CN103852547B - Detecting method for 3-ethyl adenine (3-HOEtA) in urine - Google Patents
Detecting method for 3-ethyl adenine (3-HOEtA) in urine Download PDFInfo
- Publication number
- CN103852547B CN103852547B CN201410095001.9A CN201410095001A CN103852547B CN 103852547 B CN103852547 B CN 103852547B CN 201410095001 A CN201410095001 A CN 201410095001A CN 103852547 B CN103852547 B CN 103852547B
- Authority
- CN
- China
- Prior art keywords
- urine
- eta
- ethyl
- adenine
- detecting method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a detecting method for 3-ethyl adenine (3-HOEtA) in urine. The detecting method is characterized by comprising the steps of unfreezing 24-hour urine at room temperature, uniformly mixing the urine and centrifugally filtering the urine; purifying and enriching the urine through an OasisMCX solid-phase extraction column; introducing the urine to an LC-MS (liquid Chromatograph Mass Spectrometer)/MS system for analyzing after uniformly mixing to accurately detect the content of 3-EtA in the urine. The detecting method is a brand new detecting method for 3-EtA in the urine; a deuterated standard is used as a quantitative analyte of an internal standard, so that errors caused in a sample pretreatment process can be reduced; moreover, a tandem mass spectrometry is adopted so that the selectivity, accuracy and sensitivity of the method are better improved. Through selecting and optimizing a chromatographic column and an elution condition, the chromatographic separation process is well improved, the chromatographic analysis time is shortened, and the consumption of an organic solvent is reduced.
Description
technical field:
The invention belongs to the physical and chemical inspection technical field of urine specimen, relating generally to the determination techniques field of 3-ethyl adenine in urine (3-EtA), is a kind of method adopting liquid chromatography-electrospray ionisation-tandem mass spectrometer (LC-ESI-MS/MS) to measure 3-ethyl adenine in urine (3-EtA) specifically.
background technology:
3-ethyl adenine (3-EtA) is the product after the free radical of exogenous compounds internal metabolism generation is combined with adenine.Because endogenous 3-ethyl adenine background values is low in urine, the characteristic of prototype compound can be reflected again comparatively accurately, be therefore widely used in the biological monitoring of environment alkylation pollutant as index of biological monitoring.It is reported, in urine, 3-ethyl adenine (N3-ethyladenine, 3-EtA) and smoking level have stronger correlativity, can be used in the exposure level of objectionable constituent nitrosamine in evaluation of flue gas.
At present, the report about the analyzing detecting method of DNA adduct in urine is more, but the method for high-flux analysis extracting, purify, detect plurality of target thing in urine matrix is not perfect.
summary of the invention:
Object of the present invention, just based on above-mentioned prior art situation, adopts liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology to establish the assay method of 3-ethyl adenine in urine (3-EtA).The method has fast, accurate, sensitivity high, can be used for the qualitative and quantitative analysis of 3-ethyl adenine in urine (3-EtA).The present invention, by optimizing pre-treatment and testing conditions, establishes extraction, detects the method for 3-ethyl adenine in urine, be suitable for analyzing smoking and non-smokers' urine.The separation that just can complete 3-ethyl adenine in 20 min detects, and this method is sensitive, and accurately, selectivity is good.
The object of the invention is to be achieved through the following technical solutions: the assay method of 3-ethyl adenine (3-EtA) in a kind of urine, comprises following concrete steps:
A, sample pre-treatments: urine is thawed and mixed under room temperature state, get 4 mL urines, add 4mL ultrapure water, adds mark in 100 μ L, regulates temperature to 80 DEG C with water-bath.The urine of getting after ultrapure water dilution is placed in solid-phase extraction column.Deuterated-3-ethyl adenine (d is designated as in described
5-3-EtA), interior mark concentration is 100 ng/mL.
Solid-Phase Extraction Waters Oasis MCX(6 Ml, 500mg) solid-phase extraction column, use 2 mL methyl alcohol, 5 mL activated in water solution in advance, applied sample amount is 8 mL.With 2 mL 10% methanol/water solution drip washing, then use 1 mL ethyl acetate drip washing, finally use methanol/ethyl acetate/ammoniacal liquor (47.5:47.5:5) wash-out, collect eluent and blow to dry with nitrogen at 60 DEG C, then 100 μ L aqueous dissolution are used, for LC-MS/MS compartment analysis.
