CN110824077A - System and method for measuring NNK and metabolites thereof by liquid-liquid two-dimensional chromatography based on solid-phase extraction - Google Patents
System and method for measuring NNK and metabolites thereof by liquid-liquid two-dimensional chromatography based on solid-phase extraction Download PDFInfo
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- CN110824077A CN110824077A CN201911112232.5A CN201911112232A CN110824077A CN 110824077 A CN110824077 A CN 110824077A CN 201911112232 A CN201911112232 A CN 201911112232A CN 110824077 A CN110824077 A CN 110824077A
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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 relates to a system and a method for measuring NNK and metabolites thereof by liquid-liquid two-dimensional chromatography based on solid-phase extraction. The method comprises the steps of carrying out high-efficiency purification on a prepared sample through a primary chromatogram, cutting partial fractions flowing out of NNK and NNAL in the primary chromatogram, diluting the cut fractions by using a dilution pump to reduce the solvent strength of the fractions, mixing the fractions by using a mixer, and enriching the fractions by using a solid-phase extraction column. And after the solid-phase extraction enrichment is finished, conveying a mobile phase by using an analysis pump of a secondary chromatograph through valve switching, eluting the enriched NNK and NNAL on the solid-phase extraction column from a flow direction opposite to the sample enrichment, and performing separation and tandem mass spectrometry detection by using an ultra-high performance liquid chromatography column. The method can simultaneously realize efficient purification and high-multiple enrichment of the sample, greatly improve the analysis sensitivity of the method, greatly simplify the pretreatment of the analysis sample, and accurately determine the NNK and NNAL with the ng/L order of magnitude in the blood and urine samples. Provides a sensitive, accurate and reliable new method for the research of the correlation between smoking and health.
Description
Technical Field
The invention discloses a system for measuring NNK and metabolites thereof, in particular relates to a system for measuring NNK and metabolites thereof based on solid-phase extraction liquid-liquid two-dimensional chromatography, and also relates to a method for measuring NNK and metabolites thereof in blood and urine of smokers, belonging to the field of analytical instruments.
Background
Tobacco Specific Nitrosamines (TSNAs) are an important harmful component in cigarette smoke, and 8 TSNAs are reported at present, wherein 4 kinds of research of N-nitrosonornicotine (NNN), 4- (methylnitrosamine) -1- (3-pyridyl) -1-butanone (NNK), N-Nitrosoanatabine (NAT) and N-Nitrosoanabasine (NAB) are relatively deep. Especially, the national tobacco monopoly of China most characterizes the harmfulness of cigarette smoke by 7 components of carbon monoxide, hydrogen cyanide, NNK, ammonia, BaP, phenol and crotonaldehyde, and after the limited control of the content of the components in the cigarette smoke, the research of NNK draws very high attention in China.
At present, the research of NNK in China is mainly limited to the technology of detecting and reducing NNK in cigarette smoke, the measurement of NNK in blood and urine of related smokers and the metabolic research thereof are relatively few, the carcinogenicity of NNK is originated from the metabolic activation process in vivo, NNK generates a carbonyl reduction reaction to generate 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanol (NNAL), NNK and NNAL further generate an α -hydroxylation reaction, the product can react with DNA to generate a DNA adduct and has carcinogenic activity, so the accurate measurement of NNK and stable metabolite NNAL thereof is very important for the deep research of the relevance of smoking and health.
For the detection of NNK and the metabolite NNAL, 5-3H]NNK markers, HPLC analysis. In recent years, HPLC-MS/MS is widely applied to the detection of TSNAs and metabolites thereof, especially in biologically complex matrices such as microsomes, urine, blood and the like, because of its high sensitivity and selectivity. However, the TSNAs and its metabolites in the blood and urine of smokers are very low and still require high-fold enrichment and interferent separation of the sample before measurement. At present, TSNAs and metabolites thereof are commonly used for analyzing samples by pretreatment, solid phase extraction or column chromatography separation and purification, and the method needs to concentrate and transfer the purified samples for many times and is complicated to operate; but also pollutes the environment because a large amount of organic solvent is used in the open environment, and has great harm to the health of experiment operators.
Disclosure of Invention
Aiming at the technical problems, the invention constructs a system and a method for measuring NNK and metabolites thereof by liquid-liquid two-dimensional chromatography based on solid-phase extraction. In the method, the smoker blood and urine samples can be directly subjected to sample injection analysis, so that the pretreatment operation steps of the samples are greatly simplified, and the precision of the analysis result is improved; the method can also obviously reduce the consumption of the organic solvent in the pretreatment process, and greatly reduce the emission of the organic solvent to the environment in the sample pretreatment process, thereby reducing the environmental pollution in the sample treatment process.
All percentages used herein are by weight unless otherwise indicated.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a system for measuring NNK and metabolites thereof by liquid-liquid two-dimensional chromatography based on solid phase extraction comprises a primary chromatography system and a secondary chromatography system, wherein the primary chromatography system is connected with the secondary chromatography system through a six-way valve; the solid phase extraction column is arranged on the six-way valve; the primary chromatographic system comprises a sample injector, one end of the sample injector is connected with the first pump, the other end of the sample injector is connected with the purification column, one end of the purification column is connected with a detector, one end of the detector is connected with one end of a three-way valve, the end of the three-way valve is also connected with the second pump, one end of the other two ends of the three-way valve is a waste liquid outlet, and the other end of the three-way valve is connected; the second pump cuts the NNK and NNAL outflow part of fractions into solid phase extraction and enrichment, and the rest parts flow into waste liquid; and a third pump of the secondary chromatographic system is communicated with one port of the six-way valve, the other port of the six-way valve is connected with an analytical column of the secondary chromatographic system, and the analytical column is connected with a detector.
