CN104569224A - Separation, enrichment and detection method for mercapturic acid substances in urine - Google Patents
Separation, enrichment and detection method for mercapturic acid substances in urine Download PDFInfo
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- CN104569224A CN104569224A CN201410712397.7A CN201410712397A CN104569224A CN 104569224 A CN104569224 A CN 104569224A CN 201410712397 A CN201410712397 A CN 201410712397A CN 104569224 A CN104569224 A CN 104569224A
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Abstract
The invention relates to a separation, enrichment and detection method for mercapturic acid substances in urine. The method comprises the following steps: taking ionic liquid 1-octyl-3-methylimidazolium hexafluorophate immiscible with an aqueous phase as an enrichment medium, adding a minute quantity of ionic liquid [C8mim][PF6] and acetonitrile into the urine, and controlling the enrichment conditions to achieve enrichment of the mercapturic acid substances; by adopting an HPLC-MS/MS analysis platform, analyzing and detecting mercapturic acid substances such as CEMA, DHBMA, MHBMA, PMA, HPMMA and PHEMA. The method disclosed by the invention is high in enrichment speed, one-step feeding is realized by optimizing the overall chromatographic conditions, simultaneous analysis is also realized, and the working efficiency is greatly improved.
Description
Technical Field
The invention relates to a separation, enrichment and detection method of a mercapturic acid substance in urine, in particular to a rapid separation and enrichment method of 7 mercapturic acid substances in urine and an analysis method for determining the content of the 7 mercapturic acid substances in urine of a smoker by adopting a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).
Background
The cigarette burning can generate a large amount of volatile organic compounds, including acrolein, benzene, 1, 3-butadiene, styrene, crotonaldehyde and the like, and the volatile organic compounds have strong carcinogenic or mutagenic effects, can affect metabolites of a human body after entering the human body, and mainly form the substances of the class of mercapto uric acid. The substances have better specificity, are decomposition products after the combination of exogenous electrophilic compounds and glutathione, do not further react, and can be used as smoke exposure evaluation markers.
Research has shown that 7 kinds of mercapto uric acid substances formed by the volatile organic compounds respectively: s-phenylmercapturic acid (PMA), N-acetyl-S- (2-hydroxy-1-phenylethyl) -L-cysteine (PHEMA), acetyl-S- (3-hydroxypropyl-1-methyl) -L-2-amino-3-mercaptopropionate dicyclohexylamine (HPMMA), N-acetyl-S- (4-hydroxy-2-buten-1-yl) -L-cysteine (MHBMA), N-acetyl-S- (3-hydroxypropyl) cysteine dicycloethyl (3HPMA), N-acetyl-S- (2-carboxyethyl) -L-cysteine di (dicyclohexylamine) (CEMA), and N-acetyl-S- (3), 4-Dihydroxybutyl) -L-cysteine (DHBMA). Due to the low content of these substances and the complex metabolite matrix, the analysis of the thiol-uric acid substances in the urine of smokers is very difficult.
At present, the enrichment of the mercaptouric acid substances is mainly completed by Solid Phase Extraction (SPE), and then the HPLC-MS/MS analysis detection is matched. But the SPE is adopted to realize sample enrichment, the time consumption is long, the cost is high, and the use amount of the organic solvent is large during elution, so that the method is not environment-friendly. These problems greatly affect the ability to enrich the mercaptouric acid and also affect the accuracy of the results of analyzing the mercaptouric acid substances.
The ionic liquid is a novel green solvent, is salt completely consisting of ions, is in a liquid state at room temperature, and generally consists of organic cations containing nitrogen and phosphorus and inorganic anions. By changing the composition of the anions and the cations, ionic liquids with different properties can be formed. At present, compared with widely used organic solvents, the ionic liquid has the following outstanding advantages: (1) the vapor pressure is extremely low, and the volatile matter is not easy to volatilize; (2) the product has good solubility to organic matters and inorganic matters, and can be used as a good liquid extractant; (3) has structure controllability; (4) the ionic liquid has good conductivity, thermal stability and excellent oxidation resistance. Therefore, the invention utilizes the characteristics of the ionic liquid to realize the enrichment of the mercapturic acid substances on the basis of the optimized conditions. And a high-sensitivity HPLC-MS/MS analysis platform is selected, so that the 7 kinds of the mercapturic acid substances in the urine of the smoker can be quickly and accurately analyzed.
