CN105548407A - Detecting method for modified nucleoside in urine - Google Patents
Detecting method for modified nucleoside in urine Download PDFInfo
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Abstract
The invention provides a detecting method for modified nucleoside in urine. The method specifically comprises the following steps: pretreating the urine and storing the pretreated urine on a piece of filter paper, then carrying out extraction to obtain a to-be-tested sample; carrying out liquid chromatography tandem mass spectrometry detection on the to-be-tested sample, so as to obtain the variety and the content of the modified nucleoside. In the detecting method provided by the invention, the modified nucleoside in the urine can be quickly and effectively detected qualitatively and quantitatively by storing the urine on the filter paper, then carrying out extraction and using the liquid chromatography tandem mass spectrometry method; as the urine is stored on the filter paper, and the urine sample is converted into a solid form to be stored, the stability is greatly improved, and the microbial pollution to the detected sample is avoided; furthermore, the method provided by the invention is easier and more convenient than the existing freezing method.
Description
Technical field
The present invention relates to liquid spectrum mass spectrometric hyphenated technique field, the detection method of modified nucleoside in a kind of urine.
Background technology
Modified nucleoside is mainly present in RNA, is to act on polymerized nucleoside molecule and form by specific transmethylase and ligase after transcribing.RNA metabolism all needs specific enzyme to carry out catalysis, degradable or phosphatideization recycling after normal nucleotide enzymolysis, and modified nucleoside be the glycosidic bond of normal nucleotide, base, sugared hydroxyl modified or between two in conjunction with the unique structural of generation, owing to highly being modified, the enzymatic activity that degraded modified nucleoside needs is higher, high specificity, therefore, modified nucleoside discharges from cell, discharges eventually through urine.Therefore the content of modified nucleoside can be used to the nucleoside metabolism situation evaluating human body in urine.
At present, have been reported and adopt capillary electrophoresis to detect modified nucleoside in urine, but the modified nucleoside classification that the method detects is few.And, owing to containing multiple-microorganism in urine.If preserved in liquid form for a long time, microorganism will pollute urine, affect the accuracy of testing result, all freeze preservation by being stored in refrigerator and cooled after urine sampling to be detected in existing detection method, thaw again during detection, not only need refrigerating plant, step of thawing also makes detection time elongated.
In view of this, special proposition the present invention.
Summary of the invention
The object of the present invention is to provide the detection method of modified nucleoside in a kind of urine.Being subject to the shortcoming of microbial contamination for detecting sample, in detection method provided by the invention, urine being stored on filter paper, not only making its stability greatly improve, avoid the pollution of microorganism, but also there is simple and easy, advantage easily.
In order to realize above-mentioned purpose of the present invention, spy by the following technical solutions:
In urine, the detection method of modified nucleoside, comprises the steps:
(1) be stored on filter paper after urine pre-service, obtain sample to be tested by extraction afterwards;
(2) described sample to be tested is detected by LC-MS, obtain kind and the content of modified nucleoside.
In detection method provided by the invention, by urine being stored in again by extraction process on filter paper, using LC-MS method, quantitative and qualitative analysis detection can be carried out to the modified nucleoside in urine quickly and efficiently.
Owing to being stored on filter paper by urine, urine sample is converted to solid form and preserves, stability improves greatly, thus avoids microbial contamination to detect sample.And method provided by the invention is also more simpler and easy than existing freezing, convenient.
Preferably, in step (1), the step of described extraction specifically comprises: filter paper is put into methanol-water mixed solution and get supernatant as sample to be tested after ultrasonic, centrifugal.
In detection method provided by the invention, in order to the urine specimen be kept on filter paper is fully reduced, filter paper being put into methanol-water mixed solution carries out ultrasonic, each component on filter paper is fully dissolved in solution, after obtain supernatant through centrifugal again, using this supernatant as sample to be tested.
In actual testing process, can preserve the filter paper punching of urine, aperture is 3mm, and obtaining diameter is the filter paper dick of 3mm, gets appropriate filter paper dick and is placed in centrifuge tube, carry out ultrasonic, centrifugally operated after adding methanol aqueous solution.
