectrum; the one-dimensional pump is connected with a one-dimensional chromatographic column, an outlet of the one-dimensional chromatographic column is connected with the detector through a pipeline, and an outlet of the two-dimensional chromatographic column is connected with the mass spectrum through a pipeline; the three-way interface is respectively connected with the outlet of the detector, the compensation pump and the two-position multi-way valve through pipelines, the two-position multi-way valve is respectively connected with the two-dimensional pump and the inlet of the two-dimensional chromatographic column through pipelines, the multi-color spectrum column selection switching valve is communicated with the multi-way valve, and the trapping column is communicated with the multi-color spectrum column selection switching valve.
Preferably, the trap column unit comprises a first trap column, a second trap column, a third trap column, a fourth trap column, a fifth trap column, a sixth trap column and a multicolor column selection switching valve, wherein two ends of the first trap column are respectively connected with a first inlet of the multicolor column selection switching valve and a first outlet of the multicolor column selection switching valve through pipelines, and two ends of the second trap column are respectively connected with a second inlet of the multicolor column selection switching valve and a second outlet of the multicolor column selection switching valve through pipelines; two ends of the third trapping column are respectively connected with a third inlet of the multi-color spectrum column selection switching valve and a third outlet of the multi-color spectrum column selection switching valve through pipelines; two ends of the fourth trapping column are respectively connected with a fourth inlet of the multicolor spectrum column selection switching valve and a fourth outlet of the multicolor spectrum column selection switching valve through pipelines; two ends of the fifth trapping column are respectively connected with a fifth inlet of the multicolor spectrum column selection switching valve and a fifth outlet of the multicolor spectrum column selection switching valve through pipelines; and two ends of the sixth trapping column are respectively connected with the sixth inlet of the multi-chromatographic column selective switching valve and the sixth outlet of the multi-chromatographic column selective switching valve through pipelines.
The third aspect of the invention also provides a use method of the multidimensional liquid chromatography-mass spectrometry combined analysis system, which comprises the following steps:
e1 Capture stage):
e11 Adopting a trapping mode of a multidimensional liquid chromatography-mass spectrometry system, and trapping the first target component through a second trapping column;
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 When the second target component is eluted from the one-dimensional chromatographic column, switching back to the trapping mode; the multi-color spectrum column selection switching valve is synchronously switched to a third trapping column for trapping a second target component;
e14 After the trapping of the second target component is completed, the trapping mode is switched to the analysis mode, and the impurity component between the second and third target components is cut into the waste liquid;
e15 When the third target component is eluted from the one-dimensional chromatographic column, switching back to the trapping mode; the multi-color spectrum column selection switching valve is synchronously switched to a fourth trapping column for trapping a third target component;
e16 After the trapping of the third target component is completed, the trapping mode is switched to the analysis mode, and the impurity component between the third and fourth target components is cut into the waste liquid;
e17 When the fourth target component is eluted from the one-dimensional chromatographic column, switching back to the trapping mode; the multi-color spectrum column selection switching valve is synchronously switched to a fifth trapping column for trapping a fourth target component;
e18 After the trapping of the fourth target component is completed, switching the trapping mode to the analysis mode, and cutting the impurity component between the fourth target component and the fifth target component into the waste liquid;
e19 Switching back to the trapping mode when the fifth target component is eluted from the one-dimensional chromatographic column; synchronously switching the multi-color spectrum column selection switching valve to a sixth trapping column for trapping a sixth target component so as to finish trapping all the target components on the trapping column;
e2 Analysis phase):
and switching to an analysis mode, wherein the flow of the one-dimensional pump elutes impurities and rebalances the one-dimensional chromatographic column, and the flow of the two-dimensional pump (6) elutes and analyzes all target components on the trapping unit in sequence.
