CN105527368A - Method for detecting 8-hydroxydeoxyguanosine and 8-hydroxyguanosine in urine by high-performance liquid chromatography tandem mass spectrometry technology - Google Patents
Method for detecting 8-hydroxydeoxyguanosine and 8-hydroxyguanosine in urine by high-performance liquid chromatography tandem mass spectrometry technology Download PDFInfo
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Abstract
The invention discloses a method for detecting 8-hydroxydeoxyguanosine and 8-hydroxyguanosine in urine by a high-performance liquid chromatography tandem mass spectrometry technology. According to the method, 8-hydroxydeoxyguanosine and 8-hydroxyguanosine in a standard solution and a pretreated urine sample are separately detected by adopting the high-performance liquid chromatography tandem mass spectrometry technology, 8-hydroxydeoxyguanosine and 8-hydroxyguanosine are separated firstly by using a high-performance liquid chromatography technology, and then, a calibration curve is established by using a tandem mass spectrometry isotopic internal-standard quantitative method in a manner of taking a concentration ratio of a standard to an internal standard as an X axis and taking a peak area ratio of the standard to the internal standard as a Y axis, so as to figure out the content of 8-hydroxydeoxyguanosine and the content of 8-hydroxyguanosine. Compared with the prior art, the method disclosed by the invention is efficient and stable, is good in recovery ratio and stability and has the advantages of accuracy, simplicity, convenience, economical efficiency, high efficiency and the like, thereby being applicable to popularization and application in clinical detection.
Description
Technical field
The invention belongs to biochemistry detection field, be specifically related to the method for 8-OhdG and 8-hydroxyguanosine in a kind of high performance liquid chromatography series connection second order ms technology for detection urine.
Background technology
DNA and RNA oxidative damage stress product as biomarker potential use and to be worth be an importance of biomedical research.8-OhdG and 8-hydroxyguanosine, as two kinds of important oxidation labels, produce, through blood transportation, are excreted by kidney with urine in histocyte.Because two kinds of materials are comparatively stable in vivo, and urine specimen is easy to get, and can realize Non-invasive detection, is offered help by the oxidative damage that can cause for effective evaluation body various factors the monitoring of two kinds of materials in urine.In addition, by the content detection to two kinds of biomarkers in healthy human body, the influence of the latent factors such as sex, age and smoking can be assessed.Therefore, a kind of economy of exploitation, efficiently 8-OhdG and 8-hydroxyguanosine detection method are most important.
Summary of the invention
The technical problem to be solved in the present invention is to provide the method for 8-OhdG and 8-hydroxyguanosine in a kind of high performance liquid chromatography series connection second order ms technology for detection urine, to solve the method complexity that prior art exists, the problem that specificity is poor and sensitivity is low.
For solving the problems of the technologies described above, the technical scheme that the present invention will solve is as follows:
A kind of method of 8-OhdG and 8-hydroxyguanosine in high performance liquid chromatography series connection second order ms technology for detection urine, its adopts high performance liquid chromatography series connection second order ms technology examination criteria product solution and the 8-OhdG in pretreated urine sample and 8-hydroxyguanosine respectively, high-efficient liquid phase chromatogram technology is first utilized to be separated with 8-hydroxyguanosine by 8-OhdG, recycling second order ms Isotopic Internal Standard sizing technique, with the concentration ratio of standard items and internal standard compound for X-axis, the peak area ratio of standard items and internal standard compound is Y-axis, set up calibration curve, calculate the content of 8-OhdG and 8-hydroxyguanosine, concrete chromatographic condition is:
(1) high-efficient liquid phase chromatogram condition:
The aqueous formic acid of mobile phase A: 0.1%v/v;
Mobile phase B: methyl alcohol;
Chromatographic column is UltimatePolar-RP, 2.1 × 100mm, 3 μm;
Flow velocity is 0.4mL/min;
Column temperature is 30 DEG C;
Sample size is 10 μ L;
Adopt gradient elution mode, in table 1;
Table 1 liquid phase Parameters of gradient elution
(2) Mass Spectrometry Conditions:
Under electron spray ionisation positive ion detecting pattern, adopt the scanning of the mass spectrum pattern of multiple-reaction monitoring; Spray needle voltage is 5500V; Collision gas is 5psi; Gas curtain gas is 50psi; Ion gun atomization gas and heating assisted gas are 65psi; Desolventizing temperature degree is 550 DEG C.