In order to ensure that each component in filter paper can fully be dissolved in solution, in methanol aqueous solution used, the volume content of methyl alcohol is 5%-50%, and ultrasonic time is 20-90min.
After ultrasonic, not only containing modified nucleoside in solution, also containing other solid impurity, affect the accuracy of testing result, in order to be removed as much as possible by impurity, in detection method provided by the invention, control at 10000-18000rpm by centrifugal rotating speed, the time is 5-30min.
In order to ensure the stability detecting sample, in step (1), after described urine pre-service, the step be stored on filter paper specifically comprises: drip on filter paper add the mixing of 8-bromine guanosine in urine after, and air-dry more than 3 hours.
In the above-mentioned methods, the 8-bromine guanosine added as internal standard compound, in order to carry out quantitative test to modified nucleoside.
Preferably, step (2) specifically comprises:
(21) take standard items, dissolve with methyl alcohol, obtain the titer of variable concentrations;
(22) take internal standard compound, add in described titer respectively after dissolving with methanol aqueous solution, centrifugal after ultrapure water constant volume, get supernatant as titer to be measured;
(23) described sample to be tested and described titer to be measured are detected by LC-MS, obtain kind and the content of modified nucleoside.
In detection method provided by the invention, the content of internal standard method to the modified nucleoside in urine is adopted to detect.In testing process, taking standard items methyl alcohol makes it dissolve completely, obtain the titer of variable concentrations, titer to be measured is obtained through process add internal standard compound in titer after, in order to carry out LC-MS detection, carrying out linear regression with the concentration of each modified nucleoside in the comparison urine sample of the peak area of each modified nucleoside and internal standard compound peak area respectively, obtaining the linear equation of each modified nucleoside, for carrying out quantitative test to modified nucleoside each in urine.
In order to ensure the accuracy of LC-MS testing result, the present invention adopts specific liquid-phase condition and Mass Spectrometry Conditions, concrete, and in step (23), the liquid-phase condition that described LC-MS detects is: the flow velocity of mobile phase is 0.1-1.0mL/min; Sample size 1-20 μ L; Bonded-phase chromatography post; Column temperature 20-40 DEG C; A and B is as mobile phase, and wherein A is methanol aqueous solution, and B is formic acid methanol solution, and gradient elution program is as shown in table 1.
Table 1 gradient elution program
Mass Spectrometry Conditions is: adopt ESI ion gun, positive ion MRM scans, atomization gas flow velocity 8-20L/min, gas curtain gas velocity 8-20L/min, impinging air flows speed 5-15L/min, ion source voltage 2000-4000V, ion source temperature 200-400 DEG C.
After above-mentioned liquid chromatography and Mass Spectrometer Method, effectively can carry out qualitative and quantitative analysis to the modified nucleoside in urine.Further, analysis speed is fast, and completing once to analyze only needs 13 minutes.
Because modified nucleoside kind is many, in order to guarantee to be separated by the modified nucleoside in urine as much as possible, further preferably, in described liquid-phase condition, mobile phase A is 0.1-10% methanol aqueous solution, and B is 0.1-10% formic acid methanol solution.
Based on same consideration, in described liquid-phase condition, described bonded-phase chromatography post is C18/C8 chromatographic column, and specification is (50-100) mm × (1.8-3) mm, (1.8-3) μm.
Preferably; in step (21); described standard items comprise pseudouridine, cytidine, M1A, uridine, adenosine, inosine, guanosine, 1-methyl hypoxanthine nucleosides, 5-methyl-uridin, xanthosine, 3-methyluridine, M1G, 6-methyladenosine, M2G, N4-acetyl group cytidine and N2, N2-dimethylguanosine.
In detection method provided by the invention, standard items used comprise 16 kinds, can realize the qualitative and quantitative analysis of 16 kinds of modified nucleosides, and sensing range is improved.
Preferably, in step (22), described internal standard compound is 8-bromine guanosine.
In detection method provided by the invention, select 8-bromine guanosine as internal standard compound, good stability, can be used in carrying out quantitative test to the modified nucleoside in urine.
Compared with prior art, beneficial effect of the present invention is:
(1) urine is kept on filter paper by the present invention, and its stability is improved greatly, avoids microorganism to the pollution detecting sample, solves the problem that urine cannot be preserved for a long time, be transported.
(2) the present invention is by optimization to extraction process, can make the abundant stripping of each component on filter paper, can reduce the authenticity detecting sample, and the accuracy of testing result is improved greatly.
(3) the present invention is by the optimization to the liquid-phase condition in LC-MS testing process and Mass Spectrometry Conditions, and realize the qualitative and quantitative analysis to modified nucleoside in urine, highly sensitive, accuracy is high, and analysis speed is fast.
(4) in detection method provided by the invention, adopt 8-bromine guanosine as internal standard compound, adopt pseudouridine, cytidine, M1A, uridine (U), adenosine, inosine, guanosine, 1-methyl hypoxanthine nucleosides, 5-methyl-uridin, xanthosine, 3-methyluridine, M1G, 6-methyladenosine, M2G, N4-acetyl group cytidine and N2, N2-dimethylguanosine this in 16 modified nucleoside as standard items, qualitative and quantitative analysis can be carried out to kind of the modified nucleoside of 16 in urine simultaneously, meet the actual needs detected.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 is the detection spectrogram of the embodiment of the present invention.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturer suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, be and can buy by commercially available the conventional products obtained.
Embodiment
Instrument and material:
Agilent company 6495 of U.S. tandem mass spectrometer, Angilent1290 liquid chromatograph, chromatographic column is AgilentC18 post, and specification is 50mm × 3.0mm, 2.7 μm.
Medicine and reagent:
Standard items: pseudouridine, cytidine, M1A, uridine, adenosine, inosine, guanosine, 1-methyl hypoxanthine nucleosides, 5-methyl-uridin, xanthosine, 3-methyluridine, M1G, 6-methyladenosine, M2G, N4-acetyl group cytidine, N2; N2-dimethylguanosine, all buys from Sigma-Aldrich company.
Internal standard compound: 8-bromine guanosine (8-Br-G), buys from Sigma-Aldrich company.
Methyl alcohol: chromatographically pure, Merck company.
Formic acid: chromatographically pure, Merck company.
Ultrapure water: Thermo company.
Urine sample: volunteer's urine sample.
Accurately take above-mentioned 16 kinds of standard items, dissolve with methyl alcohol respectively, be mixed with the solution of 10mg/mL respectively, then be mixed into the hybrid standard product storing solution that each standard concentration is 10 μ g/mL.
Accurately take a certain amount of internal standard compound 8-bromine guanosine, dissolving also dilute with water with 50% methanol-water mixed solution (namely the volume ratio of first alcohol and water is 1:1) is the storing solution of 0.3mg/mL, then is diluted with water to the interior mark liquid that content is 0.15mg/mL.
Get hybrid standard product storing solution 5 μ L, 10 μ L, 20 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L respectively in 1.5mL centrifuge tube, and add mark liquid in 20 μ L respectively, 1mL is settled to respectively with ultrapure water, put into supercentrifuge and carry out centrifugally operated, rotating speed is set to 12000rpm, and set of time is 5min.Supernatant is got as titer to be measured after centrifugal end.
Get urine 100ul, and add the interior mark liquid of 20ul, fully mix, after mixed liquor is dropped on filter paper, air-dry more than 3 hours of room temperature.
Air-dry filter paper punches, and aperture is 3mm, obtains the filter paper dick that diameter is 3mm.Get 2 filter paper dicks and be placed in 1.5mL centrifuge tube, (namely the volume ratio of first alcohol and water is 5:95) is dissolved with 5% methanol-water, and ultrasonic 20-30 minute, put into hydro-extractor afterwards again and carry out centrifugally operated, centrifugal rotating speed controls at 12000rpm, and the time is 20min.
Supernatant is got as sample to be tested after centrifugal end.
Obtained sample to be tested and titer sample introduction to be measured are detected to LC-MS instrument.
The liquid-phase condition that described LC-MS detects is: flow velocity is 0.1-1.0mL/min; Sample size 1-20 μ L; Column temperature 20-40 DEG C; A and B is as mobile phase, wherein A is 0.1% methanol aqueous solution (namely the volume ratio of first alcohol and water is 0.1:99.9), B is 0.1% formic acid methanol solution (namely the volume ratio of formic acid and methyl alcohol is 0.1:99.9), and gradient elution program is as shown in table 1.