Preferably, the trapping mode comprises the steps of:
f1 The sample solution flows into a one-dimensional chromatographic column to be subjected to preliminary separation under the driving of a mobile phase of a one-dimensional pump to obtain a plurality of target components, the first target component flows into a first interface of a three-way interface through a detector, and meanwhile, a compensation mobile phase introduced by a compensation pump flows into a second interface 2 of the three-way interface to mix the target components with the compensation mobile phase;
f2 Allowing the target component obtained IN the step F1) to flow OUT through a third interface of the three-way interface, allowing the target component to enter from a1 st inflow interface of the multi-way valve, allowing the target component to enter into a second interface of the multi-way valve through a first interface of the multi-way valve, allowing the target component to enter from an "IN" inlet of a multi-chromatographic column selection switching valve, allowing the target component to flow OUT from a second inlet of the multi-chromatographic column selection switching valve, allowing the target component to enter into a second trapping column for trapping, allowing a mobile phase to flow OUT from a second outlet of the multi-chromatographic column selection switching valve, allowing the mobile phase to flow OUT from an "OUT" outlet of the multi-chromatographic column selection switching valve, allowing the mobile phase to flow OUT from a sixth interface of the multi-way valve after the mobile phase enters into a fifth interface of the multi-way valve, and allowing the mobile phase to enter into waste liquid to trap the first target component;
f3 The mobile phase flows OUT from a third trapping column through a third outlet of the multi-chromatographic column selection switching valve, then flows OUT from an outlet of the multi-chromatographic column selection switching valve, enters a fifth interface of the multi-way valve, flows OUT from a sixth interface of the multi-way valve, enters waste liquid and finishes the trapping of a second target component;
f4 The mobile phase flows OUT from a fourth catching column through a fourth outlet of the multi-color spectrum column selection switching valve, flows OUT from an outlet of the multi-color spectrum column selection switching valve, enters a fifth interface of the multi-way valve, flows OUT from a sixth interface of the multi-way valve, enters waste liquid and finishes the catching of a third target component;
f5 The mobile phase flows OUT from a fifth interface of the multi-way valve, flows OUT from a sixth interface of the multi-way valve, enters waste liquid and finishes the trapping of a second target component;
f6 The mobile phase flows OUT from a sixth collecting column through a sixth outlet of the multi-color spectrum column selection switching valve, flows OUT from an OUT outlet of the multi-color spectrum column selection switching valve, enters a fifth interface of the multi-way valve, flows OUT from the sixth interface of the multi-way valve, enters waste liquid and finishes the collection of a second target component; and F1), collecting a plurality of target components obtained by preliminary separation in the step F1) on a collecting column.
Preferably, the analysis mode comprises the steps of:
g1 One-dimensional chromatographic column eluent is driven by a mobile phase introduced by a one-dimensional pump to flow into a first interface of a three-way interface, and meanwhile, a compensation mobile phase introduced by a compensation pump flows into a second interface of the three-way interface;
g2 The mixed liquid obtained in the step G1) flows out through a third interface of the three-way interface, enters through a first interface of the multi-way valve, and is discharged through a sixth interface of the multi-way valve (7);
g3 The mobile phase introduced by the two-dimensional pump flows IN through a third interface of the multi-way valve, flows OUT from a second interface of the multi-way valve after being switched, enters from an inlet of a multicolor spectrum column selection switching valve 'IN', flows OUT through a second inlet of the multicolor spectrum column selection switching valve, enters a second trapping column (10) for elution, flows OUT through a second outlet of the multicolor spectrum column selection switching valve, flows OUT from an outlet of the multicolor spectrum column selection switching valve 'OUT', enters a fifth interface of the multi-way valve, flows OUT from a fourth interface of the multi-way valve, enters the two-dimensional chromatographic column for further separation, and flows into mass spectrometry for determination;
g4 The mobile phase introduced by the two-dimensional pump flows IN through a third interface of the multi-way valve, flows OUT from a second interface of the multi-way valve after being switched, enters from an inlet of a multicolor column selection switching valve 'IN', flows OUT through a second inlet of the multicolor column selection switching valve, enters a second trapping column for elution, flows OUT through a second outlet of the multicolor column selection switching valve, flows OUT from an outlet of the multicolor column selection switching valve 'OUT', enters a fifth interface of the multi-way valve, flows OUT from a fourth interface of the multi-way valve, enters the two-dimensional chromatographic column for further separation, and flows into a mass spectrum for measurement;