The preparation of b, standard working solution: with 3-ethyl adenine (3-EtA) standard working solution of acetontrile variable concentrations.
C, liquid chromatography-tandem mass spectrometry (LC-MS/MS) measure, and draw the standard working solution of the 3-ethyl adenine (3-EtA) of the variable concentrations prepared, and inject LC-MS/MS system, obtain equation of linear regression.Sample liquid to be measured is measured, records the ratio analyzing thing and interior mark peak area, substitute into unary linear regression equation, try to achieve the content analyzing thing in sample liquid to be measured.
The present invention have chosen Waters HILIC (150 mm x 2.1 mm, 2.5 μm) chromatographic column, and flow visualizing chooses A: acetonitrile and B:10m mol/L ammonium formate/aqueous solution, A:B is 95:5.Elution time is 20 min, and sample size is 3 μ L.
Mass Spectrometry Conditions: electric spray ion source (ESI), positive ion scan mode; Spraying (IS) voltage is 5500 V; Atomization gas (Gasl) pressure is 344.5 kPa (50 psi); Gas curtain gas (CUR) pressure is 206.7 kPa (30 psi); Assisted atomization gas (Gas2) pressure is 310.0 kPa (45 psi); Ion source temperature (TEM) is 500 DEG C; A small bundle of straw, etc. for silkworms to spin cocoons on voltage (DP) is gone to be 100 eV; Impact energy (CE) is 30 eV; Residence time is 40 ms.Monitoring ion pair be 3-EtA:164 → 136(quota ion to), the qualitative ion pair of 164 → 119(); Interior mark d
5-3-EtA is 169 → 137.
The concrete technological process of the present invention is described in further detail as follows:
1, sample pre-treatments
Sample pre-treatments: urine is thawed and mixed under room temperature state, gets 4 mL urines, adds 4mL ultrapure water, adds mark (100ng/mL) in 100 μ L, regulates temperature to 80 DEG C with water-bath.The urine of getting after ultrapure water dilution is placed in 10 mL solid-phase extraction columns.Solid-Phase Extraction chooses Waters Oasis MCX(6 mL, 500 mg) pillar, use the methyl alcohol of 2 mL in advance, the water activation pillar of 5 mL, applied sample amount is 8 mL, with the methyl alcohol drip washing of 10% of 2 mL, then use 1 mL ethyl acetate drip washing, finally use methanol/ethyl acetate/ammoniacal liquor (47.5:47.5:5) wash-out, collect eluent and blow to dry with nitrogen at 60 DEG C, then 100 μ L aqueous dissolution are used, for LC-MS/MS compartment analysis.
2, LC-MS/MS measures
(1) LC-MS/MS condition
Chromatographic condition: Waters HILIC (150 mm x 2.1 mm, 2.5 μm) chromatographic column, elution requirement: mobile phase A: acetonitrile solution, pH 4.0, Mobile phase B: ammonium formate/aqueous solution (10mM), pH 4.0; A:B=95:5 flow velocity 0.25 mL/min; Column temperature 25 ° of C; Sample size 3 μ L.Elution time: 0 ~ 20 min.
Mass Spectrometry Conditions: electric spray ion source (ESI), positive ion scan mode; Spraying (IS) voltage is 5500 V; Atomization gas (Gasl) pressure is 344.5 kPa (50 psi); Gas curtain gas (CUR) pressure is 206.7 kPa (30 psi); Assisted atomization gas (Gas2) pressure is 310.0 kPa (45 psi); Ion source temperature (TEM) is 500 DEG C; A small bundle of straw, etc. for silkworms to spin cocoons on voltage (DP) is gone to be 100 eV; Impact energy (CE) is 326eV; Residence time is 40 ms.Monitoring ion pair and corresponding collision energy (CE) thereof are in table 1.
3-EtA and deuterated interior target part mass spectrometry parameters thereof under table 1 multiple-reaction monitoring pattern
* quota ion pair.