Wherein, described standard solution is prepared as follows and obtains:
The 8-OhdG of difference precise 5mg and 8-hydroxyguanosine, dissolve and be settled to 10mL, be mixed with storing solution A and B that concentration is respectively 0.48mg/mL and 0.485mg/mL ,-20 DEG C of preservations in methyl alcohol; Get after each 100 μ L of storing solution A and B mix and be settled to 10mL with after methanol dilution, being mixed with 8-OhdG concentration is 4.8 μ g/mL, and 8-hydroxyguanosine concentration is the hybrid standard liquid C1 of 4.85 μ g/mL ,-20 DEG C of preservations; Get 100 μ L hybrid standard liquid C1 and be settled to 5mL with after blank artificial urine diluted matrix, to obtain 8-OhdG concentration be 96ng/mL, 8-hydroxyguanosine concentration is the hybrid standard liquid C2 of 97ng/mL in preparation; Get 500 μ L hybrid standard liquid C2 and be settled to 5mL with after blank artificial urine diluted matrix, to obtain 8-OhdG concentration be 9.6ng/mL, 8-hydroxyguanosine concentration is the hybrid standard liquid C3 of 9.7ng/mL in preparation;
Precise 5mg [
13c,
15n
2]-8-OhdG, obtain the internal standard compound storing solution that concentration is 49.55 μ g/mL after being settled to 10mL with methyl alcohol dissolving, internal standard compound storing solution obtains internal standard compound working fluid D through methanol dilution to 0.991 μ g/mL; Internal standard compound storing solution and internal standard compound working fluid D all preserve at-20 DEG C;
Getting 25 μ l, 50 μ L and 100 μ L hybrid standard liquid C2 respectively with blank artificial urine diluted matrix is settled to 100 μ L as standard calibration point S5, S6 and S7; Getting 5 μ L, 10 μ L, 50 μ L and 100 μ L hybrid standard liquid C3 respectively with blank artificial urine diluted matrix is settled to 100 μ L, obtains standard calibration point S1, S2, S3 and S4; These 7 concentration point volumes are 100 μ L; Then in each group of standard calibration point, add 15 μ L0.991 μ g/mL internal standard compound working fluid D, mix rear methanol constant volume to 0.5mL, then vortex vibrates after 20 seconds 10min, gets 10 μ L supernatants, obtain standard solution at 4 DEG C after the centrifugal 5min of 12000g;
Wherein,
S1, S2, S3, S4, S5, S6 and S7 are as the calibration point of 8-OhdG, and S2, S3, S4, S5, S6 and S7 are as the calibration point of 8-hydroxyguanosine.
Wherein, described blank artificial urine matrix is the simulation urine of laboratory human configuration; Wherein, described blank artificial urine matrix prepares by the following method:
Get 0.5586g CALCIUM CHLORIDE DIHYDRATE, 0.6099g Magnesium dichloride hexahydrate, 4.2077g sodium chloride, 2.0596g sodium sulphate, 0.6470g Trisodium citrate dihydrate, 0.0201g sodium oxalate, 2.5449g potassium dihydrogen phosphate, 1.4388g potassium chloride, 0.9200g ammonium chloride, 2.4985g urea and 1.0520g creatinine deionized water dissolving respectively and be settled to 1L.