g5 The mobile phase introduced by the two-dimensional pump flows IN through a third interface of the multi-way valve, flows OUT from the second interface of the multi-way valve after being switched, enters from an inlet of a multicolor column selection switching valve 'IN', flows OUT through a third inlet of the multicolor column selection switching valve, enters a third trapping column for elution, flows OUT through a third outlet of the multicolor column selection switching valve, flows OUT from an outlet of the multicolor column selection switching valve 'OUT', enters a fifth interface of the multi-way valve, flows OUT from a fourth interface of the multi-way valve, enters the two-dimensional chromatographic column for further separation, and flows into a mass spectrometry for determination;
g6 The mobile phase introduced by the two-dimensional pump flows IN through a third interface of the multi-way valve, flows OUT from a second interface of the multi-way valve after being switched, enters from an inlet of a multi-color spectrum column selection switching valve 'IN', flows OUT through a fourth inlet of the multi-color spectrum column selection switching valve, enters a fourth trapping column for elution, flows OUT from a fourth outlet of the multi-color spectrum column selection switching valve, flows OUT from an outlet of the multi-color spectrum column selection switching valve 'OUT', enters a fifth interface of the multi-way valve, flows OUT from the fourth interface of the multi-way valve, enters a two-dimensional chromatographic column for further separation, and flows into a mass spectrum for determination;
g7 The mobile phase introduced by the two-dimensional pump flows IN through a third interface of the multi-way valve, flows OUT from a second interface of the multi-way valve after being switched, enters from an inlet of a multicolor column selection switching valve 'IN', flows OUT through a fifth inlet of the multicolor column selection switching valve, enters a fifth trapping column for elution, flows OUT through a fifth outlet of the multicolor column selection switching valve, flows OUT from an outlet of the multicolor column selection switching valve 'OUT', enters the fifth interface of the multi-way valve, flows OUT from a fourth interface of the multi-way valve, enters the two-dimensional chromatographic column for further separation, and flows into a mass spectrum detector for measurement;
g8 Mobile phase introduced by the two-dimensional pump flows IN through a third interface of the multi-way valve, flows OUT from a second interface of the multi-way valve after being switched, enters from an inlet of a multi-color spectrum column selection switching valve 'IN', flows OUT through a sixth inlet of the multi-color spectrum column selection switching valve, enters a fifth trapping column for elution, flows OUT from a sixth outlet of the multi-color spectrum column selection switching valve, flows OUT from an outlet of the multi-color spectrum column selection switching valve 'OUT', enters a fifth interface of the multi-way valve, flows OUT from a fourth interface of the multi-way valve, enters the two-dimensional chromatographic column for further separation, flows into a mass spectrum detector for measurement, so that target analytes trapped on each trapping column are eluted, and mass spectrum analysis is respectively carried OUT.
As described above, the present invention has the following advantageous effects:
aiming at the characteristics of complex urine sample matrix, multiple types of smoke exposure biomarkers, large property difference and the like, the invention provides a multi-center cutting two-dimensional liquid chromatography-tandem mass spectrometry method and further provides a natural isotope method to realize simultaneous quantitative analysis of the multiple types of smoke exposure biomarkers in urine. According to the invention, impurity interference is effectively removed through the separation of the first dimension, the sensitivity and accuracy of target object detection are improved, and meanwhile, pretreatment conditions, mobile phases, chromatographic columns, mass spectrum parameters and the like are comprehensively optimized, so that simultaneous analysis of acid-base compounds with large polarity differences is realized, and a more efficient and convenient high-throughput analysis method is provided for human smoke exposure evaluation.
In the prior art, the organic phase ratio of a solvent is high during the elution of a first dimension, a plurality of compounds cannot be subjected to complementary collection by using a complementary collection column, a simple processing mode of an instrument company is that a quantitative ring is used, the first dimension is divided into a plurality of sections to be transferred to the quantitative ring and stored in the quantitative ring, and the first dimension is directly transferred to a second dimension during analysis. This results in a complex sample matrix with many other compounds on the quantitation loop, interfering with the second dimension analysis, direct transfer, sometimes broad chromatographic peaks and poor sensitivity. The dosing ring system is suitable for systems with relatively high contents and relatively uncomplicated matrices. However, if the sensitivity of the compound is extremely low, the compound is particularly serious compared with the matrix interference of a sample, and the problem is difficult to solve by using a quantitative ring system at this time, so that the device provided by the invention can well solve the problem.
Drawings
FIG. 1 is a schematic diagram of a multi-center cutting two-dimensional liquid chromatography-tandem mass spectrometry system.