Disclosure of Invention
The invention aims to provide a method for separating, enriching and detecting mercapturic acid substances in urine aiming at the defects of the prior art; using an ionic liquid which is incompatible with water (1-octyl-3-methylimidazolium hexafluorophosphate, [ C ]8mim][PF6]) As an enrichment medium by applying a very small amount of ionic liquid [ C ] in urine8mim][PF6]And acetonitrile, and controlling the enrichment condition to achieve the enrichment of the mercapturic acid substances. And then, adopting an HPLC-MS/MS analysis platform to complete the analysis of 7 kinds of mercapturic acid substances, namely CEMA, DHBMA, MHBMA, PMA, HPMMA and PHEMA.
Specifically, the method for separating and enriching the mercapturic acid substances in the urine comprises the following steps:
(1) adding ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate ([ C ] into urine of smoker8mim][PF6]) And acetonitrile, mixing to form a mixed emulsion;
(2) and centrifuging to obtain the lower layer liquid enriched with the mercapturic acid substances.
The principle of enriching the mercapturic acid substances in the urine in the method provided by the invention is as follows: emulsions are generally thermodynamically stable systems of low viscosity and isotropy formed spontaneously from components such as water, oil, adjuvants, etc. in the appropriate proportions. The time to enrichment is greatly accelerated because the time to equilibrium is very short. The invention adds ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate ([ C ] C) into urine of smoker8mim][PF6]) After the extraction of the mercapturic acid, an emulsion mixed system consisting of 1-octyl-3-methylimidazolium hexafluorophosphate (ionic liquid)/acetonitrile/urine can be quickly formed, so that the aim of quickly extracting the mercapturic acid is fulfilled.
Preferably, in step (1), the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate ([ C)8mim][PF6]) The mixture ratio of the acetonitrile and the urine of a smoker is as follows: 30-100 mg: 0.5-2 mL: 2-10 mL.
Preferably, in the step (1), the mixed emulsion is formed by first vortexing for 30-120 seconds and then sonicating at 20-50 ℃ for 1-3 minutes.
Vortexes (Vortex) are sometimes also referred to as vortexes. It is a flow phenomenon that a cylinder with a small radius rotates in a static fluid to cause a surrounding fluid to make circular motion. The effect of the vortex in the experiment is to carry out sufficient material interaction between the water phase and the ionic liquid phase so as to improve the extraction efficiency and enable the system to form emulsion. The whole process must be vortex first and then ultrasonic.
Preferably, in the step (2), a high-speed centrifuge is adopted, and the centrifugation is carried out for 1-5 minutes at the rotating speed of 3000-.
Preferably, the mercaptouric acid substances comprise: s-phenylmercapturic acid (PMA), N-acetyl-S- (2-hydroxy-1-phenylethyl) -L-cysteine (PHEMA), acetyl-S- (3-hydroxypropyl-1-methyl) -L-2-amino-3-mercaptopropionate dicyclohexylamine (HPMMA), N-acetyl-S- (4-hydroxy-2-buten-1-yl) -L-cysteine (MHBMA), N-acetyl-S- (3-hydroxypropyl) cysteine dicycloethyl (3HPMA), N-acetyl-S- (2-carboxyethyl) -L-cysteine di (dicyclohexylamine) (CEMA), and N-acetyl-S- (3), 4-Dihydroxybutyl) -L-cysteine (DHBMA) and the like.
