CN105938124A - Determination method of formaldehyde-DNA adducts in saliva - Google Patents
Determination method of formaldehyde-DNA adducts in saliva Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
Provided is a determination method of formaldehyde-DNA adducts in saliva, namely the determination method of HOMe-dA and HOMe-dG in the saliva; the determination method includes the steps: collecting the saliva by using an OG-500 saliva collection tube, carrying out DNA extraction on the saliva, carrying out enzyme hydrolysis of the DNA solution, and successively adding a stable isotope internal standard, a reducing agent NaBH3CN and a DNA hydrolase; and carrying out solid-phase extraction of the hydrolysate with a Strata-X small column, collecting an eluted liquid, nitrogen-blowing to be dried at room temperature, redissolving in a methanol aqueous solution, introducing into an LC-MS/MS system, analyzing, and accurately detecting the content levels of Me-DA and Me-dG. The stable isotope is used as an internal standard quantitative analysis material, so the error caused by the sample pretreatment process can be reduced, and the selectivity, accuracy and sensitivity of the method can be improved by tandem mass spectrometry. Through selection and optimization of the chromatographic column and the elution conditions, the chromatographic separation process is relatively improved, the time of chromatographic analysis is shortened, and the consumption amount of an organic solvent is decreased.
Description
Technical field
The invention belongs to the physical and chemical inspection technical field of saliva sample, relate generally to N in saliva6-methylol-adenine
And N (HOMe-dA)2The determination techniques field of-methylol-guanine (HOMe-dG) DNA adduct, specifically one uses
Liquid chromatography-tandem mass spectrometry instrument (LC-MS/MS) measures the method for formaldehyde-DNA adduct in saliva.
Background technology
Formaldehyde is a kind of pollutant being widely present in environmental matrices, such as commercial production, motor-vehicle tail-gas and indoor dress
Pool etc.；Formaldehyde has human inheritance's toxicity, is classified as a class carcinogen by international cancer research institution (IARC).Active aldehyde radical can
With the nucleophilic group on attack DNA, form DNA adduct, wherein N6-methylol-adenine (HOMe-dA) and N2-methylol-
Guanine (HOMe-dG) is main formaldehyde-DNA adduct.Both version is unstable under monokaryon glycosides state, with also
Former dose is converted into N6-methyl-adenine (Me-dA) and N2-methyl-guanine (Me-dG) just can be detected.
Saliva is the sample collection mode of a kind of non-intrusion type, experimenter does not impact sample and easily obtains, be ready-made
Available DNA source.Oral cavity is the target organ that the harmful components such as flue gas directly expose, and correlational study shows the damage of DNA in saliva
Hinder relevant to oral cancer.In saliva, more rich cell type is mouth epithelial cells and leukocyte, because its life cycle is shorter,
In saliva, the content of DNA adduct more can show carcinogenic recent exposure, and saliva is carcinogenic at detection Medicated cigarette and food
The DNA adduct aspect of produce life has great application prospect.
Currently, with respect to DNA adduct analyzing detecting method report more, mainly have32After P-, labelling technique, enzyme connection are exempted from
Epidemic disease technology (ELISA), high performance liquid chromatography (HPLC-UV) and liquid chromatography-tandem mass spectrometry technology (LC-MS/MS).Wherein
LC-MS/MS is widely used in the detection analysis of DNA adduct due to its higher sensitivity and selectivity.At present, saliva
There is not been reported to analyze method while middle HOMe-dA and HOMe-dG.
Summary of the invention
The purpose of the present invention is based on above-mentioned prior art situation and uses liquid chromatography-tandem mass spectrometry (LC-MS/MS)
The assay method of formaldehyde-DNA adduct (HOMe-dA, HOMe-dG) in the saliva that technology is set up.The method has quick, accurate
Really, sensitivity high, can be used for the qualitative and quantitative analysis of formaldehyde-DNA adduct in saliva.