FIG. 2 is a MRM chart of the COT standard.
FIG. 3 is a MRM graph of NNAL standards.
Fig. 4 is a MRM chart of NAT standard.
FIG. 5 is a MRM chart of NAB standard.
FIG. 6 is a MRM chart of 2-NA and 1-NA standards.
FIG. 7 is a MRM chart of 3-ABP and 4-ABP standards.
FIG. 8 is a MRM chart of 2-OHNap and 1-OHNap standards.
FIG. 9 is a MRM chart of 2-OHFu and 3-OHFu standards.
FIG. 10 is a MRM chart of 1-OPhe, 2-OPhe, 3-OPhe, 4-OPhe and 9-OPhe standards.
FIG. 11 shows MRM of 1-OHPyr standards.
FIG. 12 is a TIC chromatogram of polycyclic aromatic hydrocarbons analyzed using an RP18 column and a T3 column.
FIG. 13 is a TIC chromatogram of an actual sample.
Reference numerals:
1. one-dimensional pump
2. One-dimensional chromatographic column
3. Detector
4. Three-way connector
41. First connector of three-way connector
42. Second connector of three-way connector
43. Third interface of three-way interface
5. Compensation pump
6. Two-dimensional pump
7. Multi-way valve
71. First interface of multi-way valve
72. Second interface of multi-way valve
73. Third interface of multi-way valve
74. Fourth interface of multi-way valve
75. Fifth interface of multi-way valve
76. Sixth interface of multi-way valve
8. Multi-colour spectrum column selection switching valve
81. First inlet of multi-color spectrum column selection switching valve
82. First outlet of multi-color spectrum column selection switching valve
83. Second inlet of multi-color spectrum column selection switching valve
84. Second outlet of multi-color spectrum column selection switching valve
85. Third inlet of multi-color spectrum column selection switching valve
86. Third outlet of multi-color spectrum column selection switching valve
87. Fourth inlet of multi-color spectrum column selection switching valve
88. Fourth outlet of multi-color spectrum column selection switching valve
89. Fifth inlet of multi-color spectrum column selection switching valve
810. Fifth outlet of multi-color spectrum column selection switching valve
811. Sixth inlet of multi-color spectrum column selection switching valve
812. Sixth outlet of multi-color spectrum column selection switching valve
813. Multi-color spectrum column selection switching valve IN inlet
814. OUT outlet of multi-color spectrum column selection switching valve
9. First trapping column
10. Second trapping column
11. Third trapping column
12. The fourth trap column
13. Fifth trap column
14. The sixth trap column
15. Two-dimensional chromatographic column
16. Mass spectrometry
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The reagents and test devices used in the following examples were all those conventionally used and commercially available. The reagents and instruments used were as follows:
1. reagent
Acetonitrile, methanol (HPLC grade, TEDIA corporation, usa); formic acid (. Gtoreq.98%, merck, germany); ammonium formate (sigma, U.S. a); beta-glucuronidase (Sigma, USA); artificial urine (Dongguan Chuangfeng); cotinine (COT), N-Nitrosoanabasine (NAB), N-Nitrosoneonicotinoid (NAT), 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanol (NNAL), 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), 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-OHPE), 2-hydroxyphenanthrene (2-OHPE), 3-hydroxyphenanthrene (3-OHPE), 4-hydroxyphenanthrene (4-OHPE), 9-hydroxyphenanthrene (9-OHPE), 2-hydroxyfluorene (2-OHFu), 3-hydroxyfluorene (3-OHFu), d 9 -1-hydroxypyrene (d) 9 -1-OHPyr)、 13 C 6 -3-hydroxy phenanthrene (f) 13 C 6 -3-OHPhe)、 13 C 6 -3-hydroxyfluorene (f) 13 C 6 -3-OHFlu)、d 7 -2-hydroxynaphthalene (d) 7 -2-OHNap) and the like from TRC reagent, canada.