Preferably, the invention further provides an analysis and detection method for 7 kinds of mercapturic acid substances in urine of smokers, which specifically comprises the following steps:
(I) preparing five-grade standard working solution, wherein each grade of standard working solution is methanol solution containing PMA, PHEMA, HPMMA, MHBMA, 3HPMA, CEMA and DHBMA; preparing an internal standard solution containing D5-PMA、13C6-PHEMA、D4-HPMMA、D6-MHBMA、D3-3HPMA、D3-CEMA and D7-a methanol solution of DHBMA;
(II) adding the same internal standard solution into each stage of standard working solution, and adding 1-octyl-3-methylimidazole hexafluorophosphate and acetonitrile to form mixed emulsion; then centrifugally separating to obtain lower-layer liquid; measuring the lower layer liquid by adopting a liquid chromatography tandem mass spectrum to obtain a standard chromatographic separation chart of 7 kinds of mercapturic acid substances;
(III) adding internal standard solution into urine of a smoker, and adding 1-octyl-3-methylimidazolium hexafluorophosphate and acetonitrile into the internal standard solution to form mixed emulsion; then centrifugally separating to obtain lower-layer liquid; measuring the lower layer liquid by adopting a liquid chromatography tandem mass spectrum to obtain an actual sample chromatographic separation chart of each mercapturic acid substance in the urine of the smoker;
(IV) using the prepared standard chromatographic separation chart and the actual sample chromatographic separation chart to perform qualitative and quantitative analysis.
Wherein,
preferably, in step (I), the concentrations of PMA, PHEMA, HPMMA, MHBMA, 3HPMA, CEMA and DHBMA in the first to fifth standard working solutions are 1ng/mL, 5ng/mL, 10ng/mL, 100ng/mL and 500ng/mL, respectively.
Preferably, in step (I), D is present in the internal standard solution5-PMA、13C6-PHEMA、D4-HPMMA、D6-MHBMA、D3-3HPMA、D3-CEMA and D7The concentrations of DHBMA were all 50 ng/mL.
Preferably, in the step (II), the relative addition amount of each stage of standard solution, internal standard solution, 1-octyl-3-methylimidazole hexafluorophosphate and acetonitrile is 2-10 mL: 20-100 μ L: 30-100 mg: 0.5-2 mL.
Preferably, in the step (II), the mixed emulsion is formed by firstly vortexing for 30-120 seconds and then ultrasonic processing for 1-3 minutes at 20-50 ℃.
Preferably, in the step (II), the centrifugal separation is performed by a high-speed centrifuge at 3000 and 8000 rpm for 1-5 minutes.
Preferably, in the step (iii), the relative addition amounts of the smoker urine, the internal standard solution, the 1-octyl-3-methylimidazolium hexafluorophosphate and the acetonitrile are 2-10 mL: 20-100 μ L: 30-100 mg: 0.5-2 mL.
Preferably, in the step (III), the mixed emulsion is formed by first vortexing for 30-120 seconds and then sonicating at 20-50 ℃ for 1-3 minutes.
Preferably, in step (III), the centrifugation is performed for 1-3 minutes at 3000-.
Preferably, in steps (ii) and (iii), the chromatographic conditions for the liquid chromatography tandem mass spectrometry are as follows: an anion mode EI source; the chromatographic column is as follows: a 150mm x 2.1mm i.d,3 μm SequantZIC-cHILIC chromatography column; the mobile phase A is 0.2 wt% acetic acid acetonitrile solution, and the mobile phase B is 0.2 wt% acetic acid water solution; gradient elution procedure: 0-2min 95% vA +5 v% B; 5min 90 v% A +10 v% B; 3min 60 v% A +40 v% B; 14min 50 v% A +50 v% B; 14.5min 95 v% A +5 v% B; 30min 95 v% A +5 v% B; the sample size of the liquid chromatography tandem mass spectrometry is 10 muL.