Mass Spectrometry Conditions is: adopt ESI ion gun, positive ion MRM scans, atomization gas flow velocity 10L/min, gas curtain gas velocity 10L/min, impinging air flows speed 8L/min, ion source voltage 2500V, ion source temperature 400 DEG C.
Its testing result as shown in Figure 1.In Fig. 1, the component of each numeral representative is:
1: pseudouridine; 2: cytidine; 3:1-methyladenosine; 4: uridine; 5: adenosine; 6: inosine; 7: guanosine; 8:1-methyl inosine; 9:5-methyluridine; 10: xanthosine; 11:3-methyluridine; 12:1-methylguanosine; 13:6-methyladenosine; 14:2-methylguanosine; 15:N4-acetyl group cytidine; 16:N2, N2-dimethylguanosine; The bromo-guanosine of 17:8-.
As can be seen from Figure 1; each component peak sequence is: pseudouridine, cytidine, M1A, uridine, adenosine, inosine, guanosine, 1-methyl hypoxanthine nucleosides, 5-methyl-uridin, xanthosine, 3-methyluridine, M1G, 6-methyladenosine, M2G, N4-acetyl group cytidine, N2, N2-dimethylguanosine, 8-bromine guanosine.
The concentration of each modified nucleoside in the comparison work titer of the peak area of each modified nucleoside detected in titer to be measured and the peak area of internal standard compound is carried out linear regression, obtains the typical curve of each modified nucleoside.Its result is as shown in table 2.
The typical curve of table 216 kind of modified nucleoside and the content in urine sample
According to the peak area of each modified nucleoside of urine to be measured and the ratio of internal standard compound peak area, substitute into the typical curve in table 1 respectively, obtain the content of each modified nucleoside in urine.
Detection method reappearance analyzes (recovery test)
Get 200 μ L samples to be tested, add the blank water of 100 μ L hybrid standard product storing solutions and same volume respectively in 1.5mL centrifuge tube, add mark liquid in 20 μ L more respectively, 1mL is settled to respectively with ultrapure water, put into supercentrifuge centrifugal 5min under the rotating speed of 12000rpm, make it fully mix.Get supernatant after centrifugal end and carry out LC-MS detection.The liquid-phase condition that LC-MS detects and Mass Spectrometry Conditions are with embodiment 1.
By the peak area of each modified nucleoside that detects and internal standard compound peak area than the working curve in substitution table 2, obtain the content of each modified nucleoside.
Repeat 3 operations, result relative standard deviation 1.98%-3.67%, sample recovery rate, at 81%-98.7%, shows the favorable reproducibility of detection method.
Detection method precision accuracy analysis (day interpolation difference analysis in the daytime)
Under the liquid chromatography mass condition that same embodiment 1 is identical, the hybrid standard product storing solution getting different volumes detects, continuous sample introduction 3 times in 1 day, in 3 days every day sample introduction once, result in a few days CV fluctuates at 1.98%-3.67%, and CV fluctuates at 3.51%-11.24% in the daytime, meets the requirements.
As can be seen from foregoing, detection method analysis speed provided by the invention is fast, and completing once to analyze only needs 13 minutes.Utilize detection method provided by the invention, qualitative and quantitative analysis can be carried out to kind of the modified nucleoside of 16 in urine faster and betterly.
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1. the detection method of modified nucleoside in urine, is characterized in that, comprise the steps:
(1) be stored on filter paper after urine pre-service, obtain sample to be tested by extraction afterwards;
(2) described sample to be tested is detected by LC-MS, obtain kind and the content of modified nucleoside.
2. detection method according to claim 1, is characterized in that, in step (1), the step of described extraction specifically comprises: filter paper is put into methanol-water mixed solution and get supernatant as sample to be tested after ultrasonic, centrifugal.
3. detection method according to claim 1, is characterized in that, step (2) specifically comprises:
(21) take standard items, dissolve with methyl alcohol, obtain the titer of variable concentrations;
(22) take internal standard compound, add in described titer respectively after dissolving with methanol aqueous solution, centrifugal after ultrapure water constant volume, get supernatant as titer to be measured;
(23) described sample to be tested and described titer to be measured are detected by LC-MS, obtain kind and the content of modified nucleoside.