2. Instrument for measuring the position of a moving object
The American Agilent 1290 liquid chromatograph is provided with an automatic sample injector, a quaternary mixing pump, a binary pump, a unitary pump, a column oven, a two-position multi-way valve (7), a G4234A/C quick switching valve and a diode array detector (3) (DAD); electronic balance (precision: 0.0001g, mettler Toledo, switzerland); SW12H sonicator (Sono Swiss, swiss); milli-Q water purifier (Millpore, USA); eppendorf 5810R high speed centrifuge and ultra low temperature refrigerator (Eppendorf Co., germany); API 5500 triple quadrupole mass spectrometry (16) (SCIEX corporation, usa); lyophilizer (laboconco, usa).
3. The MRM parameters for each analyte and internal standard in the mass spectrometric (16) detection are shown in table 1:
TABLE 1 MRM parameters of analytes and internal standards
Example 1
1 sample pretreatment
Thawing the collected urine sample at room temperature, putting 5mL of the sample into a 100mL beaker, sequentially adding 10mL of sodium acetate-acetic acid buffer solution (10 mM, pH 5.1), 50 mu L of internal standard solution and 50 mu L of beta-glucuronidase, uniformly mixing, sealing by a sealing membrane, and placing in a constant-temperature water bath at 37 ℃ for enzymolysis for 16h in a dark place. The sample after enzymolysis is frozen in an ultra-low temperature refrigerator at minus 80 ℃, then is frozen and dried in vacuum (the temperature of a clapboard is 30 ℃), is redissolved by 500 mu L deionized water, is centrifuged at 12000rpm for 10min, and is taken out for analysis.
Isotope target content calculation for 2COT
12 C and 13 the natural abundance of C is 98.89% and 1.11%, respectively, while COT contains 10 carbon atoms, and when MRM is used to analyze COT, the method selects m/z 178.0 as the parent ion for COT quantification, which is natural 13 [ M + H ] of C-COT] + Ion, [ M + H ] in a content of COT] + 11.1% of ions, and the content can meet the detection requirement of the method.
3 conditions of analysis
The mobile phase A of the one-dimensional pump is 10mM ammonium formate-water solution, the mobile phase B of the one-dimensional pump is methanol, an Angela Venusil XBP CN chromatographic column (2.1 mM multiplied by 100mm,5 mu m) is used as a first-dimensional separation column, the column temperature is 30 ℃, the DAD detection wavelength is 260nm, the sample injection amount is 20 mu L, the flow rate is 0.3mL/min in 0-30min, and the flow rate is 0mL/min in 31-65 min. The gradient elution conditions were: 0-3min,3% by weight of B;10-15min,95% B;21-30min,3% by weight of B. The mobile phase of the compensation pump (5) is deionized water, and the compensation flow rate is 600 mu L/min.
The mobile phase of the two-dimensional pump A was a 0.1% formic acid-water solution, B was acetonitrile, and an Xbridge C18 column (4.6 mm. Times.30mm, 5 μm) was used as a trap column and an Atlantis T3 column (2.1 mm. Times.150mm, 3.0 μm) was used as an analytical column for the second dimension. The column temperature is 30 ℃, the flow rate is 0.1mL/min in 0-11min, and the flow rate is 0.3mL/min in 11.2-65 min. Gradient eluting under 0-12min, and 5% by weight of B;15-18min,95% B;18.1-23min,5% by weight B;30-32min,95% by weight B;32.1-37min,5% by weight of B;37.1-52min,40% B;54-59min,95% by weight B;59.1-65min, 5% B.
Mass spectrometry was performed using electrospray ionization (ESI); the detection mode is multi-reaction monitoring (MRM); the ion source temperature is 600 ℃; ion pair residence monitoring time (Dwell time) of 50ms; the atomizing and assist gas pressures were 50psi; the air pressure of the air curtain is 10psi; the level of the collision gas is Medium; during positive ion mode scanning, the electrospray voltage is 5500V; the electrospray voltage was-4500V during negative ion mode scanning.
Example 2
FIGS. 2 to 11 show MRM plots for a second dimension of separation of 18 analyte standards, and it can be seen that the remaining compounds are effectively separated except for several isomers such as 3-/4-ABP, 2-/3-OHFU, 2-/3-OHPE, 1-/4-OHPE.
