CN103728379B - Method for determining acquisition quality of blood sample used for science research - Google Patents
Method for determining acquisition quality of blood sample used for science research Download PDFInfo
- Publication number
- CN103728379B CN103728379B CN201210384047.3A CN201210384047A CN103728379B CN 103728379 B CN103728379 B CN 103728379B CN 201210384047 A CN201210384047 A CN 201210384047A CN 103728379 B CN103728379 B CN 103728379B
- Authority
- CN
- China
- Prior art keywords
- sample
- blood sample
- blood
- hypoxanthine
- phosphoric acid
- 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
Abstract
The invention discloses a method for determining blood sample quality in a biological sample bank by utilizing two kinds of endogenous micromolecule metabolite, and belongs to the field of analytical chemistry application research. The group of metabolism markers is applicable to determine the quality of serum (or blood plasma) samples used for various scientific researches. The detection method is based on the metabonomics technology of high performance liquid chromatography-mass spectrometry combined technology, and after serum metabonomics data is acquired, in dependent on the relative content of hypoxanthine and sphingosine-1-phosphate in different-group samples, whether the sample quality problem caused by long-term room-temperature exposure during blood sample acquisition process exists can be determined. The method provided by the invention is beneficial to improve the quality of the blood samples in scientific research application, helps to guarantee accomplishment achieved in subsequential scientific research work and avoid manpower and material resources wastes caused by sample problems. No same kind methods are reported yet at present.
Description
Technical field
The present invention relates to analytical chemistry and metabolism group application, is a kind of method being realized judging blood sample quality by detection one group of blood Small molecular metabolic markers level.
Technical background
At present, the clinical practice of new technologies is focused in the research of life science more, thus makes the general level of the health of the mankind can benefit from the develop rapidly of science and technology.In this course, the collection of clinical sample (blood, urine, tissue etc.) is regarded as a wherein crucial ring, one of most precious resource of Ye Shi various countries development in science and technology simultaneously.But, due to hospital's (sampling) and laboratory (scientific research) normally independent work, therefore for researcher, the sample quality problem brought due to human factor in sampling process likely brings unpredictable impact to experimental result, the failure of even testing, cause the wasting of resources of manpower and materials, reduce the output of research work.Therefore, for laboratory, in a kind of sample ubiquitous and be subject to acquisition quality impact endogenous mark of crucial importance for judgement sample quality.At present, not yet have reliably, easily method can realize this detection.
Metabolism group is as technology platform important in genome times afterwards comprehensively system biology field, and main what pay close attention to is the change of the quality and quantity of metabolin in biological specimen.Metabolism group is widely used in the researchs such as disease marker screening at present.Due in small molecule metabolites ubiquity and different kind organism sample, applicant finds in the research in early stage, metabolin in blood is vulnerable to long-time room temperature and exposes, the impact such as haemolysis and multigelation, and these problems are also the factors closely-related with blood sample quality of current bibliographical information, the result for the research of proteomics, metabolism group all can have an impact.Therefore, from plasma metabolism thing, find that the metabolin relevant to the condition such as sample collection, preservation becomes possibility as the mark of judgement sample quality.
Summary of the invention
The object of the invention is to one group of endogenous small molecule metabolites to judge the method for the acquisition quality of blood sample.
Applicant finds after deliberation, and in metabolism group research, the time length be exposed to during Blood specimen collection under room temperature directly has influence on the quality of blood sample, comprises the stability of blood intracellular metabolite thing, the change etc. of kind and content.When under blood and room temperature more than 4 hours time, the metabolin more than more than 10% is had to there occurs change, in bibliographical information, the collection of blood sample and preservation process also have impact to the numerous scientific research results in the current life science fields such as proteomics.And not yet have the method that can judge Blood specimen collection quality fast at present.And with the endogenous metabolism thing in blood for mark, the quality of auxiliary judgment blood sample, by the reliability of the quality and result of study that significantly improve scientific research data, has directive significance to the research work in this field.
Based on above-mentioned research, applicant proposes with two kinds of endogenous metabolism things first, and hypoxanthine and sheath amine alcohol phosphoric acid are mark, judges the method being applicable to the quality of the blood preparation of the scientific research such as proteomics, metabolism group.