Wherein, the preprocess method of urine sample is: get 100 μ L urines in 1.5mL centrifuge tube, mixes after adding 15 μ L internal standard compound working fluid D, again by methanol constant volume to 0.5mL, vortex oscillation 1min, the centrifugal 5min of 12000g at 4 DEG C, get 10 μ L supernatants.
Wherein, precise 5mg [
13c,
15n
2]-8-OhdG, obtain the internal standard compound storing solution that concentration is 49.55 μ g/mL after being settled to 10mL with methyl alcohol dissolving, internal standard compound storing solution obtains internal standard compound working fluid D through methanol dilution to 0.991 μ g/mL; Internal standard compound storing solution and internal standard compound working fluid D all preserve at-20 DEG C.
Beneficial effect:
Compared with prior art, the inventive method efficient stable, the recovery and have good stability that (withinrun precision is between 3.63% ~ 6.39%, betweenrun precision is between 3.88% ~ 9.35%, batch in and batch between RSD all within 10%, average recovery rate is between 91.90% ~ 116.76%), there is the advantages such as accurate, easy, economic, efficient, be suitable for applying in clinical detection.
Accompanying drawing explanation
Fig. 1 a is the Selective ion mode flow chromatography figure that in standard items, 8-OhdG quota ion is right;
Fig. 1 b is the Selective ion mode flow chromatography figure of the qualitative ion pair of 8-OhdG in standard items;
Fig. 1 c is the Selective ion mode flow chromatography figure that in standard items, 8-hydroxyguanosine quota ion is right;
Fig. 1 d is the Selective ion mode flow chromatography figure of the qualitative ion pair of 8-hydroxyguanosine in standard items;
Fig. 1 e be in standard items [
13c,
15n
2the Selective ion mode flow chromatography figure of]-8-OhdG;
Fig. 1 f be in standard items [
13c,
15n
2the Selective ion mode flow chromatography figure of]-8-OhdG;
Fig. 2 a is the Selective ion mode flow chromatography figure that in urine sample, 8-OhdG quota ion is right;
Fig. 2 b is the Selective ion mode flow chromatography figure of the qualitative ion pair of 8-OhdG in urine sample;
Fig. 2 c is the Selective ion mode flow chromatography figure that in urine sample, 8-hydroxyguanosine quota ion is right;
Fig. 2 d is the Selective ion mode flow chromatography figure of the qualitative ion pair of 8-hydroxyguanosine in urine sample;
Fig. 2 e be in urine sample [
13c,
15n
2the Selective ion mode flow chromatography figure of]-8-OhdG;
Fig. 2 f be in urine sample [
13c,
15n
2the Selective ion mode flow chromatography figure of]-8-OhdG.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1
1, material
The sample of methodological study experiment comes from the urine specimen of Dean, Shanghai medical test institute employee.
(1) instrument: LC-20AD liquid chromatograph (Shimadzu, Japan), API4000
+triple quadrupole mass spectrometer (ABSciex, the U.S.), excellent general ultrapure water machine (ultrapure, Chengdu), multitube vortex mixed instrument (Hangzhou Ao Sheng Instrument Ltd.); Quick vortex mixer (new health, Jiangsu), high speed freezing centrifuge (Bai Yang, Beijing), chromatography column is UltimatePolar-RP3 μm 2.1 × 100, adjustable pipette (ThermoFisher, 0.5 ~ 10 μ L, 10 ~ 100 μ L, 100 ~ 1000 μ L) and glass apparatus, beaker, graduated cylinder etc.
(2) reagent consumptive material: methyl alcohol, acetonitrile are chromatographically pure, purchased from American world company; Formic acid is chromatographically pure, purchased from Tianjin Zhi Yuan chemical reagent company limited; 1.5mL centrifuge tube (AXYGEN, the U.S.); CALCIUM CHLORIDE DIHYDRATE, Magnesium dichloride hexahydrate, sodium chloride, sodium sulphate, Trisodium citrate dihydrate, sodium oxalate, potassium dihydrogen phosphate, potassium chloride, ammonium chloride, urea, creatinine etc. are all purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
(3) standard items: 8-OhdG (purity 96%), 8-hydroxyguanosine (purity 97%) and Isotopic Internal Standard thing [
13c,
15n
2]-8-OhdG (purity 99.1%) is purchased from Canadian TorontoResearchChemical company.