It is an object of the invention to be achieved through the following technical solutions:
The assay method of the assay method of formaldehyde-DNA adduct, i.e. HOMe-dA, HOMe-dG in a kind of saliva, including following tool
Body step:
A, saliva gather: be standardized gathering to human saliva with Oragene DNA spittle collector (OG-500).
B, the extraction of saliva DNA: Oragene saliva mixed liquor, after at least 1h is hatched in 50 DEG C of water-baths, adds Oragene
Scavenging solution, vortex oscillation, ice bath hatches 10 min；It is centrifuged at room temperature；Pipette the supernatant in a new microcentrifugal tube,
Add straight alcohol, shake the several seconds gently；Standing makes DNA molecular precipitate completely, is centrifuged and removes supernatant；Add the ethanol water of 70%
Solution cleans DNA agglomerate.DNA is redissolved with ultra-pure water.Detect at 260nm with NanoDrop 2000 ultramicrospectrophotometer
The content of DNA, is equivalent to 50 μ g/mL for one absorbance units (OD) of double-stranded DNA.
The enzyme hydrolysis of c, DNA and purification enrichment: above-mentioned DNA is dissolved in the 10 mM Tris-HCl/5 mM of 500 μ L
MgCl2In buffer (pH=7), add 30 mg NaBH at DNA solution3CN, 20 μ L mixing internal standard and 30U DNA (deoxyribonucleic acid)
Enzyme I, hatches 2h in 37 DEG C, is subsequently added 0.008U Phosphodiesterase I and 15U alkali phosphatase continues to hatch in 37 DEG C
2h.Hydrolyzed solution, through Strata-X Solid-Phase Extraction, is collected eluent liquid nitrogen and is blown to do, redissolve in 100 μ L 30% methanol aqueous solutions,
It is sample liquid to be measured.The purpose of this process is to make double-stranded DNA enzymolysis become mononucleotide state, by Solid-Phase Extraction and nitrogen
Blow the DNA adduct concentrated and purified needed for separating and being enriched with.Be designated as in described mixing 50 ng/mL [15N5] Me-dA and 10
Ng/mL [13C10 15N5]Me-dG。
D, the preparation of standard working solution: prepare Me-dA and the Me-dG standard working solution of variable concentrations with methanol respectively.
E, liquid chromatography-tandem mass spectrometry (LC-MS/MS) measure, and draw the variable concentrations hybrid standard work prepared molten
Liquid, injects LC-MS/MS system, obtains equation of linear regression, and liquid to be measured to sample is measured, and records analyte and internal standard peak
The ratio of area, substitutes into unary linear regression equation, tries to achieve the content of analyte in sample liquid to be measured.
Chromatographic condition: choose Thermo AcclaimTMPolar Advantage II C18 chromatographic column (4.6 × 150
Mm, 3 μm), flow visualizing chooses A:0.01% formic acid methanol and B:10 mM ammonium acetate solution, and elution requirement is that gradient is washed
De-: 0-2 min:25% A, 2-17 min:35% A, 17-25 min:25% A；Flow velocity is 0.38 mL/min.Analysis time is
25 min, sample size is 5 μ L.
Mass Spectrometry Conditions: electric spray ion source (ESI), multiple-reaction monitoring cation scan mode；Spray voltage: 5500 V,
Ion source temperature: 550 DEG C, the amount of gas curtain gas is 20 psi, and atomization gas is 70 psi, and dry gas is 75 psi, and collision gas is 9
Psi, injects voltage: 10 V, impact energy: 9 eV, residence time: 100 ms.Monitoring ion pair be Me-dA:266.1 →
150.2 quota ions pair, 266.1 → 117.2 qualitative ion pairs；Internal standard [15N5] Me-dA is 271.2 → 155.2；Me-dG:
282.2 → 166.2 quota ions pair, 282.2 → 117.1 qualitative ion pairs；Internal standard [13C10 15N5] Me-dG is 297.2 →
176.2。
Each analyte and interior target MRM parameter are shown in Table 1.Whole analysis process is by Applied Biosystems Analyst
Version 1.5.1 software controls.
Analyte and interior target MRM parameter thereof under table 1 multiple-reaction monitoring pattern
* it is quota ion
The range of linearity of the inventive method and detection limit:
Series standard working solution is injected LC-MS/MS, obtains standard working curve, equation of linear regression and correlation coefficient.
The point of Me-dA standard curve is 0.02,0.05,0.5,1.0,2.0,5.0,10,20 and 50 ng/mL, Me-dG standard curve
Point is 0.02,0.05,0.1,0.2,0.3,0.5,1.0 and 2.0 ng/mL, object linear good, and correlation coefficient is all higher than
0.9991.Utilizing mark-on dilution to obtain quantitative limit and the detection limit of method, concentration corresponding to ten times of signal to noise ratios of target peak is quantitative
Limit, concentration corresponding to three times of signal to noise ratios is detection limit.The standard curve of analyte and quantitative limit and detection limit are shown in Table 2.
The standard working curve of table 2 object and LOD and LOQ
The inventive method recovery of standard addition and repeatability:
Take saliva DNA sample adds the recovery of standard addition of calibration method preparation method, altogether have chosen three pitch-based sphere, often
Individual pitch-based sphere replication 3 times, the response rate of the method obtained and repeatability, the results are shown in Table 3.The response rate of object exists
Between 78.4%-109.0%, RSD is less than 5%, it was demonstrated that the accuracy of method and repeatability result are preferable.
The recovery of standard addition of table 3 analyte and repeatability
The method of the present invention overcomes the deficiency of prior art sample treatment, for the color of formaldehyde-DNA adduct in saliva
Spectral condition is optimized, and is optimized the coherent detection condition of LC-MS/MS.Side the most of the present invention
Method has a following excellent results:
1. compare with traditional liquid-phase chromatography method, owing to have chosen tandem mass spectrum so that selectivity and the sensitivity of method carry
Height, is more beneficial for the mensuration of the formaldehyde-DNA adduct of low content in saliva.
2. this method has easy and simple to handle, quick, accurate, highly sensitive and reproducible advantage.
3. the inventive method uses stable isotope can reduce in sample pretreatment process as uantitative analytical thing
The error caused, tandem mass spectrometry preferably improves selectivity and accuracy and the sensitivity of method.By chromatographic column and
The selection and optimization of elution requirement, preferably improves chromatographic separation process, shortens the time of chromatography, decreases organic
The consumption of solvent.
Accompanying drawing explanation
The total ion current figure of Fig. 1 formaldehyde-DNA adduct.
Detailed description of the invention
The present invention is described further below in conjunction with example, but is not to limit the present invention.
The assay method of formaldehyde-DNA adduct in a kind of saliva, its test process is to collect with OG-500 saliva collection tube
Saliva, carries out DNA extraction to saliva, and DNA solution carries out enzyme hydrolysis and is sequentially added into stable isotope internal standard, reducing agent
NaBH3CN and DNA hydrolytic enzyme.Hydrolyzed solution, through the little column solid phase extraction of Strata-X, collects eluent, and under room temperature, nitrogen is blown to do, multiple
It is dissolved in 30% methanol aqueous solution of 100 μ L, introduces LC-MS/MS system and be analyzed.
Example 1:
1. instrument and reagent:
AB SCIEX triple quadrupole rods tandem mass spectrometry instrument (California, USA Applied Biosystems, Inc.), Agilent 1200 is efficient
Liquid chromatograph (Agilent company of the U.S.), NanoDrop 2000 ultramicrospectrophotometer (Sai Mofei scientific & technical corporation of the U.S.), permanent
Temperature incubator (Sai Mofei scientific & technical corporation of the U.S.), nitrogen concentrate drying instrument (Bai Taiqi company of Sweden), Milli-Q water purification system
(Merck KGaA group), Analyst 1.5.1 data acquisition and procession software.
Deoxyribonuclease Ⅰ, alkali phosphatase (U.S.'s knob Great Britain biotech company), Phosphodiesterase I, acetic acid
Ammonium, formic acid and calf thymus DNA (Sigma of the U.S.), NaBH3CN(Shanghai Aladdin Reagent Company), methanol (Korea S's moral
Mountain Reagent Company), Strata-X polymer posts (33 μm, 30 mg/1mL, Guangdong Féraud door scientific instrument company limited),
Oragene DNA spittle collector (OG-500, DNA Genotek company of Canada).Standard substance Me-dA, Me-dG and internal standard
Thing [15N5]Me-dA、[13C10 15N5]Me-dG.Reagent is chromatographically pure.
2. sample treatment:
Saliva gathers: be standardized gathering to human saliva with Oragene DNA spittle collector (OG-500).
The extraction of saliva DNA: Oragene saliva mixed liquor, after at least 1h is hatched in 50 DEG C of water-baths, adds Oragene clean
Changing liquid, vortex oscillation, ice bath hatches 10 min；It is centrifuged at room temperature；Pipette the supernatant in a new microcentrifugal tube, add
Enter straight alcohol, shake the several seconds gently；Standing makes DNA molecular precipitate completely, is centrifuged and removes supernatant；The ethanol of addition 70% is water-soluble
Liquid cleans DNA agglomerate.DNA is redissolved with ultra-pure water.At 260nm, DNA is detected with NanoDrop 2000 ultramicrospectrophotometer
Content, 50 μ g/mL are equivalent to for one absorbance units of double-stranded DNA.
The enzyme hydrolysis of DNA and purification enrichment: above-mentioned DNA is dissolved in the 10 mM Tris-HCl/5 mM MgCl of 500 μ L2
In buffer (pH=7), add 30 mg NaBH at DNA solution3CN, 20 μ L mixing internal standard and 30U deoxyribonuclease Ⅰs,
In 37 DEG C, hatch 2h, be subsequently added 0.008U Phosphodiesterase I and 15U alkali phosphatase continues to hatch 2h in 37 DEG C.Water
Solution liquid, through Strata-X Solid-Phase Extraction, is collected eluent liquid nitrogen and is blown to do, redissolve in 100 μ L 30% methanol aqueous solutions as treating
Survey liquid.Described mixing internal standard 50 ng/mL [15N5] Me-dA and 10 ng/mL [13C10 15N5]Me-dG。
3. assay method: each 5 L of saliva DNA sample after the standard solution of absorption variable concentrations and pre-treatment, injects
LC-MS/MS system carries out separating to be analyzed, and records the content of formaldehyde-DNA adduct in sample.
Example 2: as described in Example 1, utilizes the method that this research is set up to formaldehyde-DNA adduct in 5 saliva samples
Carry out detecting (each sample Parallel testing three times).Establishing criteria curve and calculated by peak area obtain DNA in each sample and add
The concentration of compound, according to formula
In calculating each sample of acquisition, DNA adduct is relative to the content of nucleotide, and result is multiplied by 108Be converted into DNA adduct number/
108Individual nucleotide.Experimental result is shown in Table 4.
Table 4 analyte content in actual saliva sample
Claims (5)
1. N in an assay method for formaldehyde-DNA adduct, i.e. saliva in saliva6-methylol-adenine (HOMe-dA) and
N2The assay method of-methylol-guanine (HOMe-dG), it is characterised in that: include step in detail below:
A, saliva gather: be standardized gathering to human saliva with Oragene DNA spittle collector (OG-500)；
B, the extraction of saliva DNA: Oragene saliva mixed liquor, after at least 1h is hatched in 50 DEG C of water-baths, adds Oragene and purifies
Liquid, vortex oscillation, ice bath hatches 10 min；It is centrifuged at room temperature；Pipette supernatant in a new microcentrifugal tube, add pure
Ethanol, shakes the several seconds gently；Standing makes DNA molecular precipitate completely, is centrifuged and removes supernatant；The ethanol water of addition 70% is clear
Wash DNA agglomerate；With ultra-pure water redissolution DNA and at 260nm, detect DNA's with NanoDrop 2000 ultramicrospectrophotometer
Content, is equivalent to 50 μ g/mL for one absorbance units (OD) of double-stranded DNA；
The enzyme hydrolysis of c, DNA and purification enrichment: above-mentioned DNA is dissolved in the 10 mM Tris-HCl/5 mM MgCl of 500 μ L2Buffering
In liquid, add 30 mg NaBH at DNA solution3CN, by N6-methylol-adenine (HOMe-dA) and N2-methylol-guanine
(HOMe-dG) it is reduced into N6-methyl-adenine (Me-dA) and N2-methyl-guanine (Me-dG) detects, and adds 20 μ L
Mixing internal standard, adds 30U deoxyribonuclease Ⅰ, hatches 2h, be subsequently added 0.008U Phosphodiesterase I and 15U in 37 DEG C
Alkali phosphatase continues to hatch 2h in 37 DEG C；Hydrolyzed solution, through Strata-X Solid-Phase Extraction, is collected eluent liquid nitrogen and is blown to do, multiple
It is dissolved in 100 μ L 30% methanol aqueous solutions, is sample liquid to be measured；
D, the preparation of standard working solution: with methanol preparation variable concentrations Me-dA and Me-dG standard working solution；
E, liquid chromatography-tandem mass spectrometry (LC-MS/MS) measure, and draw the variable concentrations hybrid standard working solution prepared, note
Entering LC-MS/MS system, obtain equation of linear regression, liquid to be measured to sample is measured, and records analyte and internal standard peak area
Ratio, substitutes into unary linear regression equation, tries to achieve the content of analyte in sample liquid to be measured.
The assay method of formaldehyde-DNA adduct in saliva the most according to claim 1, it is characterised in that: institute in step c
Be designated as in the mixing stated 50 ng/mL [15N5] Me-dA and 10 ng/mL [13C10 15N5]Me-dG。
The assay method of formaldehyde-DNA adduct in saliva the most according to claim 1, it is characterised in that: step e is selected
Take Thermo AcclaimTMPolar Advantage II C18 chromatographic column, specification 4.6 × 150 mm, 3 μm, flow phase
System chooses A:0.01% formic acid methanol, B:10 mM ammonium acetate solution, gradient elution, and analysis time is 25 min, sample size
It is 5 μ L.
The assay method of formaldehyde-DNA adduct in saliva the most according to claim 3, it is characterised in that: gradient elution journey
Sequence: 0-2 min:25% A, 2-17 min:35% A, 17-25 min:25% A；Flow velocity is 0.38 mL/min.
The assay method of formaldehyde-DNA adduct in saliva the most according to claim 1, it is characterised in that: step e is gone here and there
The condition of connection mass detector: electric spray ion source ESI, multiple-reaction monitoring cation scan mode；Spray voltage: 5500 V,
Ion source temperature: 550 DEG C, the amount of gas curtain gas is 20 psi, and atomization gas is 70 psi, and dry gas is 75 psi, and collision gas is 9
Psi, injects voltage: 10 V, impact energy: 9 eV, residence time: 100 ms；Monitoring ion pair be Me-dA:266.1 →
150.2 quota ions pair, 266.1 → 117.2 qualitative ion pairs；Internal standard [15N5] Me-dA is 271.2 → 155.2；Me-dG:
282.2 → 166.2 quota ions pair, 282.2 → 117.1 qualitative ion pairs；Internal standard [13C10 15N5] Me-dG is 297.2 →
176.2。
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Cited By (1)
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| WO2020102808A3 (en) * | 2018-11-16 | 2020-09-24 | Decimadx, Llc | Oral wash for oral fluid collection and analysis and methods of use thereof |
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| US20140017672A1 (en) * | 2007-10-31 | 2014-01-16 | Rebecca HOLMBERG | Method and kit for purifying nucleic acids |
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| WO2020102808A3 (en) * | 2018-11-16 | 2020-09-24 | Decimadx, Llc | Oral wash for oral fluid collection and analysis and methods of use thereof |
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