The present invention discloses the method extracting the relative concentration information of endogenous small molecule metabolites mark from metabolism spectrum, and judge the standard of anomalous mass serum sample.Concrete steps are as follows,
1) metabonomic analysis of blood sample
Blood sample is Diagnostic Value of Fasting Serum blood (blood plasma or serum) 10-50 μ L before the meal in early morning, after organic solvent (acetonitrile, methyl alcohol etc.) albumen precipitation, gets supernatant 4 μ L with the analysis of liquid chromatography mass combined system.What liquid-phase chromatographic column adopted is reverse-phase chromatographic column, mass spectrophotometry can adopt quadrupole time-of-flight mass spec-trometry, the current general mass spectrums such as flight time mass spectrum and Fourier's Ion cyclotron Resonance Mass Spectrometry, mass resolution is best more than 10000, and quality testing error is within 5ppm.
2) workstation software of the extraction and application liquid chromatography mass combined system of serum blood small molecule metabolites metabolic markers extracts the peak area of Small molecular metabolic markers from raw data.Specific as follows:
Hypoxanthine: under positive ion mode, karyoplasmic ratio is the quasi-molecular ion of 137.045 ± 0.01, typical second order ms characteristic ion should comprise following one or more: 119.04,110.04,94.04,82.04,55.03.Sheath amine alcohol phosphoric acid: under positive ion mode, karyoplasmic ratio is the quasi-molecular ion of 380.255 ± 0.01, typical second order ms characteristic ion should comprise following one or more: 264.27,82.06
3) abnormal blood sample criterion: when being provided with standard control group when blood sample is sampled, the standard that arranges of this standard control group is: during sampling, the blood room temperature time is less than 1 hour, sample size >=10 in control group, three times of standard deviations are added for judgment value in control group sample average with two kinds of metabolins, the arbitrary ion of single sample exceedes this value, namely this sample can be considered exceptional sample, should get rid of in subsequent experimental.Standard deviation S(or SD) computing formula is formula (1):
When not having control group, it is judgment value that the average often can organizing two kinds of metabolins in sample adds twice standard deviation, and the arbitrary ion of single sample exceedes this value, and namely this sample can be considered exceptional sample; To have 1/3rd samples exceed mxm. in single group, Resurvey answered by this group sample.
In formula, N is Sample size χ
ifor single sample value,
for organizing interior sample average;
The present invention has the following advantages: for metabolism group research, and without the need to separately testing, above-mentioned two kinds of marks can directly from blood serum metabolic group extracting data, and method is simple.For other research, this detection method consumes sample few (minimum 10 microliters of blood), and speed is fast, according to different liquid chromatographic systems, can realize the detection of this mark the soonest in 5 minutes.
Meaning of the present invention is: China has a large amount of scientific research funds to invest clinical and relevant life science field at present every year, and the quality of biological specimen has decisive role for follow-up research work.The judgement of Blood specimen collection quality contributes to researcher and obtains reliable achievement in research, and the waste reducing human and material resources resource is significant.
Accompanying drawing explanation
Fig. 1: the relative content distribution of hypoxanthine in 50 routine samples.Controls: control group average, 2h: room temperature places 2 hours, 4h: room temperature places 4 hours 8h: room temperature places 8 hours 24h: room temperature places 24 hours.Mean+3SD: average adds three times of standard deviations (criterion line)
Fig. 2: the relative content distribution of sheath amine alcohol phosphoric acid in 50 routine samples.Controls: control group average, 2h: room temperature places 2 hours, 4h: room temperature places 4 hours 8h: room temperature places 8 hours 24h: room temperature places 24 hours.Mean+3SD: average adds three times of standard deviations (criterion line)
Embodiment
Embodiment
1 sample collection:
Normal adults (10 people) plasma sample A group is provided by German Tuebingen University Medical College.Every part of specimen sample 500 μ L, is undertaken by clinical criteria program, and after sampling, sample divides 5 groups, and often group comprises 10 routine samples.Sample packet comprises 0 hour standing time of room temperature, 2 hours, 4 hours, 8 hours and 24 hours 5 groups.
2 sample analyses
What stratographic analysis adopted is that Agilent 1200 series differentiates liquid chromatography (RapidResolution Liquid Chromatography, RRLC) fast, and that reverse-phase chromatographic column adopts is 10cm × 2.1mm 1.8 μm of ZORBAX
tMsB-AQ C
18post, column temperature remains on 35 ° of C.Mobile phase A is the water containing 0.1% formic acid, and Mobile phase B is acetonitrile.Gradient is that 2%B is initial, keeps in 4 minutes, rising to 30% after 1.5 minutes, reaches 100% immediately at 25 minutes.Keep carrying out column equilibration 5 minutes after 4 minutes.Flow is 0.35mL/min, and sampling volume is 4 μ L.That mass spectrophotometry adopts is Agilent 6510 quadrupole time-of-flight mass spec-trometry (Q-TOF MS, Agilent, USA).Mass spectrum carries out data acquisition in the positive-ion mode.Mass spectrum capillary voltage is set to 4000V, and dry gas (nitrogen) flow is 11L/min, and temperature is 350 DEG C.Atomisation pressure is set to 45psig.Fragmentor voltage and skimmer voltage are set to 230V and 65V respectively.The precision that the mixture solution (solvent is: acetonitrile: water (95:5, v/v)) of purine (0.605mg/l) and six phosphine piperazines (1.15mg/l) is used for keeping mass number to measure as correcting fluid and stability.They produce the ion that mass-to-charge ratio is 121.0508 and 922.0097 respectively in the positive-ion mode.Data acquisition range is mass-to-charge ratio 80-1000, with barycenter type collection.Acquisition rate is 500 milliseconds and often opens spectrogram.When carrying out automatic second mass analysis, collision voltage is set to 30V respectively.
3 metabolism group data are extracted
Molecular Feathers Extraction(MFE, Agilent) software in this research in order to extract the relative concentration values (peak area) of target metabolic mark in raw data.Extracting method is as follows:
Hypoxanthine: under positive ion mode, karyoplasmic ratio is the quasi-molecular ion of 137.0464, its second order ms characteristic ion comprises: 119.04,110.04,94.04,82.04,55.03.
Sheath amine alcohol phosphoric acid: under positive ion mode, karyoplasmic ratio is the quasi-molecular ion of 380.2554, and second order ms fragmention comprises: 264.27,82.06.
When metabolin extracts, mass number error range can be set to 0.0005-0.0015 dalton voluntarily according to the difference of instrument.
4 sample judged results
This experiment adopts standard sample group (0 hour group) in contrast, in this group, the average of two kinds of marks adds three times of standard deviations as the maximal value (judgment standard line) with reference to scope, all two kinds of metabolins one of them or whole relative concentrations are greater than this maximal value, and this sample can be determined as inefficacy sample.
Concrete numerical value: the hypoxanthic average of control group is: 13712.6, and standard deviation is: 4785.6; Within 2 hours, class value is: 0,55600.66,33896.04,16788.584,30228.97,36404.383,59543.754,34761.562,18031.14,45141.406; The value of 4 hours groups is: 113831.445,109004,74815.44,43566.566,60843.03,104976.695,68364.5,60329.24,34668.797,74803.234; The value of 8 hours groups is: 151869.53,220538.48,58983.773,73269.38,166564.3,123049.055,113528.55,74728.29,39727.586,105272.234; The value of 24 hours groups is: 185459.27,315184.34,317599,67743.8,43382.418,270372.7,317390.53,34709.305,48486.703,47966.36.
The average of control group sheath amine alcohol phosphoric acid is: 13712.6, and to be 9478.6,2 hours class values be standard deviation: 17161.514,66178.7,29025.361,17372.748,14705.517,17743.975,80747.28,29574.342,21397.24,15811.551; The value of 4 hours groups is 41077.14,81860.586,121224.23,17009.684,27130.152,44572.312,41262.88,89210.45,26746.09,87093.11; The value of 8 hours groups is: 29510.852,26613.918,33329.39,72667.125,73225.016,59446.664,135271.89,38471.168,48664.246,27452.441; The value of 24 hours is: 27976.729,53826.938,169145.9,59675.957,44460.133,232455.7,267194.5,68927.11,31419.387,66588.34.
As shown in Figure 1, when adding three times of standard deviations for judgment standard line with hypoxanthine in the average of control group level, had 4 routine samples in group in the room temperature preservation time in range of normal value at 2 hours, and other 30 routine samples are completely outside range of normal value.
As shown in Figure 2, when adding three times of standard deviations for judgment standard line with sheath amine alcohol phosphoric acid in the average of control group level, had 4 routine samples in group in the room temperature preservation time in range of normal value at 2 hours, and other 29 routine samples are outside range of normal value.The result of two metabolic markers is comprehensive, and only having the 2 routine room temperature preservation sample of 2 hours to be judged as does not affect by room temperature preservation.And other 38 routine sample standard deviations can be judged as the sample being subject to room temperature influence.
5 conclusions: in the clinical acquisitions process of actual sample, are usually difficult to accomplish that fresh sample is directly put into ultra low temperature freezer and preserved, and within 1-2 hour, are usually study acceptable standard.Utilize our these two kinds of metabolic markers disclosed, can be easy to realize the control to sample collection quality, when there being contrast, can 100% Quick room temperature open-assembly time the sample more than 4 hours, and the blood sample of room temperature open-assembly time more than 2 hours of 95% (2/40).When not contrasting, also can by utilizing average in this mark group and standard deviation, find out the sample that in sample, in sampling process, room temperature open-assembly time is long, thus provide reference, to ensure the reliability of follow-up study result for the rejecting of exceptional sample.
Claims (5)
1. one kind judges the method for the Blood specimen collection quality being used for scientific research, it is characterized in that: with the isolated blood of people for analytic target, adopt HPLC-MS technology, measure specific metabolic markers information in blood, realize the judgement to sample quality;
Described specific metabolic markers is 2 plasma metabolism marks, comprising: hypoxanthine (Hypoxanthine) and sheath amine alcohol phosphoric acid (Sphingosine 1-phosphate);
Described information to refer in 2 plasma metabolism marks the relative content of or two;
Deterministic process is: by hypoxanthine in the blood sample of said extracted and/or sheath amine alcohol phosphoric acid relative content, calculate its average in each group, screen according to single individual blood sample hypoxanthine or the relation between sheath amine alcohol phosphoric acid relative content value and average; Blood sample quantity in described each group is for being no less than 20 examples;
Or, abnormal blood sample criterion is: be provided with standard control group when blood sample is sampled, the standard that arranges of this standard control group is: during sampling, the blood room temperature time is less than 1 hour, sample size >=10 in control group, by extract blood sample in hypoxanthine and/or sheath amine alcohol phosphoric acid relative content, with two kinds of metabolins in control group sample average for benchmark screens; The blood sample blood sample quantity of described sampling is for being no less than 20 examples.
2. method according to claim 1, it is characterized in that: its detection method is characterized as, under the positive ion detecting pattern of liquid chromatography mass combined system, karyoplasmic ratio is 137.045 0.01 (hypoxanthine), the quasi-molecular ion of 380.255 0.01 (sheath amine alcohol phosphoric acid), its second order ms is characterized as, hypoxanthine: 119.04,110.04,94.04,82.04,55.03; Sheath amine alcohol phosphoric acid: 264.27,82.06.
3. method according to claim 1 or 2, it is characterized in that: liquid chromatography mass combined system can comprise high performance liquid chromatography or Ultra Performance Liquid Chromatography and mass spectrometry system, and mass spectrum can comprise single level Four bar mass spectrum, QQ-TOF mass spectrometry, flight time mass spectrum, level Four bar flight time mass spectrum, ion trap mass spectrometry or Ion cyclotron Resonance Mass Spectrometry.
4. method according to claim 1, is characterized in that:
Standard control group is to carry out the relative content of hypoxanthine and/or the sheath amine alcohol phosphoric acid analyzed immediately for normative reference or judgment value after blood sample sampling, or namely standard control group to be positioned over the relative content of hypoxanthine that blood sample that in refrigerator ,-70 to-80 DEG C are preserved carries out analyzing and/or sheath amine alcohol phosphoric acid in latter one hour for normative reference or judgment value to sample, with the blood sample in the sample group of room temperature open-assembly time the unknown after judging to sample, whether it is long inefficacy sample or exceptional sample room temperature standing time.
5. method according to claim 1, is characterized in that:
Described inefficacy sample or exceptional sample refer to that blood sample is in the blood sample being not suitable for continuing in follow-up scientific research to use being not suitable for temperature or room temperature length standing time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210384047.3A CN103728379B (en) | 2012-10-11 | 2012-10-11 | Method for determining acquisition quality of blood sample used for science research |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210384047.3A CN103728379B (en) | 2012-10-11 | 2012-10-11 | Method for determining acquisition quality of blood sample used for science research |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103728379A CN103728379A (en) | 2014-04-16 |
| CN103728379B true CN103728379B (en) | 2015-06-10 |
Family
ID=50452543
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210384047.3A Active CN103728379B (en) | 2012-10-11 | 2012-10-11 | Method for determining acquisition quality of blood sample used for science research |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103728379B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105760708B (en) * | 2016-01-25 | 2018-12-14 | 哈尔滨工业大学深圳研究生院 | A kind of mass spectrographic simulation generation method of metabolism mixture M S/MS and system |
| CN110361495B (en) * | 2019-08-21 | 2021-07-27 | 上海交通大学 | Method for detecting content of target metabolite in biological matrix |
| CN111239283A (en) * | 2020-02-18 | 2020-06-05 | 深圳脉图精准技术有限公司 | Application of pyroglutamic acid as quality inspection indicator for judging quality of metabonomics blood sample |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2721129A1 (en) * | 2007-04-12 | 2008-10-23 | Carolyn Marie Slupsky | Urine based detection of a disease state caused or effected by pneumococcal infection |
| US20120040383A1 (en) * | 2010-08-12 | 2012-02-16 | Wei Jia | Methods and Kits Relating To Metabolite Biomarkers For Colorectal Cancer |
-
2012
- 2012-10-11 CN CN201210384047.3A patent/CN103728379B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103728379A (en) | 2014-04-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mills et al. | Quantitative determination of trimethylamine in urine by solid-phase microextraction and gas chromatography–mass spectrometry | |
| Fr. Schröder et al. | Anabolic, doping, and lifestyle drugs, and selected metabolites in wastewater—detection, quantification, and behaviour monitored by high-resolution MS and MS n before and after sewage treatment | |
| Orfanidis et al. | Determination of drugs of abuse and pharmaceuticals in skeletal tissue by UHPLC–MS/MS | |
| Yi et al. | Tissue-specific metabolite profiling of alkaloids in Sinomenii Caulis using laser microdissection and liquid chromatography–quadrupole/time of flight-mass spectrometry | |
| CN102901790A (en) | Determination method of urine metabolic marker for early diagnosis of diabetic nephropathy. | |
| Stephanson et al. | Method validation and application of a liquid chromatography–tandem mass spectrometry method for drugs of abuse testing in exhaled breath | |
| Rodziewicz et al. | Rapid determination of chloramphenicol residues in milk powder by liquid chromatography–elektrospray ionization tandem mass spectrometry | |
| CN111208229B (en) | Screening method of serum metabolic marker for low bone density joint diagnosis of laying hens and application of serum metabolic marker | |
| CN107941980A (en) | The remaining ultra performance liquid chromatography tandem mass spectrum rapid assay methods of rifampin in aquatic products | |
| Watterson et al. | Relative distribution of ketamine and norketamine in skeletal tissues following various periods of decomposition | |
| CN103728379B (en) | Method for determining acquisition quality of blood sample used for science research | |
| Chiang et al. | Simultaneous derivatization and extraction of amphetamine and methylenedioxyamphetamine in urine with headspace liquid-phase microextraction followed by gas chromatography–mass spectrometry | |
| Fan et al. | Design and application of Hadamard-injectors coupled with gas and supercritical fluid sample collection systems in Hadamard transform-gas chromatography/mass spectrometry | |
| Kim et al. | A fit-for-purpose LC–MS/MS method for the simultaneous quantitation of ATP and 2, 3-DPG in human K2EDTA whole blood | |
| CN108693280A (en) | The method for quantitative determining the Sino-German paddy insulin content of biological sample by UPLC-MS/MS | |
| CA2906589A1 (en) | Method for determining derivatized analytes in a separated biological fluid | |
| Luo et al. | Determination of twenty herbicides in blood by ultrapressure liquid chromatography–tandem mass spectrometry | |
| CN103604875A (en) | Method for measuring serum metabolism markers in methylamphetamine abusers | |
| Zhang et al. | Determination and pharmacokinetics of gastrodin and p-hydroxybenzylalcohol after oral administration of Gastrodia elata Bl. extract in rats by high-performance liquid chromatography–electrospray ionization mass spectrometric method | |
| CN103197006A (en) | Method for determining serous metabolic biomarker of heroin abuse crowd | |
| CN106537139B (en) | Pass through mass spectrum standard measure tamoxifen and its metabolin | |
| CN111458417A (en) | Method and kit for combined detection of multiple antibiotics in sample to be detected | |
| CN105486775A (en) | Method for detecting content of various components in pills for treating kidney-yang deficiency | |
| CN103163225A (en) | High-sensitivity quantitative detection kit for cyclosporine A in whole blood and preparation method thereof | |
| CN105628835A (en) | Method for testing contents of multiple components of medicinal material inula cappa |
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 |