(4) quality-control product: the blank artificial urine matrix solution containing 8-OhdG and 8-hydroxyguanosine, point high, normal, basic three concentration are respectively QC (L), QC (M), QC (H), are shown in Table 1.
Table 18-hydroxy-Guanine and 8-hydroxyguanosine quality-control product corresponding concentration (unit ng/mL)
2, method
(1) chromatographic condition
Mobile phase A: the aqueous formic acid of 0.1% (v/v); Mobile phase B: acetonitrile.Flow velocity is 0.4mL/min chromatographic column is UltimatePolar-RP, 2.1 × 100mm, 3 μm, column temperature 30 DEG C, and sample size is 10 μ L, and adopt gradient elution mode, design parameter is in table 2.
Table 2 liquid phase Parameters of gradient elution
(2) Mass Spectrometry Conditions: adopt electric spray ion source (ElectrosprayIonization, ESI) and multiple-reaction monitoring (MultipleReactionMonitoring, MRM) pattern to carry out scanning of the mass spectrum.Source parameters: heat air (GS1) and auxiliary heating gas (GS2) are 65psi, and desolventizing temperature degree is 550 DEG C; Gas curtain gas (CurtainGas, CUR) is 50psi, and collision gas (CollisionGas, CAD) is 5psi; Spray needle (Ionspray, IS) voltage is 5500V.Object 8-OhdG quota ion is respectively m/z284.2>168.2 and m/z284.2>140.2 to qualitative ion pair, 8-hydroxyguanosine quota ion is respectively m/z300.1>140.1 and m/z300.1>168.1 to qualitative ion pair, and internal standard compound monitoring ion pair is m/z287.2>171.2 and m/z287.2>143.2.In order to obtain good stability and sensitivity, each compound monitoring ion pair remove a bunch voltage (DeclusteringPotential, DP) and the collision voltage parameter such as (CollisionEnergy, CE) all through system optimization, design parameter is in table 3.
Table 38-hydroxy-Guanine, 8-hydroxyguanosine and internal standard compound mass spectrum multiple-reaction monitoring ion channel parameter
Note:
afor quota ion pair,
bfor qualitative ion pair.
(3) blank artificial urine substrate preparation: get 0.5586g CALCIUM CHLORIDE DIHYDRATE, 0.6099g Magnesium dichloride hexahydrate, 4.2077g sodium chloride, 2.0596g sodium sulphate, 0.6470g Trisodium citrate dihydrate, 0.0201g sodium oxalate, 2.5449g potassium dihydrogen phosphate, 1.4388g potassium chloride, 0.9200g ammonium chloride, 2.4985g urea, 1.0520g creatinine deionized water dissolving be settled to 1L respectively.
(4) standard items preparation: quality is the 8-OhdG of 5mg with methyl alcohol and 8-hydroxyguanosine standard items are settled to 10mL respectively, is mixed with storing solution A, B that concentration is respectively 0.48mg/mL and 0.485mg/mL ,-20 DEG C of preservations.Get each 100 μ L of A, B storing solution and be settled to 10mL after methanol dilution, be mixed with the hybrid standard liquid C1 that concentration is respectively 4.8 μ g/mL and 4.85 μ g/mL ,-20 DEG C of preservations.Get 100 μ L hybrid standard liquid C1 and be settled to 5mL with after blank artificial urine diluted matrix, obtain the hybrid standard liquid C2 that concentration is respectively 96ng/mL and 97ng/mL, then get 500 μ L hybrid standard liquid C2 and be settled to 5mL through blank artificial urine diluted matrix and be mixed with the hybrid standard liquid C3 that concentration is respectively 9.6ng/mL and 9.7 μ g/mL.Be settled to 10mL after dissolving with the internal standard compound that quality is 0.5mg by methyl alcohol, obtain the internal standard compound storing solution that concentration is 49.55 μ g/mL, storing solution is through methanol dilution to 0.991 μ g/mL as internal standard compound working fluid D, and storing solution and working fluid are all in-20 DEG C of preservations.
(5) quality-control product preparation: get 10 μ L, 60 μ L respectively, the blank artificial urine matrix of 120 μ L hybrid standard product C1 is settled to 10mL.
(6) sample preparation
I calibration point process: pipette hybrid standard liquid C225 μ l respectively, the blank artificial urine matrix of 50 μ L, 100 μ L is settled to 100 μ L as standard calibration point S5, S6, S7; Get 5 μ L, 10 μ L respectively, 50 μ L, the blank artificial urine matrix of 100 μ L hybrid standard liquid C3 is settled to 100 μ L, obtain standard calibration point S1, S2, S3, S4, these 7 concentration point volumes are 100 μ L, wherein S1, S2, S3, S4, S5, S6, S7 are as the calibration point of 8-OhdG, and S2, S3, S4, S5, S6, S7 are as the calibration point of 8-hydroxyguanosine.Then respectively add 15 μ L0.991 μ g/mL internal standard compound working fluid D wherein, after mixing by methanol constant volume to 0.5mL, then vortex vibrates after 20 seconds 10min, gets 10 μ L supernatants and enter HPLC-MS/MS and analyze at 4 DEG C after the centrifugal 5min of 12000g.
II urine sample pre-treatment: get in urine 100 μ L to 1.5mL centrifuge tube, adds in 15 μ L and mixes after mark liquid D, then by methanol constant volume to 0.5mL, vortex oscillation 1min, the centrifugal 5min of 12000g at 4 DEG C, get supernatant 10 μ L and enter HPLC-MS/MS analysis.
III quality-control product pre-treatment: get quality-control product solution QC (L) respectively, each 100 μ L of QC (M), QC (H) are in 1.5mL centrifuge tube, and other is consistent with urine sample pre-treating method, repeats no more herein.
3, the Establishment and optimization of method
In order to reach higher stability and sensitivity, experiment adopt MRM pattern monitor simultaneously 8-OhdG, 8-hydroxyguanosine and [
13c,
15n
2the ion pair (Q1/Q3) of]-8-OhdG, use " Ramp " to different ions right remove a bunch voltage (DeclusteringPotential, and collision voltage (CollisionEnergy DP), etc. CE) parameter has carried out system optimization, from optimizing chromatogram the optimum can seen corresponding to ion highest signal strength, record optimization the results are shown in Table 5.In addition, other parameter such as IS, CAD, CUR, GS1 and GS2 etc. select suitable numerical value according to the reference range that instrument provides, to ensure stable signal intensity.
Table 58-hydroxy-Guanine, 8-hydroxyguanosine and [
13c,
15n
2]-8-OhdG mass spectrometry parameters optimum results
4, method validation
(1) Selective ion mode flow graph: 8-OhdG in standard items and urine sample, 8-hydroxyguanosine and [
13c,
15n
2the Selective ion mode flow chromatography figure (XIC) of]-8-OhdG is shown in Fig. 1 and Fig. 2, in standard items, 8-OhdG and 8-hydroxyguanosine concentration are 25ng/mL, and 8-OhdG and 8-hydroxyguanosine concentration are respectively 9.33ng/mL and 12.3ng/mL in urine sample, no matter be that standard items or urine sample all disturb without assorted peak, illustrate that the method condition is practical, can testing requirement be met.
(2) calibration curve: adopt Isotopically labelled internal standard, utilize Analyst software with the concentration ratio of reference material and internal standard compound for X-axis, reference material and internal standard compound peak area ratio are Y-axis, set up calibration curve, then obtain its testing concentration of correspondence and ratio of internal standard compound concentration on calibration curve according to the ratio of object in urine sample to be measured and internal standard compound peak area, the concentration of object in urine to be measured can be calculated in conjunction with known internal standard compound concentration.Analyze things to 2 kinds and carry out linear fit in respective concentration range, the linear equation related coefficient obtained all more than 0.998, linear good (table 6).Meanwhile, kit has also investigated the minimum quantitative limit LLOQ of 2 kinds of target analytes, is not less than 10 and the Cmin point that RSD is less than 20% is minimum quantitative limit LLOQ with signal to noise ratio (S/N ratio); The above RSD of quantitative limit is less than 15%, regression coefficient R
2the least concentration value of >0.98 and maximum concentration value are as the range of linearity of detection method.Parameters all can meet the detection demand of 8-OhdG and 8-hydroxyguanosine in human urine sample.
Table 68-hydroxy-Guanine and the 8-hydroxyguanosine range of linearity, related coefficient and minimum quantitative limit
(3) precision test: get more than 10 urine specimens and mix, doing interpolation recovery and precision checking to mix urine sample for adding matrix, adding standard items (8-OhdG: 4.8ng/mL, 28.8ng/mL and 57.6ng/mL respectively according to high, normal, basic three concentration; 8-hydroxyguanosine: 4.85ng/mL, 29.1ng/mL, 58.2ng/mL), each concentration samples parallel processing 6 times, try to achieve withinrun precision and the recovery, test findings is in table 7.Process each one of high, medium and low concentration samples every day, continuously process 6 days, try to achieve betweenrun precision, test findings is in table 8.
Table 7 withinrun precision test findings
Table 8 betweenrun precision test findings
4, discuss
Current 8-OhdG and the more common detection method of 8-hydroxyguanosine two kinds of biomarkers have high performance liquid chromatography electrochemical (HPLC-ECD), gas chromatography tandem mass spectrometry (GC-MS), liquid chromatography tandem mass spectrometry (LC-MS/MS), euzymelinked immunosorbent assay (ELISA) etc.Wherein euzymelinked immunosorbent assay (ELISA) specificity is poor, and HPLC-ECD method is comparatively complicated, and GC-MS method needs derivatization treatment, and the oxidation existed in derivatization process may cause 8-OhdG content artificially to increase.Compare with above several detection means, LC-MS/MS is less to sample size demand, and sample pre-treatments is comparatively simple, and mass spectroscopy has high specificity, highly sensitive advantage.
Because 8-OhdG (lgP=-1.7, pKa=10.09, water-soluble is 14.8mg/mL) is the stronger alkali compounds of polarity with 8-hydroxyguanosine two kinds of materials.To compound stronger for these polarity be separated more difficult from urine specimen matrix.Conventional liquid-liquid extraction method extraction efficiency is too low, can not meet testing requirement.And the C18 chromatographic column of routine is weak to two kinds of material retention, can not effective separate targets thing, cause matrix interference effect stronger, and there are some researches show 2-deoxyguanosine (2-deoxyguanosine, 2-dG) 8-OhdG can be oxidized in electric spray ion source (ESI) Ionized process, therefore want the content of 8-OhdG in accurate quantitative analysis urine, need 2-deoxyguanosine to come with 8-OhdG chromatographic resolution.
Up to now, the report detecting two kinds of materials about LC-MS/MS method in document adopts SPE solid phase extraction to purify and enrichment urine specimen more.But solid phase extraction complex steps, requires high to the operative skill of operating personnel, wastes time and energy, detect flux low, and the recovery and stability all have larger limitation.In addition, solid phase extraction is large to reagent and consumptive material consumption, and cost is higher, is unfavorable for promotion and application widely.And the existing method C18 chromatographic column of routine that adopts carries out chromatographic resolution to object more, because the anti-phase retention of two kinds of materials is poor, therefore often need to strengthen stick effect by adopting a high proportion of aqueous phase or increasing chromatogram column length, but the aqueous phase at high proportion continued can cause conventional C18 chromatographic column filler collapse phenomenon, affects separating effect.