In the step (IV): by retention time and massThe numbers were analyzed qualitatively. Calculating according to the ratio of the peak area of each mercapturic acid substance to the peak area of the corresponding internal standard substance, the mass of the corresponding internal standard substance and the corresponding standard working curve to obtain the content (m) of 7 mercapturic acid substancesx) The specific calculation formula is as follows:
mx＝f×Ax/(As/ms)；
wherein f is a response factor, AxAnd AsPeak areas of the analyte and the internal standard, msIs the amount of internal standard.
The detection method can be used for measuring the content of the smoking exposure marker after the urine measurement of smokers.
The invention has the technical effects and advantages that:
1. the urine has low content of mercapturic acid substances, and the pretreatment is mostly completed by Solid Phase Extraction (SPE). However, the whole operation is long in time consumption and high in cost, and the organic solvent with high volatility is used, so that the environment is influenced to a certain extent. The invention realizes the rapid enrichment of the mercapto uric acid in the urine by using the ionic liquid emulsion which is difficult to volatilize, has good stability and achieves the thermodynamic stability and the rapidness.
2. The invention realizes one-time sample injection and analysis by optimizing the integral chromatographic condition, and greatly improves the working efficiency.
Drawings
FIG. 1: ionic liquid [ C8mim][PF6]Schematic diagram of process for enriching mercapturic acid in urine;
FIG. 2: ionic liquid [ C8mim][PF6]Schematic diagram of addition amount and extraction recovery rate of the substance to be detected;
FIG. 3: the acetonitrile adding amount and the extraction recovery rate of the object to be detected are shown schematically;
FIG. 4: HPLC-MS/MS chromatographic isolation assay of 7 Standard Mercapturic acids (1, PMA; 2, PHEMA; 3, HPMMA; 4, MHBMA; 5, 3 HPMA; 6, CEMA and 7, DHBMA)
FIG. 5: HPLC-MS/MS separation chromatogram of HPMMA in urine;
FIG. 6: HPLC-MS/MS separation chromatogram of 3HPMA in urine;
FIG. 7: HPLC-MS/MS separation chromatogram of CEMA in urine;
FIG. 8: HPLC-MS/MS separation chromatogram of DHBMA in urine;
FIG. 9: HPLC-MS/MS separation chromatogram of MHBMA in urine.
Detailed Description
The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
The invention provides separation, enrichment, analysis and detection of 7 kinds of mercapto uric acid substances in urine, wherein ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate ([ C ] is added into urine of a smoker8mim][PF6]) And acetonitrile, mixing to form a mixed emulsion; centrifuging to obtain a lower layer liquid enriched with the mercapto-uric acid substances; namely as shown in fig. 1: by means of [ C8mim][PF6]The ionic liquid, acetonitrile and the urine of a smoker form an opacification system, and enrichment and separation of 7 kinds of mercapto uric acid substances are realized through substance exchange and high-speed centrifugation; and then, the HPLC-MS/MS is adopted to realize the analysis and detection of 7 kinds of mercapto uric acid substances in the urine of smokers.
The 7 kinds of mercapto uric acid substances are respectively: s-phenylmercapturic acid (PMA), N-acetyl-S- (2-hydroxy-1-phenylethyl) -L-cysteine (PHEMA), acetyl-S- (3-hydroxypropyl-1-methyl) -L-2-amino-3-mercaptopropionate dicyclohexylamine (HPMMA), N-acetyl-S- (4-hydroxy-2-buten-1-yl) -L-cysteine (MHBMA), N-acetyl-S- (3-hydroxypropyl) cysteine dicycloethyl (3HPMA), N-acetyl-S- (2-carboxyethyl) -L-cysteine di (dicyclohexylamine) (CEMA), and N-acetyl-S- (3), 4-Dihydroxybutyl) -L-cysteine (DHBMA).
The analysis and detection process comprises the following steps:
preparing five-stage standard working solution, wherein each stage of standard working solution is methanol solution containing PMA, PHEMA, HPMMA, MHBMA, 3HPMA, CEMA and DHBMA; preparing an internal standard solution containing D5-PMA、13C6-PHEMA、D4-HPMMA、D6-MHBMA、D3-3HPMA、D3-CEMA and D7-a methanol solution of DHBMA;
as a preferred implementation: the concentrations of PMA, PHEMA, HPMMA, MHBMA, 3HPMA, CEMA and DHBMA in the first-stage to fifth-stage standard working solutions are respectively 1ng/mL, 5ng/mL, 10ng/mL, 100ng/mL and 500 ng/mL;
as a preferred implementation: d in the internal standard solution5-PMA、13C6-PHEMA、D4-HPMMA、D6-MHBMA、D3-3HPMA、D3-CEMA and D7-DHBMA concentrations were all 50 ng/mL;
(II) adding the same internal standard solution into each stage of standard working solution, and adding 1-octyl-3-methylimidazole hexafluorophosphate and acetonitrile to form mixed emulsion; then centrifugally separating to obtain lower-layer liquid; measuring the lower layer liquid by adopting a liquid chromatography tandem mass spectrum to obtain a standard chromatographic separation chart of 7 kinds of mercapturic acid substances;
in a preferable implementation case, in the step (II), the relative addition amount of each stage of the standard solution, the internal standard solution, the 1-octyl-3-methylimidazole hexafluorophosphate and the acetonitrile is 2-10 mL: 20-100 μ L: 30-100 mg: 0.5-2 mL;
in a preferred implementation, in the step (II), the mixed emulsion is formed by firstly swirling for 30-120 seconds and then performing ultrasonic treatment at 20-50 ℃ for 1-3 minutes; in the step (II), the centrifugal separation adopts a high-speed centrifuge, and the centrifugation is carried out for 1-5 minutes at the rotating speed of 3000-.
(III) adding internal standard solution into urine of a smoker, and adding 1-octyl-3-methylimidazolium hexafluorophosphate and acetonitrile into the internal standard solution to form mixed emulsion; then centrifugally separating to obtain lower-layer liquid; measuring the lower layer liquid by adopting a liquid chromatography tandem mass spectrum to obtain an actual sample chromatographic separation chart of each mercapturic acid substance in the urine of the smoker;
in a preferred implementation, in the step (iii), the relative addition amounts of the smoker urine, the internal standard solution, the 1-octyl-3-methylimidazolium hexafluorophosphate and the acetonitrile are 2-10 mL: 20-100 μ L: 30-100 mg: 0.5-2 mL;
in a preferred embodiment, in the step (III), the mixed emulsion is formed by firstly vortexing for 30-120 seconds and then ultrasonic processing at 20-50 ℃ for 1-3 minutes; in the step (III), the centrifugal separation adopts a high-speed centrifuge, and the centrifugation is carried out for 1 to 3 minutes at the rotating speed of 3000-
(IV) carrying out qualitative and quantitative analysis by using the prepared standard chromatographic separation chart and the actual sample chromatographic separation chart; that is, qualitative by retention time and mass number; calculating by using the ratio of the peak area of each mercapturic acid substance to the peak area of the corresponding internal standard substance, the mass of the corresponding internal standard substance and the corresponding standard working curve to obtain 7 mercapturic acid substancesContent (m)x) The specific calculation formula is as follows:
mx＝f×Ax/(As/ms)；
wherein f is a response factor, AxAnd AsPeak areas of the analyte and the internal standard, msIs the amount of internal standard.
The related apparatus and chromatographic column configuration is as follows:
agilent 1200 liquid chromatograph equipped with G1329A autosampler, G1311A quaternary mixing pump, and G1316A column oven (agilent, usa);
eppendorf 5810R high speed centrifuge (Eppendorf, Germany);
API4000 triple quadrupole tandem mass spectrometer, electrokinetic spray ion source (ESI) and analyst1.6 software data processing system (applied biosystems, usa).
The analysis conditions of the HPLC-MS/MS instrument are as follows: an anion mode EI source; the chromatographic column is as follows: a 150mm x 2.1mm i.d,3 μm Sequant ZIC-cHILIC chromatography column; the mobile phase A is 0.2 wt% acetic acid acetonitrile solution, and the mobile phase B is 0.2% acetic acid water solution. Gradient elution procedure: 0-2min 95% vA +5 v% B; 5min 90 v% A +10 v% B; 3min 60 v% A +40 v% B; 14min 50 v% A +50 v% B; 14.5min 95 v% A +5 v% B; 30min 95 v% A +5 v% B; the sample size of the liquid chromatography tandem mass spectrometry is 10 muL.
As shown in fig. 2: by fixing urine volume, acetonitrile volume, gradually changing the ionic liquid [ C8mim [ ]][PF6]The addition amount is examined, the experimental process of the extraction recovery rate of 7 kinds of mercapturic acid substances is examined, and the ionic liquid [ C ] in the emulsion system is determined8mim][PF6]The optimum addition amount of (b) and the extraction recovery rate; namely 5ml of urine, 500 mu L of acetonitrile system is added with 60mg of ionic liquid [ C8mim][PF6]And the extraction recovery rate of 7 kinds of mercapto uric acid substances is highest.
As shown in fig. 3: through the experiment process of fixing the urine volume and the adding amount of the ionic liquid [ C8mim ] [ PF6], changing the acetonitrile volume and investigating the recovery rates of 7 kinds of the mercapto uric acid substances, the relation between the optimal adding amount of the acetonitrile in the emulsion system and the extraction recovery rate is determined, namely, 500 mu L of acetonitrile is added into the system of 5ml of urine and 60mg of the ionic liquid [ C8mim ] [ PF6], and the extraction recovery rate of the 7 kinds of the mercapto uric acid substances is the highest.
The technical scheme of the invention is explained in detail by the specific embodiment as follows:
the specific experimental methods and parameters are as follows:
the method comprises the following steps: preparing five-stage methanol solution containing PMA, PHEMA, HPMMA, MHBMA, 3HPMA, CEMA and DHBMA as standard working solution; the concentrations of PMA, PHEMA, HPMMA, MHBMA, 3HPMA, CEMA and DHBMA in the first-stage to fifth-stage standard working solutions are respectively 1ng/mL, 5ng/mL, 10ng/mL, 100ng/mL and 500 ng/mL;
is formulated to contain D5-PMA、13C6-PHEMA、D4-HPMMA、D6-MHBMA、D3-3HPMA、D3-CEMA and D7-methanol solution of DHBMA as internal standard solution, wherein D5-PMA、13C6-PHEMA、D4-HPMMA、D6-MHBMA、D3-3HPMA、D3-CEMA and D7The concentrations of DHBMA were all 50 ng/mL.
Step two: taking 5ml of each grade of standard solution, adding 50 mu L of internal standard solution and 60mg of [ C ]8mim][PF6]The ionic liquid was vortexed with 500. mu.L acetonitrile for 30 seconds, and then sonicated at room temperature for 2 minutes to form a mixed emulsion. Then placing the mixture in a high-speed centrifuge (5000 r/min), centrifuging for 3min to obtain lower layer liquid, and measuring the lower layer liquid by using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) (with the same detection conditions) to obtain 7 standard chromatographic separation graphs of the mercapturic acid, as shown in FIG. 4;
step three: taking 5ml urine sample of smoker, adding 50 μ L internal standard solution, 60mg [ C8mim][PF6]Mixing ionic liquid with 500 mul acetonitrile, vortexing for 30 seconds, and ultrasonic treating at room temperature for 2minAn emulsion. Then placing the urine sample in a high-speed centrifuge (5000 r/min), centrifuging for 3min to obtain lower layer liquid, and measuring the lower layer liquid by using HPLC-MS/MS (detection conditions are the same as above) to obtain a chromatographic separation chart of each actual mercapto uric acid sample in the urine sample, as shown in FIGS. 5-9 and Table 1;
TABLE 1 mean content of 7 mercapturic acid substances in urine of smokers (ND: not detected)
| Substance(s) | Average content (μ g/L) |
| CEMA | 477.4 |
| 3HPMA | 1568 |
| PMA | ND |
| DHBMA | 325.3 |
| MHBMA | 48.77 |
| HPMMA | 1555 |
| PHEMA | ND |
It can be seen that the mean levels of HPMMA, MHBMA, 3HPMA, CEMA and DHBMA in the urine of smokers were 1555. mu.g/L, 48.77. mu.g/L, 1568. mu.g/L, 477.4. mu.g/L and 325.3. mu.g/L, respectively, whereas both metabolites PMA and PHEMA were not detected.
The above is only an example of the detection method of the present invention for detecting the assay of 7 kinds of mercapturic acid substances (PMA, PHEMA, HPMMA, MHBMA, 3HPMA, CEMA and DHBMA) in the urine of smokers, and the detection method of the present invention can also realize the enrichment and assay detection of other mercapturic acid substances.
Claims (10)
1. A method for separating and enriching mercaptouric acid substances in urine of smokers comprises the following steps:
(1) adding ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate and acetonitrile into urine of a smoker, and mixing to form mixed emulsion;
(2) and centrifuging to obtain the lower layer liquid enriched with the mercapturic acid substances.
2. The method for separating and enriching the mercapturic acid substances in the urine as claimed in claim 1, wherein in the step (1), the mixture ratio of the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate, acetonitrile and the urine of the smoker is as follows: 30-100 mg: 0.5-2 mL: 2-10 mL.
3. The method for separating and enriching the mercapturic acid substances in the urine as claimed in claim 1, wherein in the step (1), the mixed emulsion is formed by vortexing for 30-120 seconds and then sonicating at 20-50 ℃ for 1-3 minutes.
4. The method as claimed in claim 1, wherein in the step (2), the high-speed centrifuge is used to centrifuge at 3000-.
5. The method for separating and enriching the mercapturic acid substances in the urine as claimed in claim 1, wherein the mercapturic acid substances comprise: s-phenylmercapturic acid (PMA), N-acetyl-S- (2-hydroxy-1-phenylethyl) -L-cysteine (PHEMA), acetyl-S- (3-hydroxypropyl-1-methyl) -L-2-amino-3-mercaptopropionate dicyclohexylamine (HPMMA), N-acetyl-S- (4-hydroxy-2-buten-1-yl) -L-cysteine (MHBMA), N-acetyl-S- (3-hydroxypropyl) cysteine dicycloethyl (3HPMA), N-acetyl-S- (2-carboxyethyl) -L-cysteine di (dicyclohexylamine) (CEMA), and N-acetyl-S- (3), 4-Dihydroxybutyl) -L-cysteine (DHBMA).
6. An analysis and detection method for 7 kinds of mercapturic acid substances in urine of smokers specifically comprises the following steps:
(I) preparing five-grade standard working solution, wherein each grade of standard working solution is methanol solution containing PMA, PHEMA, HPMMA, MHBMA, 3HPMA, CEMA and DHBMA; preparing an internal standard solution containing D5-PMA、13C6-PHEMA、D4-HPMMA、D6-MHBMA、D3-3HPMA、D3-CEMA and D7-a methanol solution of DHBMA;
(II) adding the same internal standard solution into each stage of standard working solution, and adding 1-octyl-3-methylimidazole hexafluorophosphate and acetonitrile to form mixed emulsion; then centrifugally separating to obtain lower-layer liquid; measuring the lower layer liquid by adopting a liquid chromatography tandem mass spectrum to obtain a standard chromatographic separation chart of 7 kinds of mercapturic acid substances;
(III) adding internal standard solution into urine of a smoker, and adding 1-octyl-3-methylimidazolium hexafluorophosphate and acetonitrile into the internal standard solution to form mixed emulsion; then centrifugally separating to obtain lower-layer liquid; measuring the lower layer liquid by adopting a liquid chromatography tandem mass spectrum to obtain an actual sample chromatographic separation chart of each mercapturic acid substance in the urine of the smoker;
(IV) using the prepared standard chromatographic separation chart and the actual sample chromatographic separation chart to perform qualitative and quantitative analysis.
7. The method according to claim 6, wherein in step (I), the concentrations of PMA, PHEMA, HPMMA, MHBMA, 3HPMA, CEMA and DHBMA in the first to fifth standard working solutions are 1ng/mL, 5ng/mL, 10ng/mL, 100ng/mL and 500ng/mL respectively; d in the internal standard solution5-PMA、13C6-PHEMA、D4-HPMMA、D6-MHBMA、D3-3HPMA、D3-CEMA and D7The concentrations of DHBMA were all 50 ng/mL.
8. The method for analyzing and detecting 7 kinds of mercapturic acid substances in urine of smokers according to claim 7, further comprising any one or more of the following characteristics:
(a) in the step (II), the relative addition amount of each grade of standard solution, internal standard solution, 1-octyl-3-methylimidazole hexafluorophosphate and acetonitrile is 1-5 mL: 20-100 μ L: 30-100 mg: 0.5-2 mL;
(b) in the step (II), the mixed emulsion is formed by firstly whirling for 30-120 seconds and then carrying out ultrasonic treatment for 1-3 minutes at the temperature of 20-50 ℃;
(c) in the step (II), the centrifugal separation adopts a high-speed centrifuge, and the centrifugation is carried out for 1-5 minutes at the rotating speed of 3000-.
9. The method for analyzing and detecting 7 kinds of mercapturic acid substances in urine of smokers according to claim 7, further comprising any one or more of the following characteristics:
(a) in the step (III), the relative addition amounts of the urine of the smoker, the internal standard solution, 1-octyl-3-methylimidazolium hexafluorophosphate and acetonitrile are 1-5 mL: 20-100 μ L: 30-100 mg: 0.5-2 mL;
(b) in the step (III), the mixed emulsion is formed by firstly swirling for 30-120 seconds and then carrying out ultrasonic treatment for 1-3 minutes at the temperature of 20-50 ℃;
(c) in the step (III), the centrifugal separation adopts a high-speed centrifuge, and the centrifugation is carried out for 1-5 minutes at the rotating speed of 3000-.
10. The method for analyzing and detecting 7 kinds of mercapto uric acid substances in urine of a smoker according to claim 6, wherein in steps (II) and (III), the chromatographic conditions in the liquid chromatography tandem mass spectrometry are as follows: an anion mode EI source; the chromatographic column is as follows: 150mm x 2.1mm i.d,3 μmSequant ZIC-cHILIC chromatography column; the mobile phase A is 0.2 wt% acetic acid acetonitrile solution, and the mobile phase B is 0.2 wt% acetic acid water solution; a gradient elution procedure was used: 0-2min 95% vA +5 v% B; 5min 90 v% A +10 v% B; 3min 60 v% A +40 v% B; 14min 50 v% A +50 v% B; 14.5min 95 v% A +5 v% B; 30min 95 v% A +5 v% B; the sample size of the liquid chromatography tandem mass spectrometry is 10 muL.
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| WO2020089797A1 (en) | 2018-10-29 | 2020-05-07 | Universidade De Aveiro | Process for extracting, concentrating and purifying an antigen from a biological sample, composition and uses thereof |
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| CN109270205A (en) * | 2018-09-30 | 2019-01-25 | 常州合全药业有限公司 | Detection method that is a kind of while detecting zwitterion |
| WO2020089797A1 (en) | 2018-10-29 | 2020-05-07 | Universidade De Aveiro | Process for extracting, concentrating and purifying an antigen from a biological sample, composition and uses thereof |
| CN110208406A (en) * | 2019-05-31 | 2019-09-06 | 浙江大学 | The mercapturic acids adduction object detecting method and application that the chloro- 1,2- propylene glycol of evaluation 3- and its ester expose in short term |
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