Furthermore, seven ports are arranged on the six-way valve and respectively from the first port to the seventh port, one end of the mixer is communicated with the seventh port, and two ends of the solid phase extraction column are communicated with the first port and the fourth port.
Furthermore, during sample analysis, sample introduction is carried out through a sample injector of the primary chromatographic system, the outflow time of the component to be detected is indicated by a detector, the component to be detected is cut through a three-way valve, the cut component is mixed through a mixer and enters from a seventh port of a six-way valve, the component to be detected flows through a solid phase extraction column through a fourth port, the component to be detected is enriched on the solid phase extraction column, meanwhile, the cut component is diluted through a second pump, the elution strength of the mobile phase is reduced, the component to be detected is ensured not to be eluted on the solid phase extraction column and is completely enriched on the solid phase extraction column, and the mobile phase which is collected through solid phase extraction flows out through the first port and the sixth port and enters waste liquid.
The method for measuring NNK and NNAL in blood and urine of smokers based on the system comprises the following steps
(a) Injecting a sample prepared, wherein the injection volume is 1.0-2.0 mL, 30% acetonitrile containing 0.01% ammonia acetate is used as a mobile phase, an Agilent Zorbax SB-C18, the sample is purified at the flow rate of 2.0mL/min by using a half-preparation chromatographic column of 5.0μm as a stationary phase, and the fraction flowing out after cutting for 14.2-18.6 min enters into solid phase extraction;
(b) delivering water to dilute the cut fraction by a second pump at the flow rate of about 2.0mL/min, mixing the diluted fraction by a mixer, and enriching the diluted fraction by a solid phase extraction column;
(c) after the solid phase extraction and enrichment are finished, acetonitrile and 0.01 mol/L ammonium acetate solution are used for gradient elution as a mobile phase by switching the six-way valve, NNK and NNAL enriched on the solid phase extraction column are eluted in a flow direction opposite to that of the solid phase extraction and enrichment, and the NNK and the NNAL in the blood and urine of a smoker can be determined by separation of an analytical column, detection and determination of liquid chromatography-mass spectrometry and quantification of an internal standard method.
Further, the solid phase extraction material is Oasis HLB, and the particle size is5.0 µm。
Further, in step (1), the sample preparation process is as follows:
after blood and urine samples are collected, accurately transferring a certain amount of samples, adding internal standard working solution, diluting with methanol, fully shaking up, and keeping the proportion of methanol in the actual sample to be detected to be 50% and the concentration of the internal standard to be 5 ng/L; the sample is quickly centrifuged at a high speed of 20000r/min for 10 min; and taking the supernatant, filtering and directly analyzing the sample.
Further, the liquid chromatography conditions were as follows:
the chromatographic column is a Waters Acquity UPLC BEH C18 chromatographic column, 2.1mm i.d. x 100 mm, 1.7 μm; the gradient conditions were: acetonitrile A, 0.01 mol/L ammonium acetate solution B, mobile phase A is changed from 10% to 80% in 0-5 min, A is changed from 80% to 10% in 6min, and the mobile phase is balanced for 5min after returning to 10% of A, so that the next sample can be carried out;
the mass spectrometry conditions were as follows:
electrospray ionization source: ESI, detection mode: multi-reaction monitoring, MRM, scanning mode: positive ion scan, electrospray voltage: 5000V, ion source temperature: 600 ℃, assist Gas1 pressure: 0.345MPa, assist Gas2 pressure: 0.483 MPa.
Further, the working curves, linear ranges, correlation coefficients, detection limits and quantitation limits for NNK and NNAL are shown in the following table:
the device of the invention is formed by connecting two sets of chromatographic systems through a six-way valve with a sample inlet. The first-stage chromatography is a semi-preparative chromatography with an on-line solid phase extraction column for cut fraction capture and a dilution pump for reducing the solvent strength of the cut fraction. The secondary chromatogram in the device is an analytical high performance liquid chromatogram which is used for further separating the components to be detected after the purification of the primary chromatogram and the enrichment of solid phase extraction, and the chromatographic system is provided with a tandem mass spectrum detector and is used for the high-sensitivity detection of NNK and NNAL.
In the sample analysis, the sample is injected in large volume through a sample injector of a primary chromatographic system. Analyzing the sample with semi-preparative chromatographic column, detecting the outflow time of the component to be detected with a detector, and cutting the component to be detected to a solid phase extraction column through a three-way valve; the rest part is directly discharged into waste liquid.
The components cut by the three-way valve are mixed by the mixer, enter from the seventh port of the six-way valve, flow through the solid phase extraction column, and are enriched on the solid phase extraction column, and meanwhile, the cut components are diluted by the second pump, so that the elution strength of the mobile phase is reduced, the components to be detected are ensured to be completely enriched on the solid phase extraction column without being eluted on the solid phase extraction column, and the mobile phase collected by the solid phase extraction flows out from the first port and the sixth port and enters the waste liquid. In this operating state, the mobile phase of the secondary chromatography system enters the waste stream directly.
After the components to be detected are cut and enriched, the flowing phase of the primary chromatographic system enters waste liquid through a three-way valve by valve switching. Meanwhile, the mobile phase of the analysis system enters from the second port of the six-way valve, passes through the solid phase extraction column, elutes the component to be detected enriched on the solid phase extraction column from the flow direction opposite to the enrichment, flows out from the fourth port and the third port, enters the analysis column of the secondary chromatographic system for separation, and is detected by the tandem mass spectrometry detector to determine the content of the component to be detected.
In the device, because the mobile phase of the primary chromatographic system is removed through the solid phase extraction column, the mobile phase of the primary chromatographic system does not enter the secondary system any more, so that the compatibility of the mobile phase between the primary system and the secondary chromatographic system is greatly improved. Compared with direct cutting, the method adopts a solid phase extraction column enrichment and reverse elution mode, does not cause chromatographic peak broadening even if large-volume fractions are cut into secondary chromatogram, and can greatly improve the sensitivity by increasing the sample introduction amount of a sample. For ultra-low content samples, the components to be detected purified by the primary chromatography can be accumulated to a sufficient amount on a solid phase extraction column (accumulation and enrichment by multiple sample injection), and then are eluted and separated by a secondary chromatography system, so that ultra-high-sensitivity analysis is realized.
The primary chromatographic system is provided with the three-way valve, the NNK and NNAL outflow part of fractions can be cut to enter the solid phase extraction and enrichment, and the rest parts of fractions flow into the waste liquid, so that the high-efficiency purification of the sample is realized. The first-stage chromatographic system is provided with a second pump, the elution strength of the first-stage chromatographic effluent fraction on the solid-phase extraction column can be reduced through the second pump, and the NNK and the NNAL which are detected can be completely reserved on the solid-phase extraction column. The first-stage chromatographic system is provided with a solvent mixer, so that the cutting fraction and the diluted mobile phase can be uniformly mixed and then pass through the solid-phase extraction column after being mixed. The solid phase extraction material used in the present invention is oasisch, particle size is50 mu m, the material has good enrichment effect on hydrophilic polar compounds.
For ultra-low content samples, the components to be detected purified by a primary chromatographic system can be accumulated and enriched to a sufficient amount on a solid phase extraction column (accumulated and enriched by multiple sample injection), and then the ultra-low content samples are eluted, separated and detected by an analysis system so as to realize ultra-high sensitivity analysis.
The method adopts deuterated d4-NNK and d3-NNAL as internal standard substances, and the internal standard substances are added when the sample is sampled, so that errors introduced in the processes of sample storage, transportation, pretreatment and the like can be deducted.
The invention adopts a method of adding a mark in an actual blood or urine sample to manufacture a working curve, keeps the matrix of the working curve completely consistent with the matrix of the actual sample, and effectively avoids the influence of the sample matrix on the ionization efficiency of a mass spectrum.
The method adopts the elution in the flow direction opposite to the sample enrichment to realize the shortening of the elution path and further avoid the broadening of chromatographic peaks; by adopting ultra-high performance liquid chromatography, the analysis time of the sample can be effectively shortened, and the analysis sensitivity is improved.
The method of the invention comprises the following aspects:
A. preparation of standard working solution
The standards NNK and NNAL used in the present invention, both having a purity of 98% or more, were purchased from Toronto Research Chemicals; the deuteron internal standards d4-NNK and d3-NNAL used, both 99% pure, were purchased from Torto Research Chemicals.
The standard substance and the internal standard substance are prepared into a standard stock solution and an internal standard stock solution with the concentrations of 0.2 mug/mL respectively by using methanol. And diluting the standard stock solution and the internal standard stock solution step by using actual blood or urine containing 50% methanol (blood is used for diluting when the measured object is blood, and urine is used for diluting when the measured object is urine) to prepare a series of standard working solutions with standard substance concentrations of 0.5, 2.0, 10, 20, 50 and 100 ng/L and internal standard substance concentrations of 5.0 ng/L for a working curve. And diluting the internal standard working solution into 500 ng/L internal standard working solution by using methanol for adding the internal standard into the actual sample.
Analytical sample preparation
Samples of blood and urine from smokers were collected and provided by Kunming medical university. Because a large amount of active enzyme exists in blood and urine samples, the component to be detected can be continuously metabolized and converted or decomposed under the action of the enzyme, and the stability of the component to be detected in the samples is poor. We find that the addition of methanol to blood and urine samples has a good sample stabilizing effect, the addition of methanol can inactivate enzymes in the samples, and components to be detected can not be converted and decomposed continuously. The influence of methanol with different proportions in a sample medium on the stability of the sample is tested, and the test result shows that: when the proportion of methanol in the sample medium is around 50%, NNK and NNAL in the sample can be kept stable, and the change rate in 1 week is not more than 5%. Therefore, in this experiment, a medium-stabilized sample of methanol adjusted to 50% immediately after sampling was selected.
The specific operation is as follows: after blood and urine samples are collected, accurately moving 5.0mL of samples, adding 100 muL of internal standard working solution, diluting to 10mL with methanol, fully shaking up, and keeping the proportion of methanol in the actual sample to be detected to be 50% and the internal standard concentration to be 5 ng/L. Rapidly taking the sample back to the laboratory, and centrifuging at a high speed of 20000r/min for 10 min; and taking the supernatant, filtering and directly analyzing the sample.
First-stage chromatography system purification of sample
Reverse phase solid phase extraction and column chromatography purification are considered to be the most common purification modes for analysis of TSNAs and their metabolites. Therefore, the first-stage chromatography in the invention adopts a reverse-phase chromatography separation mode. The sample introduction volume is 1.0-2.0 mL, and Agilent Zorbax SB-C is adopted18And (9.4 multiplied by 150mm, 5.0 mu m) separating under the chromatographic conditions that a half-preparation chromatographic column is a purifying column, 30% acetonitrile (containing 0.01% ammonium acetate) is a mobile phase and the flow rate is 2.0 mL/min. The NNK and NNAL efflux times under this chromatographic condition were previously labeled with standards for NNK and NNAL. When the sample injection volume is 2.0mL, the peak time of NNK and NNAL is 14.4-18.2 min (figure-2), and the collection time is selected to be 14.2-18.6 min in order to ensure that the high-concentration sample can be completely collected in consideration of certain drift after the retention time.
The NNK and the NNAL are cut through a three-way valve in the graph-1, namely, part of distillate flowing out in 14.2-18.6 min enters a solid phase extraction column, and the rest part of distillate is directly discharged into waste liquid. The result shows that the actual sample has good purifying effect under the condition, the components to be detected can be completely cut, and the recovery rate of the added standard substance is over 96 percent.
On-line solid phase extraction and enrichment
The sample fraction after the first-stage purification needs to be enriched by on-line solid-phase extraction, the component to be detected is enriched on a solid-phase extraction column, and the mobile phase of a first-stage chromatographic system is discharged through the solid-phase extraction column, so that the sample fraction can enter the second-stage chromatographic analysis. Because the components NNK and NNAL to be enriched are polar compounds, the enrichment effect of the reversed-phase hydrophilic chromatographic stationary phase is good, the reversed-phase hydrophilic chromatographic stationary phases of different manufacturers are screened, and the reversed-phase hydrophilic chromatographic stationary phase of Oasis HLB (Waters Co. ltd, USA) has the best enrichment effect. Earlier literature also shows that Oasis HLB has a good enriching effect on polar compounds such as melamine, streptomycin, thioproline, hydrophilic pesticides and the like.
The specifications of the solid phase extraction column selected in the present invention were (4.6 x 10 mm,5mum), because the cut fraction flowing out of the primary chromatography is 30% acetonitrile medium, the elution strength of the solid phase extraction column is strong, and the NNK and the NNAL can be completely reserved on the solid phase extraction column only by reducing the elution strength of the solvent by diluting with water. In this experiment, a second pump was selected to deliver water at a flow rate of 2.0mL/min, the mixture was mixed in a mixer and passed through a solid phase extraction column to reduce the mobile phase of the solid phase extraction column to 15% acetonitrile, so that both NNK and NNAL could be well retained on the solid phase extraction column. On-line solid phase extraction recovery experiments were performed using a NNK and NNAL mixed standard. The results show that: the solid phase extraction recovery rate of the two components to be detected is 94.3-101.6%, and the solid phase extraction recovery rate is very high.
Liquid chromatography, mass spectrometry
Liquid chromatography of NNK and NNAL typically employs ammonium acetate buffer and acetonitrile (or methanol) as mobile phases, reverse phase chromatography column separation; in the invention, a Waters Acquity UPLC BEH C18 chromatographic column (2.1 mm i.d.. times.100 mm, 1.7 mu m) is selected, and a mobile phase is obtained by gradient elution of 0.01 mol/L ammonium acetate solution and acetonitrile. Under the condition of 0.01 mol/L ammonium acetate aqueous solution, ammonium acetate provides protons required by NNK and NNAL protonation due to pyrolysis after entering a mass spectrum, so that the positive ion mode detection has higher sensitivity. Compared with isocratic elution, gradient elution can obtain better separation effect and shorten analysis time.
The invention optimizes different gradient conditions. The gradient conditions selected were: acetonitrile A, ammonium acetate solution B of 0.01 mol/L, mobile phase A of 0 to 5min is changed from 10% to 80%, A of 6min is changed from 80% to 10%, and the mobile phase is balanced for 5min after returning to 10% of A, so that next sample analysis can be carried out. Under the condition, the test can be fully and completely separated, and the analysis time is short. The column temperature had no significant effect on the separation, and the chromatographic column temperature was selected to be 30 ℃ in this experiment.
The mass spectrum conditions are optimized by needle pump sample injection, and the mass spectrum analysis conditions are selected as follows: electrospray ionization source (ESI), detection mode: multi-reaction monitoring, MRM, scanning mode: positive ion scan, electrospray voltage: 5000V, ion source temperature: 600 ℃, assist Gas1 pressure: 0.345MPa, assist Gas2 pressure: 0.483MPa, and the quantitative ion pair, cone-hole voltage (CV) and Collision Energy (CE) for each analyte are shown in Table 1.
TABLE 1 Mass Spectrometry parameters for the Compounds
| Compound (I) | Ion pair (m/z) | Taper hole electricity (V) | Collision energy (eV) |
| NNK | 208.1/122.1 | 16 | 16 |
| NNAL | 210.1/180.1 | 18 | 15 |
| NNK-d4 | 212.1/126.0 | 16 | 14 |
| NNAL-d3 | 213.1/183.1 | 20 | 15 |
F. Determination of sample analysis procedure
On the basis of the optimization of the conditions, the analysis process of the sample is determined. The sample injection volume is 1.0-2.0 mL, 30% acetonitrile (containing 0.01% ammonium acetate) is used as a mobile phase, an Agilent Zorbax SB-C18(9.4 x 150mm, 5.0 mu m) chromatographic column is used as a stationary phase, and the flow rate is 2.0 mL/min. And cutting the fraction flowing out for 14.2-18.6 min and performing solid phase extraction.
The cut fraction was diluted with water by a dilution pump at a flow rate of 2.0mL/min, mixed by a mixer, and then concentrated by a solid phase extraction column. After the solid phase extraction enrichment is finished, acetonitrile and 0.01 mol/L ammonium acetate solution are used for gradient elution as a mobile phase through valve switching, NNK and NNAL enriched on the solid phase extraction column are eluted in a flow direction opposite to that of the solid phase extraction enrichment, and the NNK and the NNAL are separated by an analytical column and detected and measured by mass spectrometry. And (5) quantifying by an internal standard method.
For ultra-low content samples, the components to be detected purified by the primary chromatography can be accumulated and enriched to a sufficient amount on a solid phase extraction column (accumulated and enriched by multiple sample injection), and then the ultra-low content samples are eluted, separated and detected by an analysis system, so that ultra-high sensitive analysis is realized.
Working curve, detection limit and quantification limit
In order to further eliminate the interference of matrix effect, besides using isotope internal standard, the working curve is also made by using the method of adding standard in the actual urine sample. Adding component standards to be measured with different concentration gradients into an actual urine sample, subtracting a blank peak area from a standard sample peak area, and performing regression with the standard peak area and the corresponding concentration to prepare a working curve; in the process of manufacturing the working curve, the matrix of the working curve is kept completely consistent with the actual sample matrix, and the interference of the sample matrix is effectively avoided.
The serial standard working solutions prepared by diluting actual blood or urine samples are tested, the concentration ratio and the peak area ratio of NNK and NNAL to the internal standard d4-NNN, d4-NNK and d3-NNAL in each standard solution are respectively calculated, the concentration ratio is used as the abscissa (x, ng/mL), the peak area ratio is used as the ordinate (y) to prepare the standard working curve, the lowest concentration standard solution is repeatedly measured for 10 times, the LOD is used as the standard deviation of 3 times, and the LOQ is used as the standard deviation of 10 times, and the results are shown in the table 2. As can be seen from Table-2, the correlation coefficients of the NNK regression equation and the NNAL regression equation are both above 0.999, and the linear relationship is good. The detection limit of NNK reaches 0.025 ng/L, and the quantification limit reaches 0.08 ng/L; the detection limit of NNAL reaches 0.040 ng/L, and the quantification limit reaches 1.22 ng/L; the method has high sensitivity.
TABLE 2 working curves, Linear Range, correlation coefficients, detection limits and quantitation limits
H. Recovery and precision of the process
The recovery and precision of the method were further tested in the present invention. The precision in the day is as follows: blood or urine samples were measured 7 times in the same day under the same conditions and the Relative Standard Deviation (RSD) of the results of the 7 replicates was calculated, giving RSD of NNK and NNAL in the range of 3.2% -3.8%; indicating that the method is accurate. The daytime precision is as follows: the blood or urine sample is measured 1 time per day for 7 days, and the relative standard deviation of the 7 measurement results is calculated, so that the RSD of NNK and NNAL is within the range of 3.5-4.1%; indicating that the method still has good precision when measured at different times.
And during the recovery rate measurement, 4 parts of the same sample are taken from each sample, wherein 1 part of the sample is not added with a standard substance to be measured, the other 3 parts of the sample are respectively added with NNK and NNAL with known amounts (three addition amounts of 2 ng/L, 4 ng/L and 10 ng/L), the sample is subjected to sample injection analysis according to selected experimental conditions, the amount measured by adding a standard sample is subtracted from the amount measured by adding the standard sample, and the recovery rate is calculated by dividing the amount added by the standard. The recovery rate of the obtained NNK and NNAL is 91.2-93.4%, which shows that the recovery rate of the method is very high.
Compared with the prior art, the invention has the following beneficial effects:
A. the invention realizes the high-efficiency purification of the sample and the high-multiple enrichment of the component to be detected by the online liquid phase-liquid phase two-dimensional chromatogram with the solid phase extraction function, thereby greatly improving the detection sensitivity of the method; can realize accurate determination of the ultra-low content of NNK and NNAL in blood and urine samples of smokers.
B. For ultra-low content samples, the components to be detected purified by the primary chromatography can be accumulated and enriched to a sufficient amount on a solid phase extraction column (accumulated and enriched by multiple sample injection), and then the ultra-low content samples are eluted, separated and detected by an analysis system, so that ultra-high sensitive analysis is realized.
C. In order to better retain the fraction cut by the primary chromatogram on the solid-phase extraction column, the dilution pump is also designed in a targeted manner, the elution strength of the fraction flowing out of the primary chromatogram on the solid-phase extraction group is reduced through the dilution pump, so that the NNK and the NNAL to be detected can be completely retained on the solid-phase extraction column, and the technical problem of high-efficiency capture of the component to be detected in the fraction cut by the primary chromatogram is effectively solved.
D. In the invention, the solid phase extraction column is eluted from the flow direction opposite to the sample enrichment, and the NNK and the NNAL are analyzed by the ultra-high performance liquid chromatography. Elution in a flow direction opposite to the sample enrichment is adopted, so that an elution path can be effectively shortened, and the chromatographic peak is prevented from widening; the ultra-high performance liquid chromatography is adopted for analysis, so that the analysis time of the sample can be effectively shortened, and the analysis sensitivity is improved.
E. In the invention, deuterated NNK and NNAL are used as internal standard substances, the internal standard substances are added when the sample is sampled, and the internal standard substances are added when the sample is sampled, so that errors caused in the processes of sample storage, transportation, pretreatment and the like can be effectively deducted, and the obtained analysis result is more reliable.
F. The invention also adopts a method of adding a mark in the actual blood or urine sample to make a working curve. Adding component standards to be measured with different concentration gradients into an actual blood or urine sample, subtracting a blank peak area from a standard sample peak area, and performing regression with the standard peak area and the corresponding concentration to prepare a working curve. The matrix of the working curve is kept completely consistent with the actual sample matrix in the process of manufacturing the working curve, the influence of the sample matrix on the ionization efficiency of a mass spectrum is effectively avoided, and a more accurate and reliable analysis result can be obtained.
Drawings
FIG. 1 is a schematic diagram of the apparatus of the present invention in a sample purification and enrichment state;
FIG. 2 is a schematic view of the apparatus according to the present invention in an analysis state;
FIG. 3 is a chromatogram of NNK and NNAL efflux from a primary chromatography system;
FIG. 4 is a typical NNK and NNAL selective ion detection chromatogram of example 1, wherein: 1. NNK; 2. NNAL.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
As shown in fig. 1, the system for measuring NNK and its metabolites by liquid-liquid two-dimensional chromatography based on solid-phase extraction of the present embodiment includes a primary chromatography system 1 and a secondary chromatography system 5, wherein the primary chromatography system 1 and the secondary chromatography system 5 are connected to a solid-phase extraction column 3 through a six-way valve 2; the gel purge effluent dilution pump 4 is connected to the three-way valve 1.5 of the gel purge system.
First-order chromatographic system 1 includes injector 1.2, injector 1.2 one end is connected with first pump 1.1, the other end is connected with purifying column 1.3, purifying column 1.3 one end detector 1.4 is connected, detector 1.4 one end is connected with three-way valve 1.5 one end, this end of three-way valve 1.5 still is connected with the second pump, the second pump is gel purification outflow fraction dilution pump 4, other both ends one end of three-way valve 1.5 is the waste liquid export, blender 1.6 is connected to the other end.
Seven ports are arranged on the six-way valve 2 and are respectively a first port to a seventh port, and one end of the mixer is communicated with the seventh port. The third pump 5.3 of the liquid chromatography system 5 is in communication with the second port. The third port is connected to an analytical column 5.2 of the secondary chromatography system 5, which analytical column 5.2 is connected to a detector 5.1. Two ends of the solid phase extraction column 3 are communicated with the first port and the fourth port.
As shown in fig. 2, during sample analysis, a sample is injected by a sample injector 1.2 of a primary chromatographic system 1, a detector 1.4 is used to indicate the outflow time of a component to be detected, the component to be detected is cut by a three-way valve 1.5, the cut component is mixed by a mixer, enters from a seventh port of a six-way valve 2, flows through a solid phase extraction column 3 through a fourth port, enriches the component to be detected on the solid phase extraction column 3, and is diluted by the dilution pump 4 to reduce the elution strength of a mobile phase, so that the component to be detected is ensured to be completely enriched on the solid phase extraction column 3 without being eluted, and the mobile phase which is collected by solid phase extraction flows out through the first port and the sixth port to enter waste liquid. In this operating state, the mobile phase of the liquid chromatography system 5 enters the waste liquid directly.
After the components to be detected are cut and enriched, the components are switched by the six-way valve 2, and the mobile phase of the primary chromatographic system 1 enters waste liquid through the three-way valve 2. Meanwhile, the mobile phase of the secondary chromatographic system 5 enters from the second port of the six-way valve, passes through the solid-phase extraction column 3, elutes the component to be detected enriched on the solid-phase extraction column 3 in the opposite flow direction to the enrichment, flows out from the fourth port and the third port, enters the analytical column of the secondary chromatographic system 5 for separation, and is detected by a detector to determine the content of the component to be detected.
The method of measuring NNK and NNAL in the blood and urine of smokers of this example was performed as follows:
the test sample in this example is a blood sample from a mixed cigarette heavy smoker.
Accurately transferring 5.0mL of sample after collection, adding 100 muL of internal standard working solution, diluting to 10mL of working solution with methanol, fully shaking up, and keeping the proportion of methanol in the actual sample to be detected at 50% and the concentration of the internal standard at 5 ng/L.
Rapidly taking the sample back to the laboratory, and centrifuging at a high speed of 20000r/min for 10 min; the supernatant was filtered and used for sample analysis. The sample injection volume is 1.0mL, 30% acetonitrile containing 0.01% ammonium acetate is used as a mobile phase, an Agilent Zorbax SB-C18 (9.4X 150mm, 5.0 mu m) chromatographic column is used as a stationary phase, and the flow rate is 2.0mL/min to purify the sample.
And cutting the fraction flowing out for 14.2-18.6 min and performing solid phase extraction. The cut fraction was diluted with water by a dilution pump at a flow rate of 2.0mL/min, mixed by a mixer, and then concentrated by a solid phase extraction column. After the solid phase extraction enrichment is finished, acetonitrile and 0.01 mol/L ammonium acetate solution are used for gradient elution as a mobile phase through valve switching, NNK and NNAL enriched on a solid phase extraction column are eluted in a flow direction opposite to that of the solid phase extraction enrichment, and then the NNK and the NNAL are separated by an analytical column, detected and measured by mass spectrometry, and quantified by an internal standard method.
The NNK content of the sample is 6.86 ng/L, and the NNAL content of the sample is 11.9 ng/L.
The chromatogram for the efflux of NNK and NNAL from the primary chromatography system is shown in FIG. 3, and the chromatogram for the selective ion detection of NNK and NNAL is shown in FIG. 4.
Example 2
The apparatus of this example is the same as example 1.
The method of measuring NNK and NNAL in the blood and urine of smokers of this example was performed as follows:
the test sample in this example is a urine sample from a mixed cigarette heavy smoker.
Accurately transferring 5.0mL of sample after collection, adding 100 muL of internal standard working solution, diluting to 10mL by using methanol, fully shaking up, and keeping the proportion of the methanol in the actual sample to be detected to be 50% and the concentration of the internal standard to be 5 ng/L. Rapidly taking the sample back to the laboratory, and centrifuging at a high speed of 20000r/min for 10 min; the supernatant was filtered and used for sample analysis. The sample injection volume is 1.0mL, 30% acetonitrile (containing 0.01% ammonia acetate) is used as a mobile phase, an Agilent Zorbax SB-C18 (9.4X 150mm, 5.0 mu m) chromatographic column is used as a stationary phase, and the flow rate is 2.0 mL/min.
And cutting the fraction flowing out for 14.2-18.6 min and performing solid phase extraction. The cut fraction was diluted with water by a dilution pump at a flow rate of 2.0mL/min, mixed by a mixer, and then concentrated by a solid phase extraction column. After the solid phase extraction enrichment is finished, acetonitrile and 0.01 mol/L ammonium acetate solution are used for gradient elution as a mobile phase through valve switching, NNK and NNAL enriched on a solid phase extraction column are eluted in a flow direction opposite to that of the solid phase extraction enrichment, and then the NNK and the NNAL are separated by an analytical column, detected and measured by mass spectrometry, and quantified by an internal standard method.
The NNK content of the sample is 4.46 ng/L, and the NNAL content is 8.87 ng/L. The chromatogram of the NNK and NNAL efflux in the primary chromatography system is shown in FIG. 3.
Example 3
The apparatus of this example is the same as example 1.
The method of measuring NNK and NNAL in the blood and urine of smokers of this example was performed as follows:
the test sample in this example is a mixed cigarette moderate smoker urine sample.
Accurately transferring 5.0mL of sample after collection, adding 100 muL of internal standard working solution, diluting to 10mL of working solution with methanol, fully shaking up, and keeping the proportion of methanol in the actual sample to be detected at 50% and the concentration of the internal standard at 5 ng/L.
Rapidly taking the sample back to the laboratory, and centrifuging at a high speed of 20000r/min for 10 min; the supernatant was filtered and used for sample analysis. The sample injection volume is 2.0mL, 30% acetonitrile (containing 0.01% ammonium acetate) is used as a mobile phase, an Agilent Zorbax SB-C18 (9.4X 150mm, 5.0 mu m) chromatographic column is used as a stationary phase, and the flow rate is 2.0mL/min to purify the sample.
And cutting the fraction flowing out for 14.2-18.6 min and performing solid phase extraction. The cut fraction was diluted with water by a dilution pump at a flow rate of 2.0mL/min, mixed by a mixer, and then concentrated by a solid phase extraction column.
After the solid phase extraction enrichment is finished, acetonitrile and 0.01 mol/L ammonium acetate solution are used for gradient elution as a mobile phase through valve switching, NNK and NNAL enriched on a solid phase extraction column are eluted in a flow direction opposite to that of the solid phase extraction enrichment, and then the NNK and the NNAL are separated by an analytical column, detected and measured by mass spectrometry, and quantified by an internal standard method.
The NNK content of the sample is 3.26 ng/L, and the NNAL content is 4.27 ng/L. The chromatogram of the NNK and NNAL efflux in the primary chromatography system is shown in FIG. 3.
Example 4
The apparatus of this example is the same as example 1.
The method of measuring NNK and NNAL in the blood and urine of smokers of this example was performed as follows:
the test sample in this example is a blood sample from a heavy smoker of a cigarette of the flue-cured type.
Accurately transferring 5.0mL of sample after collection, adding 100 muL of internal standard working solution, diluting to 10mL of working solution with methanol, fully shaking up, and keeping the proportion of methanol in the actual sample to be detected at 50% and the concentration of the internal standard at 5 ng/L.
Rapidly taking the sample back to the laboratory, and centrifuging at a high speed of 20000r/min for 10 min; the supernatant was filtered and used for sample analysis. The sample injection volume is 2.0mL, 30% acetonitrile (containing 0.01% ammonia acetate) is used as a mobile phase, and Agilent Zorbax SB-C18 (9.4)
150mm, 5.0 μm) chromatographic column as stationary phase, the flow rate is 2.0 mL/min.
And cutting the fraction flowing out for 14.2-18.6 min and performing solid phase extraction. The cut fraction was diluted with water by a dilution pump at a flow rate of 2.0mL/min, mixed by a mixer, and then concentrated by a solid phase extraction column. The solid phase extraction enrichment was accumulated 5 times.
After enrichment is finished, acetonitrile and 0.01 mol/L ammonium acetate solution are used for gradient elution as a mobile phase through valve switching, NNK and NNAL enriched on a solid phase extraction column are eluted in a flow direction opposite to that of solid phase extraction enrichment, and then separation is carried out through an analytical column, mass spectrometry detection and determination are carried out, and quantification is carried out through an internal standard method.
The NNK content of the sample is 1.12 ng/L, and the NNAL content is 1.45 ng/L. The chromatogram of NNK and NNAL efflux from a primary chromatography system is shown in FIG. 3
Example 5
The apparatus of this example is the same as example 1.
The method of measuring NNK and NNAL in the blood and urine of smokers of this example was performed as follows:
this example assay sample is a non-smoker sample.
Accurately transferring 5.0mL of sample after collection, adding 100 muL of internal standard working solution, diluting to 10mL of working solution with methanol, fully shaking up, and keeping the proportion of methanol in the actual sample to be detected at 50% and the concentration of the internal standard at 5 ng/L.
Rapidly taking the sample back to the laboratory, and centrifuging at a high speed of 20000r/min for 10 min; the supernatant was filtered and used for sample analysis. The sample injection volume is 2.0mL, 30% acetonitrile (containing 0.01% ammonium acetate) is used as a mobile phase, an Agilent Zorbax SB-C18 (9.4X 150mm, 5.0 mu m) chromatographic column is used as a stationary phase, and the flow rate is 2.0mL/min to purify the sample.
And cutting the fraction flowing out for 14.2-18.6 min and performing solid phase extraction. The cut fraction was diluted with water by a dilution pump at a flow rate of 2.0mL/min, mixed by a mixer, and then concentrated by a solid phase extraction column. The solid phase extraction enrichment was accumulated 5 times.
After enrichment is finished, acetonitrile and 0.01 mol/L ammonium acetate solution are used for gradient elution as a mobile phase through valve switching, NNK and NNAL enriched on a solid phase extraction column are eluted in a flow direction opposite to that of solid phase extraction enrichment, and then separation is carried out through an analytical column, mass spectrometry detection and determination are carried out, and quantification is carried out through an internal standard method.
Finally, the NNK and NNAL content of the sample is measured to be lower than the quantitative limit of the method. The chromatogram of the NNK and NNAL efflux in the primary chromatography system is shown in FIG. 3.
Summary of actual sample analysis
The results of using the method of the present invention for NNK and NNAL in the blood and urine of smokers are shown in Table-3. The results show that: non-smokers and e-smokers did not detect NNK and NNAL in blood and urine; NNK and NNAL are detected in blood and urine of both flue-cured cigarette heavy smokers and mixed cigarette smokers. The NNK content in the blended cigarette is obviously higher than that of a flue-cured cigarette, and the NNK and NNAL content in blood and urine of a smoker and the NNK exposure of cigarette smoke show positive correlation. The lower levels of NNK and NNAL in urine samples compared to blood samples may be associated with the constant metabolic breakdown of NNK and NNAL after inhalation into smokers.
TABLE 3 TSNAs content determination results (ng/L) in representative samples
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A system for measuring NNK and metabolites thereof by liquid-liquid two-dimensional chromatography based on solid-phase extraction is characterized in that: the system comprises a primary chromatographic system and a secondary chromatographic system, wherein the primary chromatographic system is connected with the secondary chromatographic system through a six-way valve; the solid phase extraction column is arranged on the six-way valve; the primary chromatographic system comprises a sample injector, one end of the sample injector is connected with the first pump, the other end of the sample injector is connected with the purification column, one end of the purification column is connected with a detector, one end of the detector is connected with one end of a three-way valve, the end of the three-way valve is also connected with the second pump, one end of the other two ends of the three-way valve is a waste liquid outlet, and the other end of the three-way valve is connected; the second pump cuts the NNK and NNAL outflow part of fractions into solid phase extraction and enrichment, and the rest parts flow into waste liquid; and a third pump of the secondary chromatographic system is communicated with one port of the six-way valve, the other port of the six-way valve is connected with an analytical column of the secondary chromatographic system, and the analytical column is connected with a detector.
2. The system for the liquid-liquid two-dimensional chromatographic determination of NNK and its metabolites based on solid phase extraction according to claim 1, characterized in that: seven ports are arranged on the six-way valve and are respectively from the first port to the seventh port, one end of the mixer is communicated with the seventh port, and two ends of the solid phase extraction column are communicated with the first port and the fourth port.
3. The system for the liquid-liquid two-dimensional chromatographic determination of NNK and its metabolites based on solid phase extraction according to claim 2, characterized in that: when a sample is analyzed, a sample is injected through a sample injector of a primary chromatographic system, the outflow time of a component to be detected is indicated by a detector, the component to be detected is cut through a three-way valve, the cut component is mixed through a mixer and enters from a seventh port of a six-way valve, the component to be detected flows through a solid phase extraction column through a fourth port, the component to be detected is enriched on the solid phase extraction column, meanwhile, the cut component is diluted through a second pump, the elution strength of a mobile phase is reduced, the component to be detected is ensured not to be eluted on the solid phase extraction column and is completely enriched on the solid phase extraction column, and the mobile phase which is collected through solid phase extraction enters waste liquid through the outflow of a first port and a sixth port.
4. A method for measuring NNK and NNAL in the blood and urine of smokers based on the system according to any of claims 1 to 3, characterized in that: the method comprises the following steps:
(a) injecting a sample prepared, wherein the injection volume is 1.0-2.0 mL, 30% acetonitrile containing 0.01% ammonia acetate is used as a mobile phase, an Agilent Zorbax SB-C18, the sample is purified at the flow rate of 2.0mL/min by using a half-preparation chromatographic column of 5.0μm as a stationary phase, and the fraction flowing out after cutting for 14.2-18.6 min enters into solid phase extraction;
(b) delivering water to dilute the cut fraction by a second pump at the flow rate of about 2.0mL/min, mixing the diluted fraction by a mixer, and enriching the diluted fraction by a solid phase extraction column;
(c) after the solid phase extraction and enrichment are finished, acetonitrile and 0.01 mol/L ammonium acetate solution are used for gradient elution as a mobile phase by switching the six-way valve, NNK and NNAL enriched on the solid phase extraction column are eluted in a flow direction opposite to that of the solid phase extraction and enrichment, and the NNK and the NNAL in the blood and urine of a smoker can be determined by separation of an analytical column, detection and determination of liquid chromatography-mass spectrometry and quantification of an internal standard method.
5. A method of measuring NNK and NNAL in the blood and urine of a smoker according to claim 4, wherein: the solid phase extraction material is Oasis HLB, and the particle diameter is5.0 µm。
6. A method of measuring NNK and NNAL in the blood and urine of a smoker according to claim 4, wherein: in the step (1), the sample preparation process is as follows:
after blood and urine samples are collected, accurately transferring a certain amount of samples, adding internal standard working solution, diluting with methanol, fully shaking up, and keeping the proportion of methanol in the actual sample to be detected to be 50% and the concentration of the internal standard to be 5 ng/L; the sample is quickly centrifuged at a high speed of 20000r/min for 10 min; and taking the supernatant, filtering and directly analyzing the sample.
7. A method of measuring NNK and NNAL in the blood and urine of a smoker according to claim 4, wherein: in the step (3), the liquid chromatography conditions were as follows:
the chromatographic column is a Waters Acquity UPLC BEH C18 chromatographic column, 2.1mm i.d. x 100 mm, 1.7 μm; the gradient conditions were: acetonitrile A, 0.01 mol/L ammonium acetate solution B, mobile phase A is changed from 10% to 80% in 0-5 min, A is changed from 80% to 10% in 6min, and the mobile phase is balanced for 5min after returning to 10% of A, so that the next sample can be carried out;
the mass spectrometry conditions were as follows:
electrospray ionization source: ESI, detection mode: multi-reaction monitoring, MRM, scanning mode: positive ion scan, electrospray voltage: 5000V, ion source temperature: 600 ℃, assist Gas1 pressure: 0.345MPa, assist Gas2 pressure: 0.483 MPa.
8. A method of measuring NNK and NNAL in the blood and urine of a smoker according to claim 4, wherein: the working curves, linear ranges, correlation coefficients, detection limits and quantitation limits for NNK and NNAL are shown in the following table:
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