The present invention develops the chromatography of 8-OhdG and 8-hydroxyguanosine quick pretreatment method and excellence in a kind of human urine just based on the above shortcomings and deficiencies of the prior art part, and carries out quantitative test in conjunction with Quadrupole Mass Spectrometry to two kinds of materials.
The inventive method adopts direct dilution method to detect 8-OhdG and 8-hydroxyguanosine simultaneously, only need 100 μ l urine specimens, sample pretreatment process that need not be complicated, be separated through the Polar-RP chromatographic column that polarity is embedded after Sample Dilution, effectively eliminate the interference in matrix interference and ionization process, and post collapse phenomenon can not be produced, only need 5min can complete first separation and Detection task, more existing method flux improves more, and the recovery and have good stability that (withinrun precision is between 3.63% ~ 6.39%, betweenrun precision is between 3.88% ~ 9.35%, batch in and batch between RSD all within 10%, average recovery rate is between 91.90% ~ 116.76%), have accurately, easy, economical, the advantage such as efficient, be suitable for applying in clinical detection.Although adopt the detection limit of direct dilution method method to raise to some extent, but the 101 routine human urine testing results display 8-OhdGs inspected by random samples according to existing document and this kit batch and the content range of 8-hydroxyguanosine are respectively 0 ~ 20.37ng/mL and 0 ~ 24.99ng/mL, and detection limit of the present invention is respectively 0.24ng/mL and 0.485ng/mL, the testing requirement of two kinds of materials in human urine can be met.
Claims (5)
1. the method for 8-OhdG and 8-hydroxyguanosine in high performance liquid chromatography series connection second order ms technology for detection urine, it is characterized in that, adopt high performance liquid chromatography series connection second order ms technology examination criteria product solution and the 8-OhdG in pretreated urine sample and 8-hydroxyguanosine respectively, high-efficient liquid phase chromatogram technology is first utilized to be separated with 8-hydroxyguanosine by 8-OhdG, recycling second order ms Isotopic Internal Standard sizing technique, with the concentration ratio of standard items and internal standard compound for X-axis, the peak area ratio of standard items and internal standard compound is Y-axis, set up calibration curve, calculate the content of 8-OhdG and 8-hydroxyguanosine, concrete chromatographic condition is:
(1) high-efficient liquid phase chromatogram condition:
The aqueous formic acid of mobile phase A: 0.1%v/v;
Mobile phase B: acetonitrile;
Chromatographic column is UltimatePolar-RP, 2.1 × 100mm, 3 μm;
Flow velocity is 0.4mL/min;
Column temperature is 30 DEG C;
Sample size is 10 μ L;
Adopt gradient elution mode, in table 1;
Table 1 liquid phase Parameters of gradient elution
(2) Mass Spectrometry Conditions:
Under electron spray ionisation positive ion detecting pattern, adopt the scanning of the mass spectrum pattern of multiple-reaction monitoring; Spray needle voltage is 5500V; Collision gas is 5psi; Gas curtain gas is 50psi; Ion gun atomization gas and heating assisted gas are 65psi; Desolventizing temperature degree is 550 DEG C.
2. the method for 8-OhdG and 8-hydroxyguanosine in high performance liquid chromatography according to claim 1 series connection second order ms technology for detection urine, it is characterized in that, described standard solution is prepared as follows and obtains:
The 8-OhdG of difference precise 5mg and 8-hydroxyguanosine, dissolve and be settled to 10mL, be mixed with storing solution A and B that concentration is respectively 0.48mg/mL and 0.485mg/mL ,-20 DEG C of preservations in methyl alcohol; Get after each 100 μ L of storing solution A and B mix and be settled to 10mL with after methanol dilution, being mixed with 8-OhdG concentration is 4.8 μ g/mL, and 8-hydroxyguanosine concentration is the hybrid standard liquid C1 of 4.85 μ g/mL ,-20 DEG C of preservations; Get 100 μ L hybrid standard liquid C1 and be settled to 5mL with after blank artificial urine diluted matrix, to obtain 8-OhdG concentration be 96ng/mL, 8-hydroxyguanosine concentration is the hybrid standard liquid C2 of 97ng/mL in preparation; Get 500 μ L hybrid standard liquid C2 and be settled to 5mL with after blank artificial urine diluted matrix, to obtain 8-OhdG concentration be 9.6ng/mL, 8-hydroxyguanosine concentration is the hybrid standard liquid C3 of 9.7ng/mL in preparation;
Precise 5mg [
13c,
15n
2]-8-OhdG, obtain the internal standard compound storing solution that concentration is 49.55 μ g/mL after being settled to 10mL with methyl alcohol dissolving, internal standard compound storing solution obtains internal standard compound working fluid D through methanol dilution to 0.991 μ g/mL; Internal standard compound storing solution and internal standard compound working fluid D all preserve at-20 DEG C;
Getting 25 μ l, 50 μ L and 100 μ L hybrid standard liquid C2 respectively with blank artificial urine diluted matrix is settled to 100 μ L as standard calibration point S5, S6 and S7; Getting 5 μ L, 10 μ L, 50 μ L and 100 μ L hybrid standard liquid C3 respectively with blank artificial urine diluted matrix is settled to 100 μ L, obtains standard calibration point S1, S2, S3 and S4; These 7 concentration point volumes are 100 μ L; Then in each group of standard calibration point, add 15 μ L0.991 μ g/mL internal standard compound working fluid D, mix rear methanol constant volume to 0.5mL, then vortex vibrates after 20 seconds 10min, gets 10 μ L supernatants, obtain standard solution at 4 DEG C after the centrifugal 5min of 12000g;
Wherein,
S1, S2, S3, S4, S5, S6 and S7 are as the calibration point of 8-OhdG, and S2, S3, S4, S5, S6 and S7 are as the calibration point of 8-hydroxyguanosine.
3. the method for 8-OhdG and 8-hydroxyguanosine in high performance liquid chromatography according to claim 2 series connection second order ms technology for detection urine, it is characterized in that, described blank artificial urine matrix prepares by the following method:
Getting 0.5586g CALCIUM CHLORIDE DIHYDRATE, 0.6099g Magnesium dichloride hexahydrate, 4.2077g sodium chloride, 2.0596g sodium sulphate, 0.6470g Trisodium citrate dihydrate, 0.0201g sodium oxalate, 2.5449g potassium dihydrogen phosphate, 1.4388g potassium chloride, 0.9200g ammonium chloride, 2.4985g urea and 1.0520g creatinine respectively by water-soluble solution is settled to 1L.
4. the method for 8-OhdG and 8-hydroxyguanosine in high performance liquid chromatography according to claim 1 series connection second order ms technology for detection urine, it is characterized in that, the preprocess method of urine sample is: get 100 μ L urines in 1.5mL centrifuge tube, mix after adding 15 μ L internal standard compound working fluid D, again by methanol constant volume to 0.5mL, vortex oscillation 1min, the centrifugal 5min of 12000g at 4 DEG C, get 10 μ L supernatants.
5. the method for 8-OhdG and 8-hydroxyguanosine in high performance liquid chromatography according to claim 4 series connection second order ms technology for detection urine, is characterized in that, precise 5mg [
13c,
15n
2]-8-OhdG, obtain the internal standard compound storing solution that concentration is 49.55 μ g/mL after being settled to 10mL with methyl alcohol dissolving, internal standard compound storing solution obtains internal standard compound working fluid D through methanol dilution to 0.991 μ g/mL; Internal standard compound storing solution and internal standard compound working fluid D all preserve at-20 DEG C.
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