Example 3
And analyzing the prepared standard working solution, and performing linear regression by taking the concentration ratio of the standard sample to the internal standard as a horizontal ordinate and the peak area ratio as a vertical coordinate to obtain a standard working curve of each target compound. Taking the minimum concentration standard working solution of each target object to perform parallel measurement for 10 times, calculating the standard deviation, and taking 10 times of standard deviation as the quantitative limit of the method and 3 times of standard deviation as the detection limit of the method. As a result, the method showed good linearity (R) as shown in Table 2 2 >0.993 ); the detection limit of COT is 87.0pg/mL, and the quantification limit is 290.0pg/mL; the detection limit of other target analytes is 0.8-13.1pg/mL, and the quantification limit is 2.7-43.7 pg/mL, which all meet the requirement of quantitative detection.
TABLE 2 Linear equation, correlation coefficient, detection limit and quantitation limit of the target
| Name (R) | Linear equation of equations | R 2 | Detection limit (pg/ml) | Limit of quantitation (pg/ml) |
| NNAL | Y＝1.4e -2 X+1.8e -2 | 0.9999 | 2.5 | 8.4 |
| COT | Y＝9.5e -2 X-7.6e -1 | 0.9999 | 87.0 | 290.0 |
| NAT | Y＝7e -3 X-9e -3 | 0.9988 | 1.0 | 3.3 |
| NAB | Y＝9e -3 X+7e -3 | 0.9998 | 0.9 | 3.0 |
| 2-NA | Y＝5e -3 X+4e -3 | 0.9995 | 1.4 | 4.7 |
| 1-NA | Y＝4.5e -1 X+1.8e 0 | 0.9994 | 2.7 | 9.0 |
| 3/4-ABP | Y＝2e -3 X+2e -3 | 0.9998 | 0.8 | 2.7 |
| 2-OHNap | Y＝5e -5 X+1e -3 | 0.9998 | 11.3 | 37.7 |
| 1-OHNap | Y＝4e -5 X-8e -5 | 0.9989 | 13.1 | 43.7 |
| 2/3-OHFlu | Y＝2e -6 X-8e -5 | 0.9985 | 7.9 | 26.5 |
| 2/3-OHPhe | Y＝8e -5 X+1e -3 | 0.9997 | 5.3 | 17.5 |
| 9-OHPhe | Y＝1e -4 X+4e -4 | 0.9938 | 3.1 | 10.3 |
| 1/4-OHPhe | Y＝6e -5 X-9e -5 | 0.9986 | 3.4 | 11.2 |
| 1-OHPyr | Y＝5e -5 X-3e -4 | 0.9995 | 2.9 | 9.7 |
Example 4
And (3) selecting a urine sample of a non-smoker, respectively adding three levels of standard solutions which are 1/2 (low), 1 (medium) and 2 (high) times of the average content of the target analyte in the smoker, quantifying by an internal standard method, and determining the recovery rate of the added standard. And for target objects with lower average content, such as NAT, NAB and the like, adding a scaling quantity to ensure that the scaling concentration of a low level is greater than a quantitative limit, and meeting the requirement of accurate quantification. As shown in Table 3, the recovery rates of the fractions other than 9-OHPE, 1-/4-OHPE and 1-OHPyr were about 80%, and the recovery rates of the low, medium and high three levels of the large fraction of the target analytes were between 90 and 110%. The precision of the target analyte in the day and the precision of the target analyte in the day are respectively 1.00-5.41% and 2.35-5.99% when the urine sample is subjected to 5 times of parallel measurement in the day and the day respectively. The method has better recovery rate and precision, meets the detection requirement, and can be used for quantitative analysis of trace smoke exposure biomarkers in urine.
TABLE 3 spiked recovery, in-day and in-day precision of targets in urine samples
Example 5
1 sample pretreatment
Thawing the collected urine sample at room temperature, putting 5mL of the sample into a 100mL beaker, sequentially adding 10mL of sodium acetate-acetic acid buffer solution (10mM, pH 5.1), 50 mu L of internal standard solution and 50 mu L of beta-glucuronidase, uniformly mixing, sealing by a sealing membrane, and placing in a constant-temperature water bath at 37 ℃ for enzymolysis for 16 hours in a dark place. The sample after enzymolysis is frozen in an ultra-low temperature refrigerator at minus 80 ℃, then is frozen and dried in vacuum (the temperature of a clapboard is 30 ℃), is redissolved by 500 mu L deionized water, is centrifuged at 12000rpm for 10min, and is taken out for analysis.