(2) the 3-ethyl adenine (mensuration of 3-EtA content
Draw 3-ethyl adenine (3-EtA) the standard working solution 3 μ L of the variable concentrations prepared, inject LC-ESI-MS/MS.Obtain the equation of linear regression of 3-ethyl adenine (3-EtA).Same method detects actual sample, tries to achieve the content of 3-ethyl adenine (3-EtA) in actual sample.
(3) range of linearity of the inventive method and detection limit:
With acetontrile series standard curve, the point of the typical curve of 3-ethyl adenine (3-EtA) is 0.1,0.2,0.5,1,1.5,2,2.5,3,3.5 and 5 ng/mL, compound linear good, and related coefficient is greater than 0.999.Utilize mark-on to dilute and obtain quantitative limit and the detection limit of method, concentration corresponding to target peak ten times of signal to noise ratio (S/N ratio)s is quantitative limit, and concentration corresponding to three times of signal to noise ratio (S/N ratio)s is detection limit, and it detects and is limited to 0.02 μ g/L.
(4) the inventive method recovery of standard addition and stability:
Take sample to add the recovery of standard addition of calibration method preparation method, altogether have chosen three Pitch-based sphere, each Pitch-based sphere replication 5 times, the recovery of the method obtained and stability, the results are shown in Table 2, table 3.The recovery scope 88.3-90.0% of method.In the daytime/day internal stability all within 5%, the recovery and the stability result of method of proof are better.
The recovery (n=5) of 3-EtA in table 2 urine
Between table 3 day/day internal stability
Method of the present invention overcomes the deficiency of prior art sample treatment, is optimized, and is optimized the coherent detection condition of LC-MS/MS for the chromatographic condition of 3-ethyl adenine (3-EtA) in urine.Compared with prior art the inventive method has following excellent results:
1. compare with traditional liquid-phase chromatography method, owing to have chosen tandem mass spectrum, the selectivity of method and sensitivity are improved, is more conducive to the mensuration of 3-ethyl adenine in the urine of low content (3-EtA).
2. this method has the advantage of easy and simple to handle, quick, accurate, sensitivity and good stability.
3. the present invention adopts deuterated standard items can reduce the error caused in sample pretreatment process as uantitative analytical thing, and tandem mass spectrometry better improves the selectivity of method and accuracy and sensitivity.By the selection and optimization of chromatographic column and elution requirement, improve chromatographic separation process preferably, shorten the time of stratographic analysis, decrease the consumption of organic solvent.
Accompanying drawing explanation
The total ion current figure of 3-ethyl adenine (3-EtA) in Fig. 1 urine.
Embodiment
The present invention is described further below in conjunction with example, but is not restriction the present invention.
The assay method of 3-ethyl adenine (3-EtA) in a kind of urine, its test process is that twenty-four-hour urine liquid at room temperature thaws, and mixes centrifugal filtration, by C18 solid phase extraction column purification enrichment, introduces LC-MS/MS systematic analysis after mixing.
Example 1:
1. instrument and reagent:
Agilent 1200 liquid chromatography (Agilent company of the U.S.), is furnished with G1367D automatic sampler, G 1312B two end number mixing pump and G1316B column oven; The triple quadrupole rods tandem mass spectrometry instrument (Applied biosystems) of API 5500, is furnished with electric spray ion source (ESI) and Analyst 1.5 software data disposal system.
Ammoniacal liquor is pure for analyzing; Ammonium formate, ethyl acetate, methyl alcohol are chromatographically pure (TEDIA company of the U.S.); Oasis MCX solid-phase extraction column (6 mL, Waters, US).
3-ethyl adenine, purchased from Canadian TRC company, marks purchased from American CIL laboratory in deuterated-3-ethyl adenine.
2. sample preparation:
Urine is thawed and is mixed under room temperature state, gets 4 mL urines, adds 4mL ultrapure water, adds mark (100ng/mL) in 100 μ L, regulates temperature to 80 DEG C with water-bath.The urine of getting after ultrapure water dilution is placed in 10 mL solid-phase extraction columns.Solid-Phase Extraction chooses Waters Oasis MCX(6 mL, 500 mg) pillar, use the methyl alcohol of 2 mL in advance, the water activation pillar of 5 mL, applied sample amount is 8 mL, with the methyl alcohol drip washing of 10% of 2 mL, then 1 mL ethyl acetate solution drip washing, then uses 1 mL ethyl acetate drip washing, finally use methanol/ethyl acetate/ammoniacal liquor (47.5:47.5:5) wash-out, collect eluent and blow to dry with nitrogen at 60 DEG C, then using 100 μ L aqueous dissolution, for LC-MS/MS compartment analysis.