4. detection method according to claim 3, is characterized in that, in step (23), the liquid-phase condition that described LC-MS detects is: flow velocity is 0.1-1.0mL/min; Sample size 1-20 μ L; Bonded-phase chromatography post; Column temperature 20-40 DEG C; A and B is as mobile phase, and wherein A is methanol aqueous solution, and B is formic acid methanol solution, and gradient elution program is 0-0.5min, A:97%, B:3%; 0.5-3min, A:97 ~ 5%, B:3 ~ 95%; 3-3.01min, A:5-97%, B:95-3%; 3.01-11min, A:97%, B:3%; 11.01min, A:97%, B:3%; 13min, A:97%, B:3%;
Mass Spectrometry Conditions is: adopt ESI ion gun, positive ion MRM scans, atomization gas flow velocity 8-20L/min, gas curtain gas velocity 8-20L/min, impinging air flows speed 5-15L/min, ion source voltage 2000-4000V, ion source temperature 200-400 DEG C.
5. detection method according to claim 3; it is characterized in that; in step (21); described standard items comprise pseudouridine, cytidine, M1A, uridine, adenosine, inosine, guanosine, 1-methyl hypoxanthine nucleosides, 5-methyl-uridin, xanthosine, 3-methyluridine, M1G, 6-methyladenosine, M2G, N4-acetyl group cytidine and N2, N2-dimethylguanosine.
6. detection method according to claim 3, is characterized in that, in step (22), described internal standard compound is 8-bromine guanosine.
7. detection method according to claim 4, is characterized in that, in described liquid-phase condition, mobile phase A is 0.1-10% methanol aqueous solution, and B is 0.1-10% formic acid methanol solution.
8. detection method according to claim 4, is characterized in that, in described liquid-phase condition, described bonded-phase chromatography post is C18 or C8 chromatographic column, and specification is (50-100) mm × (1.8-3) mm, (1.8-3) μm.
9. detection method according to claim 2, is characterized in that, in step (1), described centrifugal rotating speed is 10000-18000rpm, and the time is 5-20min.
10. the detection method according to any one of claim 1-9, it is characterized in that, in step (1), after described urine pre-service, the step be stored on filter paper specifically comprises: drip on filter paper add the mixing of 8-bromine guanosine in urine after, and air-dry more than 3 hours.
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| CN108037220A (en) * | 2018-01-26 | 2018-05-15 | 上海上药第生化药业有限公司 | A kind of separation method of nucleosides and alkali radical species and its application |
| CN108344815A (en) * | 2018-01-26 | 2018-07-31 | 上海上药第生化药业有限公司 | A kind of separation method of base substance and its application |
| CN112834644A (en) * | 2020-12-31 | 2021-05-25 | 郑州大学第一附属医院 | Bladder cancer related combined marker and detection kit |
| CN112858499A (en) * | 2020-12-31 | 2021-05-28 | 郑州大学第一附属医院 | Method for simultaneously determining modified nucleoside and creatinine in urine |
| CN115487542A (en) * | 2022-09-02 | 2022-12-20 | 浙江大学 | Solid-phase extraction method for modified nucleosides in urine and application of solid-phase extraction method |
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Cited By (5)
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| CN108037220A (en) * | 2018-01-26 | 2018-05-15 | 上海上药第生化药业有限公司 | A kind of separation method of nucleosides and alkali radical species and its application |
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| CN112834644A (en) * | 2020-12-31 | 2021-05-25 | 郑州大学第一附属医院 | Bladder cancer related combined marker and detection kit |
| CN112858499A (en) * | 2020-12-31 | 2021-05-28 | 郑州大学第一附属医院 | Method for simultaneously determining modified nucleoside and creatinine in urine |
| CN115487542A (en) * | 2022-09-02 | 2022-12-20 | 浙江大学 | Solid-phase extraction method for modified nucleosides in urine and application of solid-phase extraction method |
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| CN105548407B (en) | 2017-07-14 |
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