Isotope target content calculation for 2COT
12 C and 13 the natural abundance of C is 98.89% and 1.11%, respectively, and COT contains 10 carbon atoms, and when MRM is used for analyzing COT, the method selects m/z 178.0 as the quantitative parent ion of COT, which is natural 13 [ M + H ] of C-COT] + Ion, [ M + H ] in a content of COT] + 11.1% of ions, and the content can meet the detection requirement of the method.
3 conditions of analysis
The mobile phase A of the one-dimensional pump is 10mM ammonium formate-water solution, the mobile phase B of the one-dimensional pump is methanol, an Angela Venusil XBP CN chromatographic column (2.1 mM multiplied by 100mm,5 mu m) is used as a first-dimensional separation column, the column temperature is 30 ℃, the DAD detection wavelength is 260nm, the sample injection amount is 20 mu L, the flow rate is 0.3mL/min in 0-30min, and the flow rate is 0mL/min in 31-65 min. The gradient elution conditions were: 0-3min,3% by weight of B;10-15min,95% B;21-30min,3% by weight of B. The mobile phase of the compensation pump is deionized water, and the compensation flow rate is 600 mu L/min.
The mobile phase A of the two-dimensional pump (6) was 0.1% formic acid-water solution, B was acetonitrile, and an Xbridge C18 column (4.6 mm. Times.30mm, 5 μm) was used as a Trap column and a Symmetry Shield RP18 column (2.1 mm. Times.150 mm,3.0 μm) was used as an analytical column for the second dimension. The column temperature is 30 ℃, the flow rate is 0.1mL/min in 0-11min, and the flow rate is 0.3mL/min in 11.2-65 min. Gradient eluting under 0-12min, 5%; 15-18min,95% B;18.1-23min,5% B;30-32min,95% B;32.1-37min,5% by weight of B;37.1-52min,35% B;54-59min,95% B;59.1-65min, 5% of water.
Mass spectrometry was performed using electrospray ionization source (ESI); the detection mode is multi-reaction monitoring (MRM); the ion source temperature is 600 ℃; ion pair residence monitoring time (Dwell time) of 50ms; the atomizing and assist gas pressures were 50psi; the air pressure of the air curtain is 10psi; the level of the collision gas is Medium; during positive ion mode scanning, the electrospray voltage is 5500V; the electrospray voltage was-4500V in negative ion mode scan.
4 analysis of results
The choice of the second dimension chromatography column greatly affects the sensitivity and resolution of the final target analyte. Fig. 12 is a TIC diagram of the RP18 chromatographic column and the T3 chromatographic column for separating the polycyclic aromatic hydrocarbon compound during the second-dimension chromatographic column selection verification, and it can be seen that under the same chromatographic conditions, the RP18 chromatographic column has a better separation degree of the polycyclic aromatic hydrocarbon, while the T3 chromatographic column has a higher sensitivity to the polycyclic aromatic hydrocarbon, and both of them can obtain better analysis results.
Meanwhile, in example 5, a Symmetry Shield RP18 chromatographic column is used as an analytical column in the second dimension separation, so that better retention effects are obtained for 18 compounds, and the detection results are shown in FIG. 13. FIG. 13 is a TIC chromatogram obtained from analysis of an actual sample using a multi-center cut-two-dimensional liquid chromatography-tandem mass spectrometry system. As can be seen from fig. 13, when cotinine is detected by using the method of "natural carbon isotope", even if the urine sample is enriched and concentrated, the signal response of COT can be greatly reduced, and the simultaneous detection of COT and other trace metabolites is realized, so that the method can be applied to the simultaneous detection of compounds with large content differences, thereby reducing the analysis cost and workload.
In conclusion, the invention establishes a multi-center cutting two-dimensional liquid chromatography-tandem mass spectrometry method, and realizes the simultaneous analysis of multiple classes of smoke exposure biomarkers such as nicotine, tobacco-specific nitrosamine, aromatic amine and polycyclic aromatic hydrocarbon in urine. The method has high sensitivity and good reproducibility, realizes effective removal of impurities and good separation of each component, and has wide application prospect in the aspect of simultaneous analysis and detection of various substances in a complex biological sample.
Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be accomplished by those skilled in the art without departing from the spirit and scope of the present invention as set forth in the appended claims.
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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