3. assay method: draw 0.1,0.2,0.5,1,1.5,2,2.5,3, the standard solution of 3.5 and 5 ng/mL and each 3 μ L of sample after purifying, inject LC-MS/MS system and carry out compartment analysis, record the content of 3-ethyl adenine in sample (3-EtA).
Example 2: as described in Example 1, the method utilizing this research to set up detects 3-ethyl adenine (3-EtA) content in 20 urines, and in urine, the content of 3-ethyl adenine (3-EtA) is in table 4.
The content of 3-EtA in table 4 urine
Claims (3)
1. the assay method of 3-ethyl adenine (3-EtA) in urine, is characterized in that: comprise following concrete steps:
A, sample pre-treatments: urine is thawed and mixed under room temperature state, get 4 mL urines, add 4mL ultrapure water, adds mark in 100 μ L, regulates temperature to 75-85 DEG C with water-bath, get ultrapure water be diluted to 8 mL after urine be placed in solid-phase extraction column;
Solid-Phase Extraction Waters Oasis MCX solid-phase extraction column, use 2 mL methyl alcohol, 5 mL activated in water solution in advance, applied sample amount is 8 mL, with 2 mL 10% methanol/water solution drip washing, then use 1 mL ethyl acetate drip washing, finally use methanol/ethyl acetate/ammonia water mixture wash-out, collect eluent and blow to dry with nitrogen at 60 DEG C, then 100 μ L aqueous dissolution are used, for LC-MS/MS compartment analysis;
The preparation of b, standard working solution: with 3-ethyl adenine (3-EtA) standard working solution of acetontrile variable concentrations;
C, liquid chromatography-tandem mass spectrometry (LC-MS/MS) measure, draw the standard working solution of the 3-ethyl adenine (3-EtA) of the variable concentrations prepared, inject LC-MS/MS system, obtain equation of linear regression, sample liquid to be measured is measured, record the ratio analyzing thing and interior mark peak area, substitute into unary linear regression equation, try to achieve the content analyzing thing in sample liquid to be measured; Choose Waters HILIC chromatographic column, specification 150 mm x 2.1 mm, 2.5 μm, flow visualizing and elution requirement as follows: mobile phase A: acetonitrile solution, pH 4.0, Mobile phase B: 10m mol/L ammonium formate/aqueous solution, pH 4.0, A:B is 95:5, flow velocity 0.25 mL/min; Column temperature 25 DEG C; Sample size 3 μ L, elution time: 20 min; Mass Spectrometry Conditions: electric spray ion source, positive ion scan mode; Spray voltage is 5500 V; Atomization gas pressure is 344.5 kPa; Gas curtain atmospheric pressure is 206.7 kPa; Assisted atomization atmospheric pressure is 310.0 kPa; Ion source temperature is 500 DEG C; A small bundle of straw, etc. for silkworms to spin cocoons on voltage is gone to be 85eV; Impact energy is 26eV; Residence time is 40ms, and monitoring ion pair is 3-EtA:164 → 136 quota ions pair, 164 → 119 qualitative ion pairs; Interior mark d
5-3-EtA is 169 → 137.
2. the assay method of 3-ethyl adenine (3-EtA) in urine according to claim 1, it is characterized in that: be designated as deuterated-3-ethyl adenine in described, interior mark concentration is 100 ng/mL.
3. the assay method of 3-ethyl adenine (3-EtA) in urine according to claim 1, is characterized in that: the concrete proportioning of described methanol/ethyl acetate/ammonia water mixture is 47.5:47.5:5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410095001.9A CN103852547B (en) | 2014-03-14 | 2014-03-14 | Detecting method for 3-ethyl adenine (3-HOEtA) in urine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410095001.9A CN103852547B (en) | 2014-03-14 | 2014-03-14 | Detecting method for 3-ethyl adenine (3-HOEtA) in urine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103852547A CN103852547A (en) | 2014-06-11 |
| CN103852547B true CN103852547B (en) | 2015-02-11 |
Family
ID=50860455
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410095001.9A Active CN103852547B (en) | 2014-03-14 | 2014-03-14 | Detecting method for 3-ethyl adenine (3-HOEtA) in urine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103852547B (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993016198A1 (en) * | 1992-02-06 | 1993-08-19 | Albert Einstein College Of Medicine Of Yeshiva University | Method of measuring dna adducts |
| CN101706500A (en) * | 2009-11-06 | 2010-05-12 | 中国科学院生态环境研究中心 | Method for analyzing quantum dot-enhanced high-sensitivity DNA adduct |
| TW201335376A (en) * | 2012-02-29 | 2013-09-01 | Nat Univ Chung Cheng | Method for detecting DNA adducts in saliva |
-
2014
- 2014-03-14 CN CN201410095001.9A patent/CN103852547B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103852547A (en) | 2014-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103063791A (en) | Method of simultaneously determining contents of 1-OHP ,3-OHB[a]P and 3-OHB[a]A in urine | |
| CN104807906A (en) | Method for detecting piperazine residue in poultry with high efficiency | |
| CN102680565B (en) | Analysis method for quickly detecting urine creatinine by isotopic dilution, extractive electrospray ionization (EESI) and tandem mass spectrometry (MS/MS) | |
| CN102980968A (en) | Liquid chromatogram tandem mass spectrum measuring method for creatinine in urine | |
| CN110824077A (en) | System and method for measuring NNK and metabolites thereof by liquid-liquid two-dimensional chromatography based on solid-phase extraction | |
| CN103175921B (en) | Method for analyzing four benzene and toluene metabolites in urine through liquid chromatography-tandem mass spectrometry | |
| CN105424853A (en) | LC-MS/MS kit for detecting nicotine and its metabolites in saliva | |
| CN102944635B (en) | Method for determining tris (2,3-dibromopropyl) phosphate content of water | |
| CN103823010B (en) | Method for measuring 3-methyl adenine (3-MeA) in urine | |
| CN103336080A (en) | Method for simultaneously detecting tetracycline antibiotics in water | |
| CN103808836B (en) | Determination method of 3-alkylated adenine DNA adducts in urine | |
| CN103852547B (en) | Detecting method for 3-ethyl adenine (3-HOEtA) in urine | |
| CN103852548B (en) | Detecting method for 3-hydroxyethyl adenine (3-HOEtA) in urine | |
| CN103823009B (en) | Application of liquid chromatography tandem mass spectrogram method in biological monitoring of 3-alkylation adenine in mass smokers | |
| CN113030343B (en) | Liquid chromatography tandem mass spectrometry detection method for pyrroloquinoline quinone in blood plasma | |
| KR101159064B1 (en) | Quantification of active androgens in urine and blood by isotope dilution-mass spectrometry | |
| CN103822988B (en) | Application of LC (liquid chromatogram) tandem mass spectrum method in biological monitoring of N3-(2-hydroxyethyl)-adenine in large-scale smoker crowd samples | |
| CN103822991B (en) | Application of liquid chromatography tandem mass spectrometry method in biological monitoring on 3-ethyl adenines among samples of large-scale smokers | |
| CN103869037B (en) | Application of liquid chromatography-tandem mass spectrometry to biological monitoring of 3-methyladenine in large-scale smoker samples | |
| CN105628847A (en) | Method for measuring acetaldehyde-DNA adducts in saliva | |
| CN111157666A (en) | Method for simultaneously and quantitatively analyzing sulfite and sulfate ions in amine solution | |
| CN103512991A (en) | Automatic on-line analysis method for acrylamide mercapt uric acid metabolites in urine | |
| CN109324140A (en) | Ribosylzeatin Solid Phase Extraction-liquid chromatography-tandem mass spectrometry measuring method in a kind of tobacco leaf | |
| CN109324139A (en) | Ribosylzeatin liquid-liquid extraction-liquid chromatography-tandem mass spectrometry measuring method in a kind of tobacco leaf | |
| CN105628848A (en) | Method for measuring six aldehyde-DNA adducts in saliva |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |